PROTEINS WITH DIAGNOSTIC AND THERAPEUTIC USES
20220009974 · 2022-01-13
Inventors
Cpc classification
C07K14/472
CHEMISTRY; METALLURGY
A61P7/00
HUMAN NECESSITIES
A61L29/16
HUMAN NECESSITIES
A61L31/16
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61P13/02
HUMAN NECESSITIES
A61K9/48
HUMAN NECESSITIES
C07K14/3156
CHEMISTRY; METALLURGY
A61L27/54
HUMAN NECESSITIES
International classification
A61K38/16
HUMAN NECESSITIES
A61K9/48
HUMAN NECESSITIES
A61L27/54
HUMAN NECESSITIES
A61L29/16
HUMAN NECESSITIES
A61L31/16
HUMAN NECESSITIES
Abstract
A method of treating one or more medical complications associated with aberrant complement regulatory activity in a subject includes the steps of: providing a pharmaceutical composition comprising a recombinant protein capable of binding to complement factor H (CFH) and inducing increased binding of C3d and C3b by bound CFH compared to unbound CFH; and administering to a subject an effective amount of the pharmaceutical composition.
Claims
1. A method of treating one or more medical complications associated with aberrant complement regulatory activity in a subject, comprising the steps of: providing a pharmaceutical composition comprising a recombinant protein capable of binding to complement factor H (CFH) and inducing increased binding of C3d and C3b by bound CFH compared to unbound CFH; and administering to a subject an effective amount of the pharmaceutical composition.
2. The method of claim 1, wherein the recombinant protein is capable of binding to wild type CFH to form a complex with a K.sub.D of 1×10−10 M or lower.
3. The method of claim 1, wherein the recombinant protein comprises a functional fragment or a functional variant of a bacterial virulence factor.
4. The method of claim 2, wherein the recombinant protein comprises a functional fragment or a functional variant of a bacterial virulence factor.
5. The method of claim 1, wherein the recombinant protein comprises a functional fragment or a functional variant of a pneumococcal surface protein.
6. The method of claim 5, wherein the recombinant protein comprises a functional fragment or a functional variant of pneumococcal surface protein C (PspC) from Streptococcus pneumoniae.
7. The method of claim 5, wherein the recombinant protein comprises a functional fragment or a functional variant of PspC of strain D39 (NCTC no 7466) of S. pneumoniae or PspC of strain TIGR4 (NCTC no 7465) of S. pneumoniae.
8. The method of claim 5, wherein the recombinant protein comprises a functional fragment of PspC, or a functional variant thereof, which is from 70 to 150 amino acids in length.
9. The method of claim 2, wherein the recombinant protein comprises a functional fragment or a functional variant of a pneumococcal surface protein.
10. The method of claim 9, wherein the recombinant protein comprises a functional fragment or a functional variant of pneumococcal surface protein C (PspC) from Streptococcus pneumoniae.
11. The method of claim 9, wherein the recombinant protein comprises a functional fragment or a functional variant of PspC of strain D39 (NCTC no 7466) of S. pneumoniae or PspC of strain TIGR4 (NCTC no 7465) of S. pneumoniae.
12. The method of claim 9, wherein the recombinant protein comprises a functional fragment of PspC, or a functional variant thereof, which is from 70 to 150 amino acids in length.
13. The method of claim 1, wherein the recombinant protein comprises the sequence: ATENEGSTQAATSSNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRKHTQNVALNIKL SAI KTKYLRELNVLEEKSKDELPSEIKAKLDAAFEKFKKDTLKPGEK (SEQ ID NO 1), or a functional variant or fragment thereof; or KQVVDEYIEKMLREIQLDRRKHTQNVALNIKLSAIKTKYLRELNVLEEKSKDELPSEIK AKLDAAFEKFKKDTLKPGEK (SEQ ID NO 2), or a functional variant or functional fragment thereof; or ATENEGATQVPTSSNRANESQAEQGEQPKKLDSERDKARKEVEEYVKKIVGESYA KSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAVSKFEKDSSSSS SSDSSTKPEASDTAKPNKPTEPGEK (SEQ ID NO 9), or a functional variant or functional fragment thereof.
14. The method of claim 2, wherein the recombinant protein comprises the sequence: ATENEGSTQAATSSNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRKHTQNVALNIKL SAI KTKYLRELNVLEEKSKDELPSEIKAKLDAAFEKFKKDTLKPGEK (SEQ ID NO 1), or a functional variant or functional fragment thereof; or KQWDEYIEKMLREIQLDRRKHTQNVALNIKLSAIKTKYLRELNVLEEKSKDELPSEIK AKLDAAFEKFKKDTLKPGEK (SEQ ID NO 2), or a functional variant or functional fragment thereof; or ATENEGATQVPTSSNRANESQAEQGEQPKKLDSERDKARKEVEEYVKKIVGESYA KSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAVSKFEKDSSSSS SSDSSTKPEASDTAKPNKPTEPGEK (SEQ ID NO 9), or a functional variant or functional fragment thereof.
15. The method of claim 5, wherein the recombinant protein comprises the sequence: ATENEGSTQAATSSNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRKHTQNVALNIKL SAIKTKYLRELNVLEEKSKDELPSEIKAKLDAAFEKFKKDTLKPGEK (SEQ ID NO 1), or a functional variant or functional fragment thereof; or KQVVDEYIEKMLREIQLDRRKHTQNVALNIKLSAIKTKYLRELNVLEEKSKDELPSEIK AKLDAAFEKFKKDTLKPGEK (SEQ ID NO 2), or a functional variant or functional fragment thereof; or ATENEGATQVPTSSNRANESQAEQGEQPKKLDSERDKARKEVEEYVKKIVGESYA KSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAVSKFEKDSSSSS SSDSSTKPEASDTAKPNKPTEPGEK (SEQ ID NO 9), or a functional variant or functional fragment thereof.
16. The method of claim 6, wherein the recombinant protein comprises at least amino acids 68-136 of PspC, or a functional variant thereof.
17. The method of claim 1, wherein the recombinant protein binds to CFH within complement control proteins (CCPs) 8-10 of CFH.
18. The method of claim 5, wherein the recombinant protein binds to CFH within complement control proteins (CCPs) 8-10 of CFH.
19. The method of claim 9, wherein the recombinant protein binds to CFH within complement control proteins (CCPs) 8-10 of CFH.
Description
BRIEF DESCRIPTION OF THE FIGURES
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SPECIFIC DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0217] The present inventors have discovered that proteins of the present invention have the ability to attach to the key regulator of the alternative pathway of complement activation, human complement factor H (CFH), in a manner that is effectively irreversible on the biological timescale (hours to days). Furthermore the PspCN-bound CFH has at least a three-fold higher affinity for its natural ligand C3b and has a very much greater affinity for C3d (an important degradation product of C3b).
[0218] The affinity between CFH and PspCN is so strong that it effectively permanently couples PspCN to Factor H. Presumably this explains the employment by S. Pneumoniae of PspC to protect itself against complement-mediated destruction (see below). The ability of PspC to activate CFH makes it an even more effective means of self-protection.
[0219] Thus PspCN and other proteins according to the present invention represent an elegant way of capturing CFH in a highly bioactive form. If PspCN were immobilised it could be used for isolating CFH from complex mixtures such as serum.
[0220] This ability of PspCN to both bind CFH and also to enhance its affinity for its ligands, and hence its regulatory activity, also suggests its potential use as a therapeutic agent for conditions where the primary aetiology is underactivity of CFH. Such conditions include atypical haemolytic uremic syndrome (aHUS), dense deposit disease (DDD) and age-related macular degeneration (AMD).
[0221] Modifications to this sequence could be made to facilitate the attachment of labelling/detection tags or for covalent surface attachment.
Example 1—an “Active” Conformation of Human Factor H is Stabilized Upon its Irreversible Capture by Streptococcus pneumoniae Protein PspC
Introduction
[0222] The complement system is the name given to a set of 40-50 blood proteins in vertebrates that cooperate amongst themselves, and with other immune components, to recognize, tag and eliminate potentially dangerous materials such as foreign cells and the products of apoptosis and oxidative damage.sup.(1). Strategies employed by bacteria to evade the complement system are potential targets for novel antibiotics and vaccines, while therapeutic modulation of the complement system is being actively explored in a wide range of clinical contexts.sup.(2).
