Use of galerina marginata genes and proteins for peptide production
09932571 ยท 2018-04-03
Assignee
Inventors
- Heather E. Hallen-Adams (Lincoln, NE)
- John S. Scott-Craig (East Lansing, MI)
- Jonathan D. Walton (East Lansing, MI)
- Hong Luo (East Lansing, MI)
Cpc classification
C12Y304/21026
CHEMISTRY; METALLURGY
C12P21/06
CHEMISTRY; METALLURGY
C07K7/64
CHEMISTRY; METALLURGY
International classification
C12P21/06
CHEMISTRY; METALLURGY
C07K7/64
CHEMISTRY; METALLURGY
Abstract
The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.
Claims
1. A cell comprising a recombinant prepropeptide nucleic acid encoding a proline-containing peptide operably linked to a promoter, and a heterologous fungal prolyl oligopeptidase nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 236, 237, 348, 716, and 722.
2. The cell of claim 1, which is a prokaryotic cell.
3. The cell of claim 1, which is a eukaryotic cell.
4. The cell of claim 1, which is an insect cell.
5. The cell of claim 1, which is a mammalian cell.
6. A bacterial, fungal, or algal organism comprising a cell comprising a recombinant prepropeptide nucleic acid encoding a proline-containing peptide operably linked to a promoter, and a heterologous fungal prolyl oligopeptidase nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 236, 237, 348, 716, and 722.
7. The organism of claim 6, which is a eukaryotic organism.
8. The organism of claim 6, which is a fungus.
9. A method of making a synthetic cyclized peptide, comprising: a) providing, i) a cell comprising a fungal prolyl oligopeptidase with an amino acid sequence selected from the group consisting of SEQ ID NOs: 236, 237, 348, 716, and 722; ii) a recombinant prepropeptide nucleic acid comprising a nucleic acid sequence encoding a proline-containing prepropeptide, and iii) amino acids for making the synthetic cyclized peptide; and b) growing the cell under conditions for expressing said prepropeptide and thereby making the synthetic cyclized peptide.
10. The method of claim 9, wherein the amino acids for making the synthetic cyclized peptide comprise D-amino acids.
11. The method of claim 9, wherein the peptide is at least six amino acids in length.
12. The method of claim 9, wherein the peptide is up to fifteen amino acids in length.
13. The method of claim 9, wherein the peptide is a bicyclic peptide.
14. A method of making a peptide from a recombinant prepropeptide sequence, comprising: (a) providing a fungal prolyl oligopeptidase comprising an amino acid sequence selected from SEQ ID NO: 236, 237, 348, 716, and 722, and a proline-containing prepropeptide; and (b) amino acids for making the synthetic cyclized peptide comprising D-amino acids; and (c) contacting the fungal prolyl oligopeptidase with the prepropeptide and the amino acids to make the peptide.
15. The method of claim 14, wherein the peptide is at least six amino acids in length.
16. The method of claim 14, wherein the peptide is up to fifteen amino acids in length.
17. The method of claim 14, wherein the peptide is a bicyclic peptide.
18. A composition comprising a proline-containing prepropeptide, amino acids for making a synthetic cyclized peptide comprising D-amino acids, and a fungal prolyl oligopeptidase comprising an amino acid sequence with in a sequence selected from amino acid SEQ ID NO: 236, 237, 348, 716, and 722, or optionally a fungal prolyl oligopeptidase comprising an amino acid sequence with a sequence selected from amino acid SEQ ID NO: 236, 237, 348, 716, and 722 with one amino acid change.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DESCRIPTION OF THE INVENTION
(44) The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.
(45) The present invention also relates to compositions and methods comprising genes and peptides associated with cyclic peptide toxins and toxin production in mushrooms. In particular, the present invention relates to using genes and proteins from Amanita species encoding Amanita peptides, specifically relating to amatoxins and phallotoxins. In a preferred embodiment, the present invention also relates to methods for detecting Amanita peptide toxin genes for identifying Amanita peptide-producing mushrooms and for diagnosing suspected cases of mushroom poisoning. Further, the present inventions relate to providing kits for diagnosing and monitoring suspected cases of mushroom poisoning in patients.
(46) The present inventions further relate to compositions and methods associated with screening a genomic library in combination with 454 pyro-sequencing for obtaining sequences of interest. In particular, the present invention relates to providing and using novel PCR primers for identifying and sequencing Amanita peptide genes, including methods comprising RACE PCR primers and degenerate primers for identifying Amanita mushroom peptides. Specifically, the present inventions relate to identifying and using sequences of interest associated with the production of small peptides, including linear peptides representing cyclic peptides, for example, compositions and methods comprising Amanita amanitin toxin sequences.
(47) The present inventions further relate to compositions and methods associated with conserved genomic regions of the present inventions, in particular those conserved regions located upstream and downstream of small peptide encoding regions of the present inventions. Specifically, degenerate PCR primers based upon these conserved regions are used to identifying Amanita peptide-producing mushrooms.
(48) Unlike genetically based disease susceptibility, every human is susceptible to lethal mushroom toxins due to the direct action of toxins, primarily amatoxins, on ubiquitous cellular organelles. Furthermore, unlike poisonous plants, poisonous mushroom species are ubiquitously found throughout the world. For example, mushrooms in the genus Amanita section Phalloideae are responsible for more than 90% of global (worldwide) fatal mushroom poisonings. Perspectively, there are an estimated 900-1000 species of Amanita wherein the majority do not produce amatoxins (or phallotoxins) of which some are actually safe for humans to eat (
(49) Expert identification opinions are necessary due to the large number of look-alike mushrooms, such as exemplary mushroom in the following Table 1. For example, the Early False Morel Gyromitra esculenta is easily confused with the true Morel Morchella esculenta, and poisonings have occurred after consumption of fresh or cooked Gyromitra. Gyromitra poisonings have also occurred after ingestion of commercially available morels contaminated with G. esculenta. The commercial sources for these fungi (which have not yet been successfully cultivated on a large scale) are field collection of wild morels by semi-professionals. Cultivated commercial mushrooms of whatever species are almost never implicated in poisoning outbreaks unless there are associated problems such as improper canning (which lead to bacterial food poisoning).
(50) TABLE-US-00001 TABLE 1 Poisonous Mushrooms and their Edible Look-A-likes.* Mushrooms Containing Amatoxins Poisonous species Appearance Mistaken for Amanita tenuifolia pure white Leucoagaricus naucina (Slender Death Angel) (Smoothcap Parasol) Amanita bisporigera pure white Amanita vaginata (Grisette), (Death Angel) Leucoagaricus naucina (Smoothcap Parasol), white Agaricus spp. (field mushrooms), Tricholoma resplendens (Shiny Cavalier) Amanita verna pure white A vaginata, L. naucina, white (Fool's Mushroom) Agaricus spp. T. resplendens Amanita virosa pure white A vaginata, L. naucina, (Destroying Angel) Agaricus spp., T. resplendens Amanita phalloides pure white Amanita citrina (False (Deathcap) variety Deathcap), A. vaginata, L. naucina, Agaricus spp., T. resplendens Buttons of pure white Buttons of white forms of A. bisporigera., Agaricus spp. Puffballs such A. verna, as Lycoperdon perlatum, etc. A. virosa Amanita phalloides green = Russula virescens (Green (Deathcap) normal Brittlegill), Amanita cap color calyptrodermia (Hooded Grisette), Amanita fulva (Tawny Grisette), Tricholoma flavovirens (Cavalier Mushroom), Tricholoma portentosum (Sooty Head) Amanita phalloides yellow Amanita caesarea (Caesar's (Deathcap) variety Mushroom) Amanita brunnescens na Amanita rubescens (Blusher), (Cleft Foot Deathcap) Amanita pantherina (Panthercap) Galerina autumnalis LBM Little Brown Mushrooms, (Autumn Skullcap) including Gymnopilus spectabilis (Big Laughing Mushroom) and other Gymnopilus spp., Armillaria mellea (Honey Mushroom) Leucoagaricus brunnea LBM Lepiota spp., Leucoagaricus (Browning Parasol) spp., Gymnopilus spp. and other Parason Mushrooms and LBM's Lepiota josserandii, LBM Lepiota spp., Leucoagaricus L. helveola, spp., Gymnopilus spp. and other L. subincarnata Parasol Mushrooms and LBM's *Na = not available
(51) Mushrooms that produce mild gastroenteritis are too numerous to list here, where exemplary examples are shown which include members of many of the most abundant genera, including Agaricus, Boletus, Lactarius, Russula, Tricholoma, Coprinus, Pluteus, and others. The Inky Cap Mushroom (Coprinus atrimentarius) is considered both edible and delicious, and only the unwary who consume alcohol after eating this mushroom need be concerned. Some other members of the genus Coprinus (Shaggy Mane, C. comatus; Glistening Inky Cap, C. micaceus, and others) and some of the larger members of the Lepiota genus such as the Parasol Mushroom (Leucocoprinus procera) do not contain coprine and do not cause this effect. The potentially deadly Sorrel Webcap Mushroom (Cortinarius orellanus) is not easily distinguished from nonpoisonous webcaps belonging to the same distinctive genus.
(52) TABLE-US-00002 TABLE 2 Mushrooms Producing Severe Gastroenteritis. Mushrooms Producing Sever Gastroenteritis Chlorophyllum molybdites Leucocoprinus rachodes (Shaggy Parasol), (Green Gill) Leucocoprinus procera (Parasol Mushroom) Entoloma lividum Tricholomopsis platyphylla (Broadgill) (Gray Pinkgill) Tricholoma pardinum Tricholoma virgatum (Silver Streaks), (Tigertop Mushroom) Tricholoma myomyces (Waxygill Cavalier) Omphalotus olearius Cantharellus spp. (Chanterelles) (Jack O'Lantern Mushroom) Paxillus involutus Distinctive, but when eaten raw or (Naked Brimcap) undercooked, will poison some people *Bad Bug Book published by the U.S. Food & Drug Administration Center for Food Safety & Applied Nutrition Foodborne Pathogenic Microorganisms and Natural Toxins Handbook website at cfsan.fda.govt/~mow/table3.html herein incorporated by reference.
(53) Individual specimens of poisonous mushrooms are characterized by individual variations in toxin content based on mushroom genetics, geographic location, and growing conditions. For example, mushroom intoxications may be more or less serious, depending not on the number of mushrooms consumed, but of the total dose of toxin delivered. In addition, although most cases of poisoning by higher plants occur in children, toxic mushrooms are consumed most often by adults. Adults who consume mushrooms are more likely to recall what was eaten and when, and are able to describe their symptoms more accurately than are children. Occasional accidental mushroom poisonings of children and pets have been reported, but adults are more likely to actively search for and consume wild mushrooms for culinary purposes.
(54) In part because of their smaller body mass, children are usually more seriously affected by normally nonlethal mushroom toxins than are adults and are more likely to suffer very serious consequences from ingestion of relatively smaller doses. Similar to the elder population and debilitated persons who are more likely to become seriously ill from all types of mushroom poisoning, even those types of toxins which are generally considered to be mild.
(55) Recently, in addition to humans, see,
(56) I. Dangers of Mushroom Poisoning.
(57) Mushroom poisoning in subjects, particularly humans, is caused by the consumption of raw or cooked fruiting bodies of toxin producing mushrooms, also known as toadstools (from the German Todesstuhl, death's stool) to distinguish toxic from nontoxic mushrooms. There is no general rule of thumb for distinguishing edible mushrooms from toxic mushrooms (poisonous toadstools). There are generally no easily recognizable differences between poisonous and nonpoisonous species to individuals who are not experts in mushroom identification (mycologists).
(58) Toxins involved in and responsible for mushroom poisoning are produced naturally by the fungi, with each individual specimen within a toxic species considered equally poisonous. Most mushrooms that cause human poisoning cannot be made nontoxic by cooking, canning, freezing, or any other means of processing. Thus, the only way to completely avoid poisoning is to avoid consumption of the toxic species. Mushroom poisonings are almost always caused by ingestion of wild mushrooms that have been collected by nonspecialists (although specialists have also been poisoned). Most cases occur when toxic species are confused with edible species, and a useful question to ask of the victims or their mushroom-picking benefactors is the identity of the mushroom they thought they were picking. In the absence of a well-preserved specimen, the answer to this question could narrow the possible suspects considerably. Poisoning has also occurred when reliance was placed on some folk method of distinguishing poisonous and safe species. Outbreaks have occurred after ingestion of fresh, raw mushrooms, stir-fried mushrooms, home-canned mushrooms, mushrooms cooked in tomato sauce (which rendered the sauce itself toxic, even when no mushrooms were consumed), and mushrooms that were blanched and frozen at home. Cases of poisoning by home-canned and frozen mushrooms are especially insidious because a single outbreak may easily become a multiple outbreak when the preserved toadstools are carried to another location and consumed at another time.
(59) Poisonings in the United States occur most commonly when hunters of wild mushrooms (especially novices) misidentify and consume a toxic species, when recent immigrants collect and consume a poisonous American species that closely resembles an edible wild mushroom from their native land, or when mushrooms that contain psychoactive compounds are intentionally consumed by persons who desire these effects.
(60) A. Symptoms of Poisoning.
(61) Mushroom poisonings are generally acute and are manifested by a variety of symptoms and prognoses, depending on the amount and species consumed. Because the chemistry of many of the mushroom toxins (especially the less deadly ones) is unknown and positive identification of the mushrooms is often difficult or impossible, mushroom poisonings are generally categorized by their physiological effects. There are four categories of mushroom toxins: protoplasmic poisons (poisons that result in generalized destruction of cells, followed by organ failure); neurotoxins (compounds that cause neurological symptoms such as profuse sweating, coma, convulsions, hallucinations, excitement, depression, spastic colon); gastrointestinal irritants (compounds that produce rapid, transient nausea, vomiting, abdominal cramping, and diarrhea); and disulfuram-like toxins. Mushrooms in this last category are generally nontoxic and produce no symptoms unless alcohol is consumed within 72 hours after eating them, in which case a short-lived acute toxic syndrome is produced.
(62) In one embodiment, the inventors provide herein compositions and methods for providing molecular biology based diagnostic tests for accurately and reproducibly identifying DNA sequences encoding lethal fungal toxins. Thus accurate identification of mushroom toxins may be made from samples of uneaten mushrooms, including raw, cooked, frozen, dried, samples, and patient samples of undigested and partially digested, as in gastric contents, such as from human and dogs.
(63) For comparison, current methods for diagnosing mushroom poisonings are briefly described below.
(64) B. Current Diagnostic Methods.
(65) Symptoms of potentially toxic mushroom poisoning may mimic other types of diseases, such as abnormal conditions or ingestion of other types of toxins which would trigger different and likely less drastric treatments. Exemplary differentials include, Adrenal Insufficiency and Adrenal Crisis, Alcohol and Substance Abuse Evaluation, Anorexia Nervosa, Delirium Tremens, Gastroenteritis, Hepatitis, Methemoglobinemia, Pediatrics, Dehydration, Pediatrics, Gastroenteritis, Salmonella Infection, Toxicity. Anticholinergic, Toxicity, Antihistamine, Disulfuram, Disulfuramlike Toxins, Gyromitra, Mushroom Hallucinogens, Mushroom-Orellanine, Organophosphate, and Carbamate, Theophylline, etc. In addition, an idiosyncratic reaction mimics toxin poisoning when patients with trehalase deficiency who are unable to break down trehalose, a disaccharide found in mushrooms present with diarrhea after ingestion. Further patients with an immune reaction (Paxillus syndrome) may develop an acquired hypersensitivity-type reaction after repeated ingestions of specific mushrooms. This may result in hemolytic crisis and most commonly involves ingestion of Paxillus involutus. Suillus luteus also has been implicated in a psychosomatic syndrome where some patients were reported to develop anxiety-related symptoms after learning that they ate wild mushrooms. Mushroom-drug interaction-symptoms may occur with ingestion of mushrooms contaminated with bacteria, sprayed with pesticides, or supplemented with drugs such as phencyclidine. Thus, in one embodiment, genes and proteins of the present inventions may find use in identifying the presence or lack of toxin producing mushrooms, i.e. their genes related to toxin production, for example using PCR primers for amplifying genes, peptides related to toxins, for example, using antibodies which recognize toxins, and kits comprising PCR primers or antibodies.
(66) As described above, the protoplasmic poisons are the most likely to be fatal or to cause irreversible organ damage. In the case of poisoning by the deadly species of Amanita and other mushrooms that produce the Amanita peptides, important laboratory indicators of liver (elevated LDH, SGOT, and bilirubin levels) and kidney (elevated uric acid, creatinine, and BUN levels) damage will be present.
(67) Unfortunately, in the absence of dietary history, these signs could be mistaken for symptoms of liver or kidney impairment as the result of other causes (e.g., viral hepatitis). It is important that this distinction be made as quickly as possible, because the delayed onset of symptoms will generally mean that the organ has already been damaged. The importance of rapid diagnosis is obvious: victims who are hospitalized and given aggressive support therapy almost immediately after ingestion have a mortality rate of only 10%, whereas those admitted 60 or more hours after ingestion have a 50-90% mortality rate.
(68) 1. Intact Mushrooms.
(69) Ideally, once a mushroom poisoning is suspected, identification of suspect toxic mushroom, identical to the one ingested, should be made by a local medical toxicologist (certified through the American Board of Medical Toxicology or the American Board of Emergency Medicine) or at a regional poison control center.
(70) If a pre-digested mushroom sample is available, the following information would be helpful to a mycologist or physician with mushroom poisoning experience for determining the mushroom's identity: Provide any available information, for example, size, shape, and color of the mushroom including a description of the surface and the underside of the cap, the stem, gills, veil, ring, spores and the color and texture of the flesh. It would be helpful to know the location and conditions in which the mushroom grew (e.g., wood, soil). Further, it is suggested that any mushroom samples saved for mycological examination are wrapped in foil or wax paper and stored in a paper bag in a cool dry place, pending transport to the mycologist or other professional. Moreover it is discouraged to store mushroom samples for mycological identification in a plastic bag or container where the mushroom's features may be altered due to moisture condensation and further freezing which is likely to alter or destroy any distinguishing identification features of the mushroom. Alternative methods for identifying mushrooms may be done by referring to the Poisindex or a mycology handbook.
(71) Currently there are several research laboratory tests used for identifying Amanita peptides and toxins, examples of which are briefly described as follows. The Meixner test also known as the Weiland Test assay is qualitative assay used to detect amatoxins (eg, alpha-amanitin, beta-amanitin) in the mushroom. It is not recommended for use with stomach contents nor to determine edibility of a mushroom because false-positive and false-negative results have been described. Kuo, M. (2004, November). Meixner test for amatoxins. Retrieved from the MushroomExpert.Com Web site: mushroomexpert.com/meixner; herein incorporated by reference).
(72) Further, an intact or partial undigested mushroom may be analyzed for actual toxic peptides, using chemical methods such as reverse-phase HPLC. In order to rule out other types of food poisoning and to conclude that the mushrooms eaten were the cause of the poisoning, it must be established that everyone who ate the suspect mushrooms became ill and that no one who did not eat the mushrooms became ill. Wild mushrooms eaten raw, cooked, or processed should always be regarded as prime suspects. After ruling out other sources of food poisoning and positively implicating mushrooms as the cause of the illness, further diagnosis is necessary to provide an early indication of the seriousness of the disease and its prognosis.
(73) Therefore, an initial diagnosis is based entirely on symptomology and recent dietary history. Despite the fact that cases of mushroom poisoning may be broken down into a relatively small number of categories based on symptomatology, positive taxonomic identification of the mushroom species consumed remains the only means of unequivocally determining the particular type of poisoning involved, and it is still vitally important to obtain such accurate identification as quickly as possible. Cases involving ingestion of more than one toxic species in which one set of symptoms masks or mimics another set are among many reasons for needing this information.
(74) 2. Post-Ingested and Pre-Digested Mushroom Samples.
(75) If the actual mushroom is unavailable, which is frequent in post-ingestion cases with delayed onset of symptoms, the following information may be helpful for determining the mushroom's identity. Save emesis or gastric lavage fluid for microscopic examination for spores. If mushroom fragments are available, they can be stored in a 70% solution of ethyl alcohol, methanol, or formaldehyde and placed in the refrigerator. Otherwise, emesis can be centrifuged and the heavier layer on the bottom can be examined under a microscope for the presence of spores.
(76) Despite the availability of laboratory tests for identifying toxins, diagnosing a mushroom poisoning remains primarily limited to taxonomic identification of the mushroom that was eaten. Accurate post-ingestion analyses for specific toxins when no taxonomic identification is possible is essential for cases of suspected poisoning by toxin containing mushrooms, such as species of Amanita, since prompt and aggressive therapy (including lavage, activated charcoal, and plasmapheresis) can greatly reduce the mortality rate.
