YEAST O-MANNOSE NUCLEOCYTOPLASMIC GLYCOSYLATION
20170218032 · 2017-08-03
Inventors
- Adnan HALIM (Malmo, SE)
- Hiren J. JOSHI (Copenhagen, DK)
- Ida Signe Bohse LARSEN (Copenhagen, DK)
- Yanqui YUAN (Copenhagen, DK)
- Henrik Clausen (Copenhagen, DK)
Cpc classification
C07K14/00
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure provides, inter alia, compositions and methods for modulating and/or utilizing nucleocytoplasmic alpha-mannosyltransferase enzyme activity in yeast, e.g., to modulate the performance of the yeast in fermentation, respiration, bioproduction and/or bioprocessing. The disclosure also provides, inter alia, modified eukaryotic organisms comprising modulated nucleocytoplasmic alpha-mannosyltransferase enzyme activity, and uses of such modified eukaryotic organisms.
Claims
1-27. (canceled)
28. A method of producing a polypeptide comprising expressing a recombinant polypeptide in a yeast cell comprising a modulated nucleocytoplasmic alpha-mannosyltransferase enzyme activity.
29. The method of claim 28, wherein the yeast cell exhibits improved bioprocessing due to the modulated nucleocytoplasmic alpha-mannosyltransferase activity.
30. The method of claim 28, wherein the nucleocytoplasmic alpha-mannosyltransferase activity is modulated by modulating the cells' nutrient supply.
31. The method of claim 28, wherein the cell is a yeast cell selected from Saccharomyces cerevisiae or Schizosaccharomyces pombe.
32. The method of claim 28, wherein the nucleocytoplasmic alpha-mannosyltransferase activity is modulated by one or more of: A. increasing the cell's available pool of GDP-Man; B. decreasing the cell's available pool of GDP-Man C. modulating the transport of GDP-Man to the nucleocytoplasmic compartment of the cell; D. contacting the cell with an inhibitor or an activator of a nucleocytoplasmic alpha-mannosyltransferase enzyme; E. overexpressing a nucleocytoplasmic alpha-mannosyltransferase enzyme; and/or F. knocking down or knocking out a native nucleocytoplasmic alpha-mannosyltransferase enzyme.
33.-37. (canceled)
38. The method of claim 28, wherein the method comprises modulating nucleocytoplasmic O-Man glycosylation found on a polypeptide selected from the group consisting of one or more (i) carbohydrate transporter; (ii) enzyme involved in the catalysis pentose phosphate pathway; (iii) enzyme involved in the catalysis of the citric acid cycle; (iv) enzyme involved in the catalysis of the fermentation; (v) enzyme catalyzing glycolysis, gluconeogenesis, fermentation, the pentose phosphate pathway the citric acid cycle, or combinations thereof; (vi) enzyme involved in the catalysis of oxidative phosphorylation; (vii) polypeptide comprised in a plasma membrane ATPase protein complex, a vacuolar ATPase protein complex, or a combination thereof; (viii) enzyme involved in the catalysis of nitrogen metabolism; (ix) enzyme involved in the catalysis of lipid metabolism; (x) carbohydrate sensors; (x) enzyme involved in the catalysis of the biosynthesis of glycosyl-phosphatidylinositol (GPI) membrane anchors; (xii) enzyme that control levels of secondary messenger molecule cyclic adenosine monophosphate (cAMP); (xiii) enzyme involved in the catalysis of glycolysis, gluconeogenesis; and (xiv) enzyme involved in the catalysis of glycogen metabolism, trehalose metabolism.
39. The method of claim 38, wherein the polypeptide is selected from one or more of AGT1, GAL2, HXT1-17, MAL61, HXK1, HXK2, GLK1, PGI1, PFK1, PFK2, FBP1, FBA1, TPI1, GPD1, GPD2, GUT2, GPP1, GPP2, GUT1, THD1-3, PGK1, GPM1, ENO1, ENO2, PYK1, PYK2, PDA1, PDB1, LAT1, LPD1, PDX1, PCK1, PYC1, PYC2, TAL1, CIT1-3, ACO1, IDH1, IDH2, IDP1, IDP2, KGD1, KGD2, ACS1, ACS2, LPD1, LSC1, LSC2, SDH1-4, FUM1, MDH1-3, ICL1, MLS1, MLS2, PDC1, PDC5, ACS1, ACS2, PDC6, ALD4, ALD6, ADH1-5, SDHA, SDHB, SDHC, SDHD, ISP, COB, CYT1, COR1, QCR2, QCR6, QCR7, QCR8, QCR9, QCR10, COX1-4, COX5A, COX5B, COX6A, COX6B, COX7A, COX7C, COX10, COX11, COX15, ATP1-9, ATP14-21, PMA1, VMA1-8, VMA10, VMA13, STV, GSY1, GSY2, GPH1, GLT1, GDH1-3, GLN1, GPI1-3, GPI15, GPI12, YJR013w, GPI10, SMP3, GPI13, GPI17, GPI11, GAA1, GPI8, GPI15, GPI17, YLR459w, SNF3, RGT2, RAS1, RAS2, CDC25, IRA1, IRA2, and CYR1.
40. The method of claim 39, wherein the cell exhibits modulated metabolism, carbon metabolism, growth on non-fermentable compounds, levels of downstream carbon metabolites, ethanol production; nicotinamide adenine dinucleotide [NAD(H)] levels, nicotinamide adenine dinucleotide phosphate [NAD(H)] levels, flavin adenine dinucleotide [FAD(H2)] levels, energy homeostatis, proton gradients, adenosine triphosphate (ATP) levels, intracellular and/or vacuolar pH levels, levels of storage carbohydrates, downstream carbon metabolites, nitrogen metabolism, downstream nitrogen metabolites, GPI-anchor synthesis, GPI-anchored protein synthesis, and/or downstream cellular signalling.
41.-76. (canceled)
77. The method of claim 28, wherein the cell exhibits improved production of the exogenous polypeptide as compared to a corresponding yeast cell lacking any such modulation of its nucleocytoplasmic alpha-mannosyltransferase enzyme activity.
78. The method of claim 77, wherein the improved production comprises an increase in production yield of the polypeptide.
79.-104. (canceled)
105. A composition comprising a recombinant polypeptide with one or more mutation in its amino acid sequence as compared to its native polypeptide sequence, wherein the mutation modulates the O-mannosylation of the polypeptide.
106. The composition of claim 105, wherein the mutation results in decreased O-mannosylation of the polypeptide.
107. The composition of claim 105, wherein the mutation results in increased O-mannosylation of the polypeptide.
108. The composition of claim 105, comprising one or more mutation of an O-Man glycosite.
109. The composition of claim 108, wherein the mutated O-Man glycosite is any one of the O-Man glycosites identified in Table 1.
110. A yeast cell comprising one or more gene encoding one or more polypeptide that has been mutated to produce proteins with one or more amino acid substitutions that eliminates one or more O-Man glycosylation sites.
111. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES
[0034]
[0035]
[0036]
[0037]
[0038]
[0039]
[0040]
[0041] TABLE 1 lists the identified nucleocytoplasmic O-Man glycoproteins from S. cerevisiae and S. pombe. The table includes protein identifiers (Uniprot ID and Systematic ID), gene names (Gene) together with ambiguously and unambiguously identified O-Man glycosylation sites numbered according to protein sequence (Sites).
DETAILED DESCRIPTION OF THE INVENTION
[0042] The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of molecular biology, recombinant DNA techniques, protein expression, and protein/peptide/carbohydrate chemistry within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2000); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed., 2004); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Freshney, R.I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th Ed. Hoboken N.J., John Wiley & Sons; B. Perbal, A Practical Guide to Molecular Cloning (3rd Edition 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd Edition 2005). Poly(ethylene glycol), Chemistry and Biological Applications, ACS, Washington, 1997; Veronese, F., and J. M. Harris, Eds., Peptide and protein PEGylation, Advanced Drug Delivery Reviews, 54(4) 453-609 (2002); Zalipsky, S., et al., “Use of functionalized Poly(Ethylene Glycols) for modification of polypeptides” in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications. The publications discussed above are provided solely for their disclosure before the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Definitions
[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated. With regard to this specification, any time a definition of a term as defined herein, differs from a definition given for that same term in an incorporated reference, the definition explicitly defined herein is the correct definition of the term.
[0044] The words “a” and “an” denote one or more, unless specifically noted.
[0045] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In any embodiment discussed in the context of a numerical value used in conjunction with the term “about,” it is specifically contemplated that the term about can be omitted.