[0223] Central to all the pathways that activate the complement system is a rare example of a positive-feedback loop in which the opsonin and pro-inflammatory protein molecule, C3b, is amplified through formation of the C3b.Bb complex that enzymatically cleaves molecules of C3 to form the anaphylatoxin C3a plus additional C3b (
[0224] Complement factor H.sup.(5-8), which is composed of 20 CCPs (9, is an important soluble regulator of this key event since it competes with complement factor B (CFB, the precursor of Bb) for binding to C3b, accelerates irreversible dissociation (also known as decay) of the C3b.Bb complex, and is a cofactor for complement factor I (CFI)-catalysed cleavage of C3b to form iC3b that cannot bind to CFB but (like the further sequential surface-bound proteolytic degradation products, C3dg and C3d) continues to function as an opsonin. The final cleavage product, C3d, equating to the TED of C3b (
[0225] The importance of CFH for modulation of C3b amplification is underlined by the links between allelic variations in the CFH gene and the risk of a range of diseases associated with an improperly regulated complement system.sup.(12). The common (one out of every three alleles) “haplotype 2” of CFH is responsible for an estimated 50% of the risk of age-related macular degeneration.sup.(13); it includes the substitution of histidine for tyrosine at position 402 in CCP 7 that contributes to the recognition by CFH of self-surface-specific molecular markers (such as GAG chains and sialic acid clusters).sup.(4-17). Mutations linked to the potentially fatal kidney disease, atypical haemolytic uraemic syndrome (aHUS) occur throughout CFH, but are clustered in CCPs 19 and 20.sup.(18) that likewise contribute to surface recognition but also contain a binding site for C3b and its degradation products.sup.(5). The N-terminal four CCPs of CFH bind C3b (but not iC3b or C3d), are required for both cofactor and decay-acceleration activity.sup.(5), and harbour mutations and SNPs linked to age-related macular degeneration (AMD), aHUS and membranoproliferative glomulonephritis type II.sup.(20). Potentially therapeutic proteins designed to incorporate CCPs 1-4.sup.(21), or both CCPs 1-4 and CCPs 19-20.sup.(22, 23) are at various stages of development.
[0226] As the chief soluble regulator of the complement system, CFH is inevitably susceptible to microbial hijack. It is not surprising that many bacterial surface proteins bind to plasma CFH.sup.(24, 25). For example, the sub-capsular protein PspC from the nasopharyngeal commensal bacterium Streptococcus pneumoniae (also called SpsA. CbpA, and Hic) captures host-derived CFH on the capsid surface.sup.(26-28). Invasion of the lower respiratory tract or blood by S. pneumoniae can cause pneumonia, meningitis and septicaemia. There is a growing and worrying recognition that vaccine-resistant strains may colonise the nasopharyngeal tract, taking the place of strains whose capsular serotypes are present in vaccines [Weinberger D M, Malley R, Lipsitch M. Serotype replacement in disease after pneumococcal vaccination. Lancet. 2011 Dec. 3; 378(9807):1962-73. doi:10.1016/S0140-6736(10)62225-8]. It is thus of great interest that serotype invasiveness seems to be determined mainly by the ability to bind CFH. Differences in production levels, or allelic variation, of the PspC are likely responsible for these variations in CFH capture.sup.(29).
[0227] An intriguing aspect of CFH is that its regulatory properties are modulated according to C3b context. In the fluid-phase, CFH restricts C3b production to a level required for immune surveillance (via the transient ability of nascent C3b to bind covalently to any local surface) but—importantly—prevents runaway activation that would deplete plasma C3 and factor B.sup.(3, 30) On the surfaces of healthy host tissue, CFH collaborates with membrane-bound regulators.sup.(31) to shut down C3b amplification almost entirely. On the other hand, moderate levels of C3b deposition and iC3b formation are facilitated by CFH in the case of senescent or damaged cells and cell debris, leading to non-inflammatory clearance.sup.(32-34). Finally, CFH does not suppress C3b amplification on bacterial cells (lacking CFH-binding proteins or other counter-complement measures) thereby permitting a full-blown onslaught by the complement system on a microbial invader involving very rapid opsonisation, the release of pro-inflammatory C3a and C5a, and cytolysis.
[0228] Why does CFH activity vary according to the context of C3b (and C3b.Bb)? This seems unlikely to be merely a matter of selective enrichment of CFH concentrations at those surfaces requiring protection, since such a mechanism would leave CFH highly vulnerable to being exploited by microbes. The inventors set out to test whether an evolutionary arms race between host and pathogens could shed light on the little-understood relationship between the structure of CFH and its function as a self-surface specific regulator of C3b amplification. According to one hypothesis, the fact that only six or seven out of a total of 20 CCPs in the CFH molecule—located towards both its N and C termini—engage with ligands directly.sup.(15) is readily explained if most or all of the remaining 13 or 14 CCPs act to make CFH less susceptible to successful hijacking by bacterial proteins. Thus, according to this idea, mere enrichment at the bacterial surface (or the host surface) would be insufficient for effective bacterial (or host cell) protection. Instead, the full regulatory potential of CFH could only be revealed following a conformational rearrangement brought about via interaction with specific self-surface molecular signatures, or their mimics on bacterial surfaces. To test this hypothesis the inventors explored the interaction between CFH and PspC. This revealed that PspC binds extremely tightly to CFH and stabilizes a rearrangement of CCPs that reveals the previously occluded C3b/C3d-binding site located close to its C-terminus. The inventors suggest that other bacterial proteins, and indeed host-surface molecular markers, may also act to Stabilize this “Activated” Form of CFH.
MATERIALS and METHODS
Preparation of Proteins
[0229] Human complement proteins—purified from pooled plasma—were obtained from Complement Technologies Inc. (Tyler, Tex.), stored according to the supplier's instructions, and freshly thawed before being diluted and used without further purification. A library of recombinant CFH module-truncation mutants (“fragments”, see
[0230] Full-length recombinant human CFH, with a wild-type sequence (CFH(wt)), was produced in recombinant P. pastoris grown in a fermenter and purified in an enzymatically deglycosylated form as described previously.sup.(35). Expression-optimised DNA encoding the D1119G mutant of CFH (CFH(D1119G)) was synthesised by Geneart-LifeTech and sub-cloned into the P. pastoris expression vector pPICZaB. Production and purification of CFH(D1119G) was subsequently performed as described previously for CFH(wt).
[0231] A gene representing residues 37-140 of PspC (D39) was optimised for expression in Escherichia coli (see SEQ ID NO 4 below) and purchased from GeneArt-LifeTech. The resulting construct was then cloned into the pE-SumoProKan E. coli expression vector (LifeSensors, Malvern, Pa., see
[0232] For most experiments, the catalytic domain from the SUMO-specific protease, ULP1, was used to remove the hexaHis-SUMO tags from sPspCN (or sPspCNR1) (with no vector-derived residues), following which the tag was removed by a second Ni.sup.2+-affinity chromatographic step. The cleaved material—PspCN (or PspCNR1)—was then purified further on a HiPrep Superdex 75 column in PBS as above. Proteins were judged to be homogeneous by SDS-PAGE and the integrity and identity of the proteins was confirmed by mass spectrometry (not shown).
SPR-Derived Measurement of CFH Binding to sPspCN
[0233] All surface plasmon resonance experiments were performed at 25° C. on a Biacore T200 instrument (GE Healthcare) using 10 mM HEPES, 150 mM NaCl, 0.05% v/v surfactant P20, pH 7.4 (HBS-P+) supplemented with 50 μM ethylenediaminetetra-acetic acid (EDTA). Ni.sup.2+ (51 RU) and subsequently sPspCN (144) (via its N-terminal hexaHis-SUMO tag) were immobilised on a Biacore NTA sensorchip. Aliquots from a solution of full-length CFH (from plasma or from recombinant P. pastoris), or CFH fragments, were flowed over the chip at the concentrations shown for 180 s at 100 μl/min, followed by a dissociation phase of 1500 s. Due to irreversible binding by CFH, the sensor chip was regenerated between measurements by two 30 s injections of 350 mM EDTA in 1 M NaCl, followed by 30 s injections of 50 mM NaOH, then 0.5 (w/v) % sodium dodecylsulfate (SDS). Data were analysed using the Biacore evaluation software, the curves were fitted and kinetic parameters estimated on the basis of a 1:1 binding model.
[0234] The use of an NTA sensorchip to measure the affinity of CFH for sPspCN avoided the need for amine coupling with its associated potential for heterologous deposition of the protein and the danger of masking ligand-recognition sites. Moreover, it allowed for relatively straightforward regeneration of the SPR sensorchip surface between measurements. The disadvantages of this method were gradual leaching of the hexaHis-tagged analyte from the chip surface, and the potential for CFH and its fragments to bind directly to Ni.sup.2+ (Nan R, Gor J, Lengyel I, Perkins S J. Uncontrolled zinc- and copper-induced oligomerisation of the human complement regulator factor H and its possible implications for function and disease. J Mol Biol. 2008 Dec. 31; 384(5):1341-52.). Indeed we observed (data not shown) weak binding of CFH to the Ni.sup.2+-loaded NTA sensorchip surface (no PspCN present) and tight binding of CFH 6-8 to this surface. These difficulties were largely circumvented by background subtraction.
SPR Measurement of C3b, C3c and C3d Binding Affinities
[0235] Experiments were performed as above except C3b (272 RU); C3c (166 RU); and C3d (69 RU) were immobilized, using standard amine coupling, on three different flow cells of a Biacore C1 sensorchip (the fourth flow cell was used for background subtraction). Factor H (pre-incubated with or without a twofold molar excess of PspCN) was injected over the chip at the concentrations shown (90 s, 30 μl/min), followed by a dissociation phase of 400 s. The sensor chip was regenerated by 2×30 s injections of 1 M NaCl. Data were analysed using the Biacore evaluation software and equilibrium affinity parameters were calculated using a 1:1 binding affinity model.