(77) Samples of actual mushroom toxins may be recovered from poisonous fungi, cooking water of poisonous fungi, stomach contents with poisonous fungi, serum, and urine from poisoned patients. Procedures for extraction and quantitation of toxins are generally elaborate and time-consuming. In the case of using toxin based diagnostic procedures the patient will in most cases either have recovered or died by the time an analysis is made on the basis of toxin chemistry. However even with toxin chemistry, the exact chemical natures of many toxins, including toxins that produce milder symptoms are unknown. Lethal toxins are identified using chromatographic techniques (TLC, GLC, HPLC) for amanitins, orellanine, muscimol/ibotenic acid, psilocybin, muscarine, and the gyromitrins. Recently, amanitins were determined by commercially available .sup.3H-RIA kits. Amanitin ELA Kit from Alpco Diagnostics of American Laboratory Products Company PO Box 451 Windham, N.H. 03087 Sample Type Urine, Serum, Plasma .alpha.- and .gamma.-amanitin present in human urine, serum and plasma. A polyclonal antibody (Ab) specific for alpha- and gamma-Amanitin Diagnostic Accuracy of Urinary Amanitin in Suspected Mushroom Poisoning: A Pilot Study Butera et al., Clinical Toxicology, Volume 42, Issue 6 December 2004, pages 901-912; herein incorporated by reference).
(78) II. Mushroom Toxins.
(79) A large variety of toxins are produced by mushrooms, including amatoxins, phallotoxins, virotoxins, phallolysins, ibotenic acid/muscimol, alkaloids, cyclopeptides, coumarins, etc. Many of these compounds are active at extremely low concentrations and have a rapid effect including death. Milder toxins such as ibotenic acid and muscimol bind to glutamic acid and GABA receptors, respectively, and thereby interfere with CNS receptors.
(80) Amatoxins, phallotoxins, and virotoxins are found in A. bisporigera, A. ocreata, A. phalloides, A. phalloides var. alba, A. suballiacea, A. tenuifolia, A. virosa, and some other mushrooms. The phallolysins are a recently discovered group of toxins as yet observed only in A. phalloides. Many of the cyclic and noncyclic peptides found in Amanita and other toxin producing genera are toxic to humans and other mammals, ranging from mild symptoms to death.
(81) A. Amanitin Peptide Toxins.
(82) Several mushroom species, including the Death Cap or Destroying Angel (Amanita phalloides, A. virosa), the Fool's Mushroom (A. verna) and several of their relatives, along with the Autumn Skullcap (Galerina marginata, formerly called Galerina autumnalis) and some of its relatives, produce a family of cyclic octapeptides called amanitins. Because of taxonomic revisions, amanatin-producing fungi with different names might actually be the same species. Galerina marginata=G. autumnalis=G. venenata=G. unicolor (G. beinrothii, G. sulciceps, G. fasciculata, G. helvolicepsmay all actually be the same species as G. marginata). Amanitins are lethal toxins. A human LD.sub.50 for -amanitin is approximately 0.1 mg/kg (see,
(83) Amatoxins are a member of a family of related molecules of which at least 9 members are known. Alpha-amanitin is one of the principal amatoxins, comprising approximately 50% of the amatoxin content of some amatoxin-producing mushrooms. Beta-amanitin and gamma-amanitin) are toxic in addition to other types of amatoxins, including but not limited to epsilon-Amanitin, Amanin, Amanin amide, Amanullin, Amanullinic acid, and Proamanullin. Members of this toxin family differ in whether they have asparagine (the position 1 amino acid) or aspartic acid, and in the degree of hydroxylation of the position 3 isoleucine and the tryptophan, and at the Cys-Trp cross-bridge.
(84) Amatoxins can be responsible for fatal human poisonings. After ingestion, amatoxins are taken up by the liver where they begin to cause damage. They are then secreted by the bile into the blood where they are taken up by the liver again, causing a cycle of damage and excretion. In the liver, amatoxins inhibit RNA-polymerase II. The liver is slowly destroyed and is unable to repair itself due to the inactivation of the RNA-polymerase. Thus, the liver slowly dissolves with no hope of repair. Thus, one of the few effective treatments is liver transplantation (Enjalbert et al., (2002) (Treatment of Amatoxin Poisoning: 20-Year Retrospective Analysis, review of poisonings) J. Toxicol. Clin. Toxicol. 40:715; Fabrizio, et al., (2006) Transplant International 19(4):344-345; all of which are herein incorporated by reference).
(85) Poisoning by amanitins is clinically characterized by a long latent period (range 6-48 hours, average 6-15 hours) during which the patient shows few or no symptoms. Symptoms appear at the end of the latent period in the form of sudden, severe seizures of abdominal pain, persistent vomiting and watery diarrhea, extreme thirst, and lack of urine production which lasts for about 24 hours. If this early phase is survived, the patient may appear to recover for a short time, 2-3 days, during which liver damage is ongoing. This second latent period will generally be followed by a rapid and severe loss of strength, prostration, and pain-caused restlessness. During the last stages, hepatic and renal damage becomes clinically evident typically resulting in a coma. Death usually follows a period of comatose condition and occasionally is accompanied by convulsions. If recovery occurs, it generally requires at least a month and is accompanied by enlargement of the liver. Autopsy will usually reveal fatty degeneration and necrosis of the liver and kidneys.
(86) Amatoxins are particularly deadly because they are taken up by cells lining the gut where protein synthesis is immediately inhibited. The toxins are then released into the blood stream and transported to the liver. Once inside the liver cells, amatoxins inhibit RNA-polymerase II, which slows or stops new protein production which begins to cause cellular damage. Bushnell et al., (2002) Proc. Natl. Acad. Sci. USA 99:1218; Kroncke et al., (1986) J. Biol. Chem., 261:12562; Letschert et al., (2006) Toxicol Sci. 91:140; Lindell et al., (1970) Science 170:447; all of which are herein incorporated by reference). The liver secretes excess toxins into bile and into the blood stream where they are taken up by the liver again, causing a cycle of damage and excretion. Thus the liver is slowly destroyed and is unable to repair itself Amanitin toxins are excreted in the urine and evacuated from the body within hours of ingestion. However, if sufficient liver tissue is affected, liver failure will ensure death.
(87) In 50-90% of the cases, death occurs from progressive and irreversible liver, kidney, cardiac, and skeletal muscle damage. The course from ingestion to death may occur in 48 hours (large dose), but effects typically lasts 6 to 8 days in adults and 4 to 6 days in children.
(88) A dose that is likely to kill an average adult human is in the range of 6-7 mg, easily found in the cap of one mature A. phalloides. However, like other fungal toxins, the concentration which is fatal for individuals differs and relates to the concentration in different specimens and environment influences on concentration of toxin produced in one basidiocarp. These examples clearly show that any fungus collected from the field should be properly identified before it is consumed.
(89) B. Phallotoxins.
(90) In addition to bicyclic octapeptide amatoxins, mushrooms naturally produce several bicyclic heptapeptides. In particular, members of Amanita sect. Phalloideae produce bicyclic heptapeptides specifically called phallotoxins (
(91) Phallotoxins, such as phalloidin and phallacidin, are poisonous when administered parenterally, for example, when administered in a manner other than through the digestive tract, such as by inhalation, intravenous or intramuscular injection. However, because they do not appear to be absorbed by the mammalian digestive tract, they are unlikely to play a primary role in clinical mushroom poisonings.
(92) Biochemically, there are at least seven different naturally occurring phallotoxins: phalloin, phalloidin, phallisin, prophalloin, phallacin, phallacidin, and phallisacin. There are two groups of phallotoxins, neutral and acidic. The neutral phallotoxins, such as phalloidin, contain D-threonine, while the acidic ones contain D-beta-hydroxy-Aspartic acid. Phallacidin (AWLVDCP (SEQ ID NO:69)) also includes Valine whereas phalloidin contains Alanine.
(93) Phallotoxin was once thought to be responsible for the usual symptoms of fatal mushroom poisoning. The compound acts to inhibit F actin in the cell cytoskeleton. It acts immediately, and probably does not move beyond the lining of the gut.
(94) C. Virotoxins.
(95) Although they have the same toxicological effects as and appear to be derived from the phallotoxins, the virotoxins are monocyclic heptapeptides, not bicyclic peptides.
(96) There are at least six virotoxins, viroidin desoxoviroidin, alal-viroidin, alal-desoxoviroidin, viroisin, and desoxoviroisin.
(97) D. Other Types of Mushroom Toxins.
(98) Phallolysins There are at least three phallolysins that are hemolytically active proteins, but, as previously stated, they are heat and acid labile and do not pose a threat to humans.
(99) Ibotenic acid/Muscimol. Ibotenic acid is an Excitatory Amino Acid (EAA) and muscimol is its derivative. These toxins act by mimicking the natural transmitters glutamic acid and aspartic acid on neurons in the central nervous system with specialized receptors for amino acids. These toxins may also cause selective death of neurons sensitive to EAAs. However these are not known to be peptides.
(100) III. Amanita Toxin Peptides in Relation to Other Peptides.
(101) Small, modified, and biologically active peptides synthesized on ribosomes were previously identified from several sources, including bacteria, spiders, snakes, cone snails, and amphibian skin (Escoubas, 2006; Olivera, 2006; Simmaco et al., 1998). Like the Amanita peptide toxins, these peptides are synthesized as precursor proteins and often undergo post-translational modifications, including hydroxylation and epimerization. Circular proteins were discovered in microorganisms, plants and mammals, (for an exemplary review, see, Trabi and Craik, 2002).
(102) Lantibiotics. Lantibiotics, such as nisin, subtilin, and cinnamycin, are produced by species of Lactobacillus, Streptococcus, and other bacteria. They contain 19-38 amino acids. They are characterized by the presence of lanthionine, which is formed biosynthetically by dehydration of an Ala residue followed by intramolecular addition of Cys (Willey and van der Donk, 2007). The lantibiotics are similar to the Amanita peptide toxins in containing a modified, cross-linked Cys residue. However, instead of Ala in the case of lantibiotics, the Cys in the Amanita peptides is cross-linked to a Trp residue. Furthermore, thorough BLAST searching of the genome of Amanita and of all other fungi whose genomes have been sequenced (available in GenBank NR or the DOE Joint Genome Institute) did not identify any orthologs of any of the known lantibiotic dehydratases or cyclases (Willey and van der Donk, 2007).
(103) Cone snail toxins. Cone snail toxins (conotoxins) are 12-40 amino acids. They are linear peptides but are cyclized by multiple disulfide bonds (Bulaj et al., 2003). Like the Amanita peptides, the cone snail toxins exist as gene families, the members of which have hypervariable regions, corresponding to the amino acids present in the mature toxins, and conserved regions found in all members (Olivera, 2006; Woodward et al., 1990, all of which are herein incorporated by reference).
(104) Conotoxins and Amanita peptides differ in many key respects. First, the Amanita peptides are smaller (7-10 amino acids vs. 12-40 for the conotoxins) (Bulaj et al., 2003). Second, the mature conotoxins are at the carboxy termini of the preproproteins and are predicted to be cleaved by a protease that cuts at basic amino acids (Arg or Lys). In contrast, the mature Amanita peptide toxin sequences are internal to the proprotein and are predicted to require two cleavages by one or more prolyl peptidases. Third, the conotoxins are cyclized only by multiple disulfide bonds, whereas the Amanita peptides are cyclized by N-terminus to C-terminus (head-to-tail) peptide bonds and do not have disulfide bonds. Fourth, the conotoxin preproproteins have signal peptides to direct secretion into the venom duct, whereas the Amanita peptides are not secreted (Zhang et al., 2005, herein incorporated by reference) and their proproteins lack predicted signal peptides (
(105) Amphibian, snake, and spider toxins. Like the conotoxins, these peptides are synthesized on ribosomes as preproproteins, undergo posttranslational modifications, and contain multiple disulfide bonds. None of them are truly cyclic nor and all are much bigger than the Amanita peptide toxins.
(106) Cyclotides. Cyclotides such as kalata are 28-37 amino acids in size (Trabi and Craik, 2002; Craik et al., 2007, all of which are herein incorporated by reference). The precursor structure contains an N-terminal signal peptide followed by a proprotein region and a conserved N-terminal repeat region containing a highly conserved domain of 20 amino acids, one to three cyclotide domains, and a short C-terminal sequence. An Asn-endopeptidase is responsible for removing the C-terminal peptide from the proprotein and cyclizing the peptide (Saska et al., 2007), but the protease that cuts the N-terminus is apparently not known. The mature cyclotides are true head-to-tail cyclic peptides but, like the conotoxins, also have multiple disulfide bonds.
(107) Bacterial auto-inducing peptides (AIPs). Quorum sensing by certain pathogenic Gram-positive bacteria, such as species of Staphylococcus, involves the secretion and recognition of small (7-9 amino acid) ribosomally-encoded peptides called AIPs (Novicku and Geisinger, 2008). AIPs are posttranslationally cyclized by formation of a thiolactone between the carboxyl group of the C-terminal amino acid and an internal Cys. AIP proproteins are processed at the C-terminus by agrB with simultaneous condensation to form the thiolactone ring (Lyon and Novick, 2004). The inventors determined that there are no proteins related to agrB in the genomes of Amanita, Galerina, or any fungus in GenBank.
(108) Microcin and related molecules. Microcin J25 is a 21-amino acid peptide cyclized between an N-terminal Gly or Cys residue and an internal Glu or Asp residue. It is produced by E. coli; other enterobacteria produce related peptides. Processing of the primary translation product (58 amino acids) involves cleavage of a 37-residue leader peptide and cyclization. Cyclization requires two genes, mcjA and mcjB, which are part of the microcin operon (Duquesne et al., 2007). The maturation reaction requires ATP for amide bond formation. The inventors did not find any orthologs of mcjA or mcjB by BLAST searching of all available fungal genomes, including Amanita bisporigera and Galerina marginata.
(109) Another example of cycle peptides are thiazolyl peptides, highly rigid trimacrocyclic compounds consisting of varying but large numbers of thiazole rings. The backbone amino acids undergo numerous posttranslational modifications while thiazolyl peptide genes are clustered into operons in bacteria. Derivatives of thiazolyl peptides are sometimes used as antibiotics. Because thiazolyl peptides were synthesized on ribosomes by bacteria such as Streptomyces and Bacillus, the inventors' searched for homologous genes. No homologs of any of the thiazolyl peptide genes were found in the genomes of A. bisporigera, G. marginata, or other fungi in GenBank.
(110) In conclusion, comparison of the Amanita peptide toxins to other known small cyclic peptides indicates that they are unique among microbial natural products in regard to their chemistry, modes of action, and biosynthesis.
(111) A summary of several unique characteristics of Amanita peptide toxins and peptides, linear and cyclic, includes but is not limited to: (1) The Amanita peptide toxins are true head-to-tail cyclic peptides, unlike antibiotics, cone snail toxins, microcins, or AIPs. (2) The tryptathionine moiety (Trp-Cys cross-bridge) is not found in any other natural molecule (May and Perrin, 2007, herein incorporated by reference). (3) The Amanita toxins are the only known ribosomally synthesized cyclic peptides from the Kingdom Mycota (Fungi), the source of many important secondary metabolites that affect human health. (4) The known Amanita peptide toxins have unique modes of action, which contributes to their toxicity and also makes them widely used tools for basic biomedical research. The interaction of alpha-amanitin with pol II is understood in detail (Bushnell et al., 2002, herein incorporated by reference). It is therefore possible that other linear or cyclic ribosomally-synthesized peptides known or predicted to be made by species of Amanita, Galerina, Lepiota, Conocybe, etc. (for example, see, might also have biologically significant modes of action that would make them useful as pharmaceutical agents or research reagents. (5) Amatoxins are not secreted (Zhang et al., 2005, herein incorporated by reference). Consistent with this the proproteins do not have predicted signal peptides. In this regard they differ from conotoxins, lantibiotics, snake and spider venoms, amphibian peptides, or microcins. (6) The Amanita peptide toxins are among the smallest known ribosomally synthesized peptides. Their proproteins (34 and 35 amino acids) are also very small by the standards of typical ribosomally synthesized proteins. (7) No other known peptides are predicted to be processed from their proproteins by a Pro-specific peptidase, and (8) Galerina marginata has advantages over other eukaryotic synthesizers of small peptides. Snakes, amphibians, cone snails, and spiders are difficult to obtain or cultivate and their peptide toxins are made only in small venom ducts.
(112) As described herein the inventors discovered the presence of conserved and hypervariable regions in genes encoding small peptide mushroom toxins. After the inventors compared the Amanita peptide toxin genes of the present inventions to known conotoxin genes they discovered that genomic sequences of both organisms are characterized by the presence of conserved and hypervariable regions, however with notable significant differences in the size and structure of the coding regions. Cone snails appear to have the capacity to synthesize a large number of peptides on the same fundamental biosynthetic scaffold (Richter et al., (1990) Proc. Nat. Acad. Sci. USA 87:4836; Woodward et al. (1990), EMBO J. 9:1015; all of which are herein incorporated by reference). However, in contrast to the conotoxins (Olivera, (2006) J. Biol. Chem. 281:31173; herein incorporated by reference), the Amanita peptide toxin genes encode smaller peptides from shorter regions of conserved and hypervariable regions in addition to showing other significant differences, Benjamin, Denis R. 1995. Mushrooms. Poisons And Panaceas. (W.H. Freeman, New York). xxvi+422 pp; herein incorporated by reference).
(113) IV. Contemplated Role of Prolyl Oligopeptidase Family (POP) in Mushroom Peptide Toxin Production.
(114) Prolyl oligopeptidase family (POPs) from other organisms are known to cleave several classes of Pro-containing peptides including mammalian hormones such as vasopressin (Brandt et al., 2007; Cunningham and O'Connor, 1997; Garcia-Horsman et al., 2007; Polgar, 2002; Shan et al., 2005, all of which are incorporated by reference). Changes in human blood serum levels of POP have been associated with depression, mania, schizophrenia, and response to lithium (Williams, 2005, herein incorporated by reference). A POP inhibitor reverses scopolamine-induced amnesia in rats (Brandt et al., 2007, herein incorporated by reference). Mutation of a POP gene in Drosophila melanogaster results in resistance to lithium (Williams et al., 1999, herein incorporated by reference). POPs have been proposed as a treatment for celiac-sprue disease, which is caused by failure to properly digest Pro-rich peptides in gluten (Shan et al., 2002, 2005, all of which are herein incorporated by reference). Despite the demonstration that POP will cleave many small peptides, such as mammalian hormones, apparently the native, endogenous substrates of POPs are not definitively known in any biological system (Brandt et al., 2007, herein incorporated by reference).
(115) The Amanita peptide toxin system is contemplated to represent the first time a native substrate of a POP was identified, as shown during the development of the present inventions (see below and
(116) The inventors further identified sequences distantly related to human POP (GenBank accession no. NP002717) (SEQ ID NO:150) in the genome survey sequences of A. bisporigera. Orthologs of human POP (POP-like genes) were also found in every other basidiomycete for which whole genome sequences were available, for example, a POP-like gene was characterized from the mushroom Lyophyllum cinerascens. In contrast, orthologs of human POP are rare or nonexistent in fungi outside of the basidiomycetes. Thus, it appeared that at least one component of the biochemical machinery necessary for the biosynthesis of the Amanita toxins is both widespread in, and restricted to, the basidiomycetes.
(117) V. Genomic Structure of Amanita Peptide Encoding Genes of the Present Inventions.
(118) The inventors discovered the genes encoding the Amanita peptide toxins and the translated peptides relating to Amanita peptide toxins during the development of the present inventions. In particular, the inventors discovered a genomic structure of Amanita peptide toxins, AMA1 and PHA1, relating to amatoxin and phallotoxin toxins. Both types of peptides comprise a conserved stretch (A) of about 9 homologous amino acids, followed by a hypervariable region of 6 to 10 amino acids that are specific for either the two types of toxin peptides, a-amanitin or phallacidin, in addition to longer peptides. These hypervariable regions were followed by an additional conserved stretch (B) of approximately 17 homologous amino acids. The inventors contemplate that the coding sequences of the toxins are part of a larger preproprotein, of approximately 35 amino acids, that is translated and then undergoes post-translational processing to release the active peptide, similar to processing mechanisms of neuropeptides and other small peptide toxins (e.g., conotoxins).
(119) The genome of A. bisporigera contains at least 30 copies of genes coding for the first highly conserved stretch of amino acids (A), followed by a hypervariable region (P), then the second conserved region (B). The primary sequences derived from the cDNA encode peptides AWLVDCP (SEQ ID NO: 69) and IWGIGCNP (SEQ ID NO: 50), which are contemplated to be capable of cyclization into phallacidin and alpha or gamma amanitin, respectively. Neither of these peptides were found after searching the entire GenBank NR database. Therefore, by statistical coincidence they are unlikely to be present in A. bisoporigera; however, experimental results shown herein demonstrate that nucleic acid sequences are present that may encode these peptides.
(120) The Amanita peptide toxins differ from the other known naturally occurring small peptides in several ways. First, the animal peptides are not cyclized by peptide bonds known to be present in Amanita peptide toxins but acquire their essential rigidity by extensive disulfide bonds. Ribosomally synthesized cyclic peptides are known from bacteria, plants, and animals, e.g., the cyclotides and microcin J25 (Craik, (2006) Science 311:1563, Rosengren, et al., (2003), J. Am. Chem. Soc. 125:12464; all of which are herein incorporated by reference), but to the best of the inventor's knowledge all other fungal cyclic peptides are synthesized by nonribosomal peptide synthetases (Walton, et al., (2004) in Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine, J. S. Tkacz, L. Lange, Eds. (Kluwer Academic/Plenum, N.Y., pp. 127-162; Finking, et al., (2004) Annu. Rev. Micro biol. 58:453; all of which are herein incorporated by reference). Second, the Amanita peptide toxins are not secreted, and consistent with this they lack predicted signal peptides in their sequences (
(121) Sequencing of the genome of A. bisporigera to 20coverage should also yield all of the other members of the Amanita peptide toxin family, which is characterized by MSDIN as the first five amino acids of the predicted proproteins. Furthermore, other species of Amanita that make Amanita peptide toxins, such as A. phalloides and A. ocreata, should yield more members of this family. Furthermore, sequencing of additional specimens of these species of Amanita should yield more members. The inventors calculate that there are >30 MSDIN sequences in one isolate of A. bisporigera alone.