[0046] As used herein, the term “amino acid” is intended to mean both naturally-occurring and non-naturally-occurring amino acids, as well as amino acid analogs and chemical mimetics. Naturally-occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as other amino acids formed during post-translational modification such as, for example, 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine. Non-naturally-occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally-occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid. Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine (Arg or R) would have a positive charge moiety located in similar three-dimensional molecular space and having the same degree of mobility as the ε-amino group of the side chain of the naturally-occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.
[0047] Reference to the term “e.g.” is intended to mean “e.g., but not limited to” and thus it should be understood that whatever follows is merely an example of a particular embodiment, but should in no way be construed as being a limiting example. Unless otherwise indicated, use of “e.g.” is intended to explicitly indicate that other embodiments have been contemplated and are encompassed by the present invention.
[0048] Reference throughout this specification to “embodiment” or “one embodiment” or “an embodiment” or “some embodiments” or “certain embodiments” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” or “in certain embodiments” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0049] By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence (e.g., a amino acid sequence that can be compared to published sequences using the Basic Local Alignment Search Tool or BLAST algorithm; Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL (2008) NCBI BLAST: a better web interface. Nucleic Acids Res. 36:W5-W9, incorporated herein) of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
[0050] An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) an amount or level described herein. Similarly, a “decreased” or “reduced” or “lesser” amount is typically a “statistically significant” amount, and may include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or level described herein.
[0051] As used herein, “native sequence” or “native polynucleotide” or “native polypeptide” refers polynucleotide or polypeptide that is found in nature. In some embodiments, “native sequence” or “native polypeptide” refers to any primary accepted reference sequence according to the UniProt Knowledgebase database (UniProt Consortium. Update on activities at the Universal Protein Resource (UniProt) in 2013. Nucleic Acids Res. 2013 41:D43-7, incorporated herein) or the UniGene database (Pontius J U, Wagner L, Schuler G D. UniGene: a unified view of the transcriptome. In: The NCBI Handbook. Bethesda (Md.): National Center for Biotechnology Information; 2003, incorporated herein). The UniProt database can be found at the world wide web address uniprot.org, the contents of which are incorporated herein. The UniGene database can be found at the world wide web address ncbi.nlm.nih.gov/unigene, the contents of which are incorporated herein.
[0052] “Nucleocytoplasmic O-mannosylation” refers to the process of attaching an alpha-linked mannose to a polypeptide. In various embodiments, nucleocytoplasmic O-mannosylation refers to the enzymatically-catalysed process of adding an alpha-linked mannose to a polypeptide.
[0053] “Nucleocytoplasmic alpha-mannosyltransferase enzyme activity” refers to the enzyme activity that catalyzes nucleocytoplasmic O-mannosylation, e.g., the attachment of an alpha-linked mannose to a polypeptide.
[0054] “Optional” or “optionally” means that the subsequently described event, or circumstances, may or may not occur and that the description includes instances where said event or circumstance occurs and instances in which it does not.
[0055] The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may include non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
[0056] The terms “polypeptide” or “peptide” or “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, hydroxylation, or any other manipulation, such as conjugation with a labelling component.
Overview
[0057] All eukaryotes except nematodes and plants have a well-characterized O-Man glycosylation machinery for proteins trafficking the secretory pathway, and the enzymes involved in this process are multi-transmembrane spanning Dol-P-Man:protein O-mannosyltransferases (PMTs) utilizing the membrane associated dolichol phosphate β-D-Man (Dol-P-Man) donor substrate and transferring a single Man residue to selected Ser and Thr residues of proteins. These enzymes are located in the ER with their catalytic domains oriented into the lumen (Lommel and Strahl 2009, Loibl and Strahl 2013). Higher eukaryotic cells have two PMT isoenzymes (POMT1 and POMT2) and the initial O-Man glycans are elongated, branched and capped by sialic acids by a series of glycosyltransferases. Deficiencies in many of the enzymes involved in protein O-mannosylation in humans underlie a group of congenital muscular dystrophies (Endo 2015). Yeast in contrast have a family of at least six PMTs, and the initial Man monosaccharide is extended only by additional mannose residues through the actions of Golgi resident mannosyltransferases that use guanosine diphosphate α-D-Man (GDP-Man) as donor substrate (Loibl and Strahl 2013). The O-Man glycosylation and the action of multiple PMTs are essential for yeast (Gentzsch and Tanner 1996), and O-Man plays major roles in maintaining yeast cell wall integrity (Arroyo, Hutzler et al. 2011).
[0058] Current knowledge of the proteins undergoing O-Man glycosylation in yeast and the specific sites of glycosylation is limited (Lommel and Strahl 2009, Loibl and Strahl 2013), but recently we developed a glycoproteomics strategy to probe the O-Man glycoproteome of human cells using genetic engineering to simplify the O-glycan structures, the so-called “SimpleCell” strategy, in combination with Concanavalin A (ConA) lectin chromatography for enrichment of glycopeptides and mass spectrometric sequencing (Vester-Christensen, Halim et al. 2013). This resulted in identification of a large number of O-Man glycoproteins and O-Man glycosites demonstrating for example that cadherins and protocadherins are major carriers of O-Man glycans. In the present invention we modified this strategy to probe the yeast O-Man glycoproteome from total cell lysates. In striking contrast to our studies with human cell lines, we in addition to proteins entering the secretory pathway, also identified a large number of O-Man proteins annotated as classical nuclear, cytosolic or mitochondrial proteins that are not expected to be exposed to the known O-Man glycosylation machinery in the secretory pathway. The nucleocytoplasmic O-Man glycosites were located on proteins and in positions resembling that of the O-GlcNAcylation process in higher eukaryotes. We characterized the O-Man glycoproteome and found evidence to support that the nucleocytoplasmic O-Man modifications in yeast represents the missing equivalent to the O-GlcNAcylation process of higher eukaryotes.
[0059] The present invention relates, generally, to compositions and methods related to nucleocytoplasmic O-mannosylation. In particular embodiments, the present invention relates, generally, to compositions and methods related to nucleocytoplasmic O-mannosylation in yeast.
[0060] In some embodiments, the present invention provides nucleocytoplasmic O-Man glycoproteins and O-Man glycosites as targets for modulation of the biological functions of O-mannosylation in yeast.
[0061] In some embodiments this entails mutagenesis of an individual or a plurality of O-Man glycosites. Methods of mutagenesis are known in the art. In some embodiments, such mutagenesis results in modulation of yeast growth, nutrient sensing, fermentation, and/or bioproduction.
[0062] In some embodiments the present invention provides a modified polypeptide comprising a mutation in one or more O-Man glycosites. In some embodiments, suitable mutations include deletions, additions, or substitutions of any one or more native amino acid sequence in a native O-mannosylated nucleocytoplasmic polypeptide, wherein the mutation results in modulation of nucleocytoplasmic O-mannosylation of the polypeptide. Methods of making deletions, additions, and substitutions in a polypeptide sequence, e.g., via genetic engineering techniques such as site directed mutagenesis are known in the art. In some embodiments the present invention provides a modified polypeptide listed on Table 1, wherein the polypeptide is modified to comprise a mutation in one or more of the O-Man glycosites identified in Table 1.
[0063] As used herein, reference to “the modulation of nucleocytoplasmic O-mannosylation” or “modulated nucleocytoplasmic O mannosylation” refers to increasing or decreasing the O-Man occupancy of one or more O-Man glycosite on one or more polypeptide expressed in the nucleocytoplasmic compartment.
[0064] In certain embodiments, the mutation comprises a deletion or substitution of an O-Man glycosite, wherein the deletion or substitution ablates O-mannosylation of the glycosite. In certain embodiments, the mutation comprises a deletion or substitution of an amino acid residue that controls O-mannosylation of the O-Man glycosite, such that the deletion or substitution ablates or reduces O-mannosylation of the glycosite. An amino acid that “controls O-mannosylation” is an amino acid in an O-mannosylated nucleocytoplasmic protein, which is not itself an O-Man glycosite, but the presence or absence of which amino acid dictates whether, or to what extent, an O-Man glycosite is mannosylated. The skilled artisan will fully appreciate that glycosylation, like phosphorylation, is to some extent influenced by the peptide backbone sequence of the target polypeptide. The presence or absence of preferred or non-preferred sequences near or adjacent to a potential glycosite influences the efficiency at which a glycosyltransferase can glycosylate the glycosite. For example, as shown in
[0065] In certain embodiments, the mutation comprises an addition or substitution of an O-Man glycosite or an amino acid that controls O-mannosylation, wherein the addition or substitution increases or induces O-mannosylation of a glycosite present in a polypeptide expressed in the nucleocytoplasmic compartment or wherein the addition or substitution introduces a non-native amino acid into a polypeptide expressed in the nucleocytoplasmic compartment such that the non-native amino acid is O-mannosylated.