Decay-Accelerating Assay
[0236] The decay acceleration activity of CFH or CFH:PspCN was measured in an SPR-based assay as described previously. In brief, 535 RU of C3b was immobilised on a Biacore CM5 sensorchip via standard amine coupling. Subsequently, C3 convertase (C3b.Bb) was assembled on the chip by a 120 s at 10 μl/min injection of CFB plus CFD (500 nM+50 nM respectively). Formation of C3b.Bb (to 145 RU) was observed (by SPR), followed by an initial dissociation phase (200 s) to allow monitoring of the rate of intrinsic C3 convertase decay (departure of Bb). After this, CFH or CFH:PspCN complex were injected (120 sat 10 μl/min) at the concentrations shown followed by a further dissociation phase. The chip was regenerated between cycles by a 30 s injection of 0.2 μM CFH, followed by two 30 s injections of 1 M NaCl.
Haemolytic Assay
[0237] Sheep and rabbit whole blood were purchased from TCS Biosciences (Buckingham, UK) and CFH-depleted serum was purchased from Complement Technology. The haemolysis assay was performed in 96-well plates, based on a method described by Pangburn.sup.(36) except HEPES was used in place of the more traditional veronal buffer.sup.(37). Erythrocytes from 5 ml of whole blood were washed three times with 20 mM HEPES; 145 mM NaCl; 10 mM EDTA; 0.1% (w/v) gelatin (porcine skin, Fluka), pH 7.3 (Buffer H1), and a further three times with 20 mM HEPES; 145 mM NaCl; 100 μM EDTA; 0.1% (w/v) gelatin, pH 7.3 (Buffer H2). CFH-depleted serum was reconstituted with CFH construct to 2 μM and used at the final concentrations indicated. Erythrocytes were used at a concentration that gave a final A.sub.412 of 1 (rabbit) or 1.2 (sheep) when lysed completely with water. Serum reconstituted with CFH was then combined on ice, to the final concentrations indicated, with erythrocytes and Buffer H2 to yield a final volume of 45 μl. Lysis was initiated by the addition of 5 μl of a solution of 50 mM Mg.sup.2+, 50 mM ethylene glycol tetraacetic acid, pH 7.3. The reaction was incubated at 37° C. for 30 minutes whereupon it was quenched by the addition of 200 μl of Buffer H1. Unlysed cells were subsequently pelleted by centrifugation at 1500 g for 10 minutes at 4° C., then 100 μl of supernatant was removed and the A.sub.412 recorded.
Heparin-Affinity Chromatography
[0238] Protein samples (˜0.5 μM) were applied to a 1-ml HiTrap Heparin chromatography column (GE Healthcare) in 20 mM potassium phosphate, pH 7.5, and eluted with a linear gradient of 1 M NaCl over 20 column volumes.
Nuclear Magnetic Resonance Spectroscopy
[0239] NMR spectra were acquired on an Avance II 800 MHz spectrometer (Bruker) equipped with a 5-mm TCI CryoProbe (Bruker). NMR data were processed using TopSpin 3.1 (Bruker) and analysed using the CCPNMR Analysis software suite [Vranken W F, Boucher W, Stevens T J, Fogh R H, Pajon A, Llinas M, Ulrich E L, Markley J L, lonides J, Laue E D. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins. 2005 Jun. 1; 59(4):687-96.]. Samples of .sup.15N-labeled FH 8-9 and .sup.15N-labeled PspCN were used to optimize conditions by varying NaCl concentrations from 0-150 mM, pH from 4-6.5, and temperature from 15° C.-70° C. Subsequently, a sample of 40 μM .sup.15N-labeled FH 8-9 in 20 mM potassium phosphate, pH 6.2, was used to record a .sup.1H,.sup.15N HSQC spectrum at 310 K and then PspCN was added to a final concentration of 100 μM and the spectrum was re-recorded. In a separate experiment, a sample of 30 μM .sup.15N-labeled PspCN in 20 mM potassium phosphate, pH 6.0, was used to record a .sup.1H,.sup.15N HSQC spectrum at 298 K and then FH 8-9 was added to a final concentration of 40 μM and the spectrum re-recorded.
Cross-Linking and Mass Spectrometry (MS)
[0240] Freshly thawed samples of either plasma-purified CFH (20 μg) or plasma-purified CFH plus PspCN (20 μg CFH, a 1:1.15 molar ratio to PspCN) were cross-linked with bis[sulfosuccinimidyl] suberate (BS3) using a 1:4 mass ratio of protein-to-BS3, and a final [CFH]=0.56 μg/μl in the reaction mixtures. The products of cross-linking were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [(NuPAGE 4-12%, Bis-Tris gel with MOPS running buffer (Invitrogen)]. Gel bands were stained with Instantblue (Expedeon), and bands corresponding to five different cross-linking products (see
[0241] Cross-linked peptides were identified using in-house written software. In total 50 linkages were identified from these five samples. Label-free quantitation was carried out at linkage level as follows. For each unique linkage, mass spectrometric chromatographic signal intensities of all corresponding cross-linked peptides were retrieved using Pinpoint software (Thermo Fisher Scientific) and summed. To ensure that a fair comparison across samples was achieved, the signal intensity of individual linkage pairs were normalized using the summed signal intensity of 23 cross-linkages and 29 non-cross-linked CFH peptides that were observed in all five samples. The intensity variation of a cross-linkage across the five samples was thereby reflected in its relative intensity within each sample, which was calculated as the logarithm to base 5 of the quotient of the observed intensity and the average intensity of this cross-link in five samples. Furthermore, using Cluster 3.0 [de Hoon M J, Imoto S, Nolan J, Miyano S. Open source clustering software. Bioinformatics. 2004 Jun. 12; 20(9):1453-4], the 50 observed cross-links were clustered, based on their relative intensities in the five samples, in order to elucidate the correlation between their intensity variation and locations. These cross-links were not evenly distributed along the sequence of CFH and the bias in their locations afforded valuable insight into the conformation of full-length CFH and its conformational changes upon interaction with PspCN.
Results
The N-Terminal Region of PspCN Binds Irreversibly to CFH
[0242] The N-terminal region of PspC from S. pneumoniae (strain D39) (
TABLE-US-00004 TABLE 1 PspCN (D39 37-140) PspCN (TIGR4 37-179) PspCN (TIGR4 68-148) Construct k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D Plasma CFH 9.7e+6 4.4e−7 4.5e−14 1.0e+6 1.3e−10 1.4e−16 5.5e+5 4.3e−10 7.8e−16 rCFH 1.7e+11 8.8e−3 5.3e−14 rCFH-D1119G 4.1eE+6 4.7e−5 1.2e−11 CFH 8-15 1.2e+5 1.5e−4 1.3e−9 CFH 8-9 — — 2.6e−8
TABLE-US-00005 TABLE 2 C3b C3d Construct K.sub.D/M SE(K.sub.D) Chi.sup.2/RU.sup.2 K.sub.D/M SE(K.sub.D) Chi.sup.2/RU.sup.2 Plasma CFH 4.9e−7 6.0e−8 9.1 N/A N/A N/A Plasma CFH:PspCN 1.6e−7 1.8e−8 40.3 2.3e−6 1.6e−7 0.4 rCFH 5.8e−7 9.3e−8 29.8 N/A N/A N/A rCFH:PspCN 1.6e−7 2.2e−8 81.4 2.1e−6 2.0e−7 4.2 rCFH-D1119G 5.4e−7 9.3e−8 9.4 N/A N/A N/A rCFH-D1119G:PspCN 3.2e−7 4.2e−8 12.6 N/A N/A N/A
PspCN Binds to a Central Site in CFH
[0243] PspC was previously reported to bind to various sites of CFH including CCPs 8-11, 13-15 and 19-20 (Dave S, et al. 2004 Mayand Hammerschmidt S, et al. 2007); Duthy T G, et al. Infect Immun. 2002 October; 70(10):5604-11). We revisited this issue using sPspCN with relevant recombinant CFH fragments from our library.sup.(15) (
[0244] We found (
[0245] That PspCN interacts tightly with CCPs 8-9 suggested burial of a substantial intermolecular interface upon complex formation. To investigate further, we recorded a .sup.1H,.sup.15N HSQC NMR spectrum of .sup.15N CFH 8-9 with and without unlabeled PspCN. This revealed the expected very large number of .sup.1H and .sup.15N chemical-shift perturbations (
[0246] To interrogate the structure of the complex formed by PspCN and full-length CFH we used a combination of chemical cross-linking and mass spectrometry (MS). To samples of plasma-purified CFH mixed with PspCN we added BS3, which is homobifunctional and forms cross-links between primary amines that are up to 11.4 Å apart. We then resolved cross-linked products by SDS-PAGE, excised the bands and subjected them to tryptic digestion. Then we applied MS/MS to identify pairs of cross-linked peptides. Within the cross-linked product that migrated on SDS-PAGE as though it had a molecular weight (MW) corresponding to a 1:1 PspCN:CFH complex (Band s4,
[0247] While these experiments do not exclude the direct participation of other CCPs, taken together they imply that CCPs 8, 9 and 10 are primarily responsible for interactions between CFH and PspCN. Although PspCN—commensurate with its small size (12 kDa)—may bind directly to just a few central CCPs (of ˜7 kDa each), its remarkably tight complex with CFH may be favoured by a cooperative spatial rearrangement of other CCPs, stabilized by new intramolecular intermodular interactions. To test this hypothesis we compared chemical cross-linking data within CFH before and after PspCN addition.