(122) Further, the inventors contemplate that genes for Amanita peptide toxin biosynthesis will be clustered within the Amanita genome. As shown herein, an example of genomic organization of PHA1 (for phallacidin) genes in relation to adjacent genes encoding potential enzymes.
(123) VI. Contemplated Role of P450 Homologs in Mushroom Peptide Toxin Production.
(124) Many of the Amanita peptide toxins are hydroxylated at isoleucine, tryptophan, proline, and/or aspartic acid. Hydroxylation of the Amanita peptide toxins might be catalyzed by cytochrome P450 monooxygenases, which are known to catalyze hydroxylation of many other fungal secondary metabolites (e.g., Malonek et al., 2005; Tudzynski et al., 2003). Filamentous fungi differ widely in their numbers of P450's. Whereas some filamentous fungi have >100, the Basidiomycete Ustilago maydis has only about 17 (drnelson.utmem.edu/CytochromeP450.html). The inventors found three P450 genes clustered with two copies of PHA1 (
(125) In terms of identifying new P450 genes contemplated to be involved in Amanita peptide toxin biosynthesis, three candidates in the three P450's were found on a lambda clone clustered with two copies of PHA1 (
(126) VII. Galerina Mushrooms for Use in the Present Inventions.
(127) Further, the present invention relates to using genes and proteins from Galerina species encoding mushroom peptide toxins, specifically amatoxins. Galerina sequences and Galerina mushrooms are particularly contemplated for use in the present inventions because Galerina, unlike Amanita, is a culturable fungus that produces amanitins in the laboratory. Amatoxins are induced in cultured Galerina, by several methods, for example, Benedict R G, V E Tyler Jr., L R Brady, L J Weber (1966) Fermentative production of amanita toxins by a strain of Galerina marginata. J Bacteriol 91:1380-1381; and preferably using methods described in Muraoka S, T Shinozawa (2000) Effective production of amanitins by two-step cultivation of the basidiomycete, Galerina fasciculata GF-060. J Biosci Bioeng 89:73-76, herein incorporated by reference.
(128) Thus the present inventions further relate to compositions and methods associated with creating and screening genomic libraries from Galerina for sequences of interest. In particular, the present invention relates to providing and using PCR primers for identifying and sequencing Galerina genes, including methods comprising RACE PCR primers. Specifically, the present inventions relate to identifying and using sequences of interest, i.e. sequences encoding proteins associated with the production of small peptides, including cyclic peptides, for example, compositions and methods comprising Galerina POP homologs and amatoxins.
(129) Examples of procedures used to ligate the DNA construct of the invention, the promoter, terminator and other elements, respectively, and to insert them into suitable cloning vehicles containing the information necessary for replication, are well known to persons skilled in the art (see, e.g., Sambrook et al., 1989; herein incorporated by reference).
(130) The polypeptide may be detected using methods known in the art that are specific for the polypeptide. These detection methods may include use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or SDS-PAGE gel blotted onto membranes for immunoblotting. For example, an enzyme assay may be used to determine the activity of the polypeptide. Procedures for determining enzyme activity are known in the art for many enzymes.
(131) A. Peptide Toxin Genes in Galerina Mushrooms.
(132) The inventors' were surprised to discover that sequences of the peptide toxin genes in Galerina marginata is quite different compared to A. bisporigera. See
(133) Using AMA1 from A. bisporigera as the search query, two orthologs of AMA1 were identified in the partial genome survey sequence of G. marginata and designated as GmAMA1-1 and GmAMA1-2.
(134) PCR primers unique to GmAMA1- and GmAMA1-2 were designed. For GmAMA1-1, the unique primers were 5-CTCCAATCCCCCAACCACAAA-3 (forward, SEQ ID NO:682) and 5-GTCGAACACGGCAACAACAG-3 (reverse, SEQ ID NO:683). For GmAMA1-2, the primers were: 5-GAAAACCGAATCTCCAATCCTC-3 (forward, SEQ ID NO:684), and 5-AGCTCACTCGTTGCCACTAA-3 (reverse, SEQ ID NO:685). PCR primers for each gene were designed based on the partial sequences and used to amplify full-length copies. The amplicons were cloned into E. coli DH5 and sequenced.
(135) The genomic DNA sequences were used for primer design to obtain full-length cDNAs by Rapid Amplification of cDNA Ends (RACE) using the GeneRacer kit (Invitrogen, Carlsbad, Calif.). A cDNA copy of GmAMA1-1 was obtained using primers 5-CCAACGACAGGCGGGACACG-3 (5-RACE, SEQ ID NO:686) and 5-GACCTTTTTGCTTTAACATCTACA-3 (3-RACE, SEQ ID NO:687), and of GmAMA1-2 with primers 5-GTCAACAAGTCCAGGAGACATTCAAC-3 (5-RACE, SEQ ID NO:688) and 5-ACCGAATCTCCAATCCTCCAACCA-3 (3-RACE, SEQ ID NO:689).
(136) Alignments of genomic and cDNA copies were done using Spidey located at (ncbi.nlm.nih.gov/spidey/) and Splign located at ncbi.nlm.nih.gov/sutils/splign.cgi.
(137) GmAMA1-1 contained three introns while GmAMA1-2 contained two introns (
(138) The translational start site of a gene is typically contemplated as the first in-frame ATG, SEQ ID NO:711 after the transcriptional start site, SEQ ID NO:710. When this criterion was applied to GmAMA1-1, a start site was indicated that was analogous to AMA1 of A. bisporigera. This start site resulted in a predicted preproprotein, SPIPQPQTHLTKDLFALTSTMFDTNATRLPIWGIGCNPWT AEHVDQTLASGNDIC, SEQ ID NO:690, and proprotein, SEQ ID NO: 704. However, when this criteria was applied to GmAMA1-2, there was an in-frame ATG that is 78 nucleotides upstream of the ATG, indicated in
(139) GmAMA1-1 and GmAMA1-2 were both predicted to encode 35-amino acid proproteins, the same size as the proprotein of AMA1 in A. bisporigera. The toxin-encoding region (IWGIGCNP) was in the same relative position as it was in AMA1. There were 31 nucleotide differences between GmAMA1-1 and AMA1 in the coding region of 108 nucleotides (ATG through the stop codon). This resulted in a low level of amino acid conservation outside the toxin region and the amino acids immediately upstream of the toxin region (NATRLP, SEQ ID NO:754 (
(140) The sequenced proproteins were added to a family of genes including and related to AMA1 and PHA1 in A. bisporigera, A. phalloides, and A. ocreata, a group of genes that started with MSDIN. In contrast, when a start codon was contemplated in the same location between GmAMA1-1 and GmAMA1-2 the first five amino acids of the two G. marginata -amanitin genes were MFDTN, SEQ ID NO: 675. Searching the inventors' G. marginata database with the upstream and downstream regions of GmAMA1-1 and GmAMA1-2 did not reveal any additional related sequences. Conversely, searching with the conserved regions of GmAMA1-1 and GmAMA1-2 did not reveal any related sequences in A. bisporigera beyond the known MSDIN family members described herein.
(141) Distribution of -amanitin Genes in the Genus Galerina. Within the genus Amanita, AMA1 and PHA1 are known to be present in section Phalloideae, which contains the known amatoxin- and phallotoxin-producing species in this genus. To explore the distribution of the -amanitin genes in relation to toxin production in Galerina, four species of Galerina were compared by DNA blotting (also known as Southern blotting) and RNA blotting (also known as Northern blotting).
(142) Recent taxonomic revision of this genus indicates that G. marginata and G. venenata are synonyms, whereas G. hybrida and G. badipes are considered as separate species (Enjalbert et al., 2004; Gulden et al., 2001, 2005, all of which are herein incorporated by reference). In Southern blots, a GmAMA1-1 probe [a genomic DNA sequence made with primers (5-ATGTTCGACACCAACTCCACT-3, SEQ ID NO:672) and (5-CGCTACGTAACGGCATGACAGTG-3, SEQ ID NO:673) hybridized to all three -amanitin producers (G. marginata, G. badipes, and G. venenata) but not to the toxin nonproducer, G. hybrida (lane 3) (
(143) Regulation of GmAMA1 by Low Carbon. Successful amplification of GmAMA1-1 and GmAMA1-2 by reverse transcriptase PCR with gene-specific probes indicated that both genes are transcribed in culture. Expression was further studied by RNA blotting. Muraoka and Shinozawa (2000, herein incorporated by reference) showed that -amanitin production in G. fasciculata was upregulated on low glucose medium (carbon starvation). The inventors' found that expression of GmAMA1-1 and/or GmAMA1-2 were also up-regulated by carbon starvation in G. marginata and G. badipes (
(144) Discovering that G. marginata peptide toxin genes differed from those of A. bisporigera was surprising in several ways. First, the proproteins share little overall amino acid identity except in the toxin region itself (IWGIGCNP) with the exception of short regions outside of the toxin sequence. For example, whereas the A. bisporigera peptide toxin proproteins start with MSDIN, SEQ ID NO:674, (or with only a single amino acid difference), the two copies of AMA1 in G. marginata started with MFDTN, SEQ ID NO:675. Additionally, the inventors found conservations in the four amino acids after MSDIN, which were also found after MFDTN, and the start of the peptide toxin coding region (IWGIGCNP). These conserved motif sequences were found as ATRLP, SEQ ID NO:676, or STRLP, SEQ ID NO:677, in the proproteins of both the A. bisporigera peptide toxins and the G. marginata peptide toxins. The complete conservation of the Pro residue immediately upstream of the peptide toxin coding region was believed to be significant because Pro is believed to be required for processing of the proprotein by a prolyl oligopeptidase. The inventors further contemplated that upstream conserved region of amino acids in G. marginata peptide toxin sequences (i.e. N[A/S]TRL, SEQ ID NO:678) is important for recognition of the proproteins by Gm POPB. There was little conservation between the downstream conserved regions of the A. bisiporigera and the G. marginata genes. For example, MFDTNATRLP SEQ ID NO:679, was unexpectedly found in place of MSDIN.
(145) Second, G. marginata was discovered to contain two nearly identical copies of the -amanitin gene with at least one variant of each whereas one copy of the -amanitin gene was found in A. bisporigera. Conversely, A. bisporigera has at least two copies of genes encoding phallacidin (PHA1) while none were found in the sequenced isolate of G. marginata, and phallacidin or other phallotoxins have not been reported from G. marginata.
(146) Third, the inventors were surprised to find two sequences related to the -amanitin genes in the genome of G. marginata whereas a large family of related sequences (>30 members), which encode predicted, but chemically unknown, cyclic peptides was discovered in the A. bisporigera genome. These predicted peptides were discovered by translating the A. bisporigera genes contained 7 to 10 amino acids where the majority lacked Trp and Cys predicted to be used to form tryptathionine, which was a characteristic of the amatoxins and phallotoxins of A. bisporigera peptides.
(147) G. marginata and other species of Galerina were known to make -amanitin (Enjalbert et al., 2004; Muraoka et al., 1999; Muraoka and Shinozawa, 2000, all of which are herein incorporated by reference). However phallotoxins were not found in Galerina species however some species were reported to make -amanitin. -amanitin differs from -amanitin in having Asp in place of Asn. The difference between these two forms of amanitin was predicted to be genetically encoded and not catalyzed by, e.g., a transamidase, because the genome of A. phalloides contains a gene that was predicted to directly encode -amanitin.
(148) The inventors confirmed that the isolate of G. marginata prepared and used herein did not synthesize -amanitin and the genome lacks a gene for -amanitin. In other isolates, traces of -amanitin from G. marginata grown in culture were detected i.e. Benedict et al. (1966, 1967, all of which are herein incorporated by reference). Further, -amanitin was not detected in several wild North American specimens of Galerina. Therefore, some species and/or isolates of Galerina do make -amanitin and others do not, therefore each isolate must be tested. Other forms of amanitin, such as -amanitin and -amanitin, differ from -amanitin and -amanitin in their pattern of hydroxylation. This chemical difference was not found in encoding DNA.
(149) B. Full Length POP Gene Production.
(150) The G. marginata partial genome survey was discovered to contain two orthologs of the POP genes of A. bisporigera. Genomic PCR, reverse transcriptase PCR, and RACE were used, as described herein, to isolate full-length copies of these two genes and determine their intron/exon structures (
(151) During the development of the present inventions, two orthologs were found in the G. marginata genome sequences corresponding to the two A. bisporigera prolyl oligopeptidases (AbPOPA and AbPOPB) described herein. The G. marginata genes with closest identity to AbPOPA or AbPOPB were designated as GmPOPA and GmPOPB, respectively.
(152) Sequences hybridizing to AbPOPA were found to be present in amatoxin and phallotoxin-producing and non-producing species of Amanita, whereas AbPOPB was found present only in the toxin-producing species. By DNA blotting GmPOPA was present in all four specimens of Galerina, however GmPOPB was not present in the amanitin non-producing species G. hybrida (
(153) For isolating and cloning full-length cDNA sequences for GmPOPA and GmPOPB, PCR primers that corresponded to the amino and carboxyl termini of both genes (which were present on different contigs) were designed from the genome survey sequence. The forward primers were 5-TTTAGGGCAGTGATTTCGTGACA-3, SEQ ID NO: 692, and 5-AACAGGGAGGCGATTATTCAAC-3, SEQ ID NO: 693, and the reverse primers were 5-GAACAATCGAACCCATGACAAGAA-3, SEQ ID NO: 694, and 5-CCCCCATTGATTGTTACCTTGTC-3, SEQ ID NO: 695. The primer pairs were used in both combinations and successful amplification indicated the correct pairing of 5 and 3 primers. The resulting amplicons were cloned into E. coli DH5 and sequenced.
(154) The RACE primers for GmPOPA were 5-CGGCGTTCCAAGGCGATGATAATA-3 (5-RACE), SEQ ID NO: 696, and 5CATCTCCATCGACCCCTTTTTCAGC-3 (3-RACE), SEQ ID NO: 697, and for GmPOPB 5-AGTCTGCCGTCCGTGCCTTGG-3 (5-RACE), SEQ ID NO: 698, and 5-CGGTACGACTTCACGGCTCCAGA-3 (3-RACE), SEQ ID NO: 699. Sequences generated from the RACE reactions were used to assemble full-length cDNAs of two genes, GmPOPA and GmPOPB (see
(155) Alignments of genomic and synthetic cDNA copies (see,
(156) GmPOPA and POPB were predicted to encode exemplary polypeptides as shown in
(157) The inventors' contemplate that POP proteins encoded by the G. marginata POP sequences (known as GmPOP) of the present inventions are capable of enzymatic activity. There are three critical amino acids that constitute the active site in other POP proteins (Szeltner et al., (2008) Current Protein and Peptide Science 9:96-107, herein incorporated by reference). In a crystallized POP protein, the active site residues were Ser554, Asp641, and His680. The location of these active site residues in POPA are: Ser581, Asp665, and His 701. In POPB they are Ser571, Asp661, and His698. Thus the GmPOP genes of the present inventions are contemplated to be capable of encoding POP proteins with these active site amino acids in analogous positions for a protein capable of enzymatic activity.
(158) The inventors showed that isolated prolyl oligpeptidase (POP) proteins of other mushroom species were capable of initial processing of the proproteins of amatoxins and phallotoxins. First, in the extended MSDIN family of Amanita, discovered by the inventors and now shown to correspond to an MFDTN, SEQ ID NO:675, family of -amanitin genes of G. marginata, flanking Pro residues are completely conserved. One Pro remains in the mature toxin while the other is removed with the flanking sequence. Second, an enzyme that proteolytically cleaves a synthetic phalloidin proprotein, isolated from the phalloidin-producing fungus Conocybe albipes, was identified during the development of the presence inventions as a POP protein. The same enzyme cleaves at both Pro residues to release the mature linear peptide (AWLATC in the case of phalloidin). Third, toxin-producing species of Amanita have two POP genes, whereas all other sequenced basidiomycetes have one. One of the Amanita POP genes, AbPOPB, was found during the development of the presence inventions restricted to toxin-producing species, like AMA1 and PHA1 themselves. Fourth, the distribution of AbPOPB and -amanitin overlap in mushroom tissues was found during the development of the presence inventions, indicating a cytological connection between -amanitin biosynthesis and accumulation. G. marginata was discovered to have two POP genes, like Amanita but unlike other, toxin non-producing species of mushrooms. GmPOPB is absent from species such as G. hybrida that do not make toxins. Thus, AbPOPB and GmPOPB are believed to be involved in the biosynthesis of the amatoxins and/or phallotoxins in their respective species.
(159) VIII. Recombinant Polypeptide Products of Amanita and Galerina Genes.
(160) A desired end product, i.e., the polypeptide of interest, such as a POP enzyme, may be expressed by a host cell, such as a bacterium, i.e. E. coli, as a heterologous protein or peptide. Thus the polypeptide may be any polypeptide heterologous to the bacterial cell. The term polypeptide is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. The heterologous polypeptide may also be an engineered variant of a polypeptide. The term heterologous polypeptide is defined herein as a polypeptide, which is not native to the host cell. Preferably, the host cell is modified by methods known in the art for the introduction of an appropriate cloning vehicle, i.e., a plasmid or a vector, comprising a DNA fragment encoding the desired polypeptide of interest. The cloning vehicle may be introduced into the host cell either as an autonomously replicating plasmid or integrated into the chromosome. Preferably, the cloning vehicle comprises one or more structural regions operably linked to one or more appropriate regulatory regions.
(161) The structural regions are regions of nucleotide sequences encoding the polypeptide of interest. The regulatory regions include promoter regions comprising transcription and translation control sequences, terminator regions comprising stop signals, and polyadenylation regions. The promoter, i.e., a nucleotide sequence exhibiting a transcriptional activity in the host cell of choice, may be one derived from a gene encoding an extracellular or an intracellular protein, preferably an enzyme, such as an amylase, a glucoamylase, a protease, a lipase, a cellulase, a xylanase, an oxidoreductase, a pectinase, a cutinase, or a glycolytic enzyme.
(162) The resulting polypeptide may be isolated by methods known in the art. For example, the polypeptide may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. The isolated polypeptide may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989, herein incorporated by reference).
(163) IV. Compositions and Methods for Expressing Small Linear Peptides and Cyclic Peptides Using Transformed Galerina Marginata and Lysates.
(164) The inventors grew G. marginata in the laboratory and collected mycelium for use in the following transformation procedure. The inventors show herein the successful transformation of the alpha-amanitin-producing fungus Galerina marginata with a test construct. Thus the inventors' contemplate producing commercial levels of amanatin in addition to novel, non-natural analogs of amanitin. Further, the inventors' contemplate making novel linear and cyclic peptides from synthetic prepropeptides.
(165) The following are exemplary methods for making buffers and reagents for us in the present inventions. Galerina culture methods: Vegetative mycelial stocks were prepared by culturing aseptic fragments of fruiting bodies on HSVA plates. Fungal colonies were transferred and reisolated until pure cultures were obtained. The stocks were subcultured every 6 months. HSV-2C (1 L): 1 g yeast extract, 2 g glucose, 0.1 g NH.sub.4Cl, 0.1 g CaSO.sub.4.5H.sub.2O, 1 mg thiamine.HCl, and 0.1 mg biotin, pH 5.2 (Muraoka and Shinozawa, 2000, herein incorporated by reference). Agar medium (HSVA) for subculture contained 2% agar in HSV. Protoplasting Buffer: In 20 ml of 1.2 M KCl add 500 mg Driselase (Sigma), 1 mg chitinase (Sigma), and 300 mg lysing enzyme from Aspergillus sp. Sigma #L-3768. Stir for 30 min and filter sterilize in a 0.45 um filter. Sorbitol Tris-HCl Ca (STC) buffer: Solution a) 1.2 M sorbitol, 10 mM Tris-HCl (pH8.0), 50 mM CaCl.sub.2, autoclaved. Solution b) 30% PEG Solution Mix: 300/(W/V) polyethylene glycol/STC buffer. Filter sterilize in a 0.45 um filter. Regeneration medium (RM): a) HSV-2C (1 L) and b) sucrose 273.5 g/500 ml of water. Autoclave solutions a) and b) separately and combine after autoclaving.