[0066] In another embodiment the invention relates to methods to modulate the nucleocytoplasmic O-Man glycosylation process for example by nutrient deprivation.
[0067] In some embodiments, the invention relates to methods of modulating the nucleocytoplasmic O-Man glycosylation process for example by altering access to the donor substrate GDP-Man.
[0068] In some embodiments, the invention relates to methods of modulating the nucleocytoplasmic O-Man glycosylation process for example by altering access to glucose.
[0069] In some embodiments, nutrient deprivation, e.g., restricted access to glucose or GDP-Man, results in an increase in the O-mannosylation of a polypeptide.
[0070] In other embodiments, nutrient deprivation e.g., restricted access to glucose or GDP-Man, results in an increase in the O-mannosylation of a polypeptide.
[0071] In another embodiment the inventions relates to methods to modulate the O-Man glycosylation process for example by inhibitors or activators of the O-Man glycosyltransferase activity catalyzing the transfer of Man to polypeptides in the nuclear and cytoplasmic space found in yeast.
EXAMPLES
[0072] The purpose of the following examples are given as an illustration of various embodiments of the invention and are thus not meant to limit the present invention in any way. Along with the present examples the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
Example 1
Determining the O-Man Glycoproteome of S. cerevisiae and S. pombe
[0073] Since yeast is known to produce heterogeneous elongated poly-mannose structures, we initially used the kre2zΔktr1Δktr3Δ mutant strain (Mut), lacking the α1,2-mannosyltransferase (kre2) involved in the second biosynthetic step of O-Man glycans and additional gene family members (ktr1 and ktr3), to enable analysis of the O-Man glycoproteome with more simplified O-Man glycan structures (
[0074] We used total cell lysates obtained by vortexing with glass beads in Rapigest detergent for trypsin digestion. N-linked glycans were removed by PNGase F digestion and the digests were subjected to Concanavalin A (ConA) lectin weak affinity chromatography (LWAC) for enrichment of O-Man glycopeptides. Enriched O-Man glycopeptides were further fractionation by isoelectric focusing (IEF) and analyzed by nLC-MS/MS (
Methods
Yeast Strains and Culture Conditions.
[0075] The following Saccharomyces cerevisiae strains were used: wild-type (BY4741, MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), the KTR/MNT triple mutant (BY4741 except kre2Δ::His3-GFP, ktr1Δ::SAT, ktr3Δ::KanMX4, a gift of H. Bussey) and BY5457 loxp::pep4Δ::loxp (wild type BY5457 (MATα ura3-52::p1785 leu2-3,112 his4-519 trp1 pho3-1 pho5-1 canr) with the additional loxp::pep4Δ::loxp modification, a gift from Rosa Laura Lopez Marques). WT cells (BY4741) were grown in rich medium (YPD) containing 2% glucose, 2% peptone, and 1% yeast extract under aerobic conditions at 30° C. with constant shaking at 170 rpm in a rotary shaker incubator (I Series 26, New Brunswick Scientific Co., Inc., Edison, N.J., USA). Cell growth was determined by measuring the optical density at a wavelength of 600 nm (OD600). S. cerevisiae cultures were grown to mid-log phase (OD600 0.8 to 1.1) and harvested by centrifugation at 3,000×g for 5 min. Growth conditions and harvesting of BY5457 cells is described below. The Schizosaccharomyces pombe WT (no-marker h−) strain, a derivative of the WT heterothallic strains 972h- and 975h+, was used. The fission yeast cells were propagated at 30° C. in YES media (5 g/L yeast extract, 30 g/L glucose, 225 mg/L adenine, 225 mg/L leucine, 225 mg/L uracil) and harvested in the late exponential phase by centrifugation (3000 g, 2 min) and lysed using glass beads as described below.
LWAC Isolation of O-Man Glycopeptides.
[0076] Cell extracts were prepared from a total of 100 OD600 units of packed yeast cells with acid washed 5 mm glass beads in ice-cold 0.1% Rapigest (Waters Corp., Milford, Mass., USA) in 50 mM ammonium bicarbonate by vortexing in reciprocal shaker (Hybaid RiboLyser) for 4×25 s with 1 min intervals at 4° C. The bottom of the tube was punctured, and the lysate was collected. Unbroken cells and larger cell debris were removed by a low-speed centrifugation step at 1,500×g for 5 min at 4° C. The cleared lysates were heated at 80° C., 10 min, followed by reduction in 5 mM dithiothreitol at 60° C., 30 min and alkylation in 10 mM iodoacetamide at room temperature (RT), in darkness and 30 min. Samples were digested with 25 μg trypsin (Roche) over night (ON), heat-inactivated by incubation at 95° C., 20 min and treated with 8 U PNGase F ON at 37° C. An additional 4 U PNGase F was added and incubated at 37° C. for 4 h. The digests were acidified with 12 μl TFA, incubated at 37° C., 20 min, cleared by centrifugation at 10,000×g, 10 min and purified by Sep-Pak C18 (Waters) columns. The LWAC protocol for isolation of O-Man glycopeptides was as previously described (Vester-Christensen, Halim et al. 2013). Sep-pak purified peptides were concentrated by evaporation and the reduced solution was diluted with an equal volume of 2× Con A buffer A (40 mM Tris HCl, pH 7.4, 300 mM NaCl, 2 mM CaCl2/MgCl2/MnCl2/ZnCl2, 1 M urea) before loading in a 2.8 m long Con A lectin agarose column. The column was washed with 10 column volumes (CV) ConA buffer A at 100 μl/min before elution with 5 CV's ConA buffer B (20 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM CaCl2/MgCl2/MnCl2/ZnCl2, 0.5M methyl-α-D-glucopyranoside/methyl-α-D-mannopyranoside) at 50 μl/min. Fractions containing glycopeptides were purified by in-house packed Stage tips (Empore disk-C18, 3M) and further fractionated by isoelectric focusing (IEF) as previously described (Vakhrushev, Steentoft et al. 2013). For each IEF fraction, 50% was analysed by nLC/MS/MS as described below. The remaining 50% of each IEF fraction was digested at 37° C. ON with 30 U/ml Jack bean α-mannosidase (Prozyme) in 100 mM sodium acetate, 2 mM Zn2+, pH 5 prior to analysis by nLC/MS/MS.
nLC/MS/MS and Data Analysis.
[0077] Mass spectrometric analyses were performed essentially as previously described (Vester-Christensen, Halim et al. 2013). Samples were analyzed on a set up composed of an EASY-nLC 1000 (Thermo Fisher Scientific) interfaced via a nanoSpray Flex ion source to an LTQ-Orbitrap Velos Pro hybrid spectrometer (Thermo Fisher Scientific). The EASY-nLC 1000 was equipped with a polar end-capped C18-silica column; 21 cm length, 75 μm inner diameter and 1.9-μm particle size. A data-dependent mass spectral acquisition routine, HCD and subsequent ETD scan, was used for all runs. Briefly, a precursor MS1 scan (m/z 355-1,700) of intact peptides was acquired in the Orbitrap at a resolution setting of 30,000, followed by Orbitrap HCD-MS2 and ETD-MS2 of the five most abundant multiply charged precursors in the MS1 spectrum; a minimum MS1 signal threshold of 50,000 ions was used for triggering data-dependent fragmentation events; MS2 spectra were acquired at a resolution of 15,000. Data processing was carried out using Proteome Discoverer 1.4 software (Thermo Fisher Scientific) as previously described (Vester-Christensen, Halim et al. 2013) with minor modifications as outlined below. Raw data files (.raw) were processed using the Sequest HT node and searched against the canonical S. cerevisiae proteome (7225 entries) downloaded from the Uniprot database (October, 2013) or the canonical S. pombe proteome (5092 entries; September 2015). Spectral assignments at the medium confidence level (p>0.01) and below were resubmitted to a second Sequest HT node using semi-specific tryptic cleavage. Final results were filtered for high-confidence (p<0.01) identifications only. Spectra matched to peptides with nucleocytoplasmic O-Man modifications were inspected manually to verify the accuracy of the assignments. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino, Deutsch et al. 2014) via the PRIDE partner repository with the dataset identifier PXD002924. Signal peptides were retrieved manually from Uniprot or through the SignalP tool (server version 4.1); transmembrane domains were predicted with TMHMM (server version 2.0); both tools available at http://www.cbs.dtu.dk/services/.