Analysis of CFH Architecture by Cross-Linking and Mass Spectrometry
[0248] In the absence of cross-linker, a solitary band was detected following SDS-PAGE of a plasma-purified CFH sample, as expected. After a 60-minute incubation of the same CFH sample (0.56 μg/μL) with 3.9 mM BS3, two SDS-PAGE bands appeared that were both candidates for monomeric CFH (
[0249] Upon addition of PspCN at a 1.15:1 molar ratio to 0.56 μg/μL CFH, followed by incubation with 3.9 mM BS3, these two bands collapsed into one (
[0250] We surmised from these studies that binding of PspCN to central CFH facilitates adoption of a new stable conformation of CFH, helping to explain the negligible dissociation rate of the bimolecular complex. The PspCN-stabilized conformation of CFH may have nascent exposed self-association sites that would explain increased dimer capture by cross-linker. But analysis (in the absence of cross-linker) by size-exclusion chromatography-multiple angle light scattering (SEC-MALS) showed a predominantly 1:1 complex of plasma-purified CFH:PspCN and did not detect any CFH dimer before (loaded at 710 ug/ml) or after (loaded at 520 ug/ml) PspCN addition. Unexpectedly, a small quantity of dimeric PspCN had formed in the sample submitted for SEC-MALS before mixing with CFH, and there was a corresponding appearance of FH:PspCN.sub.2 complex. But these species were present in small amounts and are almost certainly artefacts. More importantly, we hypothesized that there are newly exposed ligand-binding sites in the PspCN-stabilized conformation of CFH and we set out to test this hypothesis through functional assays.
New Binding Site for TED/C3d Exposed by PspCN Binding to CFH
[0251] Commercially sourced plasma-purified human C3b, C3c and C3d were immobilized using standard amine coupling to different channels of the same C1 SPR chip (Biacore). Subsequently, having demonstrated that PspCN does not bind to C3b, C3c or C3d (data not shown), the affinities of CFH, or the CFH:PspCN complex, for C3b along with its various degradation products were measured in the same experiment (
[0252] We found that CFH (alone) did not bind well to C3c or C3d on the SPR chip (
Functional Assays of CFH:PspCN
[0253] Given that PspCN has evolved the capability to bind very tightly to CFH and modify its conformation, the question arises, what effect does PspCN binding have on the functional properties of CFH as an inhibitor of C3b amplification. We tested its effect on decay-accelerating activity by assembling the convertase on an SPR chip and watching it dissociate into its components before and after added CFH (or CFH:PspCN) (
PspCN Reveals Cryptic Effects of Disease-Linked Mutation in CFH
[0254] We produced in P. pastoris the mutant CFH(D1119G) (
[0255] We made the PspCN:CFH(D1119G) complex and tested its binding to C3b and C3d. The complex showed some improvement, compared to CFH(D1119G) alone, in C3b binding (Table 2) but—unlike the CFH(wt):PspCN complex—it showed no enhancement of C3d binding. Thus PspCN binding has revealed a striking difference between CFH(wt) and CFH(D1119G) that was not evident from a C3b/C3d-binding study performed in the absence of PspCN on an SPR chip. This reaffirms our hypothesis that PspCN can disinter the partially or completely buried C-terminal C3d/C3b-binding site of CFH.
[0256] We next asked whether the latent deficit in the ability of CFH(D1119G) to bind to the TED domain of C3b, which can be unmasked by PspCN, has consequences for the ability of this mutant to accelerate the decay of C3b.Bb. We found that CFH(D1119G), by itself, accelerates the disassociation of C3b.Bb about as well as does CFH(wt) (
[0257] Next we investigated the extent to which the D1119G mutation perturbs CFH from acting on a surrogate host-cell surface. Plasma-purified human CFH, when added to CFH-deficient human serum, protects sheep erythrocytes (
[0258] Finally we attempted to partially recreate the scenario of a heterozygous individual with the CFH(D1119G) mutation by performing the same haemolysis assays using a mixture of 1 μM CFH(wt) and 1 μM CFH(D1119G) (
Discussion
[0259] CFH is key to the control of C3b amplification that is the pivotal event in the operation of the complement system. It is essential that CFH distinguishes between C3b on host cells and on foreign ones. This is unlikely to be solely a matter of selective enrichment of CFH at those surfaces requiring protection; that would allow CFH to be very easily hijacked by microbes, rendering the complement system virtually useless as a first line of defence against infection. Thus CFH may have evolved to “hide” some of its complement-regulatory sites and reveal them only in the presence of certain molecular markers. We speculated that such a hypothetical strategy could explain why CFH has so many CCP modules that do not appear to bind directly to ligands. They could allow CFH to adopt different conformations—exposing different ligand-binding sites—in response to surface-specific molecular signatures. Our results presented here, based on an investigation of the complex formed between PspCN and CFH, strongly back such a model.
[0260] We showed, using SPR, that the N-terminal 104-amino acid residues of PspC from S. pneumoniae (D39) (residues 37-140 of the pre-processed protein) are sufficient to bind irreversibly, on the biological timescale, to recombinant human CFH as well as human CFH purified from blood. This observation is in agreement with the previously reported binding of plasma-purified CFH (Lu L, Ma Y, Zhang J R. Streptococcus pneumoniae recruits complement factor H through the amino terminus of CbpA. J Biol Chem. 2006 Jun. 2; 281(22):15464-74), to a similar-length N-terminal construct of PspCN, which was assessed in solution using isothermal titration calorimetry and found to be too tight to measure (<1 nM). Our complex of PspCN:CFH is significantly more stable than the complex observed in a previously conducted SPR-based experiment using a slightly extended PspC construct (residues 39-152) (Hammerschmidt S, Agarwal V, Kunert A, Haelbich S, Skerka C, Zipfel P F. The host immune regulator factor H interacts via two contact sites with the PspC protein of Streptococcus pneumoniae and mediates adhesion to host epithelial cells. J Immunol. 2007 May 1; 178(9):5848-58). On the other hand, we did not observe any loss or gain of affinity for a significantly longer construct (residues 39-259) and it is likely that PspCN encompasses the entirety of the CFH-binding site within full-length PspC.
[0261] Plasma-derived CFH has eight out of nine potential N-glycosylation sites, mainly in central CCPs, occupied by bi-sialylated bi-antennary glycans (Fenaille F, Le Mignon M, Groseil C, Ramon C, Riandé S, Siret L, Bihoreau N. Site-specific N-glycan characterization of human complement factor H. Glycobiology. 2007 September; 17(9):932-44). The recombinant CFH used in this study (both wild-type and D1119G) had been enzymatically deglycosylated. That all these versions of CFH have comparable affinities for PspCN shows that the N-glycans of plasma CFH do not play a significant role in PspCN binding. They also appeared to be non-essential for binding to C3b or for participation in a range of assays carried out in vitro (Jouvin M H, Kazatchkine M D, Cahour A, Bernard N. Lysine residues, but not carbohydrates, are required for the regulatory function of H on the amplification C3 convertase of complement. J Immunol. 1984 December; 133(6):3250-4).
[0262] Assuming that PspC on the bacterial surface has a similar affinity as PspCN for CFH, then the bacterium will acquire and retain—presumably for the duration of its time spent in the bloodstream—a CFH molecule attached to each of its surface-displayed PspC molecules. It has been suggested (Agarwal V, Asmat T.sub.M, Luo S, Jensch I, Zipfel P F, Hammerschmidt S. Complement regulator Factor H mediates a two-step uptake of Streptococcus pneumoniae by human cells. J Biol Chem. 2010 Jul. 23; 285(30):23486-95; Quin L R, Onwubiko C, Moore Q C, Mills M F, McDaniel L S, Carmicle S. Factor H binding to PspC of Streptococcus pneumoniae increases adherence to human cell lines in vitro and enhances invasion of mouse lungs in vivo. Infect Immun. 2007 August; 75(8):4082-7) that S. pneumoniae utilizes this molecule not only for avoiding C3b-amplification but also, preparatory to invasion, for localization at the surfaces of host cells bearing ligands for CFH. It was therefore intriguing to learn more about the structural and functional properties of this remarkably tight complex.