(166) The following is an exemplary Galerina transformation protocol for use in the present inventions. Around 20 pieces of mycelium were used to inoculate 100 ml of HSV-2C broth in a 250 ml Erlenmeyer flask. This inoculate was placed on a shaker at 150 rpm at room temperature for 9-15 days, until cloudy. The culture medium and fungus was used to begin the following steps. The cultures were: 1. Filtered through sterile Miracloth and the collected mycelia was washed thoroughly with sterile water. This fungal mycelium was placed in a sterile 250 ml Erlenmeyer flask. 20 ml Protoplasting Buffer (see recipe below) was added. 2. Digested for 8 hours on a rotary shaker at 26-30 C at 120 rpm. 3. Digestion mix was filtered through a 30 micron Nitex nylon membrane (Tetko Inc. Kansas City, Mo., U.S.A.)) into 1-2 sterile 30 ml Oakridge tubes on ice. Filtered solution was turbulent due to the presence of protoplasts when checked under the microscope. 4. This filtered solution was centrifuged in Oakridge tubes at 4 C at 2000g for 5 min. 5. Supernatant was carefully poured off and discarded. Protoplast pellet was gently resuspended in approx. 10 ml of STC buffer and resuspended by shaking gently. Solution was spun at 2000g for 5 min. 6. Repeat step 5 once. 7. Supernatant was discarded and the protoplast pellet was gently resuspended in 1 ml of STC buffer with a wide orifice pipette and transferred to a microcentrifuge tube and spun at room temperature at 4000g for 6 min. 8. Supernatant was poured off and protoplasts were resuspended in 1 ml of STC in a final volume with concentration of 10.sup.8-10.sup.9 protoplast/ml. The tube was placed on ice. 9. The following mixture was combined: 50 l protoplasts, 50 l STC buffer, 50 ul 30% PEG solution and 10 ul plasmid or PCR product (1 g) depending upon the experiment. When plasmids were used they were linearized with a restriction enzyme which cut the DNA in a noncoding region. 10. 2 ml of 30% PEG solution was added and the tubes incubated for 5 min. 11. 4 ml of STC buffer was added and gently mixed by inversion. 12. The mix was added to Regeneration Media (RM) (see below) at 47 C., and mixed by inversion then poured into Petri dishes. Each solution mixture was plated in several plates. 13. Protoplasts were regenerated for up to 20 days until tiny colonies started to appear as viewed by eye. 10 ml of RM amended with 10 g/ml Hygromycin B was overlayed onto the cultures. 14. Putative transformants were isolated from colonies that grew after the Hygromycin B overlay and eventually emerged on the surface of the overlaid agar. Examples of colonies collected for use in the present inventions are shown by arrows in
(167) After colonies were collected the presence of the inserted Hygromycin B transgene was tested by PCR. Primers specific to the hygromycin resistance gene used in
(168) The inventor's contemplate that G. marginata can be transformed with synthetic genes, using the G. marginata specific contemplated cut sites, i.e. synthetic sequences comprising nucleotides encoding MDSTN, TRIPL and Prolines in conserved positions. For examples, in one embodiment, a synthetic DNA sequence encoding an amino acid sequence of alpha-amanitin may be expressed. In one embodiment, alpha-amanitin production would be increased, for example, using a high expression promoter, transforming Galerina with multiple copies of the alpha-amanitin gene.
(169) In another contemplated embodiment, a synthetic, novel cyclic peptide is synthesized by transformed Galerina by changing specific bases of synthetic G. marginata alpha-amanitin sequences (including PCR copies of isolated peptide toxin genes and base by base construction of nucleic acid sequences) in order to make other types of peptide toxins and peptides. In one example, replacing the codon AAC (Asn) with GAC (Asp) will encode beta-amanitin instead of alpha-amanitin. Beta-amanitin production in G. marginata would be easily detected by reverse-phase HPLC because the inventor's isolate of G. marginata makes barely detectable levels of beta-amanitin.
(170) The inventors further contemplate changing other amino acids to make non-natural amanitin derivatives, as one example, replacing Gly with Ala by replacing GGT with GCT. Even further, the inventor's contemplate an embodiment for making linear and cyclic peptides of at least six, seven, eight, nine, ten or more amino acids comprising the general formula XWXXXCXP, SEQ ID NO:702, where X is any amino acid. The Pro is retained in these peptides in order for correct processing by POP, and the presence of Trp (W) and Cys (C) will result in the biosynthesis of tryptathionine, a unique hallmark of the Amanita toxin peptides. Expression of synthetic peptides and peptide toxins would be monitored by standard assays including but not limited to PCR generated fragments (as in
(171) Even further, the inventors' contemplate making new Galerina isolates that do not produce peptide toxins for use in the present inventions. In one embodiment, the inventors' contemplate knocking out genomic peptide toxin genes for making a new Galerina isolate that does not express peptide toxins. As examples for removing genomic peptide toxin genes in Galerina, i.e. test Galerina (isolates of Galerina used in the following methods) would be subject to homologous integration of transforming DNA that would be used for removing regions of DNA comprising the peptide toxin genes in transformed test Galerina, spontaneous mutants and induced mutants of test Galerina would be made then screened for loss of peptide toxin gene expression and more preferably loss of peptide toxin genes. Another method for eliminating endogenous toxin production is RNAi, which has been used in other basidiomycete fungi (Heneghan et al., Mol Biotechnol. 2007 35(3):283-96, 2007, herein incorporated by reference). Loss of toxin expression in test isolates would be monitored by standard assays including but not limited to genomic sequencing of test Galerina, PCR generated fragments of genomic sequences (as in
(172) G. marginata has numerous advantages as an experimental system for use in the present inventions. First, G. marginata is cultured under laboratory conditions, unlike most species of Amanita, which do not grow well in the laboratory (Benedict et al., 1966, 1967; Muraoka and Shinozawa, 2000; Zhang et al., 2005, all of which are herein incorporated by reference). Second, G. marginata produced -amanitin in culture and production was increased by carbon starvation. Third, genomic sequencing and genetic studies were facilitated by the availability of a peptide toxin-producing monokaryotic strain (isolate) of G. marginata. Fourth, the panoply of peptide toxin genes, estimated greater than 30 members in species of Amanita, was not found in the laboratory isolate of G. marginata, where only two genes were found during the development of the present inventions.
EXPERIMENTAL
(173) The following examples serve to illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
(174) In the experimental disclosures which follow, the following abbreviations apply: N (normal); M (molar); mM (millimolar); M (micromolar); mol (moles); mmol (millimoles); mol (micromoles); nmol (nanomoles); pmol (picomoles); g (grams); mg (milligrams); g (micrograms); ng (nanograms); pg (picograms); L and 1 (liters): ml (milliliters); l (microliters); cm (centimeters); mm (millimeters); m (micrometers); nm (nanometers); U (units); min (minute); s and sec (second); deg (degree); C. (degrees Centigrade/Celsius).
Example I
(175) Materials and Methods
(176) The following is a description of exemplary materials and methods that were used in subsequent Examples during the development of the present inventions.
(177) A. Exemplary Mushroom Species of the Present Inventions (
(178) The inventors selected the genome of Amanita bisporigera to provide sequences of interest because of reports on consistently high, albeit somewhat variable, levels of amatoxins and phallotoxins within individual fruiting bodies combined with the relative ease of obtaining exemplary wild growing mushrooms by merely identifying and harvesting the mushrooms.
(179) Exemplar Basic Molecular Biology Techniques. The inventors developed and used the following exemplary materials and methods during the development of the present inventions. During the development of the present inventions the inventors were surprised to successfully clone cDNAs encoding toxin genes from mature mushrooms in addition to mushrooms in the button stage.
(180) Genomic DNA Isolation. Although the carpophores (fruiting bodies) contain high concentrations of the toxins, like other ectomycorrhizal Basidiomycetes, species of Amanita grow slowly and do not form carpophores in culture (Muraoka et al., (1999) Appl. Environ. Microbial. 65:4207; Zhang et al., (2005) FEMS Microbial Lett. 252:223; all of which are herein incorporated by reference). Therefore, A. bisporigera mushrooms, an amatoxin and phallotoxin producing species native to North America, were harvested from the wild. Caps and undamaged stems were cleaned of soil and debris, frozen at 80 C., and lyophilized.
(181) Genomic DNA was extracted from the lyophilized fruiting bodies using cetyl trimethyl ammonium bromide-phenol-chloroform isolation (Hallen, et al., (2003) Mycol. Res. 107:969; herein incorporated by reference). For studies requiring RNA, RNA was extracted using TRIZOL (Invitrogen) (Hallen, et al., (2007) Fung. Genet. Biol., 44:1146; herein incorporated by reference in its entirety). Specifically, DNA for genomic blotting was cut with PstI and electrophoresed in 0.7% agarose.
(182) Probe Labeling, DNA Blotting, and Filter Hybridization. Standard protocols were followed for these and similar molecular biology procedures (see, Maniatis, et al., Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor, N.Y., 1982, herein incorporated by reference) and Singh, et al., (1984) Nucl. Acids Res. 12:5627, herein incorporated by reference). In general, hybridization was done overnight at 65 C. in 4SET (600 mM NaCl, 120 mM Tris-HCl, pH 7.4, 8 mM EDTA), 0.1% sodium pyrophosphate, 0.2% SDS, 10% dextran sulfate, 625 g/ml heparin. Washing: twice in 2SSPE (300 mM NaCl. 20 mM NaH.sub.2PO.sub.4, 2 mM EDTA, pH 7.4), 0.1% SDS at 21 C., then twice in 0.1.times.SSPE and 0.1% SDS at 60 Celcius.
(183) PCR Amplification of Peptide Encoding Genes. PCR primers for amanitin and phallacidin amplification from A. bigospora were based on fragments within sequences shown in
(184) TABLE-US-00003 TABLE3 PCRprimersusedformakingsyntheticamanitin (AMA1)andphallacidingenes(PHA1) Sequence Name SEQIDNO: SEQUENCE AMA1, SEQIDNO:1 5 CCATCTGGGGTATCGGTTGC3 forward AMA1, SEQIDNO:2 5 TTGGGATTGTGAGGTTTAGAG reverse GTC3 PHA1, SEQIDNO:3 5 CGTCAACCGTCTCCTC3 forward PHA1, SEQIDNO:4 5 ACGCATGGGCAGTCTA3 reverse
(185) A 551-bp fragment of the A. bisporigera -tubulin gene was amplified using primers 5-ACCTCCATCTCGTCCATACCTTCC-3 (SEQ ID NO: 5) and 5-TGTTTGCCACGCTGCATACTA-3 (SEQ ID NO: 6) then used as a control probe on DNA blots. PCR amplification was done using REDTaq ReadyMix DNA polymerase (Sigma) and appropriate reagents under 30 cycles of denaturation (94 C., 30 sec), annealing (55 C., 30 sec), and extension (72 C., 5 min).
(186) Target Genes for Sequencing. PCR target gene products were purified using Wizard SV Gel and PCR Clean-Up System (Promega) and then cloned into TOPO pCR 4 (Invitrogen) for obtaining sequence information.
(187) B. Exemplary Mushroom Species of the Present Inventions (
(188) Biological Material. Four species of Galerina were obtained from Centraalbureau voor Schimmelcultures (CBS), Utrecht, Netherlands, including G. marginata (CBS 339.88), G. badipes (CBS 268.50), G. venenata (CBS 924.72), and G. hybrida (CBS 335.88). G. marginata CBS 339.88 is monokaryotic and was confirmed to make -amanitin. G. venenata is considered synonymous with G. marginata (Gulden et al., 2001, herein incorporated by reference). The cultures were maintained on potato dextrose agar. For DNA isolation, the isolates were cultured in liquid medium for 15-30 d with rotary shaking at 120 rpm at 23 C. The medium was HSV-2C, which contains (per liter) 1 g yeast extract, 2 g glucose, 0.1 g NH.sub.4Cl, 0.1 g CaSO.sub.4.5H.sub.2O, 1 mg thiamine.HCl, and 0.1 mg biotin, pH 5.2 (Muraoka and Shinozawa, 2000). For induction experiments, the media had the same formulation, except that high carbon (HSV-5C) and low carbon (HSV-1C) media contained 5 g glucose and 1 g glucose, respectively (Muraoka and Shinozawa, 2000, herein incorporated by reference).
(189) Nucleic Acid Isolation and Genome Sequencing. Lyophilized fungal mycelia were ground in liquid nitrogen with a mortar and pestle. High molecular weight DNA was isolated using Genomic-tip 100/G (Qiagen, Germantown, Md.; catalog #10234) and RNA was extracted with TRIzol (Invitrogen, Carlsbad, Calif.), following the manufacturers' protocols.
(190) Genomic DNA was sequenced by 454 pyrosequencing at the Research Technology Support Facility (RTSF) at Michigan State University. A general library was constructed using standard protocols and sequenced on a 454 GSFLX Titanium Sequencer (Roche manual, 20th ed., herein incorporated by reference). Raw reads were assembled with Newbler and assembled into a searchable database.
(191) Cloning and Gene Characterization. AMA1 and PHA1 are the designations for the -amanitin- and phallacidin-encoding genes, respectively, of A. bisporigera; the prefix Ab is used to designate other genes from A. bisporigera. The prefix Gm is used to designate all genes from G. marginata.
(192) DNA and RNA Blotts. DNA for Southern blotting was digested with PstI and electrophoresed in 0.7% agarose. Probe labeling, blotting, and filter hybridization followed standard protocols (Scott-Craig et al., 1990, herein incorporated by reference). Hybridizations were performed for 15 hr at 65 C. Roughly 2 g of DNA were loaded per lane. Probes were made by labeling genomic DNAs of GmAMA1-1, GmPOPA, and GmPOPB with [.sup.32P]dCTP.
(193) For the GmAMA1 induction experiment, G. marginata was cultured in HSV-5C media for 30 d and then transferred to HSV-5C or HSV-1C and grown for an additional 10 d. The resulting mycelia were lyophilized and stored at 80 C. prior to RNA extraction. Full-length cDNA was prepared using the GeneRacer RACE kit, following the manufacturer's protocols. Hybridization probes were amplified using a specific 5 primer (5-ATGTTCGACACCAACTCCACT-3, SEQ ID NO:680) and GeneRacer 3 nested primer (5-CGCTACGTAACGGCATGACAGTG-3, SEQ ID NO:681). Probe labeling, RNA gel electrophoresis, and blotting followed standard protocols (Scott-Craig et al., 1990, herein incorporated by reference). Each lane was loaded with 15 g total RNA.
(194) Amanitin Extraction and Analysis. G. marginata was cultured in HSV-5C media for 30 d and then transferred to fresh HSV-1C medium for an additional 10 d. After harvest, the mycelium was lyophilized and stored in at 80 C. A portion of dried mycelium (0.2 gm) was ground in liquid nitrogen and mixed with 2 ml methanol:water:0.01 M HCl (5:4:1) (Enjalbert et al., 1992; Hallen et al., 2003, herein incorporated by reference). The suspension was incubated at 22 C. for 30 min and then centrifuged at 10,200g for 10 min at 4 C. The supernatant was collected and filtered through a 0.22 filter. Chromatographic separation was done on a C18 column (Vydac 218TP54) attached to an Agilent Model 1100 HPLC with detection at 230, 290, and 305 nm. Elution solution A was water+0.1% trifluoroacetic acid, and solution B was acetonitrile+0.075% trifluoroacetic acid. The flow rate was 1 ml/min with a gradient from 100% A to 100% B in 30 min. An -amanitin standard (Sigma A2263) was dissolved in water at a concentration of 100 g/ml. Loadings were 40 l unknown or 20 l standard.
Example II
(195) This example describes exemplary methods for providing a fungal genomic library, specifically an Amanita spp., library.
(196) The inventors initially contemplated the existence of an amatoxin synthetase gene that was a member of the class of enzyme known as nonribosomal peptide synthetases.
(197) However after extensive unsuccessful attempts to obtain amatoxin synthetase genes or gene fragments through PCR-based techniques using isolated genomic DNA, see, Example III, and biochemical methods (such as, ATP-pyrophosphate exchange assay; amino acid feeding studies, etc.), the inventors subsequently initiated a shotgun genome sequencing project for obtaining genes of interest, such as genes associated with cyclized peptide production, toxin production, peptide encoding genes, toxin encoding genes, etc. One genomic library was generated by the Genomics Technology Support Facility at Michigan State University and one was generated by Macrogen, Inc. Each library yielded genomic fragments of approximately 2-kb in length. Random clones were end sequenced by automated dideoxy sequencing.
(198) Approximately 5.7 Mb sequence was generated in approximately 10,000 unidirectional sequencing reads using dideoxy sequencing using an ABI 3730 Genetic Analyzer and an ABI Prism 3700 DNA Analyzer (sequencing performed at the Research Technologies Support Facility at Michigan State University, and by Macrogen, Inc.).
(199) The inventors originally began a public Amanita sequence database; however, after a brief posting of the above-described sequencing results, the inventors removed those sequences from public access (see, Examining amatoxins: The Amanita Genome Project. Hallen, Walton, 159. The utility of the incomplete genome: the Amanita bisporigera genome project. Mar. 15-20, 2005 Asilomar Conference Center, Pacific Grove Calif. Fungal Genetics Newsletter, Volume 52-Supplement XXIII FUNGAL GENETICS CONFERENCE; herein incorporated by reference). Moreover, to the inventors' knowledge, sequences of the present inventions were never publicly available.
(200) The inventors subsequently also completed at least four runs on a Genome Sequencer 20 from 454 Life Sciences (Margulies et al., (2005) Nature 437:376; herein incorporated by reference). This generated approximately 70 MB of sequence data, which is approximately 2 coverage of the genome of A. bisporigera, based on the known size of other Homobasidiomycetes, (Le Quere et al., Fung. Genet. Biol. 36, 234 (2002); Coprinus cinereus Sequencing Project. Broad Institute of MIT and Harvard (broad.mitedu/annotation/genome/coprincis_cinereus/Hom-e.html); all of which are herein incorporated by reference).
(201) The inventors structured and maintained the sequenced DNA in a password-protected, private BLAST-searchable format. The sequences were compared to GenBank's non-redundant database.
(202) BLASTX (translated query against protein database) was used in searching the non-redundant database (NR) at GenBank, and TBLASTX (translated query against translated database) and BLASTN (nucleotide query against nucleotide database) were used in searching the genomes of Coprinopsis cinereus (also known as Coprinus cinereus) and Phanerochaete chrysosporium, the two closest relatives to Amanita bisporigera for which complete genome sequences were available at that time. In some embodiments, BLAST results were examined, catalogued, and automatically annotated.
Example III
(203) This example describes the failure of the inventors to obtain a gene homologous to a fungal nonribosomal peptide synthetases (NRPSs) in Amanita bisporigera, which produces amatoxins, phallotoxins, and other putative Amanita peptide toxins. Details are shown in a poster entitled Examining amatoxins: The Amanita Genome Project Hallen Walton 159. The utility of the incomplete genome: the Amanita bisporigera genome project. Mar. 15-20, 2005 Asilomar Conference Center Pacific Grove Calif. Fungal Genetics Newsletter, Volume 52Supplement XXIII FUNGAL GENETICS CONFERENCE; herein incorporated by reference.
(204) Because known fungal cyclic peptides are biosynthesized by methods comprising nonribosomal peptide synthetases (NRPSs) (Walton, et al., in Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine, et al., Eds. (Kluwer Academic/Plenum, N.Y., 2004, pp. 127-162; Finking, et al., (2004) Arum Rev Microbiol 58:453-488, all of which are herein incorporated by reference), the inventors initiated an attempt to identify by PCR in the total genomic DNA of Amanita bisporigera sequences encoding an NRPS using PCR primers based on known bacterial and fungal NRPSs and total A. bisporigera DNA as template. The inventors contemplated that any NRPS genes sequences within the Amanita bisporigera genome should have been readily amplified using two or more of PCR primers. Then, from sequencing genomic DNA outward from the PCR products, they should have ultimately identified an NRPS with 8 adenylating domains containing other conserved regions present in all known NRPS-encoding sequences.