[0078] Cytoplasmic proteins (S3 fraction) were prepared as follows: Cells (BY5457 loxp::pep4Δ::loxp) were grown as above in YPAD medium (YPD supplemented with 0.01% Adenine hemisulphate w/v). Cells were pelleted by centrifugation at 800×g, 15 min, 4° C., and suspended in a 1:5 ratio in 50 ml lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) supplemented with 1 tablet complete protease inhibitor (Roche) and lysed by French press at 4° C., 40 Kpsi (S1 fraction). The S1 fraction was cleared by centrifugation at 5000×g, 4° C., 15 min. The supernatant (S2) was subjected to ultracentrifugation at 45.000×g at 4° C., 30 min, followed by 100.000×g at 4° C., 3 h, resulting in the cytoplasmic S3 (supernatant) fraction. The S3 fraction was trypsin digested, ConA enriched and purified by Stage tips as described above. For each LWAC elution fraction, 5% of total sample was injected and analyzed by HCD fragmentation only.
MALDI and GC-MS Analysis.
[0079] The S3 fraction (13 ml) was dialyzed against 6 L 50 mM NH4HCO3, pH 7.8, using 8,000 Da MWCO membranes and concentrated by evaporation. Following reduction and alkylation (described above), the cytosolic proteins were trypsin digested ON and purified by Sep-Pak C18 (Waters) columns. The tryptic peptides were dried, resolubilized in 100 mM NaOH, 1 M NaBH4 and incubated ON at 50° C. The β-elimination reaction was terminated by the addition of 8 μL glacial acetic acid and the released O-glycan alditols were separated from proteins by a second Sep-Pak C18 (Waters) purification. Reduced O-glycans were desalted by Dowex AG 50W X8 cation exchange resin (Bio-Rad) followed by repeated (×5) addition of 500 μL 1% acetic acid in methanol and evaporation over a stream of N2 gas. For MALDI analysis, released oligosaccharides were permethylated essentially as previously described (Ciucanu and Costello 2003). Briefly, released oligosaccharides were dried in glass vials to which 18 mg NaOH powder, 150 μl dimethyl sulfoxide (DMSO) with 0.1% H.sub.2O (v/v) and 30 μl methyl iodide was added; the mixture was incubated at RT, 1 h and the reaction was terminated by addition of 150 μl ice cold H.sub.2O followed by 200 μl chloroform. The organic phase was washed five times with 1 ml H.sub.2O and finally dried with a stream of N2 gas. Permethylated oligosaccharides were reconstituted in 30 μl 50% methanol in H.sub.2O (v/v) and 1 μl was co-crystallized with an equal amount of matrix (10 mg/ml 2,5-dihydroxybenzoic acid (DHB) and 2.5 mM sodium acetate in 70% acetonitrile in H2O (v/v)). MALDI TOF (Autoflex Speed, Bruker Daltonics, Bremen, Germany) was operated in the reflector mode using positive polarity and 2000 laser shots/spot. For GC-MS, reduced O-glycans were spiked with 1 μg myo-inositol (internal standard) and depolymerized by incubation in 0.5N methanolic HCL (Supelco), 80° C., 16 h. Monosaccharides were per-O-trimethylsilylated with Tri-Sil reagent (Thermo Scientific) at 80° C., 30 min. GC-MS analysis was performed using a TRACE™ GC Ultra gas chromatograph coupled to a PolarisQ ion trap mass spectrometer. Samples were injected (splitless mode) at 40° C. (1 min), oven temperature was ramped to 150° C. (25° C./min) followed by an increase to 200° C. (1° C./min) before a final ramp to 260° C. (10° C./min) where it was held for 5 min. Monosaccharides were identified by comparison of retention times and mass spectra to hexose and hexitol standards. All retention times were relative to the myo-inositol internal standard.
Example 2
Identification of the Nucleocytoplasmic O-Man Glycoproteome of Yeast
[0080] Analysis of the identified proteins using the methods described in Example 1 revealed that a large proportion (n=83, 29%) were known or predicted nuclear and/or cytosolic proteins without recognizable signal peptides. This finding was in striking contrast to our previous analysis of the O-Man glycoproteome of human cells, where all identified glycoproteins were known or predicted to traffic the secretory pathway (Vester-Christensen, Halim et al. 2013). We therefore also inspected all identified glycoproteins with signal peptides for the specific localization of O-glycosites with respect to known or predicted membrane orientation, and found sites predicted to be located in the cytosolic compartment (Table 1). Thus, more than a third of the O-Man glycoproteins identified in Mut S. cerevisiae were classical nuclear, cytosolic or mitochondrial proteins that are not exposed to the known O-Man glycosylation machinery in the secretory pathway (
[0081] To exclude the possibility that the unexpected finding was related to the kre2Δktr1Δktr3Δ mutant strain, we further explored this by analyzing total cell lysates of wild-type (WT) S. cerevisiae, which resulted in identification of 261 O-Man glycoproteins of which a similar fraction of 91 (35%) glycoproteins were from nuclear, cytoplasmic or mitochondrial compartments. Also, we sought to enrich for cytoplasmic proteins by analyzing a crude cytoplasmic fraction (S3) of fractionated WT yeast, and this resulted in identification of a number of unique nucleocytoplasmic glycoproteins (
[0082] We also explored the O-Man glycoproteome of the fission yeast S. pombe (WT) using the same experimental approach as above (
[0083] We focussed mainly on the deepest nucleocytoplasmic O-Man glycoproteome data obtained from S. cerevisiae (Table 1).
[0084] The nucleocytoplasmic O-glycans were shown to be based on Man-α-O-Ser/Thr. Our O-glycoproteomics strategy largely hinges on the α-Mannose specificity of the lectin ConA used for the LWAC as well as the MS identification of the mass increment for hexose residues. Thus, the approach does not reveal the absolute stereochemistry of the identified modifications. We therefore performed a series of experiments to verify the anomeric and epimeric configuration of the identified peptide-linked hexose residue. First, we released O-linked glycans by reductive β-elimination and analyzed the O-glycan profiles by MALDI-TOF mass spectrometry and found Hex.sub.2-5 oligosaccharides to be the dominating species. This glycoprofiling does not enable us to selectively quantify the composition of hexose structures on the nucleocytoplasmic glycoproteins, but we identified nucleocytoplasmic proteins with both a single and a disaccharide hexose structure showing that at least some elongation occurs. Mammalian nucleocytoplasmic O-GlcNAc is not elongated by other glycans, but in plants this type of glycosylation is elongated (Heese-Peck, Cole et al. 1995). The glycans released by reductive β-elimination were subsequently subjected to acid hydrolysis and trimethylsilyl derivatization prior to gas chromatography-mass spectrometry (GC-MS) analysis. This approach enables the reducing end hexose to be differentiated from internal or terminal hexose residues through its distinct retention time in GC-MS, and for the β-eliminated yeast O-glycans, the reducing end hexose displayed a similar retention time and fragmentation pattern as the mannitol standard, thus confirming that the peptide-linked hexose is a mannose residue (
Example 3
Nucleocytoplasmic O-Man Residues are Located in Proximity to Phosphorylation Sites
[0085] The 160 identified O-Man nucleocytoplasmic glycoproteins identified in S. cerevisiae included transcription factors, nucleoporins, histones and kinases, which are all classical proteins undergoing O-GlcNAcylation as well as phosphorylation in higher eukaryotes (Table 1). For the identified nucleocytoplasmic yeast glycoproteins, 80% are known to be phosphorylated (Bodenmiller, Campbell et al. 2008, Gnad, Gunawardena et al. 2011, Hornbeck, Kornhauser et al. 2012, Sadowski, Breitkreutz et al. 2013), and as much as 19% (221 in total) of the identified O-Man glycosites were found to be identical to previously identified phosphorylation sites (Table 1), a relatively high number considering that neither the current yeast phosphoproteome nor the O-Man glycoproteome presented here are complete.