[0263] Based on three orthogonal approaches—binding affinities of truncation mutants, NMR chemical-shift perturbations and chemical cross-linking—we found that PspCN binds very tightly towards the middle region of CFH at a site within CCPs 8-10. We obtained no evidence for significant direct participation of other CCPs. Nonetheless full-length CFH binds to PspCN far more tightly than FH 8-15 so it is clear that other CCPs play a role, albeit an indirect one, in complex formation as discussed further below. Our results concur with a flow-cytometry study (Dave S, et al. 2004) that implied some or all of CCPs 6-10 were crucial for binding of CFH, to Pneumococcal isolates of 14 different strains, via the N-terminal 225 amino acids of PspCN. Our results are also largely in agreement with Hammerschmidt et al (Hammerschmidt S, et al. 2007) who used a 250-residue segment of PspC from strain ATCC 33400 (serotype 1) that they called SH2. They showed by dot blot and by SPR (although no K.sub.D values were reported) that CCPs 8-11 binds to SH2 while FH 1-7, FH 11-15 and FH 16-20 showed no or very little binding to SH2. These authors demonstrated that heparin (that interacts with CCPs 7 and 20 of CFH, but also binds PspCN according to our observations) inhibits the SH2-CFH interaction, while a monoclonal antibody to CCPs 19-20 inhibits binding of SH2 to FH 8-20. This suggested to the authors a second direct PspCN-binding site must be located on the C-terminal modules. Our studies (although they do demonstrate a very weak FH 19-20:PspCN interaction) suggest these inhibitors are more likely to work indirectly by blocking an intramolecular component of the binary complex, as discussed further below. Another study (Duthy T G, et al. 2002) also reported binding (by ELISA) of FH 8-15 as well as FH 8-13, but not FH 1-7, FH 10-12 or FH 16-20, to PspC, which concurs with our data. They were, however, unable to detect binding for FH 8-12, and only weak binding to FH 8-14, and they thus assigned the binding site to FH 13-15. All their constructs were produced as recombinant truncation mutants in P. pastoris, like the ones in the current study, but yields were very low, so authentication would have been difficult. In sum, there are now multiple lines of evidence supporting direct engagement by PspC of modules 8-10 in CFH that are free of binding sites for C3b, GAGs or other known ligands. This would be consistent with the notion that it benefits the bacterium to leave these sites available.
[0264] The negligible off-rate and hence remarkably high affinity of the CFH:PspCN complex may derive from the adoption of stable, mutually dependent, new conformations by both protein partners upon complex formation. Thus, although its MW is just 11 kD, and hence it could only contact a few CCPs simultaneously, PspCN binds extremely tightly to CFH (155 kD) and this domain becomes a folded protein in the process (we have not investigated whether this domain is folded when it is part of the intact PspC protein).
[0265] Full-length CFH binds to PspCN approximately four orders-of-magnitude more tightly than does CFH 8-9, and three orders-of-magnitude more tightly than does CFH 8-15. This is despite the lack of evidence from our studies that CCPs other than 8, 9 and 10 participate directly in PspCN binding. Cross-linking tells us that PspCN binding causes major changes in the structure of the bigger protein. Due to its extended and flexible architecture, and the consequent potential for interactions between non-neighbouring CCP modules, free CFH has multiple conformational possibilities; our result suggest that some of these are more stable than others but are separated from one another by relatively high activation energy barriers. PspcN binding could induce the formation of a stable conformer not kinetically accessible to CFH in the absence of PspCN. The existence of such a conformation is suggestive that self-surface specific molecular markers (such as GAGs and sialic acid) might also act to facilitate transitions between CFH conformers through a similar strategy. The interesting observation that other bacterial proteins (unlike PspC) bind directly to the same regions of CFH (CCPs 7 and 20) (Ferreira V P, Pangburn M K, Cortes C. Complement control protein factor H: the good, the bad, and the inadequate. Mol Immunol. 2010 August; 47(13):2187-97) as do host-surface molecular markers (Schmidt C Q, Herbert A P, Kavanagh D, Gandy C, Fenton C J, Blaum B S, Lyon M, Uhrin D, Barlow P N. A new map of glycosaminoglycan and C3b binding sites on factor H. J Immunol. 2008 Aug. 15; 181(4):2610-9) indicates that they too might act in this way.
[0266] The results discussed so far relate to cross-links observed in a 1:1 PspCN:CFH complex. This is the dominant species in a mixture of CFH and PspCN according to analysis by SEC-MALS (loaded onto the size-exclusion column at 500 mg mL.sup.−1 or ˜3 mM). Yet the cross-linking data indicated that PspCN increased the tendency of CFH to form dimers in which CCPs 5 and 7 (presumably from different monomers) are brought close together and there is also a dimer-specific cross-link between CCPs 18 and 20. While two molecules of CFH could interact directly with the same PspCN molecule, this seems unlikely given the small size of PspCN, and its large interaction surface with CCPs 8-9 in what appeared to be the 1:1 complex observed in the NMR samples. On the other hand it is feasible that the altered CFH conformation stabilized by PspCN results in exposure of previously buried regions of CFH that participate in a weak self-association that can be captured by cross-linking; based on the observed dimer-specific cross-links these are likely to involve CCPs 5-7 and possibly CCPs 18-20. This effect may be a product of encounters (captured by cross-linking) between two soluble PspCN:CFH complexes. This result parallels a previous report that soluble GAG-surrogates, such as heparin, induced oligomerisation of soluble CFH that could not be explained by multiple CFH molecules binding to the same GAG, and that might also have arisen from soluble CFH:GAG complexes self-associating via nascent exposed sites. It is not known whether CFH/PspC dimers form on the bacterial surface.
[0267] In this study we used SPR to measure the affinity of FH and FH:PspCN for C3b chemically immobilized on a carboxymethylated surface of a sensorchip. This experimental setup is probably a very poor model of a self-surface, and hence the affinities observed in the case of SPR may be similar to those that would be observed on a “random” complement-activating surface (such as a microbe). In the current study, both recombinant and plasma-purified CFH interacted with C3b, immobilized on the surface of a SPR sensorchip, with a K.sub.D (0.6 mM) that is in line with other reported values. This is 15-times tighter and four-times tighter, respectively, compared to either FH 1-4 (K.sub.D=˜10 mM) or FH 19-20 (K.sub.D=2-3 mM) (Schmidt C Q, Herbert A P, Kavanagh D, Gandy C, Fenton C J, Blaum B S, Lyon M, Uhrin D, Barlow P N. A new map of glycosaminoglycan and C3b binding sites on factor H. J Immunol. 2008 Aug. 15; 181(4):2610-9). We also were in agreement with authors of previous studies.sup.(38 and 22) in our observation that full-length CFH binds poorly to C3d immobilized on the SPR sensorchip when compared to either CFH or CFH 19-20 binding to C3b, or CFH 19-20 binding to C3d. It is noteworthy that in previous studies, C3b was deposited on the chip in various ways, including via its thioester, and similar results were obtained.sup.(39). Thus the C3b/TED-binding site at the C-terminus of intact CFH is not fully available when CFH is presented with C3d on the SPR chip surface.
[0268] This begs the important question as to whether the C-terminal C3b/d-binding site of CFH, which is unavailable to bind C3d, is nonetheless deployed following an encounter between CFH and C3b. Interestingly, in the current study, CFH(D1119G) binds to C3b on the SPR sensorchip almost as well as does CFH(wt), despite the fact that the fragment CFH 19-20(D1119G) has virtually no affinity for C3b or C3d.sup.(39). This suggests that in SPR-based assays of CFH binding to C3b (as well as those measuring binding to C3d), the C-terminal site effectively makes no contribution. On the other hand, CFH(D1119G) (like CFH(wt)) binds fifteen-fold more tightly than does CFH 1-4. It may be that additional CCPs between CCPs 5 and 18) contribute to the intermolecular interaction and indeed it was shown previously that recombinant CFH 6-8 binds very weakly to C3b.sup.(15) and Jokiranta T S, Hellwage J, Koistinen V, Zipfel P F, Meri S. Each of the three binding sites on complement factor H interacts with a distinct site on C3b. J Biol Chem. 2000 Sep. 8; 275(36):27657-62). Alternatively, the CFH:C3b complex could be favoured by intramolecular interactions that are absent in free CFH (by analogy with the CFH:PspCN complex). In either case, we may conclude that the C-terminal C3b/C3d binding site in full-length CFH is, by default, masked or occluded.
[0269] In contrast, it is clear that the C3b/C3d binding site is available in the CFH:PspCN complex, since this is the only coherent explanation for its >50-fold better C3d binding, compared to intact CFH, which equates with the C3d-binding affinity of FH 19-20. Moreover, two-site binding of CFH:PspCN to C3b also helps to explain its relatively high affinity for C3b (60-fold higher than for FH 1-4 alone and 18-fold higher than FH 19-20 alone). Although the difference in affinities (for C3b) between CFH and CFH:PspCN is “only” four fold, this could be highly significant for the bacterium. Were CFH captured in its “latent” conformation, a K.sub.D of 0.6 μM (for the C3b:CFH complex) might be inadequate for control of C3b amplification that is a binary on-off process able to overwhelm regulators once a C3b population threshold is breached; on the other hand a K.sub.D of 0.15 μM for the CFH:PspCN-C3b complex may be sufficient to maintain the numbers of tethered C3b molecules on the surface below the threshold for run-away activation. Such a notion, together with the ten-fold improvement in decay-acceleration activity, is consistent with the significantly improved haemolysis-prevention properties of PspCN-bound CFH observed in the present study. The ability of PspCN to restore haemolysis protection in CFH-depleted serum supplemented with a 1:1 mixture of CFH(wild-type) and CFH(D1119G) is suggestive of its therapeutic potential.