(205) TABLE-US-00004 TABLE4 PCRprimersusedthatfailedtoobtainaNRPSsequence (SeeFIG.3). ForwardPrimers5-3 ReversePrimers5-3 AIxKAGxA:SEQ GCNATHTNN AIxKAGx GCNGNNCCNGCY IDNO:5 AARGCNGGN SEQIDNO: TTNNADATNGC NCNGC 6 FTSGSTG TTYACITCI na na (JA4F):SEQID GGITCI NO:7 ACIGG.sup.1 YTSGSTGI:SEQ TAYACNAGY na na IDNO:8 GGNAGYACN GG YTSGSTG2: TAYACNAGY na na SEQIDNO:9 GGNTCNACN GG YTSGSTG3: TAYACNTCN na na SEQIDNO:10 GGNTCNACN GG YTSGSTG4: TAYACNTCN na na SEQIDNO:11 GGNAGYACN GG SRGKPKG:SEQ TCTAGAGGN na na IDNO:12 AARCCNAAR GG.sup.2 TGKPKG:SEQ ACNGGNAAR TGKPKG: CCYTTNGGYTTN IDNO:13 CCNAARGG.sup.4 SEQIDNO: CCNGT 14 YGPTE:SEQ TAYGGNCCN YGPTE:SEQ TTCNGTNGGNCC IDNO:15 ACNGA.sup.4 IDNO:16 RTA YGPTE2:SEQ TACGGNCCN na na IDNO:17 ACNGAN na na GELIIGG: CCNCCNATNATN SEQIDNO: AGYTCNCC 18 ARGYX:SEQ TBGCNCGNG ARGY:SEQ GTANCCNCGNGC IDNO:19 GNTACN IDNO:20 GAN YK/RTGDL: TACARRACN YKTGDL: ARRTCNCCNGTN SEQIDNO:21 GGNGAYCT SEQIDNO: TTRTATCTAGA.sub.2 22 YRTGDLV:SEQ TAYMGIACI na na IDNO:23 GGIGAYYTI GT Y/FRTGDL/R TWYGCIACI na na G/VR(TGD): GGIGAYYKI SEQIDNO:24 GKICG.sup.3 ELGEIE:SEQ GARYTNGSN KDTQVK GGIACYTGITGR IDNO:25 GARATHGA (JA5):SEQ TCYTT.sup.1 IDNO:26 na na LLXLGGXS AWIGARKSICCI (LGG):SEQ CCIRRSIMRAAR IDNO:27 AA.sup.3 GGDSIA/T:SEQ GGNGGNGAAY GGDSIA/T GCNGYDATNSWR IDNO:28 TCNATYRCN A:SEQID TCNCCNCC NO:29 na na GGHSIA/T GCNGYRATNGAR A:SEQID TGNCCNCC NO:XX na na GDSITA CGCCGTGATCGA Cochliobolus ATCCCC victoriae: SEQID NO:30 ISGDW:SEQID CAYCAYNNN ISGDW:SEQ CCTNCCRTCNSW NO:31 ATHWSNGAY IDNO:32 NATNNNRTGRTG GGNTGG EGHGRE:SEQ GARGGNCAY EGHGRE: TCNCKNCCRTGN IDNO:33 GGNMGNGA SEQIDNO: CCYTC 34 DAYPCSC. GATGCCTAC DVYPCTP: GTKCANGSRWAN victoriae:SEQ CCATGCTCG SEQIDNO: ACRTCYTC IDNO:35 36 PCTPLQ:SEQ CCNTGYACN PCTPLQ: TGNARNGGNGTR IDNO:37 CCNYTNCA SEQIDNO: CANGG 38 na na PCTPLQ2: TGIARIGGIGTR SEQIDNO: CAIGG 39 QEGLMA(JA1): CARGARGGI QEGLMA: CGCATNAGNCCY SEQIDNO:40 YTIATGGC.sup.1 SEQIDNO: TCCTG 41 QEGLMA:SEQ KARGGNATG QEGLMA: GCNWTCATNCCY IDNO:42 AWNGC SEQIDNO: TMYTG 43 .sup.1Primer sequences that the inventors obtained from Dr. Aric Weist .sup.2Primers referenced in Panaccione, (1996) Mycological Research 100:429-436; herein incorporated by reference. .sup.3Primers referenced in Turgay & Marahiel (1994), Peptide Research 7:238-241; herein incorporated by reference. .sup.4Primers references in Nikolskaya et al. (1995) Gene 165:207-211 Abbreviations: A, adenine; T, thymine; G, guanine; C, cytosine; I, inosine, K, G or T; R, A or G; M, A or C; W, A or T; Y, C or T. Na = not available
(206) In order to find an NRPS in A. bisporigera, the inventors first contemplated that amatoxins were synthesized via a non-ribosomal peptide synthetase (NRPS) as found in other types of fungi (see, example in
(207) The inventors generated more than 70 MB of DNA sequence and searched using BLAST and more than 20 known NRPS genes and proteins from prokaryotes and eukaryotes for evidence for an NRPS in the genome of A. bisporigera. However, the inventors did not find evidence for any NRPS-like sequence in A. bisporigera. In contrast, the inventors discovered that the most closely related sequences to NRPSs were orthologs of aminoadipate reductase and acyl-CoA synthase, which, like bacterial and fungal NRPSs, are classified within the aminoacyl-adenylating superfamily (Finking et al., (2004) Annu. Rev. Microbiol. 58:453; herein incorporated by reference).
(208) Approximately 59% of the Amanita bisporigera sequences of the present inventions did not show a hit to the GenBank NR database. This is consistent with results from other fungal genome projects (see, e.g. Schulte, U (2004) Genomics of filamentous fungi. In Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine (J S Tkacz & L Lange, eds.):15-29. Kluwyer Academic/Plenum Publishers, New York; herein incorporated by reference). Little annotation is yet available for fungal genomes, so the proportion of unidentified sequences is high. Three thousand eight sequences that produced no hits to GenBank NR did yield hits to the Phanerochaete chrysosporium and/or Coprinopsis cinereus genomes. The following known genes were identified using BLAST comparisons of the novel Amanita fragments of the present inventions. The inventors found matches contemplated to be Amanita homologs to members of the aminoacyl-adenylating superfamily (Finking et al., (2004) Annu Rev Microbiol 58:453-488; herein incorporated by reference) which includes but is not limited to exemplary sequences of L-aminoadipate-semialdehyde dehydrogenase. In particular, L-aminoadipate-semialdehyde dehydrogenase is related to but is not a non-ribosomal peptide synthetase (NRPS), an enzyme originally contemplated to be responsible for Amanita peptide toxin biosynthesis. The inventors ruled out a NRPS identity of this match after they sequenced the remainder of the clone 16_c01KoreaM13Rrc, then extended the sequence by approximately 700 by using inverse PCR.
(209) Cap64 is a capsule formation protein first identified in the pathogenic basidiomycete Filobasidiella neoformans with a known homolog in the saprophytic basidiomycete Pleurotus ostreatus, of which the later does not form capsules associated with mammalian pathogenicity. The discovery of an AmanitaCap64 homologous sequence was not expected because like Pleurotus, Amanita species are not known to form capsules associated with mammalian pathogenicity.
(210) Laccases, like Cap64, were not expected even though they were previously found to be widespread in saprophytic fungi (Coprinopsis, Melanocarpus, and the white rot fungus Trametes), and in both asco- and basidiomycetes. Their role in an ectomycorrhizal fungus such as Amanita, which is expected to obtain most of its nutrients in the form of photosynthate and would therefore lack the need to degrade plant tissue, is unknown.
(211) Therefore, despite predictions to the contrary, the inventors did not find evidence of an NRPS gene that would likely be involved with synthesizing amatoxins and phallotoxins (Walton et al. (2004) Peptide synthesis without ribosomes. In: Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine. J Tkacz, L Lange, eds. Kluwer Academic, New York, pp. 127-162; herein incorporated by reference). Yet on the other hand, surprisingly, the inventors discovered other types of genes.
Example IV
(212) This example describes exemplary compositions and methods for identifying amatoxin-encoding genes. The inventors initially focused on amatoxins, in particular amanitins, bicyclic octapeptides which are more potent toxins to humans than any of the other mushroom toxins and are directly responsible for the majority of fatal human mushroom poisonings. Specifically, this example describes the discovery of an A. bisporigera gene sequence contemplated to encode alpha amanitin.
(213) An exemplary structure of -amanitin is cyclic(L-asparaginyl-4-hydroxy-L-prolyl-(R)-4,5-dihydroxy-L-isoleucyl-6-hydroxy-2-mercapto-L-tryptophylglycyl-L-isoleucylglycyl-L-cysteinyl), cyclic (4-8)-sulfide, (R)S-oxide (ChemIDplus), wherein the amino acids have the L configuration and several amino acids are modified by hydroxylation. When simplified to the 20 proteogenic amino acids, the chemical name became cyclic(NPIWGIGC) (SEQ ID NO:46) (ChemIDplus). However because this is a cyclized peptide, the order in which the amino acids are assembled biosynthetically was unknown. Moreover, the structure of -amanitin, RN: 21150-22-1 was based upon the known chemical structure of -amanitin RN: 23109-05-9 and named in a similar manner. See chem.sis.nlm.nih.gov/chemidplus/ProxyServlet?objectHandle=DBMaint&actionH-andle=default&nextPage-jsp/chemidheavy/ResultScreen.jsp&ROW_NUM=0&TX TSUPER-LISTID=023109059.
(214) Therefore, the inventors searched the DNA sequences from their A. bisporigera genome seeking DNA fragments capable of encoding amino acid sequences of amanitins, such as predicted sequences comprising a predicted sequence of NPIWGIGC (SEQ ID NO:46). Thus the inventors discovered an exemplary sequence encoding -amanitin, ECIMO1V02FKY4Z S CCCAACTAAATCCCATTCGAACCTAACTCCAAGACCTCTAAACCTCACA ATCCCAATGTCTGACATCAATGCTACCCGTCTCCCATCTGGGGTATCGG TTGCAACCCGTGCG, length=113 (SEQ ID NO:48) encoding prepropetide PTKSHSNLTPRPLNLTIPMSDINATRLPIWGIGCNPC (SEQ ID NO:49), propeptide in BOLD, underlined peptide, SEQ ID NO: 50. The inventors' exemplary sequence translated into a IWGIGCNP, SEQ ID NO: 50, which the inventors contemplate would be capable of forming a, cyclo(IWGIGCNP), SEQ ID NO: 51, wherein the inventors further contemplated several posttranslational hydroxylations and a sulfoxide crossbridge between the Trp and the Cys in order to form the bicyclic peptide known as alpha-amanitin. The inventors used the amino acid sequence and the nucleic acid sequences encoding IWGIGCNP (SEQ ID NO: 50) for searching known sequences in GenBank's non-redundant database. There was no evidence of any gene encoding or protein with IWGIGCNP (- and -amanitins) (SEQ ID NO: 50). Therefore, the inventors contemplated that these sequences are unique for A. bisporigera and further these sequence orders were unlikely to be present in an Amanita genome by statistical coincidence.
(215) The inventors also obtained a second and longer sequence comprising nucleotides encoding IWGIGCNP (SEQ ID NO: 50) using inverse PCR (AMA1 forward and reverse primers, see above) and obtained a genomic sequence contig 49252 AATCTCAGCGTTCAGTACCCAACTCCCATTCGAACCTAACTCCAAGACCT CTAAACCTCACAATCCCAATGTCTGACATCAATGCTACCCGTCTCCCCAT CTGGGGTATCGGTTGCAACCCGTGCGTCGGTGACGACGTCACTACG, length=146 (SEQ ID NO:52) encoding SQRSVPNSHSNLTPRPLNLTIPMSDINATRLPIWGIGCNPCVGDDVTT (SEQ ID NO:53), propeptide in BOLD, underlined peptide, SEQ ID NO: 50.
(216) Therefore the inventors found nucleotide sequences that encode the amino acid sequence of .alpha.-amanitin with the sequence order of IWGIGCNP, in single letter code, and further identified two larger genomic sequences encoding an IWGIGCNP amanitin peptide in the genome of A. bisporigera. The inventors contemplated that amanitins would be a cyclic permutation of linear peptides of IWGIGCNP (- and -amanitins) and IWGIGCDP (SEQ ID NO:54) (- and -amanitins).
Example V
(217) This example demonstrates using amino acid and nucleic acid information of the present inventions, inverse PCR and RACE methods to identify a cDNA and a large genomic fragment that comprises an amanitin gene as indicated in
(218) The inventors initiated a genomic survey using nucleic acid coding regions encoding the AMA1 gene, as described in the previous Example. SEQ ID NOs: 48, 49, 52, and 53, encoding an AMA1 polypeptide, were used to design AMA1 forward and reverse primers that were used in an inverse PCR reaction to obtain a larger genomic fragment of the AMA1 gene. Specifically, inverse PCR, using circularized PvuI generated genomic fragments as target (template) DNA resulted in the isolation of a 2.5-kb fragment of flanking genomic DNA.
(219) RACE (Rapid Amplification of cDNA Ends) (for example, see, Frohman et al., (1988) Proc Natl Acad Sci 85:8998-9002; herein incorporated by reference), was used to obtain a full-length cDNA copy of AMA1, SEQ ID NO:55, encoding an AMA1 polypeptide, SEQ ID NO:56. When compared to the AMA1 genomic sequence, SEQ ID NO:57, the cDNA indicated that AMA1 contains three introns (53, 59, and 58 nt in length), with canonical GT/AG boundaries. Two of the introns were in the 3 untranslated region, while the first intron was in the third codon from the end of the coding region (
(220) TABLE-US-00005 TABLE5 ExamplesofRACEprimersusedherein. SEQUENCE SEQID Name SEQUENCE NO:XX GeneRace 5 5-GCACGAGGACACUGACAUGGACUGA-3 SEQID Primer NO:6 GeneRacer 5 5-GGACACTGACATGGACTGAAGGAGTA-3 SEQID NestedPrimer NO:58 GeneRacer 3 5-GCTGTCAACGKFACGCTACGTAACG-3 SEQID Primer NO:8 3 AMA1RACE 5 CCCATTCGAACCTAACTCCAAGAC3 SEQID initialprimer NO:9 3 AMA1RACE 5 CCTCTAAACCTCACAATCCCAATG3 SEQID primer,nested NO:10 primer 5 AMA1RACE 5 GCCCAAGCCTGATAACGTCCACAACT3 SEQID cDNA,primer NO:11 5 AMA1RACE 5 TATCGCCCACTACTTCGTGTCATA3 SEQID cDNA,nested NO:12 primer 3 PHA1,initial 5 GACCTCTGCTCTAAATCACAATG3 SEQID primer NO:13 3 PHA1,nested 5 ATCAATGCCACCCGTCTTCCTG3 SEQID primer NO:14 5 PHA%initial 5 CGGATCATTTACGTGGGTTTTA3 SEQID primer NO:15 5 nested 5 AACTTGCCTTGACTAGTGGATGAGAC3 SEQID primer NO:16
(221) Thus an exemplary amino acid sequence of the proprotein of AMA1 is MSDINATRLPIWGIGCNPCIGDDVTTLLTRGEALC, SEQ ID NO:617, underlined peptide, SEQ ID NO: 50. The inventors further contemplated an exemplary structure of -amanitin, wherein Asn is replaced by Asp to provide IWGIGCDP, SEQ ID NO:54. Indeed, further investigations described below, did result in the finding of an Amanita PCR product encoding a -amanitin, sequence.
(222) An RNA blot of total RNA extracted from mushrooms of Amanita bisporigera probed with DNA fragment SEQ ID NO: 48 showed an approximately 400 nt band contemplated as an AMA1 mRNA. Minor discrepancies between the genomic and cDNA sequences are likely due to natural variation among the amatoxin genes.
Example VI
(223) This example describes the discovery of an A. bisporigera gene sequence contemplated to encode a phallotoxin, specifically a phallacidin toxin sequence.
(224) An exemplary structure of phallacidin is a cyclic(L-alanyl-2-mercapto-L-tryptophyl-4,5-di hydroxy-L-leucyl-L-valyl-er-ythro-3-hydroxy-D-alpha-aspartyl-L-cysteinyl-cis-4-hydroxy-L-prolyl)cyclic (2-6)-sulfide, RN: 26645-35-2, with predicted amino acid sequences simplified to the 20 proteogenic amino acids comprising cyclo(ATCPAWL), SEQ ID NO:70. Another phallotoxin, phalloidin, RN: 17466-45-4, is a cyclic(L-alanyl-D-threonyl-L-cysteinyl-cis-4-hydroxy-L-prolyl-L-alanyl-2-mercapto-L-tryptophyl-4,5-dihydroxy-L-leucyl), cyclic (3,6)-sulfide, which translates into the sequence cyclo(ATCPAWL), SEQ ID NO:70. Several of the phallacidin and phalloidin amino acids are hydroxylated. The Asp residue (which is replaced by Thr in phalloidin) has the D configuration at the alpha carbon.
(225) A genomic survey of A. bisporigera sequences yielded at least 2 nucleic acid sequences encoding a predicted sequence comprising a linear AWLVDCP, SEQ ID NO:69, which would encode cyclicphallacidin (SEQ ID NO:71), for example, SEQ ID NO:72, ECGK9LO01B8L63 S TGAGGAGACGGTTGACGTCGTCACCGACGCATGGGCAGTCTACAAGCCA AGCAGGAAGACGGGTGGCATTGATGTCAGACATTGTGATTTAGAGTAG, length=97 encoding LLITMSDINATRLPCVGDDVNRLL, SEQ ID NO:73, and SEQ ID NO:74, contig73170, TGAGGAGACGGTTGACGTCGTCACCGACGCATGGGCAGTCTACAAGCCA AGCAGGAAGACGGGTGGCATTGATGTCAGACATTGTGATTTAGAGTAGA GGTCTTGGGTTCGAGTTCGAATGGGAGGTAAG, length 130, encoding a prepropeptide LTSHSNSNPRPLLITMSDINATRLPAWLVDCPCVGDDVNRLL, showing the propeptide in BOLD and underlined peptide SEQ ID NO:69.
(226) Inverse PCR following PvuI and SacI digestion of whole genomic DNA and ligation was used to isolate genomic fragments of 1.6 kb and 1.9 kb, respectively, named phallacidin sequence PHA1#1-1893 bp. SacI, SEQ ID NO:76, and phallacidin-sequence PHA1#2-1613 nt. PvuI, SEQ ID NO:77, collectively named PHA1, comprising phallacidin amino acid sequences. These were two different classes of sequences, identical in the region of phallacidin, SEQ ID NO:78, but diverged approximately 135 nt upstream. These two sequences showed that A. bisporigera genome has at least two copies of the PHA1 gene, both of which encode a phallacidin toxin sequence,
(227) Nucleotide sequences of a cDNA for PHA1 are shown in
(228) Because two different phallacidin genomic sequences were obtained, the inventors contemplate that A. bisporigera has at least two copies of PHA1. Further, the inventors concluded that these two PHA1 sequences represent natural variants of the phallacidin gene because both are present in the same isolate of A. bisporigera. The inventors further contemplate that these two PHA1 genes arose as a gene duplication event.
Example VII
(229) This example describes methods and results from exemplary comparisons of AMA1 and PHA1 for obtaining exemplary consensus sequences.
(230) Based on the cDNA sequence, the inventors chose the first ATG sequence downstream of the transcriptional start site as the translational start site of the proprotein polypeptides and the first in-frame stop codon as the translational stop. AMA1 and PHA1 nucleic acid and predicted amino acid sequences were compared by alignment of each set of two target sequences using a BLAST engine for local alignment through the NCBI website, (world wide web.ncbi.nlm.nih.gov/blast/b12 seq/wblast2.cgi).