[0086] One important example was the glycogen phosphorylase (GP) enzyme, which mobilizes cellular energy by breaking down glycogen into glucose-1-phosphate (G1P). The mechanism of GP activation is conserved among eukaryotes and involves, in addition to allosteric elements, a single phosphorylation at Thr31 in yeast and Ser15 in mammals, both located in the N-terminal regulatory domain (
[0087] We also compared the proximity of all O-Man glycosites to known phosphorylation sites, which further demonstrated that the O-Man glycosites assigned to nucleocytoplasmic proteins in general tended to have phosphorylation sites closer than the O-Man glycosites assigned to proteins and protein domains exposed to the secretory pathway (
[0088] We further analysed the amino acid sequences surrounding the nucleocytoplasmic O-Man glycosites but this approach did not reveal any apparent sequence motifs for glycosylation (
[0089] Analysis of the subset of proteins for which mammalian orthologs could be reliably established (79/160) revealed that 24% of these have also been reported to be O-GlcNAcylated in rodents or humans (Wang, Torii et al. 2011, Alfaro, Gong et al. 2012, Trinidad, Barkan et al. 2012, Cao, Cao et al. 2013). This analysis is likely biased by the limited depth of the O-glycoproteome data available currently, and we expect the overlap to increase with deeper characterization of the two glycoproteomes. Thus, the identified O-Man glycosylation of nucleocytoplasmic proteins is likely to represent the equivalent to O-GlcNAcylation found in higher eukaryotes, and the discovery suggests that yeast possess a hitherto unidentified O-glycosylation machinery that operates in cytoplasmic, nuclear and mitochondrial compartments.
Example 4
O-Man Glycosites and Comparison to Mammalian O-GlcNAcylation Sites
[0090] We selected examples of orthologous proteins with known O-GlcNAcylation sites and identified O-Man glycosites for more detailed analysis. Although the existence of O-GlcNAc on histones was questioned in a recent study (Gagnon, Daou et al. 2015), O-GlcNAcylation is believed to be one of several post-translational modifications (PTMs) that constitute the histone code and regulate histone interactions with DNA and effector proteins, and this PTM has been found on histones H2A, H2B and H4 (Sakabe, Wang et al. 2010). It has further been demonstrated that O-GlcNAcylation of human histone H2B in response to glucose levels modulates the transcriptional response by promoting mono-ubiquitination of lysine residue 120 (Fujiki, Hashiba et al. 2011). A glucose-dependent response is also observed in yeast where the orthologous histone H2B.1 undergoes mono-ubiquitination at the conserved lysine residue, although the preceding step promoting this mono-ubiquitination has not been identified (Dong and Xu 2004). We found O-Man glycosites on the highly conserved yeast histone ortholog H2B.1 positioned virtually in the same region as O-GlcNAcylation on the human histone H2B (
[0091] The plasma membrane H+-ATPase (PMA1) is O-GlcNAcylated in the cytosolic region in mammals (Trinidad, Barkan et al. 2012), and we identified O-Man glycosites on the yeast PMA1 ortholog that were conserved and overlapping with the known O-GlcNAcylation sites as well as phosphorylation sites (
[0092] In summary, the O-Man glycosites on nucleocytoplasmic proteins share the characteristic feature of overlap with phosphorylation sites and are positioned similarly to O-GlcNAc sites on evolutionary conserved proteins and sites. O-mannosylation of nucleocytoplasmic proteins in yeast was therefore considered likely to serve the equivalent functions as the O-GlcNAcylation process in higher eukaryotes. The nutrient sensing role of O-GlcNAcylation is likely to be mirrored by O-mannosylation in yeast with the difference being that GDP-Man, and not UDP-GlcNAc, will likely serve the role of donor substrate and key nutrient sensor in yeast. The biosynthesis of GDP-Man would thus link glucose and nucleotide metabolism in a network node capable of integrating nutrient signals that are ultimately relayed as nucleocytoplasmic O-Man glycosylation. The apparent shift to nutrient sensing by UDP-GlcNAc in higher eukaryotes from GDP-Man, may indicate a need to evolve a more complex nutrient sensor capable of incorporating glucose, nucleotide, amino acid, and fatty acid metabolism by flux through the hexosamine biosynthetic pathway. Yeast is the only eukaryotic cell type without identifiable orthologous OGT/OGA genes involved in O-GlcNAcylation, and the invention here provides an explanation since the enzymes required for transfer and removal of α-Mannose residues are likely to have entirely different structures and be encoded by non-homologous genes.
Example 5
O-Man Nucleocytoplasmic Glycosylation in Yeast is Dynamic and can be Modulated
[0093] We probed the dynamics of nucleocytoplasmic O-Man glycosylations by subjecting WT yeast to a short burst of external stimuli. More specifically, a mid-log phase culture of WT Saccharomyces cerevisiae was divided in two and centrifuged (1000 g, 2 min). Growth media was discarded and one part of the pellet was resuspended in YPD growth media (described above) with nutrients (2% glucose) and the other part was resuspended in nutrient deprived growth media (0% glucose). Following 10 min stimuli, the yeast were lysed as described above, tryptic digests were differentially labelled with stable dimethyl isotopes and subjected to ConA LWAC, IEF and mass spectrometry. The tryptic digests of non-stimulated (2% glucose) yeast were labelled with “light” dimethyl reagent (resulting in N—(CH.sub.3).sub.2) and the tryptic digests of stimulated yeast (0% glucose) were labelled with “medium” or deuterated (D) dimethyl reagent (resulting in N—(CD.sub.3).sub.2). This labelling retains the chemico-physical properties of the peptides and O-Man glycopeptides but introduces a +4.0251 Da mass increment per label which is used to measure the relative change (quantity) between two samples. The differentially labelled peptides and glycopeptides were identified as described above and quantified using the “Precursor Ion Quantifier” node of Proteome Discoverer 1.4. In the ConA flow-through fraction, we identified >2000 peptides with quantification values. The quantification ratios (medium to light) of these non-glycosylated peptides were plotted on a log 10 scale and demonstrated a Gaussian distribution centred around 0, thereby showing that no significant changes had occurred at the proteome level (
Example 6
Identification and Characterization of an O-Man GDP-Man: Polypeptide Glycosyltransferase Activity in Yeast
[0094] In order to assay potential enzyme activity responsible for the O-Man glycosylation of nucleocytoplasmic protein, we designed a peptide substrate (LKNVPTPSPSPKPQH) based on identified glycoproteins covering the putative acceptor sites (Table 1).
Methods
[0095] Wild-type S. cerevisiae were grown in YPD media (10 g/L of BactoYeast extract, 20 g/L of BactoPeptone and 20 g/L D-glucose) by shaking at 37° C. Cells were pelleted by centrifugation at 5000 rpm for 10 min and 5 ml packed cells were lysed in 10 ml ice-cold buffer containing 150 mM NaCl, 50 mM Tris pH 7.4 and complete protease inhibitors (Roche) with acid-washed glassbeads (0.4-0.8 mm in diameter) in a beadbeater (1 min pulse, 5 times). Soluble lysate was collected by ultracentrifugation at 50,000 g for 30 min using a SS-34 rotor. Enzymatic reactions containing 50 mM HEPES pH7.4, 1 mM DTT, 10 mM MgCl2, 0.01% Tween-20, 10 μM GDP-mannose* (mannose moiety is .sup.14C isotope labeled; the specific activity is adjusted to 4000 cpm/nmol with non-labeled GDP-mannose) and 5 mM LKNVPTPSPSPKPQH were incubated with soluble lysate at room temperature for 3 hours before subjecting to weak ion exchange Dowex 1×8-100 resin for removal of un-reacted GDP-mannose. Samples were than either directed added to scintillation liquid and subject to scintillation counting or injected onto C18 RP-HPLC for further separations. Fractions were then collected and subjected to scintillation counting for demonstration of incorporation of .sup.14C-Man into peptide substrates.
