Title: Functional Characterization of the Complement Control Protein Homolog of Herpesvirus Saimiri
Abstract: Herpesvirus saimiri (HVS) is a lymphotropic virus that causes T-cell lymphomas in New World primates. It encodes a structural homolog of complement control proteins named complement control protein homolog (CCPH). Previously, CCPH has been shown to inhibit C3d deposition on target cells exposed to complement. Here we have studied the mechanism by which it inactivates complement. We have expressed the soluble form of CCPH in Escherichia coli, purified to homogeneity and compared its activity to vaccinia virus complement control protein (VCP) and human complement regulators factor H and soluble complement receptor 1. The expressed soluble form of CCPH bound to C3b (KD = 19.2 μm) as well as to C4b (KD = 0.8 μm) and accelerated the decay of the classical/lectin as well as alternative pathway C3-convertases. In addition, it also served as factor I cofactor and supported factor I-mediated inactivation of both C3b and C4b. Time course analysis indicated that although its rate of inactivation of C4b is comparable with VCP, it is 14-fold more potent than VCP in inactivating C3b. Site-directed mutagenesis revealed that Arg-118, which corresponds to Lys-120 of variola virus complement regulator SPICE (a residue critical for its enhanced C3b cofactor activity), contributes significantly in enhancing this activity. Thus, our data indicate that HVS encodes a potent complement inhibitor that allows HVS to evade the host complement attack. Herpesvirus saimiri (HVS) is a lymphotropic virus that causes T-cell lymphomas in New World primates. It encodes a structural homolog of complement control proteins named complement control protein homolog (CCPH). Previously, CCPH has been shown to inhibit C3d deposition on target cells exposed to complement. Here we have studied the mechanism by which it inactivates complement. We have expressed the soluble form of CCPH in Escherichia coli, purified to homogeneity and compared its activity to vaccinia virus complement control protein (VCP) and human complement regulators factor H and soluble complement receptor 1. The expressed soluble form of CCPH bound to C3b (KD = 19.2 μm) as well as to C4b (KD = 0.8 μm) and accelerated the decay of the classical/lectin as well as alternative pathway C3-convertases. In addition, it also served as factor I cofactor and supported factor I-mediated inactivation of both C3b and C4b. Time course analysis indicated that although its rate of inactivation of C4b is comparable with VCP, it is 14-fold more potent than VCP in inactivating C3b. Site-directed mutagenesis revealed that Arg-118, which corresponds to Lys-120 of variola virus complement regulator SPICE (a residue critical for its enhanced C3b cofactor activity), contributes significantly in enhancing this activity. Thus, our data indicate that HVS encodes a potent complement inhibitor that allows HVS to evade the host complement attack. The complement system is an integral participant in the innate mechanisms of immunity and, thus, has a burden of performing surveillance in the host and protecting it from all the pathogens including viruses (1Cooper N.R. Volanakis J.E. Frank M.M. The Human Complement System in Health and Disease. Marcel Dekker, Inc., New York1998: 393-407Google Scholar, 2Mullick J. Kadam A. Sahu A. Trends Immunol. 2003; 24: 500-507Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Earlier studies have decisively demonstrated that both acute and latent viruses are susceptible to complement-mediated neutralization (3Bernet J. Mullick J. Singh A.K. Sahu A. J. Biosci. 