(231) Alignment of the predicted proproteins, amanitin to phallacidin sequences, is shown in
(232) When the inventors compared the sequences of genomic and cDNA copies of AMA1 and PHA1, the inventors observed that both comprise 3 introns (approximately 57, 70, and 51 nt in length), in approximately the same positions. Furthermore, AMA1 and PHA1 gene sequences and their translation products were found to be similar in overall size and sequence, except strikingly in the region encoding the peptide toxins themselves (
(233) Within amino acid encoding regions (the proproteins), nucleic acid sequence regions upstream of IWGIGCNP (amatoxin) and AWLVDCP (phallotoxin (SEQ ID NO:69)) comprise 28 of 30 identical nt (93%), while regions downstream of IWGIGCNP and AWLVDCP comprise 41 of 50 identical nt (82%). However, these findings were in contrast to the amatoxin and phallotoxin-encoding regions themselves (IWGIGCNP and AWLVDCP) where merely 12 of 24 nt were identical (50%). Thus the inventors designated these proprotein areas of .alpha.-amanitin and phallacidin as being composed of three domains, one conserved upstream region (A), one conserved downstream region (B), and a hypervariable peptide region (P) encoding amatoxin and phallotoxin. In other words, proprotein sequences of the present inventions consist of an upstream conserved region (A), a downstream conserved region (B) in relation to a variable region (P), such that the variable Amanita cyclic peptide toxin region is flanked by two conserved regions, (
(234) Because amatoxins contain 8 amino acids and phallotoxins contain 7 amino acids, the inventors inserted a 3-nucleotide gap (---) in the cDNA sequence and a one-amino acid space (-) in the proprotein sequence in order to emphasize the alignment of the conserved sequences downstream of the amatoxin and phallotoxin-encoding regions (
(235) TABLE-US-00006 TABLE6 ExemplarycomparisonsbetweenAMA1andPHA1usingBLASTN. ComparisonandIdentity No.aa/No.aa SEQIDNO: Sequence (percentidentity) AMA1A. atgtctgacatcaatgct SEQIDNO: acccgtcttccc(30aa) 182 PHA1A, atgtctgacatcaatgcc AMA1Av.PHA1A SEQIDNO: acccgtcttccc(30aa) 29/30(96%), 18 AMA1B. tgcatcggtgacgacgtc SEQIDNO: actacactcctcactcgt 19 ggcgaggccctttgt(51aa) PHA1B, tgcgtcggtgacgatgtc AMA1Bv.PHA1B SEQIDNO: aaccgtctcctcactcgt 41/50(82%) 20 ggcgagagcctttgg(48aa) AMA1toxin, atctggggtatcggttgcaacccg SEQIDNO: (24aa) 21 PHA1toxin, gcttggcttgtagattgc---cca AMA1toxinv.PHA1toxin SEQIDNO: (21aa) 12/24 22 (50%)
(236) TABLE-US-00007 TABLE7A ExemplaryBLASTsearchesforAMA1andPHA1usingBLAST. Comparison andIdentity Query No.na/No. percent SEQ Hit na identity Alpha- Rhodococcussp.gb|CP000431.1| 28/32 87% Amanitin CGGGTACAACACGTGCATCGGTGACGC CGTCA ZebrafishDNAsequenceemb|CR385042.30| 28/33 84% CGACACTACCCTCACCACTCGTGCCCTT AGTTA Phallacidin Agrobacteriumtumefaciensgb|AE009415.1.| 31/35 88% TCTGTGACGATGTCATCCAGTCTC- TCACTCGTA CP000479.1Mycobacteriumavium104 28/33 84% CGTCGGTGACGATGTACACCGTCGCCA CGCTCG AC112739.5Rattusnorvegicus7BAC 26/30 86% CH230-108A12 TGTCAACCGTCTCCTCTGTCGTTTCCTTT G XM_382946.1GibberellazeaePH-1 25/28 89% chromosone1conservedhypotheticalprotein (FG02770.1)partialmRNA CGTCGGTGACGATGTCCTCCGTCTCTTC AM444890.2Vitisviniferacontig 22/23 95% TTGTAGACTGCCCATGCGTCTGT gb|AAQY01001277.1| Phytophthorasojae 21/21 100% strain P6497CGGTGACGATGTCAACCGTCT gb|AAQR01490933.1| Otolemurgarnettii 21/21 100% cont1.490932 TGTCTGACATCAATGCCACCC
(237) TABLE-US-00008 TABLE7B ExemplaryBLASTsearchesforAMA1andPHA1usingBLASTN. Compatisonand IdentityNo. percent SEQIDNO: QuerySEQ Hit na/No.na identity 524 AmanitinA ATGTCTGACATCAATGCTACCCGTCTCCCC 30/30 100% 563 ref|XM__01182437.1| PREDICTED: 19/20 95% StrongylocentrotuspurpuratussimilartoESP-1 (LOC574923),purpleseaurchin TGTCTGACATCAATGGTACC 530 dbj|AK173931.1| CionaintestinaliscDNA, 18/18 100% ATGTCTGACATCAATGCT 564 ref|XM_001365250.1| Monodelphisdomestica 17/17 100% similartotransducinbeta-3-subunitmRNAshort- tailedopossums, GTCTGACATCAATGCTA 568 ref|XM_814507.1| TrypanosomacruzistrainCL 16/16 100% Brenerkinesin AATGCTACCCGTCTCC 565 ref|XM_652576.1| AspergillusnidulansFGSCA4 16/16 100% hypotheticalprotein (AN0064.2TGTCTGACATCAATGC 537 emb|BX842594.1| NeurosporacrassaDNA 16/16 100% linkagegroupIIBACcloneB18P7 TGTCTGACATCAATGC 532 dbj|AP007162.1| AspergillusoryzaeRIB40 16/16 100% genomicDNA,SC102 CTGACATCAATGCTAC 82 PhallacidinA ATGTCTGACATCAATGCCACCCGTCTTCCC 30/30 100% 567 ref|XM_753671.1| Cornsmutisofmaizecaused 20/21 95% bythepathogenicplantfungusUstilagomaydis CATCAATGCCACCCGCCTTCC 542 gb|AC122231,2| MusmusculusBACclone 19/19 100% RP23- 135M3ATGTCTGACATCAATGCCA 536 emb|AL031736.16| HumanDNAsequencefrom 19/19 100% cloneRP4- 738P11ATGTCTGACATCAATGCCA 562 ref|NM_202010.2| ArabidopsisthalianaFUS5 18/18 100% (FUSCA5);MAPkinasekinase (FUS5)CAATGCCACCCGTCTTCC 566 ref|XM_652576.1| Aspergillusnidulans 18/18 100% FGSCA4hypotheticalprotein (AN0064.2) TGTCTGACATCAATGCCA 533 dbj|AP008214.1| Oryzasativa(japonicacultivar- 18/18 100% group)genomic TCTGACATCAATGCCACC 543 gb|EF469872.1| HelianthusannuusRFLPprobe 17/17 100% ZVG13mRNAsequence AATGCCACCCGTCTTCC 538 emb|CR619305.1| Bcells(Ramoscellline) 17/17 100% GTCTGACATCAATGCCA 538 emb|CR595196.1| Tcells(Jurkatcellline) 17/17 100% GTCTGACATCAATGCCA 538 emb|CR592893.1| NeuroblastomaofHomo 17/17 100% sapiens(human) GTCTGACATCAATGCCA 531 dbj|AK173931.1| CionaintestinalisorSeasquirt. 17/17 100% ATGTCTGACATCAATGC 525 AmanitinB TGCATCGGTGACGACGTCACTACT 45 100% CTCCTCACTCGTGCCCTTTGT 573 Strongylocentrotuspurpuratus 19/19 100% CATCGGTGACGACGTCACT 548 Ostrococcuslucimarinusunicellularcoccoid 18/18 100% greenalga CGATCGGTGACGACGTCA 529 Chaetomiumglobosumdematiaceousfilamentous 18/18 100% fungusinfectousinhumus CTCCTCACTCGTGCCCTT 546 HumanDNAsequencefromcloneXXyac-60D10 18/18 100% TCACTACTCTCCTCACTC 561 RattusnorvegicusLEA_4domaincontaining 17/17 100% protein ACGTCACTACTCTCCTC 526 AtlanticSalmon 17/17 100% CTCCTCACTCGTGCCCT 527 BurkholderiacenocepaciaGram-negative 17/17 100% bacteriaPathogen ATCGTGACGACGTCAC 547 OmithorhynchusanatinusPlatypus 17/17 100% ACGTCACTACTCTCCTC 82 PhallacidinB TGCGTCGGTGACGATGTCAACCGT 45 100% CTCCTCACTCGTAGCCTTTGG 528 ChaetomiumglobosumCBS148.51 24/26 92% GGTGACGATGACAACCGCCTCCTCAG 545 Giberellazeae 23/25 92% CGTCGGTGACGATGTCCTCCGTCTC 571 Rhizobiumleguminosarumbv.viciae 20/21 95% chromosome CGTCGGTGACGAGGTCAACCG 574 Tetraodonnigroviridis 19/19 100% GATGTCAACCGTCTCCTCA
(238) The conserved amino acid regions encoded by conserved domains A and B and consensus region B were used as query sequences for BLAST searching the GenBank public NR database. These sequences per se were not found within the database, however somewhat similar sequences were discovered, with exemplary sequences shown below.
(239) TABLE-US-00009 TABLE8 ExemplaryhomologycomparisonsusingConsensusMSDINATRLP,XWXXXCXP, andCVGDDVXXLLTRALCasquerysequencesusingBLASTP (MSDINATRLPXWXXXCXPCVGDDVXXLILTRALC,SEQIDNO:45). IdentityNo. SEQUENCE aa/matchingNo.aa GenBanksequencehit AMA1 7/10(70%), gb|EDN21666.1| predictedprotein ConservedA [BotryotiniafuckelianaB05.10] MSDINATRLP SEQIDNO:46 7/8(87%), gb|EAT86097.1| hypotheticalprotein SNOG_06266[Phaeosphaeria nodorumSN15] 7/9(77%), gb|EAK82279.1| hypotheticalprotein UM01662.1[Ustilagomaydis521] 6/9(66%) gb|EAU90435.1| predictedprotein [Coprinopsiscinereaokayama7#130] MREINSTRLP7/10 predictedprotein[Botryotinia (70%) fuckelianaB05.10].Pathogenic fungus(akaBotrytiscinerea)that causesgraymoldrotinplants MSNIAAPRLP7/10 gb|ABD10583.1| EndopeptidaseClp (70%) [Frankiasp.CcI3] MSDIAWIIPDNATR hypotheticalproteinCC1G_09232 8/13(61%) [Coprinopsiscinereaokayama7#130] SDVNAPRLP7/9 hypotheticalproteinUM01662.1 (77%) [Ustilagomaydis521] SDI-ATRLP8/9 non-ribosomalpeptidesynthetase (88%) [Saccharopolysporaerythraea NRRL2338] AMA1 8/11(72%) gb|ABF87913.1|ATP-binding Conserved protein,ClpXfamily[Myxococcus RegionB xanthusDK1622] CIGDDVTTLL TRGEALC SEQIDNO: 618 8/10(80%) emb|CAG61741.1|unnamedprotein product[CandidaglabrataCBS138] 10/16(62%) gb|EAK84527.1|hypotheticalprotein UM03624.1[Ustilagomaydis521] 11/16(68%) gb|EAU39589.1|conserved hypotheticalprotein[Aspergillus terreusNIH2624] 8/8(100%) dbj|BAE56937.1|unnamedprotein product[Aspergillusoryzae] PHA1 14/21(66%) gb|AAZ10451.1|hypotheticalprotein Conserved Tb927.3.4180[Trypanosomabrucei] RegionB CVGDDVNRL LTRGESLC SEQID NO:89 11/18(61%) gb|EAQ84320.1|hypothticalprotein CHGG_10724[Chaetomium globosumCBS148.51] 9/11(81%) gb|ABE92653.1|Peptidase,cysteine peptidaseactivesite;Aromatic- ringhydroxylase[Medicago truncatula] 9/14(64%) gb|EDN63642.1|conservedprotein [SaccharomycescerevisiaeYJM789] ConsensusB 9/14(64%) ref|XP_760134.1|hypothetical CXGDDVXXL GDDVAALLSRRVLC proteinUM03987.1[Ustilagomaydis LTRXLC 521] SEQID NO:91 8/12(66%) ref|ZP_00591779.1|ClpX,ATPase regulatorysubunit[Prosthecochloris aestuariiDSM271] greensulfur bacterium
Example VIII
(240) This example describes materials and methods for determining whether the amatoxin and phallotoxin-encoding nucleic acids are specific for Amanita mushroom species that produce amatoxins and phallotoxins.
(241) Many secondary metabolites such as mushroom peptide toxins are limited in their taxonomic distribution; for example, most species of Amanita do not make amatoxins or phallotoxins. Thus the inventors contemplated whether the lack of amatoxin and phallotoxin production among other species of Amanita was due to absence of the encoding genes or due to the absence of productive translation of the genes. The inventors tested for the presence of amatoxins such as alpha-amanitin and phallotoxins such as phallacidin and in the same mushrooms tested for the presence of DNA encoding alpha amanitin (AMA1) and phallacidin (PHA1). The inventors tested for the presence of AMA1 and PHA1 in the genomes of known amatoxin and phallotoxin-producing mushroom species and non-producing mushroom species in order to associate the AMA1 and PBA1 sequences with amatoxin and phallotoxin production.
(242) Preparation and Isolation of Amanita Genomic Sequences. DNA was extracted from a variety of species of Amanita that were either known as amatoxin and phallotoxin-producers (A. bisporigera, A. ocreata, A. aff. suballiacea and A. phalloides) or were known to not produce amatoxins (A. novinupta, A. franchetti, A. porphyria, A. velosa, A. gemmata, A. muscaria, A. flavoconia, A. section Vaginatae, and A. hemibapha). DNA was extracted from lyophilized fruiting bodies using cetyl trimethyl ammonium bromide-phenol-chloroform isolation (Hallen, (2003) Mycol. Res. 107:969; herein incorporated by reference). Following the usual preparation methods, sequences were separated by gel electrophoresis and then transferred to blotting media for subsequent probe hybridization.
(243) Southern blots of DNA were probed with AMA1 and PBA1 as described. As shown in
(244) The results showed that AMA1 and PHA1 sequences hybridized to DNA from known amatoxin and phallotoxin-producing species but did not hybridize to the species known to not produce these compounds. The inventors concluded that these genes were present in amatoxin and phallotoxin-producing species and absent in non-producers, thus providing additional evidence that the genes described herein encode amatoxins and phallotoxins.
(245) Extraction and analysis of amatoxins and phallotoxins. Variability in toxin content is known even within species of Amanita that normally produce amatoxins and phallotoxins (Beutler, et al., (1981) J. Nat. Prod. 44:422 and Tyler, et al., (1966) J. Pharm. Sci. 55:590; all of which are herein incorporated by reference in its entirety). Therefore in order to confirm that the presence of AMA1 and PHA1-encoding sequences correlates with actual production of amatoxins and phallotoxins, the inventors tested the same mushrooms that were used for extraction of DNA and Southern blotting (
(246) Each of the tested mushrooms that contain amatoxins and phallotoxins, but none of the nonproducers, hybridizes to AMA1 and PHA1. This is consistent with AMA1 and PHA1 as being responsible for alpha-amanitin and phallacidin biosynthesis and provides a molecular explanation for why Amanita species outside of sect. Phalloideae are not deadly poisonous. Some of the species of Amanita that do not make amatoxins or phallotoxins are edible, but others make toxic compounds chemically unrelated to the Amanita cyclic peptide toxins.
Example IX
(247) This Example demonstrates PCR amplification of an -amanitin gene in mushroom species known to produce -amanitin while failing to amplify DNA from species that do not produce alpha-amanatin (
(248) PCR amplification of the gene for -amanitin. Primers were based on the sequences in
(249) A. gemmata and A. muscaria are species of Amanita that do not make amatoxins (or phallotoxins) and did not yield a PCR product using these primers (
Example X
(250) This Example shows the development of conserved regions upstream and downstream of Amanita peptide encoding regions.
(251) The unexpected complex hybridization patterns shown in
(252) The conserved upstream and downstream amino acid sequences of AMA1 and PHA1 were used as queries using BLASTP to search for additional related sequences in the A. bisporigera genome sequence database. The inventors thereby found at least 12 new related DNA sequences that could encode proproteins as long or longer than the proproteins of AMA1 and PHA1 and another 10-15 partial sequences (missing the upstream or the downstream conserved sequences) see exemplary sequences, including partial sequences in
Example XI
(253) This example describes methods and results of using conserved regions of AMA1 and PHA1 for obtaining additional regions encoding potentially biologically active linear or cyclic peptides from A. bisporigera, A. phalloides, and other species of Amanita. In particular, a DNA sequence encoding amino acid sequences was found that was highly similar to -amanitin and comprising the amino acid sequence found in .beta.-amanitin, and a DNA that was highly similar to phallacidin and comprising the amino acid sequence found in phalloidin.
(254) During the course of developing the present inventions, the inventors discovered regions of conserved sequence whose use resulted in the discovery of additional sequences contemplated to encode proproteins related to amatoxin and phallotoxin proproteins, which could encode novel small linear or cyclic peptides. Degenerate primers were designed against the conserved sequences of AMA1 and PHA1. DNA extracted from A. phalloides and A. ocreata was used as template. This also shows that the AMA1 and PHA1 genes and related genes are conserved in other species of amatoxin and phallotoxin-producing Amanita species, and that PCR primers designed against one species (A. bisporigera) function to identify amatoxin and phallotoxin genes in other species of Amanita.
(255) New degenerate PCR primer sequences that the inventors developed and used on genomic DNA as a template were 5-ATGTCNGAYATYAAYGCNACNCG (forward), SEQ ID NO: 97, and 5-AAGGSYCTCGCCACGAGTGAGGAGWSKRKTGAC (reverse), SEQ ID NO: 98, W indicates A or T, S indicates C or G, K indicates G or T, R indicates A or G, and Y indicates T or C. The resulting PCR products (approximately 100 nt) were cloned and sequenced. Exemplary sequences of three amplicons are:
(256) number 1: ATGTCTGATATTAATGCAACGCGTCTTCCCTTCAATATTCTGCCATTCAT GCTTCCCCCGTGCGTCAGTGACGATGTCAATATACTCCTCACTCGTGGCG AG, SEQ ID NO: 99, translation: MSDINATRLPFNILPFMLPPCVSDDVNILLTRGE, SEQ ID NO: 110, [predicted to encode a unique linear and cyclic peptide, underlined, SEQ ID NO: 114]; number 2: ATGTCAGATATCAATGCGACGCGTCTTCCCATATGGGGAATAGGTTGCG ACCCGTGCATCGGTGACGACGTCACCATACTCCTCACTCGTGGCGAG translation, SEQ ID NO: 101, MSDINATRLPIWGIGCDPCIGDDVTILLTRGE, SEQ ID NO: 102, [predicted to encode beta-amanitin SEQ ID NO:54]; number 3: ATGTCGGATATTAATGCTACACGTCTTCCAATTATTGGGATCTTACTTCC CCCGTGCATCGGTGACGATGTCACCCTACTCCTCACTCGTGGCGAG, SEQ ID NO: 103, [translation: MSDINATRLPIIGILLPPCIGDDVTLLLTRGE, SEQ ID NO: 47, [predicted to encode a unique linear or cyclic peptide, underlined SEQ ID NO: 117]; and number 4: ATGTCAGACA TTAACGCGAC CCGTCTTCCCGCCTGGCTCGCCACCTGCCCGTGCGCCGGTGACGACGTCA ACCCTCTCCT CACTCGTGGC GAG, SEQ ID NO: 105, translation: MSDINATRLPAWLATCPCAGDDVNPLLTRGE, SEQ ID NO: 106, [predicted to encode phalloidin, underlined (SEQ ID NO:136].
(257) TABLE-US-00010 TABLE 9 Exemplary comparisons of Amanita peptide sequences. Identity No. na/ Percent Preprotprotein nucleic acid matching No. na Identity Alpha-Amanitin vs. new peptide 1 35/41 85% SEQ ID NO: 114 Alpha-Amanitin vs. new peptide 2, 79/91 86% beta-Amanitin Alpha-Amanitin vs. new peptide 3 36/41 87% SEQ ID NO: 117 Phallacidin vs. new peptide 1 SEQ 34/40 85% ID NO: 114 Phallacidin vs. new peptide 2, beta- 33/40 82% Amanitin Phallacidin vs. new peptide 3 SEQ 35/40 87% ID NO: 117
(258) The inventors then initiated a BLASTN and TBLASTN search of the Amanita bisporigera genome DNA sequences using conserved region A for identifying homologous sequences. The inventors discovered numerous nucleic acid sequences encoding MSDINVTRLP SEQ ID NO:88 or versions thereof, followed by variable short regions that were in turn followed by regions homologous to regions B of AMA1 and PHA1, see,
(259) TABLE-US-00011 TABLE10A ExemplarycomparisonstoAMA1andPHA1. Name Proprotein Identity [amanitin] MSDINATRLPIWGIGCNP 100% peptide,SEQID CVGDDVITLLTRGE NO:48 SEQIDNO:107 [phallacidin], MSDINATRLPAWLVDCP 25/32(78.1%) SEQIDNO:49 CVGDDVNRLLTRGE SEQIDNO:108 [consensus],SEQ MSDINATRLPXWXXXCAP IDNO:50 CVGDDVXXLLTRGE SEQIDNO:109 newpotential MSDINATRLPFNILPFMLPP AMA123/34 peptide1,SEQ CVSDDVNILLTRGE (67%) IDNO:51 SEQIDNO:110 PHA122/34 (64%) newpotential MSDINATRLPIWGIGCDP AMA129/32 peptide2,SEQ CIGDDVTILLTRGE (90%) IDNO:52 SEQIDNO:111 PHA124/32 (75%) newpotential MSDINATRLPIIGILLPP AMA126/32 peptide3,SEQ CIGDDVTLLLTRGE (81%) IDNO:53 SEQIDNO:112. PHA122/32 (68%) newpotential MSDINATRLPAWLATCPC AMA126/32 peptide4,SEQ AGDDVNPUTRGE (81%) IDNO:54 SEQIDNO:113 PHA122/32 (68%)
(260) TABLE-US-00012 TABLE10B ExemplarycomparisonsusingAmanitapeptidesequencesas querysequencesinGenBank(BLASTP). Alpha- IWGIGCNP 6/8(75%) gb|AAZ19981.1| conserved amanitin (8) IWGIGCVL hypotheticalprotein (AMA1) (SEQIDNO: (SEQIDNO: [Psychrobacterarcticus273-4] 50) 655)(6/8 gv|EAU82808.1| hypothetical (75%) proteinCC1G_11325 [Coprinopsiscinera okayama7#130] Alpha- IWGIGCNP 5/8(40.0%) AWLVDCP(pha1) amanitin (8) (SEQIDNO:69) (AMA1) (SEQIDNO: 50) phallacidin AWLVDCP AWLVDC(SEQ gb|EAV54171.1| simgma54 (PHA1) (7) IDNO:656) specifictranscriptional (SEQIDNO: 6/7(85.5%) regulator,Fisfamily 69) AWVVDCP [Burkholderiaambifaria (SEQID MC40-6] NO:657) gb|AAG04585.1| AE004550_1 6/7 probabletranscriptional (85.5%) regulator[Pseudomonas aeruginosaPAO1] gb|EAL84365.1| conserved hypotheticalprotein [Aspergillusfumigatus Af293] Peptide1 FNILPFMLPP 2/10(20%) AMA1PHA1 SEQID (10) 2/10(20%) ref|ZP_01047917.1| NO:114 8/10(80%) hypotheticalprotein NB311A_09386[Nitrobacter sp.Nb-311A] beta- IWGIGCDP 7/8(87%) AMA1 amanitin (8) 5/8(40.0%) PHA1 SEQIDNO: 7/8(87%) ref|YP_265415.1| 54 hypotheticalprotein Psyc_2134[Psychrobacter arcticus273-4] Peptide3 IIGILLPP(8) 4/8(50%) AMA1 SEQIDNO: 1/8(12.5%) PHA1 117 7/8(87%) gb|ABR79950.1| hypothetical IIGILLP protein[Klebsiellapneumoniae 7/7(100%) subsp.pneumoniaeMGH 78578] ref|YP_001292803.1 hypotheticalprotein [Haemophilusinfluenzae PittGG] ref|XP_001139896.1| PREDICTED:prolyl4- hydroxylase,alphaIsubunit isoform2[Pantroglodytes]
(261) TABLE-US-00013 TABLE10C ExemplarysequencesrelatedtoAMA1andPHA1.Predictedamino acidsequencesencodedbygenomicsurveysequencesofA. bisporigera(FIG.7).Spacesweresometimesinsertedbefore andafterthepeptide/toxinregions(underlined),whenthe peptide/toxinreigonhadfewerthan10predictedaminoacids, inoredertoemphasizetheconservationoftheupstreamand downstreamsequences.*indicatesstopcodon.Thesearegenomic surveysequences.BasedonthecDNAsequencesofAMA1andPHA1, anintrolincontemplatedneartheC-terminusoftheindicated proproteins. SEQIDNO: ExemplaryAmanitapeptides SEQIDNO:23 MSDINATRLPHPFPLGLQPCAGDVDNLTTKGEG SEQIDNO:111 MSDINATRLPIWGIGCDPCIGDDVTILLTRGE SEQIDNO:113 MSDINATRLPAWLATCPCAGDDVNPLLTRGE SEQIDNO:26 MSDINATRLPGFVPILFPCVGDDVNTALT SEQIDNO:27 MSDINATRLPFYQFPDFKYPCVGDDIEMVLARGER* SEQIDNO:28 MSDINATRLPFFQPPEFRPPCVGDDIEMVLTRG* SEQIDNO:29 MSDINATRLPLFLPPVRMPPCVGDDIEMVLTRGER* SEQIDNO:30 MSDINATRLPLFLPPVRLPPCVGDDIEMVLTR SEQIDNO:31 MSDINATRLPYVVFMSFIPPCVNDDIQVVLTRGEE* SEQIDNO:32 MSDINATRLPCIGFLGIPSVGDDIEMVLRH SEQIDNO:44 MSDINATRLPLSSPMLLPCVGDDILMV SEQIDNO:34 MSDINATRLPILMLAILPCVGDDIEVLRRGEG* SEQIDNO:35 MSDINATRLPIPGLIPLGIPCVSDDVNPTLTRGER* SEQIDNO:36 MSDINATRLPGAYPPVPMPCVGDADNFTLTRGEK* SEQIDNO:37 MSDINATRLPGMEPPSPMPCVGDADNFTLTRGN SEQIDNO:118 MSDINATRLPHPFPLGLQPCAGVDNLTLTKGEG*
(262) In particular, the inventors analyzed three sequences encoding short peptides and potential toxins including comparing sequence homology to -amanitin and phallacidin.