[0096] Enzymatic incorporation of .sup.14C labeled mannose transferred from GDP-mannose into the peptide substrate (LKNVPTPSPSPKPQH) was demonstrated by both direct scintillation counting of total enzyme reactions after removal of unreacted GDP-mannose by Dowex chromatography as well as by C18 RP-HPLC analysis of fractions containing the peptide and O-Man glycopeptide (
TABLE-US-00001 TABLE 1 S. cerevisiae Systematic UniProt ID ID Gene Sites D6VTK4 YFL026W STE2 363; 366; 368 O14455 YPL249C-A RPL36B 5 O14467 YOR298C-A MBF1 63; 69 O14467 YOR298C-A MBF1 50; 51; 53 P00330 YOL086C ADH1 41; 46 P00358 YJR009C TDH2 199; 201; 207; 208; 209 P00358 YJR009C TDH2 251; 252 P00358 YJR009C TDH2 139; 140 P00359 YGR192C TDH3 251; 252 P00359 YGR192C TDH3 139; 140 P00359 YGR192C TDH3 199; 201; 207; 208; 209 P00360 YJL052W TDH1 199; 201; 207; 208; 209 P00431 YKR066C CCP1 67; 68; 69; 76 P00549 YAL038W CDC19 492; 494 P00924 YGR254W ENO1 188; 196 P00925 YHR174W ENO2 188; 196 P02293 YDR224C HTB1 123; 126; 127; 128; 129 P02293 YDR224C HTB1 100 P02557 YFL037W TUB2 143; 145; 149; 153 P02994 YBR118W TEF2 267 P02994 YPR080W TEF1 267 P04037 YGL187C COX4 33; 45 P05030 YGL008C PMA1 899 P05030 YGL008C PMA1 558; 568 P05030 YGL008C PMA1 459; 464 P06169 YLR044C PDC1 514; 515; 522 P06367 YCR031C RPS14A 77 P06367 YCR031C RPS14A 22; 26; 31; 34 P06367 YCR031C RPS14A 119; 123; 125; 126 P06738 YPR160W GPH1 31 P06738 YPR160W GPH1 77; 78 P06774 YGL237C HAP2 255; 256; 257 P06776 YOR360C PDE2 139; 142; 147; 150; 159 P07246 YMR083W ADH3 68; 73 P07280 YOL121C RPS19A 97; 99 P08018 YJL128C PBS2 151; 156; 161; 164; 166; 168; 170; 172; 174 P08153 YDR146C SWI5 114; 118; 119 P08153 YDR146C SWI5 505; 510 P08153 YDR146C SWI5 488; 490; 492 P0C0X0 YLR264W RPS28B 5; 8 P0C2I5 28; 36; 38; 40 P0C2I8 28; 36; 38; 40 P14540 YKL060C FBA1 56; 62; 63 P14540 YKL060C FBA1 11; 24 P14540 YKL060C FBA1 347; 349; 352 P14907 YJL041W NSP1 580; 581; 583; 584; 589 P14907 YJL041W NSP1 298; 299; 304 P14922 YBR112C CYC8 715; 720; 725; 728 P15891 YCR088W ABP1 163; 165; 167; 169; 174 P19097 YPL231W FAS2 31 P19097 YPL231W FAS2 67; 73 P20048 YMR013C SEC59 315 P21147 YGL055W OLE1 506 P21192 YLR131C ACE2 501 P21306 YPL271W ATP15 39; 41; 46 P21306 YPL271W ATP15 52; 55; 58; 61 P21375 YJR051W OSM1 251; 252 P21576 YKR001C VPS1 559; 565; 568; 569; 570 P22133 YOL126C MDH2 357 P22133 YOL126C MDH2 372 P22211 YNL183C NPR1 326; 328; 330; 336; 338; 339; 347; 349 P23292 YNL154C YCK2 69; 70 P23585 YMR011W HXT2 29; 32; 36 P23644 YMR203W TOM40 15; 20; 23 P23797 YMR281W GPI12 205 P24784 YPL119C DBP1 375 P24813 YDR423C CAD1 337; 339; 346; 347; 350 P24813 YDR423C CAD1 312; 313 P24813 YDR423C CAD1 320; 323; 330; 332 P25297 YML123C PHO84 465; 466; 467; 477 P25297 YML123C PHO84 500; 504 P25297 YML123C PHO84 577; 578; 579; 581 P25297 YML123C PHO84 22 P25357 YCR033W SNT1 64; 69; 74; 75; 84 P25358 YCR034W ELO2 334; 336; 338 P25573 YCL044C MGR1 353 P26786 YOR096W RPS7A 133 P27895 YEL061C CIN8 984; 985; 993 P28272 YKL216W URA1 230 P29509 YDR353W TRR1 165; 174 P29509 YDR353W TRR1 237; 242; 251; 254 P32047 YNL074C MLF3 79; 87; 88 P32047 YNL074C MLF3 407; 408; 411; 414; 415; 416; 418; 420; 421 P32338 YGR044C RME1 135; 137; 138; 141; 143 P32338 YGR044C RME1 224 P32338 YGR044C RME1 109; 110 P32338 YGR044C RME1 72 P32338 YGR044C RME1 153 P32338 YGR044C RME1 138; 141; 143 P32338 YGR044C RME1 166; 167; 169; 173 P32342 YKL122C SRP21 77; 79 P32342 YKL122C SRP21 69 P32457 YLR314C CDC3 501; 503; 509; 513 P32486 YPR159W KRE6 116; 117; 122 P32599 YDR129C SAC6 102; 103; 107; 108; 111 P32614 YEL047C FRD1 31 P32790 YBL007C SLA1 454; 467 P32839 YDR375C BCS1 227; 232 P32904 YHR147C MRPL6 48; 49; 53; 57 P33336 YGR143W SKN1 196; 200; 202 P33894 YOR219C STE13 77 P35197 YDL226C GCS1 168; 170; 174; 175 P35732 YKL054C DEF1 86; 101 P35997 YKL156W RPS27A 11; 14 P36024 YKR072C SIS2 76; 77; 82; 84 P36060 YKL150W MCR1 59 P36093 YKL043W PHD1 138; 147; 150; 151 P36093 YKL043W PHD1 322 P36112 YKR016W MIC60 273; 275 P36112 YKR016W MIC60 304; 310 P37267 YGR174C CBP4 80; 85 P38073 YBR033W EDS1 665 P38166 YBL102W SFT2 37; 40; 41; 45; 48 P38236 YBR057C MUM2 162; 170 P38272 YBR130C SHE3 392; 393; 394; 396; 398; 404 P38314 YBR214W SDS24 85; 90; 92; 94; 98 P38330 127; 132; 134; 137 P38631 YLR342W FKS1 1857; 1860; 1864; 1866; 1868 P38749 YHL009C YAP3 133; 135; 146 P38797 YHR076W PTC7 38; 42; 45; 51; 52; 54; 56; 60 P38797 YHR076W PTC7 173; 174; 182 P38881 YHR195W NVJ1 65; 75 P38968 YDL195W SEC31 814; 815; 816; 817; 822 P38968 YDL195W SEC31 974; 975; 977; 980 P38968 YDL195W SEC31 940; 948 P38968 YDL195W SEC31 900; 908; 911 P38969 YOR266W PNT1 408; 414; 419 P38972 YGR061C ADE6 333; 338; 341; 348; 350 P39013 YNL084C END3 17 P39523 YMR124W EPO1 370 P39726 YAL044C GCV3 38 P39928 YIL147C SLN1 786; 791 P39935 YGR162W TIF4631 2; 5; 9; 11; 13 P39935 YGR162W TIF4631 908; 911; 912 P39940 YER125W RSP5 191; 192; 195; 197; 198; 201; 203; 205; 206; 207 P39952 YER154W OXA1 33; 36 P39960 YER155C BEM2 92; 93; 94; 95; 96; 97; 106; 108; 110 P40005 YEL003W GIM4 89; 93 P40049 59; 63; 67; 71 P40087 YER143W DDI1 378; 379; 383; 384; 386 P40087 YER143W DDI1 368; 373; 375 P40107 YGL225W VRG4 6; 16; 20; 24 P40159 47; 48; 53; 60 P40356 YGL025C PGD1 134; 135; 137; 140; 141; 144; 148; 149; 151; 152; 153; 155 P40463 YIL135C VHS2 108; 117 P40529 YIL044C AGE2 195; 196; 197; 199; 200; 202; 203; 204; 206; 207; 213 P40564 YIR004W DJP1 321; 322 P41821 YNL291C MID1 451; 452; 453; 454; 455 P42839 YNL321W VNX1 26; 28; 29; 30; 32; 33 P42845 YNL309W STB1 129; 131 P42939 YGR206W MVB12 91 P43574 YFL021W GAT1 399 P43594 YFR011C MIC19 61; 67 P43594 YFR011C MIC19 108 P43638 YJL042W MHP1 209; 213; 218 P47068 YJL020C BBC1 608; 609; 612; 613; 620; 621; 624 P47116 