2003; 28: 249-264Crossref PubMed Scopus (74) Google Scholar, 4Lachmann P.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8461-8462Crossref PubMed Scopus (31) Google Scholar). Thus, complement exerts a strong selective pressure on viruses during infection. These data suggest that for their successful survival, viruses must have developed mechanisms to subvert this system. Consistent with this premise, genome sequencing of poxviruses and herpesviruses have shown that members of these families encode for structural homologs of human regulators of the complement activation (RCA) 3The abbreviations used are: RCA, regulators of complement activation; AP, alternative pathway; CP, classical pathway; HVS, herpesvirus saimiri; CCP, complement control protein; vCCP, viral CCP; CCPH, complement control protein homolog; sCCPH, soluble CCPH of herpesvirus saimiri; VCP, vaccinia virus complement control protein; SPICE, smallpox inhibitor of complement enzymes; MOPICE, monkeypox inhibitor of complement enzymes; Kaposica, Kaposis's sarcoma-associated herpesvirus inhibitor of complement activation; sCR1, soluble complement receptor 1; EA, antibody coated sheep erythrocytes; ORF, open reading frame; RU, response unit. family (5Seet B.T. Johnston J.B. Brunetti C.R. Barrett J.W. Everett H. Cameron C. Sypula J. Nazarian S.H. Lucas A. McFadden G. Annu. Rev. Immunol. 2003; 21: 377-423Crossref PubMed Scopus (507) Google Scholar, 6Alcami A. Koszinowski U.H. Trends Microbiol. 2000; 8: 410-418Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 7Means R.E. Lang S.M. Chung Y.H. Jung J.U. Front. Biosci. 2002; 7: 185-203Crossref PubMed Google Scholar, 8Gewurz B.E. Gaudet R. Tortorella D. Wang E.W. Ploegh H.L. Curr. Opin. Immunol. 2001; 13: 442-450Crossref PubMed Scopus (50) Google Scholar, 9Liszewski M.K. Leung M.K. Hauhart R. Buller R.M. Bertram P. Wang X. Rosengard A.M. Kotwal G.J. Atkinson J.P. J. Immunol. 2006; 176: 3725-3734Crossref PubMed Scopus (89) Google Scholar). The RCA family members are formed by tandemly repeating complement control protein (CCP) domains or short consensus repeats, which fold into a bead-like structure, and multiple CCPs are separated by linkers of 2-7 residues (10Hourcade D. Holers V.M. Atkinson J.P. Adv. Immunol. 1989; 45: 381-416Crossref PubMed Scopus (394) Google Scholar, 11Kirkitadze M.D. Barlow P.N. Immunol. Rev. 2001; 180: 146-161Crossref PubMed Scopus (176) Google Scholar, 12Blom A.M. Villoutreix B.O. Dahlback B. Mol. Immunol. 2004; 40: 1333-1346Crossref PubMed Scopus (153) Google Scholar). These proteins regulate complement by two distinct mechanisms (i) by accelerating the irreversible dissociation of the classical/lectin (C4b,2a) and alternative (C3b,Bb) pathway C3-convertases and (ii) by serving as cofactors in serine protease factor I-mediated inactivation of C3b and C4b (the subunits of C3-convertases) (13Pangburn M.K. Immunopharmacology. 2000; 49: 149-157Crossref PubMed Scopus (149) Google Scholar, 14Lambris J.D. Sahu A. Wetsel R. Volanakis J.E. Frank M. The Human Complement System in Health and Disease. Marcel Dekker Inc., New York1998: 83-118Google Scholar). To date detailed characterization of all these activities has been performed for the complement control protein homologs of vaccinia virus (VCP) (15Mckenzie R. Kotwal G.J. Moss B. Hammer C.H. Frank M.M. J. Infect. Dis. 1992; 166: 1245-1250Crossref PubMed Scopus (139) Google Scholar, 16Sahu A. Isaacs S.N. Soulika A.M. Lambris J.D. J. Immunol. 1998; 160: 5596-5604Crossref PubMed Google Scholar, 17Mullick J. Bernet J. Panse Y. Hallihosur S. Singh A.K. Sahu A. J. Virol. 