[0270] These observations suggest strongly that other bacterial proteins induce the same or a similar activated conformation of CFH to that induced by binding of PspCN. The great majority of bacterial proteins bind elsewhere to CFH but they could nonetheless stabilize a similar conformation. Cross-linking shows that CCPs 7 and 20 are close together in the PspCN complex and many bacterial proteins engage both these sites, potentially bringing them together. It will therefore be important to explore the structures of other bacterial protein:CFH complexes using similar methods to those we have deployed in the current study. A tantalizing further possibility is that molecular markers on self-surfaces stabilize the same activated conformation of CFH. It is noteworthy that polyanionic markers (modelled experimentally by heparin) bind to CCPs 7 and 20.sup.(15) while more recently, oxidation-specific epitopes (thought to be important for recruiting CFH to damaged cells) also interact with these two CCPs..sup.(34) This possibility highlights the importance of using biomimetic surfaces for assessing the functional repercussions of disease-linked SNPs and mutations in CFH because otherwise some will be missed—as exemplified herein with our C3b-binding study of CFH(D1119G).
[0271] In conclusion, there is now strong evidence to suggest that CFH circulates in a latent form wherein some of its C3b-binding capability is locked away, preventing it being a highly effective regulator of C3b amplification and therefore spontaneously shutting down complement activation altogether. Factor H can also exist in an “active” form in which its C3b-binding sites are available and juxtaposed in such a way as to bind bivalently and therefore avidly to its C3b (or C3b.Bb) target. The bacterial protein PspC can induce this active conformation and it is feasible that other bacterial proteins use the same trick (although via a different form of engagement with CFH). That such a conformation exists, and is exploited by bacteria, is highly suggestive of it also being the active conformation on self surfaces. Further studies are now needed to test whether this is indeed the molecular basis for self versus non-self discrimination by the key soluble regulator of the vertebrate complement system.
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[0299] 28. Jarva, H., Janulczyk, R., Hellwage, J., Zipfel, P. F., Bjorck, L., and Meri, S. (2002) Streptococcus pneumoniae evades complement attack and opsonophagocytosis by expressing the pspC locus-encoded Hic protein that binds to short consensus repeats 8-11 of factor H, Journal of immunology 168, 1886-1894. [0300] 29. Hyams, C., Trzcinski, K., Camberlein, E., Weinberger, D. M., Chimalapati, S., Noursadeghi, M., Lipsitch, M., and Brown, J. S. (2012) Streptococcus pneumoniae capsular serotype invasiveness correlates with the degree of factor H binding and opsonisation with C3b/iC3b, Infection and immunity. [0301] 30. Bexborn, F., Andersson, P. O., Chen, H., Nilsson, B., and Ekdahl, K. N. (2008) The tick-over theory revisited: formation and regulation of the soluble alternative complement C3 convertase (C3(H2O)Bb), Mol Immunol 45, 2370-2379. [0302] 31. Kirkitadze, M. D., and Barlow, P. N. (2001) Structure and flexibility of the multiple domain proteins that regulate complement activation, Immunol Rev 180, 146-161. [0303] 32. Trouw, L. A., Bengtsson, A. A., Gelderman, K. A., Dahlback, B., Sturfelt, G., and Blom, A. M. (2007) C4b-binding protein and factor H compensate for the loss of membrane-bound complement inhibitors to protect apoptotic cells against excessive complement attack, The Journal of biological chemistry 282, 28540-28548. [0304] 33. Leffler, J., Herbert, A. P., Norstrom, E., Schmidt, C. Q., Barlow, P. N., Blom, A. M., and Martin, M. (2010) Annexin-II, DNA, and histones serve as factor H ligands on the surface of apoptotic cells, The Journal of biological chemistry 285, 3766-3776. [0305] 34. Weismann, D., Hartvigsen, K., Lauer, N., Bennett, K. L., Scholl, H. P., Charbel Issa, P., Cano, M., Brandstatter, H., Tsimikas, S., Skerka, C., Superti-Furga, G., Handa, J. T., Zipfel, P. F., Witztum, J. L., and Binder, C. J. (2011) Complement factor H binds malondialdehyde epitopes and protects from oxidative stress, Nature 478, 76-81. [0306] 35. Schmidt, C. Q., Slingsby, F. C., Richards, A., and Barlow, P. N. (2011) Production of biologically active complement factor H in therapeutically useful quantities, Protein Expr Purif 76, 254-263. [0307] 36. Pangburn, M. K. (2002) Cutting edge: localization of the host recognition functions of complement factor H at the carboxyl-terminal: implications for hemolytic uremic syndrome, Journal of immunology 169, 4702-4706. [0308] 37. Blaum, B. S., Deakin, J. A., Johansson, C. M., Herbert, A. P., Barlow, P. N., Lyon, M., and Uhrin, D. (2010) Lysine and arginine side chains in glycosaminoglycan-protein complexes investigated by NMR, cross-linking, and mass spectrometry: a case study of the factor H-heparin interaction, Journal of the American Chemical Society 132, 6374-6381. [0309] 38. Goicoechea de Jorge, E., Caesar, J. J., Malik, T. H., Patel, M., Colledge, M., Johnson, S., Hakobyan, S., Morgan, B. P., Harris, C. L., Pickering, M. C., and Lea, S. M. (2013) Dimerization of complement factor H-related proteins modulates complement activation in vivo, Proceedings of the National Academy of Sciences of the United States of America 110, 4685-4690. [0310] 39. Morgan, H. P., Schmidt, C. Q., Guariento, M., Blaum, B. S., Gillespie, D., Herbert, A. P., Kavanagh, D., Mertens, H. D., Svergun, D. I., Johansson, C. M., Uhrin, D., Barlow, P. N., and Hannan, J. P. (2011) Structural basis for engagement by complement factor H of C3b on a self surface, Nat Struct Mol Biol 18, 463-470. [0311] 40. Kazatchkine, M. D., Fearon, D. T., and Austen, K. F. (1979) Human alternative complement pathway: membrane-associated sialic acid regulates the competition between B and beta1 H for cell-bound C3b, Journal of immunology 122, 75-81.
Example 2—Further Investigation of PspCN and Related Proteins
[0312] Further work was conducted to investigate the properties of PspCN, variants of PspCN and therapeutic uses of such proteins.
Methods:
Protection Against Haemolysis of PNH-Like Erythrocyte
[0313] PNH-like human erythrocytes were prepared according to a modified version of Ezzell et al. (1991). Briefly, whole human blood was obtained from donors by phlebotomy and stored in Alsever's solution at 4° C. Cells were washed three times with RBC Wash Buffer (20 mM HEPES; 145 mM NaCl; 10 mM EDTA; 0.1% w/v gelatine, pH 7.3) and re-suspended after the final wash in PBS. After each wash step the top 10% of the cell pellet was aspirated to remove the white cell-containing buffy coat. A 1-ml aliquot of packed cells was added to an 8% w/v solution of 2-aminoethylisothiouronium bromide (AET, Sigma-Aldrich) solution that had been titrated to pH 8.0 using HCl. The cell suspension was incubated for nine minutes at 37° C. with constant agitation, after which time the cells were washed three times with RBC Wash Buffer containing no EDTA.
[0314] PNH-like cells were then washed once with 20 mM HEPES; 145 mM NaCl; 0.1% w/v gelatine, pH 6.4. These cells were then incubated at 37° C. with acidified normal human serum, 5 mM magnesium EGTA, and the appropriate concentration of either plasma-purified CFH (Complement Technology) or purified recombinant PspCN in a total reaction volume of 50 μl. After 30 minutes the reaction was quenched by the addition of 200 μl of ice-cold quench buffer (20 mM HEPES; 145 mM NaCl; 10 mM EDTA; 0.1% w/v gelatine; pH 7.3). The cells and cell debris were removed by centrifugation at 3,000×g for 10 minutes at 4° C., after which a 100-μl aliquot was removed and its absorbance at 410 nm recorded.
Determination of the Minimum PspC Sequence Necessary for Factor H Binding
[0315] Constructs containing the coding sequence for PspC(52-140), PspC(62-140), PspC(72-140), PspC(37-130), PspC(37-120) and PspC(37-110) were produced according to the method used to make the PspCN(37-140) sequence. CFH binding was then assessed using the same SPR assay that had been developed to determine the CFH-binding affinity of the PspCN(37-140) protein.
Modification of PspCN
[0316] The N and C termini of PspCN were modified to contain a cysteine and flexible peptide linker segment. In the case of the N-terminally Cys modified PspCN (Cys-PspCN) the extra residues included were: CGSGSGSGSGG-PspCN (SEQ ID NO 7), and in the case of the C-terminally Cys modified PspCN (PspCN-Cys) the extra residues included were: PspCN-GSGSGSGSGGC* (SEQ ID NO 8). These modified versions of PspCN were cloned and expressed as SUMO-fusion proteins in E. coli using the same methods as had been developed for PspCN. The purification of these proteins was also similar to that used for PspCN with the exception that tris(2-carboxyethyl)phosphine (TCEP) was included during all steps of purification in order to preserve the thiol groups of the cysteine residues.