YJR059W PTK2 722; 726; 727; 729; 730; 732; 737 P47116 YJR059W PTK2 729; 730; 732; 737 P47143 YJR105W ADO1 258; 264; 268; 269 P48015 YDR019C GCV1 58; 66; 71 P48559 YNL304W YPT11 8; 13; 14; 16; 19; 21; 23 P48562 YNL298W CLA4 311; 314; 316; 322 P49723 YGR180C RNR4 332; 334; 336 P50276 YGR055W MUP1 6; 9 P50896 YDR505C PSP1 282; 283; 286 P53075 YGL228W SHE10 290 P53075 YGL228W SHE10 196; 202; 204 P53159 YGL075C MPS2 373; 382 P53172 YGL056C SDS23 400; 402; 404; 405; 409; 410; 413; 416; 417; 420; 422; 424; 425 P53220 YGR033C TIM21 24; 28; 29; 32; 34; 35 P53297 YGR178C PBP1 230; 231; 238; 239 P53319 YGR256W GND2 479; 486; 487; 488 P53599 YNR031C SSK2 1254; 1257; 1258; 1260; 1263 P53885 YNL173C MDG1 306; 308; 310; 313 P53901 YNL152W INN1 384; 385; 386; 388; 392 P53946 YNL059C ARP5 58; 60 P54785 YMR070W MOT3 314; 315; 326; 327; 332; 339; 340 Q00246 YKR055W RHO4 264; 268; 269; 270; 273; 274; 276; 278; 280 Q03063 YPL049C DIG1 142; 148; 151 Q03088 YPL032C SVL3 689; 694; 702; 709 Q03088 YPL032C SVL3 662; 665; 667 Q03104 YML128C MSC1 52; 54; 60 Q03104 YML128C MSC1 52; 54 Q03104 YML128C MSC1 415; 417; 419; 420 Q03104 YML128C MSC1 52; 54; 60; 63 Q03104 YML128C MSC1 419; 420 Q03104 YML128C MSC1 415; 417; 419; 420; 429 Q03281 YDR458C HEH2 411 Q03281 YDR458C HEH2 607; 608 Q03640 YML072C TCB3 1279; 1285; 1291; 1295 Q03667 YMR002W MIX17 23; 24; 41; 50 Q03707 YML034W SRC1 642; 644; 652 Q03772 YDR163W CWC15 21; 23 Q04439 YMR109W MYO5 1054; 1058; 1066 Q04439 YMR109W MYO5 992; 1001; 1005 Q04947 YDR233C RTN1 246 Q04947 YDR233C RTN1 197; 206 Q04958 YML059C NTE1 21; 24; 25; 28; 29; 32; 33 Q04958 YML059C NTE1 25; 28; 29; 32; 33 Q04958 YML059C NTE1 577; 582 Q04958 YML059C NTE1 584; 589 Q04964 YML058W SML1 25; 27 Q05050 YMR031C EIS1 50; 51; 56 Q06011 YLR390W ECM19 102; 105; 108 Q06179 YLR454W FMP27 730; 734; 737 Q06315 YLR187W SKG3 977; 982 Q06608 101; 103; 105; 106 Q06616 168; 169; 170; 175; 179 Q06681 YDR326C YSP2 1375; 1379; 1382 Q06681 YDR326C YSP2 1333 Q07362 YDL053C PBP4 28; 32 Q07549 YDL123W SNA4 113; 116 Q07793 28; 36; 38; 40 Q08001 44; 45; 46; 52 Q08001 66; 67; 74; 78 Q08300 140; 141; 148; 151 Q08300 169; 170 Q08300 148; 151; 158; 161 Q12019 YLR106C MDN1 193; 200; 204; 207 Q12199 YPR040W TIP41 39; 40; 43; 44; 46; 50; 55 Q12199 YPR040W TIP41 16 Q12273 28; 36; 38; 40 Q12276 YOR227W HER1 50; 51; 56 Q12328 YDL217C TIM22 4; 12 Q12363 YOR230W WTM1 37; 39; 41 Q12427 YDR169C STB3 334; 336; 337; 340; 341 Q12443 YDL204W RTN2 318 Q12443 YDL204W RTN2 362; 363; 368; 370 Q12515 YDL173W PAR32 141; 145; 146; 147; 148 Q12515 YDL173W PAR32 85 Q12515 YDL173W PAR32 138; 141; 145; 146; 147; 148 Q12522 YPR016C TIF6 74; 76; 77 Q3E823 YPL189C-A COA2 37; 40; 42
S. pombe
TABLE-US-00002 UniProt ID Systematic ID Gene Sites A6X980 SPAC1250.07 sfc7 101; 103; 104; 105; 108; 110; 118 O13601 SPBC11B10.05c rsp1 103; 104; 108 O13702 SPAC13F5.03c gld1 442; 446 O13735 SPAC15A10.16 bud6 171; 175; 180 O13736 SPAC16E8.01 shd1 927; 928; 929; 944 O13900 SPAC22A12.09c sap114 468; 470; 475; 477 O13962 SPAC24C9.02c cyt2 34; 39; 43; 47 O14007 SPAC29A4.04c cbf5 383; 388 O14015 SPAC29A4.12c mug108 6; 7; 8; 11; 14 O14015 SPAC29A4.12c mug108 6; 7; 8; 11; 14; 23; 25 O14018 SPAC29A4.15 srs1 395; 396; 400 O14033 SPAC29B12.12 SPAC29B12.12 91 O14069 SPAC1687.06c rp144 46; 60 O14160 SPAC4A8.08c vrs2 269 O42644 SPAC10F6.03c cts1 81; 83; 89; 90 O42663 SPAC27D7.09c SPAC27D7.09c 226; 227 O42663 SPAC27D7.09c SPAC27D7.09c 222; 226; 227 O42663 SPAC27D7.09c SPAC27D7.09c 227 O42663 SPAC27D7.09c SPAC27D7.09c 222; 226; 227; 242; 246 O42665 SPAC27D7.11c SPAC27D7.11c 296; 298; 302; 304; 306 O42665 SPAC27D7.11c SPAC27D7.11c 302; 304; 306 O42665 SPAC27D7.11c SPAC27D7.11c 292; 293; 296; 298; 302; 304; 306 O42665 SPAC27D7.11c SPAC27D7.11c 304; 306 O42665 SPAC27D7.11c SPAC27D7.11c 306 O42665 SPAC27D7.11c SPAC27D7.11c 298; 302; 304; 306 O42665 SPAC27D7.11c SPAC27D7.11c 265; 266; 267; 272; 274; 276; 277; 282; 283 O42667 SPAC27D7.13c ssm4 295; 298; 301 O42700 SPCC1020.06c tal1 33; 34; 37; 43 O42917 SPBC16A3.11 eso1 623 O42976 SPBC20F10.07 SPBC20F10.07 741 O42976 SPBC20F10.07 SPBC20F10.07 761 O42980 SPBC17D11.01 nep1 371; 379; 383; 388 O43002 SPBC2G2.03c sbh1 53; 54; 56 O43024 SPBC354.10 def1 417; 429 O43024 SPBC354.10 def1 695; 703; 705; 707; 712 O43024 SPBC354.10 def1 510; 519; 524 O59667 SPBC29A3.02c his7 321; 323 O59733 SPBC3E7.13c syf2 120 O59824 SPCC965.04c yme1 120; 122; 126; 128; 130; 131; 132; 133; 136; 137; 138; 140 O60057 SPBC56F2.06 mug147 295 O60057 SPBC56F2.06 mug147 295; 302 O60069 SPBC13G1.07 swf1 308 O60073 SPBC13G1.11 ykt6 148 O60081 SPCC1494.07 SPCC1494.07 903; 904; 909 O60101 SPBC14F5.04c pgk1 373; 375 O74417 SPCC14G10.04 SPCC14G10.04 167; 174; 175 O74423 SPCC162.07 ent1 510; 518 O74423 SPCC162.07 ent1 494; 499; 510; 518 O74454 SPCC16C4.09 sts5 67; 75; 76 O74718 SPCC584.04 sup35 77; 86; 89 O74718 SPCC584.04 sup35 77; 86; 89; 95 O74840 SPCC1235.03 cue2 368; 370; 377 O74864 SPCC18.14c rpp0 24; 34; 35; 40 O74895 SPCC576.11 rpl15 183; 188 O74989 SPCC338.12 pbi2 73 O74989 SPCC338.12 pbi2 36; 39 O74989 SPCC338.12 pbi2 5 O74989 SPCC338.12 pbi2 57; 73 O94248 SPCC737.08 SPCC737.08 3903; 3905; 3910; 3912; 3916; 3924 O94259 SPBP8B7.10c utp16 62; 67; 68; 69; 71; 73 O94274 SPBP8B7.26 SPBP8B7.26 24; 25 O94274 SPBP8B7.26 SPBP8B7.26 57 O94305 SPCC5E4.05c mgl1 341; 342; 344; 345; 346; 350; 354; 355; 356 O94358 SPBC428.10 SPBC428.10 646; 647; 649; 650 O94385 SPBC29A10.07 pom152 144; 145 O94399 SPCC126.06 twf1 306; 307; 312; 313; 314; 315 O94428 SPBC660.09 mug168 79; 81; 82; 84; 88 O94428 SPBC660.09 mug168 54; 55; 58; 64; 66 O94428 SPBC660.09 mug168 58; 64; 66; 69 O94428 SPBC660.09 mug168 64; 66; 69 O94432 SPBC660.15 msi2 333 O94700 SPBC83.17 SPBC83.17 