2005; 79: 12382-12393Crossref PubMed Scopus (40) Google Scholar), variola virus (SPICE) (18Rosengard A.M. Liu Y. Nie Z. Jimenez R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8808-8813Crossref PubMed Scopus (177) Google Scholar), monkeypox virus (MOPICE) (9Liszewski M.K. Leung M.K. Hauhart R. Buller R.M. Bertram P. Wang X. Rosengard A.M. Kotwal G.J. Atkinson J.P. J. Immunol. 2006; 176: 3725-3734Crossref PubMed Scopus (89) Google Scholar), and Kaposi's sarcoma-associated herpesvirus (Kaposica/KCP) (19Mullick J. Bernet J. Singh A.K. Lambris J.D. Sahu A. J. Virol. 2003; 77: 3878-3881Crossref PubMed Scopus (57) Google Scholar, 20Spiller O.B. Blackbourn D.J. Mark L. Proctor D.G. Blom A.M. J. Biol. Chem. 2003; 278: 9283-9289Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Sequence comparison of the viral homologs of RCA (vCCPs) show that the sequence similarity between the poxvirus homologs exceeds 91%, whereas that among the herpesvirus homologs varies from 43 to 89%. These data suggest that the herpesvirus homologs are more diverse in structure compared with the poxvirus homologs. Whether this structural diversity in herpesvirus homologs is also reflected in their function is not clear, as among the herpesvirus homologs, detailed functional characterization has been performed only for the Kaposi's sarcoma-associated herpesvirus homolog (Kaposica/KCP) (19Mullick J. Bernet J. Singh A.K. Lambris J.D. Sahu A. J. Virol. 2003; 77: 3878-3881Crossref PubMed Scopus (57) Google Scholar, 20Spiller O.B. Blackbourn D.J. Mark L. Proctor D.G. Blom A.M. J. Biol. Chem. 2003; 278: 9283-9289Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 21Mullick J. Singh A.K. Panse Y. Yadav V. Bernet J. Sahu A. J. Virol. 2005; 79: 5850-5856Crossref PubMed Scopus (32) Google Scholar, 22Mark L. Lee W.H. Spiller O.B. Proctor D. Blackbourn D.J. Villoutreix B.O. Blom A.M. J. Biol. Chem. 2004; 279: 45093-45101Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Herpesvirus saimiri (HVS), the prototype of rhadinoviruses, is regularly found in its natural host, the squirrel monkey. Although it does not cause any disease in its natural host, infection in other New World primates such as tamarins, common marmosets, and owl monkey causes acute peripheral T cell lymphoma within less than 2 months (23Desrosiers R.C. Falk L.A. J. Virol. 1982; 43: 352-356Crossref PubMed Google Scholar, 24Desrosiers R.C. Bakker A. Kamine J. Falk L.A. Hunt R.D. King N.W. Science. 1985; 228: 184-187Crossref PubMed Scopus (98) Google Scholar). In addition, the virus is also capable of transforming simian and human T cells in vitro (25Biesinger B. Trimble J.J. Desrosiers R.C. Fleckenstein B. Virology. 1990; 176: 505-514Crossref PubMed Scopus (118) Google Scholar, 26Cabanillas J.A. Cambronero R. Pacheco-Castro A. Garcia-Rodriguez M.C. Martin-Fernandez J.M. Fontan G. Regueiro J.R. Clin. Exp. Immunol. 2002; 127: 366-373Crossref PubMed Scopus (11) Google Scholar). Interestingly, unlike any other viruses, the HVS harbors two homologs of complement regulatory proteins, (i) a homolog of RCA encoded by ORF4 and (ii) a homolog of the terminal complement inhibitor CD59 encoded by ORF15 (27Albrecht J.C. Nicholas J. Biller D. Cameron K.R. Biesinger B. Newman C. Wittmann S. Craxton M.A. Coleman H. Fleckenstein B. J. Virol. 1992; 66: 5047-5058Crossref PubMed Google Scholar, 28Means R.E. Choi J.K. Nakamura H. Chung Y.H. Ishido S. Jung J.U. Curr. Top Microbiol. Immunol. 