(263) TABLE-US-00014 TABLE11 ExemplaryAmanitaPetides. Peptidesequence SEQIDNumber. IWGIGCNP SEQIDNO:50 AWLVDCP SEQIDNO:69 XWXXXCXP SEQIDNO:135 FNILPFMLPP SEQIDNO:114 IWGIGCDP SEQIDNO:54 IIGILLPP SEQIDNO:117 AWLATCP SEQIDNO:136 GFVPILFP SEQIDNO:137 FYQFPDFKYP SEQIDNO:138 FFQPPEFRPP SEQIDNO:139 LFLPPVRMPP SEQIDNO:140 LFLPPVRLPP SEQIDNO:141 YVVFMSFIPP SEQIDNO:142 CIGFLGIP SEQIDNO:143 LSSMLAILP SEQIDNO:144 ILMLAILP SEQIDNO:145 IPGLIPLGIP SEQIDNO:146 GAYPPVPMP SEQIDNO:147 GMEPPSPMP SEQIDNO:148 HPFPLGLQP SEQIDNO:149
Example XII
(264) This example shows the complex hybridization patterns of Example VIII,
(265) Using the conserved upstream and downstream amino acid sequences of AMA1 and PHA1 as queries, the inventors found at least 15 new related sequences (Table 11B) and another 10-15 partial sequences in the genome survey sequence of A. bisporigera. Each of them had an upstream conserved consensus sequence MSDINATRLP (MSD, N, R, and Pare invariant), and a downstream conserved consensus CVGDDXXXXLTRGE (D is invariant). The putative peptide toxin regions, which start immediately downstream of an invariant Pro residue and end just after an invariant Pro residue, are more variable compared to the upstream and downstream sequences. The hypervariable regions contain seven to ten amino acids, while all of the twenty proteinogenic amino acids are represented at least once (
(266) TABLE-US-00015 TABLE11B AMA1andPHA1relatedsequences. FifteenadditionalAMA1andPHA1relatedsequences foundinagenomesurveyofA.bisporigerausing conservedupstreamanddownstreamaminoacid sequencesofAMA1andPHA1asqueries. SEQIDNO:XX MSDINATRLPIWGIGCN--PCVGDDVTTLLTRGE SEQIDNO:303 MSDINATRLPAWLVDC---PCVGDDVNRLLTRGE SEQIDNO:304 MSDINATRLPIWGIGCD--PCIGDDVTILLTRGE SEQIDNO:305 MSDINATRLPIIGILLP--PCIGDDVTLLLTRGE SEQIDNO:306 MSDINATRLPFNILPFMLPPCVSDDVNILLTRGE SEQIDNO:110 MSDINTARLPFYQFPDFKYPCVGDDIEMVLARGE SEQIDNO:308 MSDINTARLPFFQPPEFRPPCVGDDIEMVLTRGE SEQIDNO:309 MSDVNDTRLPFNFFRFPY-PCIGDDSGSVLRLGE SEQIDNO:310 SMDINTARLPLFLPPVRMPPCVGDDIEMVLTRGE SEQIDNO:311 MSDINTARLPYVVFMSFIPPCVNDDIQVVLTRGE SEQIDNO:312 MSDINAIRAPILMLAIL--PCVGDDIEVLRRGEG SEQIDNO:313 MSDINGTRLPIPGLIPLGIPCVSDDVNPTLTRGE SEQIDNO:314 MSDINATRLPGAYPPVPM-PCVGDADNFTLTRGE SEQIDNO:315 MSDINATRLPHPFPLGLQ-PVAGDVDNLTLTKGE SEQIDNO:316 MSDINATRLPAWLATC---PCAGDDVNPLLTRGE SEQIDNO:317
(267) Fifteen sequences listed in Table 11B were used for constructing a WebLogo graphic (Crooks et al., 2004, herein incorporated by reference) showing the relative conservation by letter size representing amino acids, such that highly conserved amino acids are represented by large letters (for example, MSDIN; positions 1-5, and P; positions 10 and 20) while less conserved amino acids have smaller letters (for example A/T, G/S; positions 6 and 23, respectively) and low areas of conserved amino acids have small letters (for example, in regions 11-18). These results showed upstream MSDINATRLP (SEQ ID NO: 88) (MSD, N, R, and P are invariant, consensus was MSDXNXXRXP) and downstream conserved consensus CVGDDXXXXLTRGE (SEQ ID NO: 239) (D is invariant).
Example XIII
(268) This example shows exemplary sequences for amanitin produced by G. marginata mushrooms.
(269) Galerina marginata (a synonym for G. autumnalis) produces amatoxins but not phallotoxins (Benedict et al., 1966). This fungus is contemplated as a potentially valuable experimental system for elucidating the biosynthesis and regulation of amatoxin biosynthesis because, unlike Amanita, it is saprophytic and grows and produces amatoxins in culture (Muraoka and Shinozawa, 2000). Galerina spp. are relatively small and rare, but they nonetheless sometimes cause mushroom poisonings (e.g., Kaneko et al, 2001, herein incorporated by reference, and
(270) Therefore, the inventors sequenced about 40 MB of G. marginata and identified two genomic sequences that could encode alpha-amanitin (GmAMA1) (
(271) An RNA blot of the Galerina marginata amanitin gene (GmAMA1) showed that the gene is expressed in two known amanitin-producing species of Galerina (G. marginata and G. badipes) and not in a nonproducer (G. hybrida), and that the gene is induced by low carbon. Lane 1: G. hybrida, high carbon. Lane 2: G. hybrida, low carbon. Lane 3: G. marginata, high carbon. Lane 4: G. marginata, low carbon. Lane 5: G. badipes, high carbon. Lane 6: G. badipes, low carbon. Each lane was loaded with 15 ug total RNA. The agarose gel was blotted to nitrocellulose by standard methods and probed with the G. marginata AMA1 gene (GmAMA1) predicted to encode alpha-amanitin. Fungi were grown in liquid culture for 30 d on 0.5% glucose (high carbon) then switched to fresh culture of 0.5% glucose or 0.1% glucose (low carbon) for 10 d before harvest. The major band in lanes 3-6 is about 300 bp. The high MW signal in lane 1 is spurious.
(272) Therefore, by RNA blotting, the inventors found that GmAMA1 is expressed in culture and is induced by carbon starvation, as has been reported for the toxin itself (Muraoka and Shinozawa, 2000, herein incorporated by reference) (
(273) Genomic DNA Isolation. Galerina marginata, an amatoxin producing species of circumboreal distribution, was harvested from the wild. Caps and undamaged stems were cleaned of soil and debris, frozen at 80 C., and lyophilized.
(274) Genomic DNA was extracted from the lyophilized fruiting bodies using cetyl trimethyl ammonium bromide-phenol-chloroform isolation (Hallen, et al., (2003) Mycol. Res. 107:969; herein incorporated by reference). For studies requiring RNA, RNA was extracted using TRIZOL (Invitrogen) (Hallen, et al., (2007) Fung. Genet. Biol., 44:1146; herein incorporated by reference in its entirety). The inventors used a Genome Sequencer FLX from 454 Life Sciences (Margulies, et al., (2005) Nature 437:376; herein incorporated by reference) for generating sequences from Galerina species genomic DNA. There was no subcloning necessary. The inventors structured and maintained the sequenced DNA in a password-protected, private BLAST-searchable format.
(275) Therefore, the inventors searched the DNA sequences from their Galerina marginata genome seeking DNA fragments capable of encoding amino acid sequences of amanitins, such as predicted sequences comprising a known predicted sequence of IWGIGCNP. Thus the inventors discovered an exemplary DNA sequence encoding either or both -amanitin and/or -amanatin (these two forms of amanitin have the same amino acid sequence because they differ only in hydroxylation, which is a posttranslational modification). The sequences were compared (BLAST) to Amanita sequences previously discovered by the inventor and disclosed in a Provisional U.S. Patent Application Ser. No. 61/002,650 (
(276) Specifically, PCR primers were designed based on the full-length (248 bp) Genome Sequencer 454 FLX read encoding IWGIGCNP and were used successfully to amplify the predicted amanitin coding region from G. marginata genomic DNA for use as probes in Southern and Northern blots. Primers were also designed for inverse PCR, in order to isolate and sequence DNA upstream and downstream of the amanitin-encoding region. Primers are as follows: A) Gal 454 start F: CCA GTG AAA ACC GAG TCT CCA; SEQ ID NO: 319, B) Gal before MFD F: CAA AGA TCT TCG CCC TTG CCT; SEQ ID NO: 320; C) Gal CDS MFD F: ATG TTC GAC ACC AAC TCC ACT, SEQ ID NO: 321; D) Gal end 454 R: ACA CAT TCA ACA AAT ACT AAC; SEQ ID NO: 322; E) Gal inverse->: GCT GAA CAC GTC GAT CAA ACT; SEQ ID NO: 323; F) Gal inverse<-: TCC ATG GGT TGC AGC CAA TAC; SEQ ID NO: 324. Primer combinations A:D, B:D, and C:D amplify unique PCR products from G. marginata of sizes 244, 201 and 169 bp, respectively; when cloned and sequenced, these PCR products are perfect matches to the Genome Technologies 454 FLX sequence.
Example XIV
(277) This Example describes identifying potential prolyl oligopeptidase (POP)like genes in fungal species.
(278) The inventors discovered during the development of the present inventions, that both sequences of the present inventions and the structurally resolved Amanita cyclic peptides (amatoxins and phallotoxins) contained conserved Prolines. In particular, the inventors found in each predicted peptide sequence a Proline was located downstream of a N-terminal conserved region where proline (Pro) was the last amino acid of the sequence, while the last amino acid in the peptide toxin region itself was always a conserved Pro (for examples,
(279) Based on the conservation of a Pro residue immediately upstream of the peptide toxin region, and of a Pro as the last amino acid in the toxin region of all Amanita peptide toxin family members the inventors contemplated that an enzyme that recognizes and cleaves peptides at the carboxy side of Pro residues catalyzes the first post-translational step in Amanita toxin biosynthesis. Further, Based on the properties of the known proline-specific peptidases (Cunningham, et al., (1997) Biochim Biophys Acta 1343:160, Polgar, (2002) Cell. Mol. Life Sci. 59:349; all of which are herein incorporated by reference), the inventors contemplated that a member of the prolyl oligopeptidase family (POP) (EC 3.4.21.26) family was the most likely to be involved in the processing of the proproteins encoded by AMA1 and PHA1.
(280) POPs are known to be widespread in animals, plants, and bacteria. However, none of the other known Pro-recognizing proteases specifically cleave at internal Pro residues of small peptides (Cunningham and O'Connor, 1997; Gass and Khosla, 2007).
(281) Thus, the inventors used a human POP sequence (GenBank NP_002717, SEQ ID NO: 150) as a query sequence to search GenBank and known fungal genomes in order to identify a candidate fungal POP (see Table 12 below). A TBLASTN search was conducted using human POP (GenBank NP_002717) as query. BLASTP (default parameters) identified no orthologs of human POP with a score >53 and E value <e-06 in any fungus outside the Basidiomycetes, except perhaps Phaeosphaeria nodorum (SNOG_1288; score=166; E value=3e-40) (
(282) Orthologs of human POP are were present in other Basidiomycetes including Coprinopsis cinereus (GenBank CC1G_09936), Ustilago maydis (UM05288), Cryptococcus neoformans (SNOG_11288 and XP_567292), Laccaria bicolor (Lacbi1|303722) hypertext transfer protocol site:genome.jgi-psf.org/Lacbi1/Lacbi1.home.html), Phanerochaete chrysosporium (Phchr1|1293) hypertext transfer protocol site:genome.jgi-psf.org/Phchr1/Phchr1.home.html), and Sporobolomyces roseus (Sporo1|33368) hypertext transfer protocol site:genome.jgi-psf.org/Sporo1/Sporo1.home.html). A POP enzyme has been previously purified from the mushroom Lyophyllum cinerascens (Yoshimoto, et al., (1988) J Biochem. 104:622; herein incorporated by reference). Surprisingly, POP orthologs (POP-like genes and proteins) are rare or nonexistent in fungi outside of the Basidiomycetes, a possible exception being one in the Ascomycete Phaeosphaeria (Septoria) nodorum (SNOG_11288). However, this single potential Ascomycete POP-like gene is much less similar to human POP than any of the POP-like genes found in Basidiomycetes.
(283) TABLE-US-00016 TABLE 12 Exemplary results using human prolyl oligopeptidase (POP; (GenBank NP_002717, SEQ ID NO: 150) as a query sequence for fungal sequences (BLAST of GenBank unless otherwise noted). Fungal sequences related to human POP found in public databanks Sequence Reference No. SEQ ID NO: XX human prolyl (GenBank NP_002717) SEQ ID NO: 150 oligopeptidase (POP). Coprinopsis (Coprinus) (GenBank CC1G_09936) SEQ ID NO: 151 cinereus Ustilago maydis (GenBank UM05288) SEQ ID NO: 152 Cryptococcus (GenBank XP_567311) SEQ ID NO: 153 neoformans Cryptococcus (GenBank XP_567292) SEQ ID NO: 154 neoformans Laccaria bicolor* (The DOE Joint Genome SEQ ID NO: 155 Institute (JGI) Lacbil|303722) Phanerochaete (The DOE Joint Genome SEQ ID NO: 156 chrysosporium * Institute (JGI) Phchrl|1293) Puccinia graminis PGTG_14822.2 na Sporobolomyces roseus* (The DOE Joint Genome SEQ ID NO: 157 Institute (JGI) 1|33368, Sporo1|33368) mushroom Lyophyllum Yoshimoto, et al., (1988) na cinerascens J. Biochem. 104: 622; herein incorporated by reference Ascomycete (GenBank SNOG_11288) SEQ ID NO: 158 Phaeosphaeria (Septoria) nodorum
(284) Based upon these discoveries the inventors contemplated that a POP-like protease was rare or nonexistent in the Ascomycota yet found widespread within the Basidiomycota.
Example XV
(285) This example describes the identification and isolation of an Amanita bisporigera orthologous to human prolyl oligopeptidase (POP). The inventors used the sequence for human POP (GenBank NP_002717) for screening their A. bisporigera genomic DNA sequence database.
(286) Genome survey sequences were identified in the A. bisporigera genome (subject) by TBLASTN using human POP (GenBank accession no. NP_002717, SEQ ID NO:150) as a query sequence (
(287) TABLE-US-00017 TABLE13 Exemplaryhomologyresultsusinghumanprolyloligopeptidase (POP)asaquerysequence(BLASTofA.bisporigeragenome). SequencesrelatedtohumanPOP foundintheAmanitagenome SEQ ofthepresentinventions SEQUENCE IDNO: ECGK9LO02JKSHRR TTGAGAGCACACAAGTCTGGTATGAGAGC SEQID AAAGACGGAACGAAAGTTCCAATGTTCAT NO:159 CGTTCGTCACAAATCAACGAAATTTGACG GAACGGCGCCGGCGATTCAAAACGG ECGK9LO02JKSHRR ESTQVWYESKDGTKVPMFIVRHKSTKFDGT SEQID APA NO:160 contig26093 CGTATATCGAACTGCCAAGGTCAAGGGTT SEQID TAAATCCGAACGATTTCGAGGCTCGACAG NO:161 GTGACTAGTTGGTTTTATATTGCATGAAA AGTGCGTCTCATGCGGTCTAGGTGTGGTA TGACAGCTACGACGGAACAAAGATTCCA ATGTTCATCGTCCGTCACAAGAATACCAA ATTTAATGGGACGGCGCCAGCTATACAAT ATGG contig26093 VWYDSYDGTKIPMFIVRHKNTKFNGTAPAI SEQID QY NO:162 ECIMO1V02I2IO5A CGACAAACAAGTAACACCTACGCGCGAA SEQID AAACTCGCGATCTCCGGCGGCAGCAACGG NO:163 CGGACTCCTCGTCGGCGCAAGCCGATTGA CCCAGCGCCCCGACCTCTTCG ECIMO1V02I2IO5A EKLAISGGSNGGLLVGASRLTQRPDLF SEQID NO:164 ECIM01V01CKHE5R ATCCTCGGATGGCACAGCCTCGCTCTCCA SEQID TGTATGATTTCTCACACTGTGGCAAATAC NO:165 TTCGCATATGGTATTTCTCTTTCCGTATGT AATTTT ECIM01V01CKHE5R SSDGTASLSMYDFSHCGKYFAYGISLS SEQID NO:166 EEISCGG02IHTSVR GGGATAATTAATTGCAGCGAGTTATGACA SEQID ACGGAAAAACCCACCTCTTCTCAGTAGAT NO:167 TTTCCTCCGCCATGCCCCCGCTTTCTTGTCT ACACGTAGCAGAAGTGGA EEISCGG02IHTSVR PLLLRVDKKAGHGGGKSTEK SEQID NO:168 ECIM01V02H2WNRS DGTKVPMFIVRHKSTK SEQID NO:169
(288) After identifying homologous fragments, the inventors used PCR to amplify two Amanita prolyl oligopeptidase (POP)-like genes, with primers shown in Tables 14A and 14B. The full genomic sequences of prolyl oligopeptidas-likeA (POPA), SEQ ID NO: 170 and prolyl oligopeptidas-likeB (POPB), SEQ ID NO: 171 are shown in
(289) TABLE-US-00018 TABLE14A PCRprimersusedtoamplifyprolyloligopeptidase-likeA(POPA) genomicsequencesandfor5 and3 RACEtoidentifyfull-length cDNAclonesofPOPA. SEQID Primer Sequence NO: PopAgenomic 5 GAAACGAGAGGCGAAGTCAAGGTG3 SEQID forwardprimer NO:172 PopAgenomic 5 AAGTGGATGACGATTATGCGGCAG3 SEQID reverseprimer NO:173 PopAgene- 5 GATTGGGTATTTGGCGCAGAAGTCACG3 SEQID specificprimer NO:174 for3 RACE (usedwith GeneRacer3 primer) PopAgene- 5 ATGTCTCGCCGAACTCGCCGCCTCCTC3 SEQID specificprimer NO:175 for5 RACE (usedwith GeneRacer5 primer)
(290) TABLE-US-00019 TABLE14B PCRprimersusedtoamplifyprolyloligopeptidase-likeB(POPB) genomicsequencesandfor5 and3 RACEtoidentifyfull-length cDNAclonesofPOPA. SEQID Primer Sequence NO: PopBgenomic 5 TCAAATGAAGTAGACGAATGGAC3 SEQID forwardprimer NO:176 PopBgenomic 5 CACACGGATGAGCAATGGATGAG3 SEQID reverseprimer NO:177 PopBgene- 5 AAAGTTCCAATGTTCATCGTTCGTCA3 SEQID specificprimer NO:178 for3 RACE (usedwith GeneRacer3 primer) PopBgene- 5 TGGGACTAAAGAATGGATCGGCTGTAAT3 SEQID specificprimer NO:179 for5 RACE (usedwith GeneRacer5 primer)
(291) The finding of a second POP gene was unexpected. Furthermore, the inventors found at least two POP genes in A. bisporigera, while the majority of other mushrooms whose genomes were examined by BLAST had only one POP (i.e., Coprinus cinerea, Laccaria bicolor, Phanerochaete chrysosporium, and Agaricus bisporus). Based on genome survey sequences, Galerina species are contemplated to contain genes for the two types of POPs (see above). By Southern blotting, POPA is present in all Amanita species (
(292) POPB fragments were not observed to hybridize to any species tested outside of sect. Phalloideae even after prolonged autoradiographic exposure. Therefore, the inventors contemplated that while POPA appears to be present in the genomes of peptide toxin producing and peptide nontoxin producing mushrooms, the presence of POPB appears to be limited to peptide toxin producing mushroom species and thus identifies an amanitin-toxin producing mushroom from a nontoxin (at least for amanitin) producing mushroom.