72; 73; 74 O94733 SPCC191.01 SPCC191.01 7; 8 P0CT53 SPCC794.09c tef101 289; 296 P19117 SPAC23C11.05 ipp1 29; 30 P28758 SPAC821.10c sod1 55; 59; 60 P36580 SPBC19C2.07 fba1 312 P36584 SPAPB8E5.06c rpl302 328 P40370 SPBC1815.01 eno101 189; 192; 195 P40372 SPAC17A5.03 rpl301 328 P78812 SPBC660.16 SPBC660.16 143 P79058 SPAC10F6.16 mug134 89; 92 P79058 SPAC10F6.16 mug134 99; 102; 108; 111 P79082 SPBC27.06c mgr2 81; 83 P87148 SPBC25H2.06c hrf1 11; 17 P87175 SPBC3D6.10 apn2 355; 357; 359; 363; 365; 367; 368; 370; 371; 373; 375 P87216 SPAC10F6.06 vip1 189; 193; 194 P87216 SPAC10F6.06 vip1 169; 175; 176; 177; 180 P87216 SPAC10F6.06 vip1 141; 144 P87216 SPAC10F6.06 vip1 115; 118 P87216 SPAC10F6.06 vip1 175; 176; 177; 180; 181 P87216 SPAC10F6.06 vip1 230; 232; 235; 236; 237; 239; 247; 250; 251; 253 P87216 SPAC10F6.06 vip1 101; 105; 109 P87216 SPAC10F6.06 vip1 122; 128; 132; 133 P87216 SPAC10F6.06 vip1 2; 8; 11; 15 P87216 SPAC10F6.06 vip1 156 Q09719 SPAC31A2.07c dbp10 133; 134; 138; 140; 146 Q09748 SPBC12C2.08 dnm1 291 Q09811 SPAC2G11.12 rqh1 1261; 1263; 1265; 1267; 1269 Q10104 SPAC18G6.04c shm2 89 Q10234 SPAC4G9.17c mrp5 186; 190; 191 Q10475 SPAC17C9.03 tif471 318; 329 Q92371 SPAC4F10.14c btf3 75; 78; 79; 84; 85 Q9HFE8 SPBP16F5.03c tra1 615; 616; 622; 626; 627; 629; 630; 633 Q9P3A9 SPAC1565.05 utp8 303 Q9P6M1 SPAC688.04c gst3 228; 232 Q9P6M1 SPAC688.04c gst3 228; 232; 240 Q9P7L2 SPBC21C3.11 ubx4 241; 243; 245; 248; 251; 253 Q9P7U1 SPAC977.15 SPAC977.15 194; 198; 202; 204; 208 Q9URZ2 SPAC869.09 SPAC869.09 37; 39; 42; 43; 45; 46 Q9URZ2 SPAC869.09 SPAC869.09 16; 21 Q9US22 SPAC1783.08c rp11502 183; 188 Q9UT74 SPAC343.16 lys2 677; 678; 680; 682; 683; 684 Q9UTC5 SPAC227.14 yfh7 43; 44 Q9UTR5 SPAC1006.06 rgf2 43; 44; 46; 48; 50; 52; 55; 56 Q9UUA1 SPCC23B6.01c SPCC23B6.01c 400; 403; 408; 409; 414 Q9UUE1 SPBC17G9.11c pyr1 13; 14; 16; 20 Q9UUG0 SPAC926.09c fas1 2055; 2057 Q9Y7K2 SPBC216.07c tor2 1327 Q9Y7R8 SPCC306.08c mdh1 146; 151; 152 Q9Y7U0 SPCC63.14 eis1 439; 440; 448 Q9Y7U0 SPCC63.14 eis1 227 Q9Y7U0 SPCC63.14 eis1 1160; 1166; 1172 Q9Y7U0 SPCC63.14 eis1 596; 597; 601; 602 Q9Y7U0 SPCC63.14 eis1 992; 996; 997 Q9Y7U0 SPCC63.14 eis1 116; 117; 118; 121 Q9Y7U0 SPCC63.14 eis1 245; 251; 252 Q9Y7X6 SPBC365.12c ish1 234 Q9Y7X6 SPBC365.12c ish1 234; 243 Q9Y7X6 SPBC365.12c ish1 437; 439; 443 Q9Y7X6 SPBC365.12c ish1 393; 395; 396; 404; 406; 407 Q9Y7X6 SPBC365.12c ish1 675 Q9Y7X6 SPBC365.12c ish1 254 Q9Y7X6 SPBC365.12c ish1 389; 393; 395; 396; 404; 406; 407 Q9Y7X6 SPBC365.12c ish1 126; 130; 132; 134 Q9Y7X6 SPBC365.12c ish1 663; 664; 667; 668; 675 Q9Y7X6 SPBC365.12c ish1 569; 575; 577; 583 Q9Y7X6 SPBC365.12c ish1 667; 668; 675 Q9Y7X6 SPBC365.12c ish1 320 Q9Y7X6 SPBC365.12c ish1 658; 660; 663; 664; 667; 668; 675 Q9Y7X6 SPBC365.12c ish1 303 Q9Y7X6 SPBC365.12c ish1 384; 389; 393; 395; 396; 404; 406; 407 Q9Y7X6 SPBC365.12c ish1 418; 421; 422; 424 Q9Y7X6 SPBC365.12c ish1 418; 421; 422; 424; 437; 439 Q9Y7X6 SPBC365.12c ish1 21; 25; 26 Q9Y7X6 SPBC365.12c ish1 86; 88; 89; 95 Q9Y7X6 SPBC365.12c ish1 437; 439 Q9Y7X6 SPBC365.12c ish1 658; 660 Q9Y7X6 SPBC365.12c ish1 225; 226; 234 Q9Y7X6 SPBC365.12c ish1 160; 163; 167 Q9Y7X6 SPBC365.12c ish1 268; 269 Q9Y7X6 SPBC365.12c ish1 337 Q9Y7X6 SPBC365.12c ish1 664; 667; 668; 675 Q9Y7X6 SPBC365.12c ish1 556; 560; 563 Q9Y7X6 SPBC365.12c ish1 393; 395; 396 Q9Y7X6 SPBC365.12c ish1 452; 456 Q9Y7X6 SPBC365.12c ish1 37; 43; 48 Q9Y7X6 SPBC365.12c ish1 197; 207; 208; 211; 212 Q9Y7X6 SPBC365.12c ish1 389; 393; 395; 396 Q9Y7X6 SPBC365.12c ish1 389; 393; 395; 396; 404 Q9Y7X6 SPBC365.12c ish1 389; 393; 395; 396; 404; 406 Q9Y7X6 SPBC365.12c ish1 634; 640 Q9Y7X6 SPBC365.12c ish1 367; 370; 378; 381; 384 Q9Y7X6 SPBC365.12c ish1 437; 439; 443; 447 Q9Y7X6 SPBC365.12c ish1 74; 80 Q9Y7X6 SPBC365.12c ish1 418; 421; 422; 424; 437; 439; 443
[0097] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent application, foreign patents, foreign patent application and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, application and publications to provide yet further embodiments. These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
[0098] Applicants incorporate by reference herein in its entirety the following article (and all related supplements, supporting information, and data depositions: Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast; by Adnan Halima, Ida Signe Bohse Larsen, Patrick Neubert, Hiren Jitendra Joshia, Bent Larsen Petersen, Sergey Y. Vakhrushev, Sabine Strahl, and Henrik Clausen. Proc Natl Acad Sci USA. 2015 Dec. 22; 112(51):15648-53. This article is available at the following world-wide-web address: pnas.org/cgi/doi/10.1073/pnas.1511743112, and the supplement for this article is also incorporated herein by reference in its entirety, and is available at the following world-wide-web address: pnas.org/content/112/51/15648?tab=ds.
[0099] Data Deposition:
[0100] The mass spectrometry proteomics data related to the abovementioned PNAS manuscript have been deposited in the ProteomeXchange Consortium via the PRoteomics IDEntifications (PRIDE) Partner Repository (accession no. PXD002924), and this data is available at the world-wide-web addresss: proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD002924). The content of this data deposition is incorporated herein by reference in its entirety.
[0101] Supporting Information:
[0102] This manuscript also contains supporting information that is available online at the world wide web address: pnas.org/lookup/suppl/doi:10.1073/pnas.1511743112/-/DCSupplemental. The content of this supporting information is incorporated herein by reference in its entirety.
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