SPR Experiments to Determine Whether the Cys-Mutated Proteins Retain FH Binding Ability
[0317] SPR was performed using His-SUMO-Cys-PspCN and His-SUMO-PspCN-Cys as the ligand in SPR experiments and commercially available plasma-purified CFH (Complement Technology, Tyler Tex.), using the same method as that used to assess the CFH-binding ability of sPspCN.
PEGylation of Cys-PspCN
[0318] Cys-PspCN was mixed with a 20-fold molar excess of methoxy-PEG-maleimide (Sigma-Aldrich) and incubated for 2.5 h at room temperature with stirring in PBS supplemented with 1 mM TCEP. Aliquots of the putatively PEGylated Cys-PspCN were subjected to SDS-PAGE under reducing conditions. Successfully PEGylated protein was judged to be that which displayed a band shift relative to the untreated protein.
Quantification of CFH Levels in Normal Human Serum
[0319] ELISA style assays to quantify CFH levels in normal human serum were carried out in standard flat-bottomed 96-well plates. Wells were coated with sPspCN (100 μl; 8 μg/ml) overnight at 4° C. and were subsequently blocked using phosphate buffered saline (PBS) supplemented with 5% w/v fat-free dried milk powder. Wells were washed three times with PBS supplemented with 0.05% v/v Tween 20. Wells were then incubated with for one hour at room temperature with a 1:800 dilution of the primary antibody (OX24-Biotin (Lifespan Biosciences LS-C62878)). Wells were then washed three times with PBS supplemented with 0.05% v/v Tween 20. Wells were then incubated with for one hour at room temperature with a 1:250 dilution of streptavidin-peroxidase polymer ultrasensitive (Sigma-Aldrich 5-2438). Wells were then washed three times with PBS supplemented with 0.05% v/v Tween 20. Wells were then incubated for 20 minutes with o-phenylenediamine dihydrochloride (OPD) substrate followed by the addition of an equal volume of 2.5 M H.sub.2SO.sub.4. The absorbance of the wells was read at 490 nm.
Results:
[0320] PspCN is able to inhibit haemolysis of ‘PNH-like’ erythrocytes by acidified serum. The results of the haemolysis assay in
The Minimum Sequence Necessary for Wild-Type Factor H Binding is 62-140
[0321] The sensorgrams shown in
Cys-Modified PspCN Binds to Factor H with a Similar Affinity to Native-Sequence PspCN
[0322] The shape of the curves shown in
[0323] Furthermore,
[0324] In addition, with reference to
Cys-PspCN can be Effectively PEGylated with Methoxy-PEG-Maleimide
[0325] Methoxy-PEG-maleimide efficiently couples to Cys-PspCN under mild conditions as can be seen in
CFH can Effectively be Quantified Using PspCN in an ‘ELISA Style’ Assay.
[0326] Plasma-purified CFH (Complement Technology), at known concentrations, was used to build up a standard curve (
Preparing the PspCN Affinity Chromatography Resin.
[0327] PspCN (37-140) modified to contain an N-terminal Cys residue and a linker comprised of the residues GSGSGSGSGG (Cys-PspCN) was coupled to Ultralink Iodoacetyl chromatography resin (Thermo Scientific) via its N-terminal Cys residue. The coupling was performed based on the manufacturer's instructions. Ultralink Iodoacetyl resin (10 ml) was washed with 50 ml of coupling buffer (50 mM Tris, 5 mM EDTA, pH 8.5) using a gravity fed chromatography column. A solution of Cys-PspCN (20 ml at 2 mg/ml) in coupling buffer supplemented with 2.5 mM TCEP was mixed with the Ultralink Iodoacetyl resin for 15 minutes at room temperature in a gravity fed chromatography column. The column was then stood upright and incubated for a further 30 minutes at room temperature. Unbound Cys-PspCN was then removed by draining off the fluid phase and washing the column with 30 ml of coupling buffer. To block unreacted iodoacetyl groups on the resin, the resin was then mixed with 20 ml of 50 mM L-cysteine.HCl in coupling buffer for 15 minutes at room temperature. The column was then stood upright and incubated for a further 30 minutes at room temperature. The resin was then washed with 60 ml of 1 M NaCl followed by 60 ml of phosphate buffered saline (PBS) supplemented with 0.05% NaN.sub.3. Using this method approximately 1-1.5 mg of Cys-PspCN was coupled to each ml of resin.
Purification of FH from Human Plasma
[0328] Human plasma (normal mixed pool) in Alsever's solution was purchased from TCS Biosciences. The PspCN resin (5 ml) was incubated with 100 ml of the human plasma at 4° C. for 1 hour. Following the incubation the mixture was applied to an empty gravity fed chromatography column and the plasma drained off. The resin was then washed with 3×50 ml of HBS (20 mM HEPES; 150 mM NaCl; pH 7.5), followed by a further wash with 2×50 ml of 4 M NaCl. Highly purified FH was eluted from the column using 0.1 M glycine buffer, pH 2.5.
[0329] The successful isolation of FH from human plasma is shown in the gels of
Use of PspCN to Protect Surfaces
[0330] With reference to
[0331] Accordingly, the presence of a PspCN coating at a surface can significantly reduce the deposition of C3b on that surface, and therefore, reduce the immunoresponse to that surface.
Use of PspCN as a Diagnostic to Detect the Presence of Anti-FH Autoantibodies:
[0332] With reference to
TIGR4 Strain
[0333] Fragments of the PspC protein of the TIGR4 strain of Streptococcus pneumonia, referred to here as TIGR4 PspC, was shown to bind tightly to CFH. TIGR4 PspC (37-179) binds CFH with a K.sub.D of 10.sup.−16 M and TIGR4 PspC (68-148) binds CFH with a K.sub.D of 10.sup.−15 M.
Ability of PspCN (CbpN) from S. pneumoniae Strain TIGR4 to Bind to and Activate CFH
[0334] With reference to
[0335] With reference to
[0336] With reference to
TABLE-US-00006 TABLE 3 C3b C3d Construct K.sub.D/M SE(K.sub.D) Chi.sup.2/RU.sup.2 K.sub.D/M SE(K.sub.D) Chi.sup.2/RU.sup.2 CFH 5.5e−7 7.8e−8 0.35 N/A N/A N/A CFH:TIGR4 (37-179) 2.8e−7 3.3e−8 1.15 1.2e−6 8.4e−8 0.10
REFERENCES FOR EXAMPLE 2
[0337] Brooks-Walter et al. (1999) Infect Immun. 1999 December; 67(12): 6533-6542. [0338] Ezzell, J L et al. (1991) Blood 77:2764-2773
RELEVANT SEQUENCES
[0339]