Example XVI
(293) This example describes the expression and isolation of prolyl oligopeptidase (POP) of the present inventions.
(294) The inventors first tried to express POP genes from A. bisporigera in a heterologous system, which has been successful with porcine and bacterial POPs (Szeltner et al., 2000; Shan et al., 2005). Exhaustive attempts were made to express these fungal proteins in E. coli or Pichia pastoris in a soluble, active form but were unsuccessful. However the inventors were able to use the inclusion bodies to raise antibodies; see below.
(295) Therefore, the inventors purified POP from the mushroom Conocybe lactea (also known as C. albipes or C. apala). Conocybe lactea was chosen as a source of POP because (1) it produces phalloidin, one of the phallotoxins; (2) it grows abundantly in the lawns of Michigan State University while Amanita mushrooms themselves are less common and more restricted in their fruiting season. Proteins isolated from Conocybe were assayed for POP activity with a standard colorimetric substrate (Z-Gly-Pro-pNA) and was inhibited by a specific POP inhibitor, Z-Pro-Prolinal.
(296) The inventors synthesized model peptides, ATRLPIWGIGCNPCVGDD (SEQ ID NO:318), MSDINATRLPAWLATCPCAGDD, and ATRLPAWLVDCPCVGDD (SEQ ID NO:249), i.e., the mature toxin peptides flanked by five amino acids on each end. Based on other successful synthetic POP substrates (e.g., Shan et al., 2005; Szeltner et al., 2000), these were contemplated as test mimics of the proproteins. The peptides IWGIGCNP (SEQ ID NO:50), AWLATCP (SEQ ID NO: 136), and AWLVDCP (SEQ ID NO:69) were also synthesized as standards.
(297) Extracts of Conocybe mushrooms catalyze the cleavage of a model phalloidin peptide to the mature heptamer. The responsible enzyme was purified. Specifically, Conocybe mushrooms were freeze-dried, ground in buffer, and the extracts concentrated by ammonium sulfate precipitation. After desalting, the proteins were fractionated by anion exchange high-performance liquid chromatography (or high pressure liquid chromatography, HPLC).
(298) Fractions containing peptides were assayed using Z-Gly-Pro-pNA and the model phallacidin substrate. Reaction products were separated by reverse phase HPLC (
(299) The inventors purified the enzyme responsible for cleaving synthetic model compounds to the linear, mature forms to a single band on an SDS-PAGE gel. Sequencing of this protein showed high sequence similarity to POPA and POPB from A. bisporigera and POP proteins from other organisms including pig and human. After incubation of the test propeptide and the isolated POPB, the inventors consistently observed the production of a mature seven-amino acid product (
(300) The results showed that purified POP cuts a synthetic phalloidin peptide precisely at the expected flanking Pro residues. The purified POP also cut a synthetic amanitin precursor and a synthetic phallacidin precursor.
(301) Further contemplated products (shown in Table 15) for alpha-amanitin; phalloidin precursors where natural or synthetic propeptide sequences will be the substrates for Conocybe POPB protein.
(302) TABLE-US-00020 TABLE15 Peptidesandtheircorrespondingmolecularmassfor useinthepresentinventions. SEQID Peptide Mr(molecular NO: No. AMA1peptides mass) 549 1 TRLPIWGIGCNPCIGD 1714.99 (substrate) 549 2 TRLPIWGIGCNPCIGD 1712.99 (substrate,Cys oxidizedto disulfide) 551 3 TRLPIWGIGCNP 1326.55 (cutonCside) 552 4 IWGIGCNPCIGD 1247.42 (cutatNside) 552 5 IWGIGCNPCIGD 1245.42 (cutatNside, oxidized) 50 6 IWGIGCNP(final 858.98 product,cutboth sides) 51 7 IWGIGCNP 840.97 (cyclized)
(303) Thus, the inventors found production of the mature heptapeptide of phalloidin by extracts of Conocybe, i.e. isolated POPB extracts (
(304) Amanita species in sect. Phalloideae, and Galerina, have two predicted POP genes (
Example XVII
(305) In this Example, POPA and POPB of A. bisporigera were expressed in inclusion bodies, purified and used to provide rat anti-POPA and POPB antibodies for use in the present inventions.
(306) E. coli were engineered for expressing POPA and POPB (in separate bacterium). Expression of recombinant POP was done by the procedures outlined in the pET handbook (Novagen). Briefly, a pET vector engineered to comprise a POP coding sequence of the present inventions was transformed into Escherichia coli AD494 cells, and cultures were grown according to the manufacturer's instructions in Luria-Bertani medium and then induced with isopropyl-D-thiogalactoside (final concentration of 1 mM) for 3 h. Pelleted cells were lysed with a French press (16,000 p.s.i.) and recentrifuged, and the pellet was extracted with B-Per II reagent (Pierce, Rockford, Ill.). The resulting purified inclusion bodies were solubilized and refolded using the Protein Refolding Kit (Novagen) according to the manufacturer's instructions.
(307) The inventors raised antibodies against POPA and POPB of A. bisporigera (POPB shown in
Example XVIII
(308) In this example, exemplary Galerina POP sequences identified using Amanita bisporigera POPA and POPB were used as query sequences for searching a library of Galerina sequences created by the inventors for their use during the development of the present inventions, and additional mushroom libraries. These Galerina sequences were obtained by the inventors from 454 sequencing (45 Mb total), see above. Not every sequence with identity to these genes are shown, merely what are considered the best examples.
(309) Galerina marginata POP sequences were identified using Amanita bisporigera POPA (
Example XIX
(310) Genes for fungal secondary metabolites are typically clustered (Walton, 2000; Keller et al., 2005). Examples include aflatoxin, penicillin, HC-toxin, fumonisin, sirodesmin, and gibberellins (Ahn et al., 2002; Gardiner et al., 2004; Tudzynski and Holter, 1998). From Basidiomycetes, an example of clustering are the genes for ferrichrome (Welzel et al., 2005).
(311) To test clustering of Amanita toxin genes, the inventors constructed a partial lambda genomic library of A. bisporigera (insert size about 15 kb) and screened it with PHA1. One exemplary lambda clone was found to contain two copies of PHA1 and three putative cytochrome P450 genes (
(312) In this example, a software program and system, FGENESH, Salamov and Solovyev, Genome Res. 2000. 10:516-522, at softberry.com, //linux 1.softberry.com/berry.phtml?topic=fgenesh& group=programs&subgroup=-gfind. was used to identify and predict novel sequences adjacent to PHA genes of a 13,254 bp lambda clone (SEQ ID NO:327). This software predicts genes (by which we mean predicting where the gene starts and stops and where intron and exons are) when the gene is pasted in as genomic sequence. In recent rice genome sequencing projects, this software was cited the most successful (gene finding) program (Yu et al. (2002) Science 296:79) and was used to produce 87% of all high-evidence predicted genes (Goff et al. (2002) Science 296:79).
(313) However, gene prediction is an inexact science, so the FGENESH software is trained with known gene structures from different organisms. That is, different organisms have different (and poorly understood) rules for gene structure. Gene structure in humans isn't the same as plants, etc. To get the best prediction, an organism on which the software has been trained that is taxonomically closest to the source of the DNA was used. Therefore, the inventors used a known Coprinus (Coprinopsis) cinerea model for their Amanita genes.
(314) Using this type of analysis as shown in
(315) This example shows that two copies of PHA1 are clustered with each other and with three P450 genes. A map of predicted genes in this lambda clone (13.4 kb), isolated using PHA1 as probe is shown in
Example XX
(316) This example shows identification of exemplary variants of two -amanitin genes identified in laboratory isolates of Galerina marginata.
(317) The inventors' were surprised to discover that sequences of the peptide toxin genes in Galerina marginata are quite different compared to A. bisporigera. See
(318) Using AMA1 from A. bisporigera as the search query, two orthologs of AMA1 were identified in the partial genome survey sequence of G. marginata and designated as GmAMA1-1 and GmAMA1-2.
(319) PCR primers unique to GmAMA1-1 and GmAMA1-2 were designed. For GmAMA1-1, the unique primers were 5-CTCCAATCCCCCAACCACAAA-3 (forward, SEQ ID NO:682) and 5-GTCGAACACGGCAACAACAG-3 (reverse, SEQ ID NO:683). For GmAMA1-2, the primers were: 5-GAAAACCGAATCTCCAATCCTC-3 (forward, SEQ ID NO:684), and 5-AGCTCACTCGTTGCCACTAA-3 (reverse, SEQ ID NO:685). PCR primers for each gene were designed based on the partial sequences and used to amplify full-length copies. The amplicons were cloned into E. coli DH5 and sequenced.
(320) The genomic DNA sequences were used for primer design to obtain full-length cDNAs by Rapid Amplification of cDNA Ends (RACE) using the GeneRacer kit (Invitrogen, Carlsbad, Calif.). A cDNA copy of GmAMA1-1 was obtained using primers 5-CCAACGACAGGCGGGACACG-3 (5-RACE, SEQ ID NO:686) and 5-GACCTTTTTGCTTTAACATCTACA-3 (3-RACE, SEQ ID NO:687), and of GmAMA1-2 with primers 5-GTCAACAAGTCCAGGAGACATTCAAC-3 (5-RACE, SEQ ID NO:688) and 5-ACCGAATCTCCAATCCTCCAACCA-3 (3-RACE, SEQ ID NO:689).
(321) Alignments of genomic and cDNA copies were done using Spidey located at (ncbi.nlm.nih.gov/spidey/) and Splign (ncbi.nlm.nih.gov/sutils/splign/splign.cgi).
(322) GmAMA1-1 contains three introns while GmAMA1-2 contains two introns (
(323) The translational start site of a gene is typically contemplated as the first in-frame ATG after the transcriptional start site. When this criterion was applied to GmAMA1-1, a start site was indicated that was analogous to AMA1 of A. bisporigera. However, when this criteria was applied to GmAMA1-2, there was an in-frame ATG that is 78 nucleotides upstream of the ATG indicated in
(324) GmAMA1-1 and GmAMA1-2 were both predicted to encode 35-amino acid proproteins, the same size as the proprotein of AMA1 in A. bisporigera. The toxin-encoding region (IWGIGCNP) was in the same relative position as it was in AMA1. There were 31 nucleotide differences between GmAMA1-1 and AMA1 in the coding region of 108 nucleotides (ATG through the stop codon). This results in a low level of amino acid conservation outside the toxin region and the amino acids immediately upstream of the toxin region (NATRLP, SEQ ID NO:754 (
(325) The sequenced proproteins were added by the inventors to form a group of a family of genes including and related to AMA1 and PHA1 in A. bisporigera, A. phalloides, and A. ocreata start with MSDIN. In contrast, when a start codon is contemplated in the same location between GmAMA1-1 and GmAMA1-2 the first five amino acids of the two G. marginata -amanitin genes are MFDTN, SEQ ID NO: 675. Searching of the G. marginata database with the upstream and downstream regions of GmAMA1-1 and GmAMA1-2 did not reveal any additional related sequences. Conversely, searching with the conserved regions of GmAMA1-1 and GmAMA1-2 did not reveal any related sequences in A. bisporigera beyond the known MSDIN family members described herein.
Example XXI
(326) This example shows identification of two exemplary full-length genes encoding orthologs of Prolyl oligopeptidase genes, i.e. POPA and POPB proteins, isolated from G. marginata.
(327) During the development of the present inventions, using a G. marginata partial genome survey, the inventors' discovered two orthologs of the POP genes of A. bisporigera. These two orthologs corresponded to the two A. bisporigera prolyl oligopeptidases (AbPOPA and AbPOPB) described herein. The G. marginata genes with closest identity to AbPOPA or AbPOPB were designated as GmPOPA and GmPOPB, respectively. Genomic PCR, reverse transcriptase PCR, and RACE were used, as described herein, to isolate full-length copies of these two genes and determine their intron/exon structures (
(328) Sequences hybridizing to AbPOPA were found to be present in amatoxin and phallotoxin-producing and non-producing species of Amanita, whereas AbPOPB was found present only in the toxin-producing species. By DNA blotting GmPOPA was present in all four specimens of Galerina, however GmPOPB was not present in the amanitin non-producing species G. hybrida (
(329) For isolating and cloning full-length cDNA sequences for GmPOPA (SEQ ID NO: 715) and GmPOPB (SEQ ID NO: 717), PCR primers that corresponded to the amino and carboxyl termini of both genes (which were present on different contigs) were designed from the genome survey sequence. The forward primers were 5-TTTAGGGCAGTGATTTCGTGACA-3, SEQ ID NO: 692, and 5-AACAGGGAGGCGATTATTCAAC-3, SEQ ID NO: 693, and the reverse primers were 5-GAACAATCGAACCCATGACAAGAA-3, SEQ ID NO: 694, and 5-CCCCCATTGATTGTTACCTTGTC-3, SEQ ID NO: 695. The primer pairs were used in both combinations and successful amplification indicated the correct pairing of 5 and 3 primers. The resulting amplicons were cloned into E. coli DH5 and sequenced.
(330) The RACE primers for GmPOPA were 5-CGGCGTTCCAAGGCGATGATAATA-3 (5-RACE), SEQ ID NO: 696, and 5-CATCTCCATCGACCCCTTTTTCAGC-3 (3-RACE), SEQ ID NO: 697, and for GmPOPB 5-AGTCTGCCGTCCGTGCCTTGG-3 (5-RACE), SEQ ID NO: 698, and 5-CGGTACGACTTCACGGCTCCAGA-3 (3-RACE), SEQ ID NO: 699. Sequences generated from the RACE reactions were used to assemble full-length cDNAs of two genes, GmPOPA and GmPOPB (see
(331) Alignments of genomic and synthetic cDNA copies (see,
(332) GmPOPA and POPB were predicted to encode exemplary polypeptides as shown in
Example XXII
(333) This example shows an exemplary successful transformation of G. marginata.
(334) The inventors grew G. marginata in the laboratory and collected mycelium for use in the following transformation procedure. The inventors show herein the successful transformation of the alpha-amanitin-producing fungus Galerina marginata with a test construct. Thus the inventors' contemplate producing commercial levels of amanatin in addition to novel, non-natural analogs of amanitin. Further, the inventors' contemplate making novel linear and cyclic peptides from synthetic prepropeptides.
(335) The following are exemplary methods for making buffers and reagents for us in the present inventions. Galerina culture methods: Vegetative mycelial stocks were prepared by culturing aseptic fragments of fruiting bodies on HSVA plates. Fungal colonies were transferred and reisolated until pure cultures were obtained. The stocks were subcultured every 6 months. HSV-2C (1 L): 1 g yeast extract, 2 g glucose, 0.1 g NH.sub.4Cl, 0.1 g CaSO.sub.4.5H.sub.2O, 1 mg thiamine.HCl, and 0.1 mg biotin, pH 5.2 (Muraoka and Shinozawa, 2000, herein incorporated by reference). Agar medium (HSVA) for subculture contained 2% agar in HSV. Protoplasting Buffer: In 20 ml of 1.2 M KCl add 500 mg Driselase (Sigma), 1 mg chitinase (Sigma), and 300 mg lysing enzyme from Aspergillus sp. Sigma #L-3768. Stir for 30 min and filter sterilize in a 0.45 um filter. Sorbitol Tris-HCl Ca (STC) buffer: Solution a) 1.2 M sorbitol, 10 mM Tris-HCl (pH8.0), 50 mM CaCl.sub.2, autoclaved. Solution b) 30, PEG Solution Mix: 30% (W/V) polyethylene glycol/STC buffer. Filter sterilize in a 0.45 um filter. Regeneration medium (RM): a) HSV-2C (1 L) and b) sucrose 273.5 g/500 ml of water. Autoclave solutions a) and b) separately and combine after autoclaving.
(336) The following is an exemplary Galerina transformation protocol for use in the present inventions. Around 20 pieces of mycelium were used to inoculate 100 ml of HSV-2C broth in a 250 ml Erlenmeyer flask. This inoculate was placed on a shaker at 150 rpm at room temperature for 9-15 days, until cloudy. The culture medium and fungus was used to begin the following steps. The cultures were: 1. Filtered through sterile Miracloth and the collected mycelia was washed thoroughly with sterile water. This fungal mycelium was placed in a sterile 250 ml Erlenmeyer flask. 20 ml Protoplasting Buffer (see recipe below) was added. 2. Digested for 8 hours on a rotary shaker at 26-30 C at 120 rpm. 3. Digestion mix was filtered through a 30 micron Nitex nylon membrane (Tetko Inc. Kansas City, Mo., U.S.A.)) into 1-2 sterile 30 ml Oakridge tubes on ice. Filtered solution was turbulent due to the presence of protoplasts when checked under the microscope. 4. This filtered solution was centrifuged in Oakridge tubes at 4 C at 2000g for 5 min. 5. Supernatant was carefully poured off and discarded. Protoplast pellet was gently resuspended in approx. 10 ml of STC buffer and resuspended by shaking gently. Solution was spun at 2000g for 5 min. 6. Repeat step 5 once. 7. Supernatant was discarded and the protoplast pellet was gently resuspended in 1 ml of STC buffer with a wide orifice pipette and transferred to a microcentrifuge tube and spun at room temperature at 4000g for 6 min. 8. Supernatant was poured off and protoplasts were resuspended in 1 ml of STC in a final volume with concentration of 10.sup.8-10.sup.9 protoplast/ml. The tube was placed on ice. 9. The following mixture was combined: 50 l protoplasts, 50 l STC buffer, 50 ul 30% PEG solution and 10 ul plasmid or PCR product (1 g) depending upon the experiment. When plasmids were used they were linearized with a restriction enzyme which cut the DNA in a noncoding region. 10. 2 ml of 30% PEG solution was added and the tubes incubated for 5 min. 11. 4 ml of STC buffer was added and gently mixed by inversion. 12. The mix was added to Regeneration Media (RM) (see below) at 47 C., and mixed by inversion then poured into Petri dishes. Each solution mixture was plated in several plates. 13. Protoplasts were regenerated for up to 20 days until tiny colonies started to appear as viewed by eye. 10 ml of RM amended with 10 g/ml Hygromycin B was overlayed onto the cultures. 14. Putative transformants were isolated from colonies that grew after the Hygromycin B overlay and eventually emerged on the surface of the overlaid agar. Examples of colonies collected for use in the present inventions are shown by arrows in
(337) After colonies were collected the presence of the inserted Hygromycin B transgene was tested by PCR. Primers specific to the hygromycin resistance gene used in
(338) The inventor's contemplate that G. marginata can be transformed with synthetic genes, using the G. marginata specific contemplated cut sites, i.e. synthetic sequences comprising nucleotides encoding MDSTN, TRIPL and Prolines in conserved positions. For examples, in one embodiment, a synthetic DNA sequence encoding an amino acid sequence of alpha-amanitin may be expressed. In one embodiment, alpha-amanitin production would be increased, for example, using a high expression promoter, transforming Galerina with multiple copies of the alpha-amanitin gene.
(339) In another contemplated embodiment, a synthetic, novel cyclic peptide is synthesized by transformed Galerina by changing specific bases of synthetic G. marginata alpha-amanitin sequences (including PCR copies of isolated peptide toxin genes and base by base construction of nucleic acid sequences) in order to make other types of peptide toxins and peptides. In one example, replacing the codon AAC (Asn) with GAC (Asp) will encode beta-amanitin instead of alpha-amanitin. Beta-amanitin production in G. marginata would be easily detected by reverse-phase HPLC because the inventor's isolate of G. marginata makes barely detectable levels of beta-amanitin.
(340) The inventors further contemplate changing other amino acids to make non-natural amanitin derivatives, as one example, replacing Gly with Ala by replacing GGT with GCT. Even further, the inventor's contemplate an embodiment for making linear and cyclic peptides of at least six, seven, eight, nine, ten or more amino acids comprising the general formula XWXXXCXP, SEQ ID NO:702, where X is any amino acid. The Pro is retained in these peptides in order for correct processing by POP, and the presence of Trp (W) and Cys (C) will result in the biosynthesis of tryptathionine, a unique hallmark of the Amanita toxin peptides. Expression of synthetic peptides and peptide toxins would be monitored by standard assays including but not limited to PCR generated fragments (as in
(341) Even further, the inventors' contemplate making new Galerina isolates that do not produce peptide toxins for use in the present inventions. In one embodiment, the inventors' contemplate knocking out genomic peptide toxin genes for making a new Galerina isolate that does not express peptide toxins. As examples for removing genomic peptide toxin genes in Galerina, i.e. test Galerina (isolates of Galerina used in the following methods) would be subject to homologous integration of transforming DNA that would be used for removing regions of DNA comprising the peptide toxin genes in transformed test Galerina, spontaneous mutants and induced mutants of test Galerina would be made then screened for loss of peptide toxin gene expression and more preferably loss of peptide toxin genes. Another method for eliminating endogenous toxin production is RNAi, which has been used in other basidiomycete fungi (Heneghan et al., Mol Biotechnol. 2007 35(3):283-96, 2007, herein incorporated by reference). Loss of toxin expression in test isolates would be monitored by standard assays including but not limited to genomic sequencing of test Galerina, PCR generated fragments of genomic sequences (as in
(342) All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in mycology, molecular biology, biochemistry, chemistry, botany, and medicine, or related fields are intended to be within the scope of the following claims.