2002; 269: 187-201PubMed Google Scholar). The ORF4 was predicted to encode a protein containing four CCP modules followed by a transmembrane domain. Analysis of posttranscriptional processing indicated that ORF4 transcript occurs as unspliced as well as single-spliced mRNA. The unspliced mRNA codes for a membrane-bound glycoprotein containing four extracellular CCPs along with a transmembrane region, whereas the spliced mRNA codes for a soluble protein that lacks transmembrane region (29Albrecht J.C. Fleckenstein B. J. Virol. 1992; 66: 3937-3940Crossref PubMed Google Scholar). Initial characterization of the RCA homolog of HVS (named complement control protein homolog, CCPH) showed that expression of the membrane form of this protein on BALB/3T3 cells inhibited C3d deposition on these cells when they were incubated with whole human serum (30Fodor W.L. Rollins S.A. Biancocaron S. Rother R.P. Guilmette E.R. Burton W.V. Albrecht J.C. Fleckenstein B. Squinto S.P. J. Virol. 1995; 69: 3889-3892Crossref PubMed Google Scholar). Although this study demonstrated the complement inhibiting activity of this protein, the mechanism by which it inactivates complement activation was not elucidated. In the present study we describe the mechanism of complement regulation by the RCA homolog of HVS. Our results show that the soluble form of the RCA homolog (sCCPH; CCP1-4 without the transmembrane domain) interacts with complement proteins C3b as well as C4b and accelerates decay of the classical/lectin and alternative pathway C3-convertases. In addition, the protein also has the ability to serve as factor I cofactor and support factor I-mediated inactivation of C3b and C4b. Importantly, we show that its factor I cofactor activity for C3b is 14-fold higher in comparison to VCP, the most completely characterized vCCP, and that Arg-118 plays a critical role in enhancing this activity. Reagents and Buffers—Antibody-coated sheep erythrocytes (EA) were made by incubating sheep erythrocytes with anti-sheep erythrocyte antibodies procured from ICN Biomedical Inc. (Irvine, CA). Veronal-buffered saline (VBS) contained 5 mm barbital, 145 mm NaCl, and 0.02% sodium azide, pH 7.4. GVB was VBS containing 0.1% gelatin, GVB2+ was GVB containing 0.5 mm MgCl2 and 0.15 mm CaCl2, and GVBE was GVB with 10 mm EDTA. MgEGTA contained 0.1 m MgCl2 and 0.1 m EGTA, and phosphate-buffered saline, pH 7.4, contained 10 mm sodium phosphate and 145 mm NaCl. Complement Proteins and Their Proteolytically Activated Products—The human complement protein C3 was purified according to Hammer et al. (31Hammer C.H. Wirtz G.H. Renfer L. Gresham H.D. Tack B.F. J. Biol. Chem. 1981; 256: 3995-4006Abstract Full Text PDF PubMed Google Scholar) with minor modifications as previously described (17Mullick J. Bernet J. Panse Y. Hallihosur S. Singh A.K. Sahu A. J. Virol. 2005; 79: 12382-12393Crossref PubMed Scopus (40) Google Scholar), and native C3 was separated from C3 (H2O) by running on a Mono S column (32Pangburn M.K. J. Immunol. Methods. 1987; 102: 7-14Crossref PubMed Scopus (56) Google Scholar). The complement factors H and I were kindly provided by Prof. Michael K. Pangburn (University of Texas Health Centre, Tyler, TX.). Human factor B was purified as follows. Human plasma was subjected to a stepwise precipitation with 11 and 26% polyethylene glycol. The 26% polyethylene glycol precipitate was dissolved in 10 mm sodium phosphate, pH 7.4, run on Source Q column (Amersham Biosciences) in the same buffer, and eluted with a linear gradient of 0-0.5 m NaCl. Fractions containing factor B were identified by Ouchterlony analysis, pooled, and loaded onto a Mono S 5/5 column (Amersham Biosciences) in 50 mm sodium phosphate, pH 6.0. Bound proteins were eluted with a linear salt gradient of 0-0.5 m NaCl and analyzed by SDS-PAGE. Homogeneous factor B fractions were pooled and concentrated. The recombinant human soluble form of complement receptor type 1 (sCR1) was a generous gift from Dr. Henry Marsh (AVANT Immunotherapeutics, Inc., Needham, MA.). C3b, the proteolytically activated form of C3, was generated by limited tryptic cleavage of C3 and purified on a Mono Q 5/5 (Amersham Biosciences) column as previously described (16Sahu A. Isaacs S.N. Soulika A.M. Lambris J.D. J. Immunol. 