TABLE-US-00007 D39 PspC gene sequence-complete CDS (SEQ ID NO 3) 1 atgcttgtca ataatcacaa atatgtagat catatcttgt ttaggacagt aaaacatcct 61 aattactttt taaatattct tcctgagttg attggcttga ccttgttgag tcatgcttat 121 gtgacttttg ttttagtttt tccagtttat gcagttattt tgtatcgacg aatagctgaa 181 gaggaaaagc tattacatga agttataatc ccaaatggaa gcataaagag ataaatacaa 241 aattcgattt atatacagtt catattgaag taatatagta aggttaaaga aaaaatatag 301 aaggaaataa acatgtttgc atcaaaaagc gaaagaaaag tacattattc aattcgtaaa 361 tttagtattg gagtagctag tgtagctgtt gccagtcttg ttatgggaag tgtggttcat 421 gcgacagaga acgagggaag tacccaagca gccacttctt ctaatatggc aaagacagaa 481 cataggaaag ctgctaaaca agtcgtcgat gaatatatag aaaaaatgtt gagggagatt 541 caactagata gaagaaaaca tacccaaaat gtcgccttaa acataaagtt gagcgcaatt 601 aaaacgaagt atttgcgtga attaaatgtt ttagaagaga agtcgaaaga tgagttgccg 661 tcagaaataa aagcaaagtt agacgcagct tttgagaagt ttaaaaaaga tacattgaaa 721 ccaggagaaa aggtagcaga agctaagaag aaggttgaag aagctaagaa aaaagccgag 781 gatcaaaaag aagaagatcg tcgtaactac ccaaccaata cttacaaaac gcttgaactt 841 gaaattgctg agttcgatgt gaaagttaaa gaagcggagc ttgaactagt aaaagaggaa 901 gctaaagaat ctcgaaacga gggcacaatt aagcaagcaa aagagaaagt tgagagtaaa 961 aaagctgagg ctacaaggtt agaaaacatc aagacagatc gtaaaaaagc agaagaagaa 1021 gctaaacgaa aagcagatgg taagttgaag gaagctaatg tagcgacttc agatcaaggt 1081 aaaccaaagg ggcgggcaaa acgaggagtt cctggagagc tagcaacacc tgataaaaaa 1141 gaaaatgatg cgaagtcttc agattctagc gtaggtgaag aaactcttcc aagctcatcc 1201 ctgaaatcag gaaaaaaggt agcagaagct gagaagaagg ttgaagaagc tgagaaaaaa 1261 gccaaggatc aaaaagaaga agatcgccgt aactacccaa ccaatactta caaaacgctt 1321 gaccttgaaa ttgctgagtc cgatgtgaaa gttaaagaag cggagcttga actagtaaaa 1381 gaggaagcta aggaacctcg agacgaggaa aaaattaagc aagcaaaagc gaaagttgag 1441 agtaaaaaag ctgaggctac aaggttagaa aacatcaaga cagatcgtaa aaaagcagaa 1501 gaagaagcta aacgaaaagc agcagaagaa gataaagtta aagaaaaacc agctgaacaa 1561 ccacaaccag cgccggctac tcaaccagaa aaaccagctc caaaaccaga gaagccagct 1621 gaacaaccaa aagcagaaaa aacagatgat caacaagctg aagaagacta tgctcgtaga 1681 tcagaagaag aatataatcg cttgactcaa cagcaaccgc caaaaactga aaaaccagca 1741 caaccatcta ctccaaaaac aggctggaaa caagaaaacg gtatgtggta cttctacaat 1801 actgatggtt caatggcaac aggatggctc caaaacaacg gttcatggta ctatctaaac 1861 gctaatggtg ctatggcgac aggatggctc caaaacaatg gttcatggta ctatctaaac 1921 gctaatggtt caatggcaac aggatggctc caaaacaatg gttcatggta ctacctaaac 1981 gctaatggtg ctatggcgac aggatggctc caatacaatg gttcatggta ctacctaaac 2041 agcaatggcg ctatggcgac aggatggctc caatacaatg gctcatggta ctacctcaac 2101 gctaatggtg atatggcgac aggatggctc caaaacaacg gttcatggta ctacctcaac 2161 gctaatggtg atatggcgac aggatggctc caatacaacg gttcatggta ttacctcaac 2221 gctaatggtg atatggcgac aggttgggtg aaagatggan atacctggta ctatcttaaa 2281 gcatcaggtg ctatgaaagc aagccaatgg ttcaaagtat cagataaatg gtactatgtc 2341 aatggctcag gtgcccttgc agtcaacaca actgtagatg gctatggagt caatgccaat 2401 ggtgaatggg taaactaaac ctaatataac tagttaatac tgacttcctg taagaacttt 2461 ttaaagtatt ccctacaaat accatatcct ttcagtagat aatataccct tgtaggaagt 2521 ttagattaaa aaataactct gtaatctcta gccggattta tagcgctaga gactacggag 2581 tttttttgat gaggaaagaa tggcggcatt caagagactc tttaagagag ttacgggttt 2641 taaactatta agccttctcc aattgcaaga gggcttcaat ctctgctagg gtgctagctt 2701 gcgaaatggc tccacggagt ttngc
[0340] Amino acid sequence transcribed from SEQ ID NO 3 (SEQ ID NO 4)—the PspCN sequence (amino acids 37 to 140) is shown in bold:
TABLE-US-00008 MFASKSERKVHYSIRKFSIGVASVAVASLVMGSVVHATENEGSTQAAT SSNMAKTEHRKAAKQVVDEYIEKMLRETQLDRRKHTQNVALNIKLSAI KTKYLRELNVLEEKSKDELPSEIKAKLDAAFEKFKKDTLKPGEKVAEA KKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAEFDVKVKEAELEL VKEEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKRK ADGKLKEANVATSDQGKPKGRAKRGVPGELATPDKKENDAKSSDSSVG EETLPSSSLKSGKKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNTYKTL DLEIAESDVKVKEAELELVKEEAKEPRDEEKIKQAKAKVESKKAEATR LENIKTDRKKAEEEAKRKAAEEDKVKEKPAEQPQPAPATQPEKPAPKP EKPAEQPKAEKTDDQQAEEDYARRSEEEYNRLTQQQPPKTEKPAQPST PKTGWKQENGMWYFYNTDGSMATGWLQNNGSWYYLNANGAMATGWLQN NGSWYYLNANGSMATGWLQNNGSWYYLNANGAMATGWLQYNGSWYYLN SNGAMATGWLQYNGSWYYLNANGDMATGWLQNNGSWYYLNANGDMATG WLQYNGSWYYLNANGDMATGWVKDGXTWYYLKASGAMKASQWFKVSDK WYYVNGSGALAVNTTVDGYGVNANGEWVN
[0341] The synthetic, codon-optimised DNA sequence that was used for the expression of PspCN is shown below (SEQ ID NO 5):
TABLE-US-00009 GCAACCGAAAATGAAGGTAGCACCCAGGCAGCAACCAGCAGCAATATG GCAAAAACCGAACATCGTAAAGCAGCCAAACAGGTTGTGGATGAGTAT ATCGAAAAAATGCTGCGTGAAATTCAGCTGGATCGTCGTAAACATACC CAGAATGTTGCACTGAACATTAAACTGAGCGCCATCAAAACCAAATAT CTGCGTGAACTGAATGTGCTGGAAGAGAAAAGCAAAGATGAACTGCCG AGCGAAATTAAAGCAAAACTGGATGCAGCCTTTGAAAAATTCAAAAAA GATACCCTGAAACCGGGTGAGAAATAA
TABLE-US-00010 Human CFH amino acid sequence-signal peptide underlined-un-processed form (SEQ ID NO 6): 10 20 30 40 MRLLAKIICL MLWAICVAED CNELPPRRNT EILTGSWSDQ 50 60 70 80 TYPEGTQAIY KCRPGYRSLG NVIMVCRKGE WVALNPLRKC 90 100 110 120 QKRPCGHPGD TPFGTFTLTG GNVFEYGVKA VYTCNEGYQL 130 140 150 160 LGEINYRECD TDGWTNDIPI CEVVKCLPVT APENGKIVSS 170 180 190 200 AMEPDREYHF GQAVRFVCNS GYKIEGDEEM HCSDDGFWSK 210 220 230 240 EKPKCVEISC KSPDVINGSP ISQKIIYKEN ERFQYKCNMG 250 260 270 280 YEYSERGDAV CTESGWRPLP SCEEKSCDNP YIPNGDYSPL 290 300 310 320 RIKHRTGDEI TYQCRNGFYP ATRGNTAKCT STGWIPAPRC 330 340 350 360 TLKPCDYPDI KHGGLYHENM RRPYFPVAVG KYYSYYCDEH 370 380 390 400 FETPSGSYWD HIHCTQDGWS PAVPCLRKCY FPYLENGYNQ 410 420 430 440 NYGRKFVQGK SIDVACHPGY ALPKAQTTVT CMENGWSPTP 450 460 470 480 RCIRVKTCSK SSIDIENGFI SESQYTYALK EKAKYQCKLG 490 500 510 520 YVTADGETSG SITCGKDGWS AQPTCIKSCD IPVFMNARTK 530 540 550 560 NDFTWFKLND TLDYECHDGY ESNTGSTTGS IVCGYNGWSD 570 580 590 600 LPICYERECE LPKIDVHLVP DRKKDQYKVG EVLKFSCKPG 610 620 630 640 FTIVGPNSVQ CYHFGLSPDL PICKEQVQSC GPPPELLNGN 650 660 670 680 VKEKTKEEYG HSEVVEYYCN PRFLMKGPNK IQCVDGEWTT 690 700 710 720 LPVCIVEEST CGDIPELEHG WAQLSSPPYY YGDSVEFNCS 730 740 750 760 ESFTMIGHRS ITCIHGVWTQ LPQCVAIDKL KKCKSSNLII 770 780 790 800 LEEHLKNKKE FDHNSNIRYR CRGKEGWIHT VCINGRWDPE 810 820 830 840 VNCSMAQIQL CPPPPQIPNS HNMTTTLNYR DGEKVSVLCQ 850 860 870 880 ENYLIQEGEE ITCKDGRWQS IPLCVEKIPC SQPPQIEHGT 890 900 910 920 INSSRSSQES YAHGTKLSYT CEGGFRISEE NETTCYMGKW 930 940 950 960 SSPPQCEGLP CKSPPEISHG VVAHMSDSYQ YGEEVTYKCF 970 980 990 1000 EGFGIDGPAI AKCLGEKWSH PPSCIKTDCL SLPSFENAIP 1010 1020 1030 1040 MGEKKDVYKA GEQVTYTCAT YYKMDGASNV TCINSRWTGR 1050 1060 1070 1080 PTCRDTSCVN PPTVQNAYIV SRQMSKYPSG ERVRYQCRSP 1090 1100 1110 1120 YEMFGDEEVM CLNGNWTEPP QCKDSTGKCG PPPPIDNGDI 1130 1140 1150 1160 TSFPLSVYAP ASSVEYQCQN LYQLEGNKRI TCRNGQWSEP 1170 1180 1190 1200 PKCLHPCVIS REIMENYNIA LRWTAKQKLY SRTGESVEFV 1210 1220 1230 CKRGYRLSSR SHTLRTTCWD GKLEYPTCAK R