1998; 160: 5596-5604Crossref PubMed Google Scholar). C4b, the proteolytically activated form of C4, was purchased from Calbiochem. Purity of all the proteins exceeded 95%, as judged by SDS-PAGE analysis. Cloning, Expression, Purification, and Refolding of the Soluble Form of Herpesvirus Saimiri Complement Control Protein Homolog (sCCPH) and the R118A Mutant—The herpesvirus saimiri CCPH gene (CCP domains 1-4) was PCR-amplified from the CCPH clone pCEX-1 (a kind gift of Drs. John Lambris, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA and Jens-Christian Albrecht, Institut für Klinische und Molekulare Virologie, Erlangen, Germany) with specific primers 5′-GGAATTCAGCTGTCCTACACGTAACCAG-3′ (the EcoRI site is underlined) and 5′-CCGCTCGAGCATACATTCAGGAATAGCTGG-3′ (the XhoI site is underlined) and cloned into the bacterial expression vector pET29 at the EcoRI and XhoI sites. The R118A mutant was constructed from this clone by using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). After verifying the fidelity of both the clones by DNA sequencing, they were transformed into Escherichia coli BL21 cells for expression. Expression of sCCPH and R118A mutant (numbering according to the mature protein sequence (29Albrecht J.C. Fleckenstein B. J. Virol. 1992; 66: 3937-3940Crossref PubMed Google Scholar)) was performed as described below. A single colony of the bacterial clone expressing sCCPH or the mutant protein was inoculated into 5 ml of LB-kanamycin media (LB media containing 30 μg/ml kanamycin) and grown overnight at 37 °C, and 2 ml of this culture was transferred into 100 ml of LB-kanamycin. The culture was grown for 2 h at 37°C, and thereafter 10 ml of the culture was transferred to 600 ml of LB-kanamycin and grown at 37 °C until the optical density reached 0.6 at A600. Protein expression was induced by the addition of 1 mm isopropyl 1-thio-β-d-galactopyranoside, and the induced culture was further grown for 4 h. The cells were harvested by centrifugation at 8000 rpm at 4 °C. For purification of the expressed proteins, frozen cell pellets (∼16 g) were gently resuspended in 48 ml of 50 mm Tris, pH 8.0, 1 mm EDTA, 100 mm NaCl, and 0.1 mm phenylmethylsulfonyl fluoride. The cell suspension was then treated with lysozyme (0.3 mg/ml), stirred for 20 min, mixed with deoxycholic acid (1.3 mg/g), and stored at 37 °C for 30 min. After incubation, the lysate was sonicated with 15 pulses of 15 s each and centrifuged at 10,000 × g for 20 min at 4 °C. The pellet containing the inclusion bodies was washed twice with 50 mm Tris, pH 8.0, 10 mm EDTA, 100 mm NaCl, and 0.5% Triton X-100 and solubilized in 50 mm Tris, pH 8.0, 1 mm EDTA, 100 mm NaCl, and 8 m urea. The sample was then centrifuged at 4 °C for 30 min at 11,000 rpm, and the supernatant obtained was loaded onto a nickel nitrilotriacetic acid-agarose column (Qiagen, Hilden, Germany) pre-equilibrated with 100 mm NaH2PO4, 10 mm Tris, 8 m urea, pH 8.0. The column was washed with the binding buffer containing 10 mm imidazole, and the bound protein was eluted with 400 mm imidazole. The purified proteins were refolded by using a rapid dilution method (1:50) described previously (33White J. Lukacik P. Esser D. Steward M. Giddings N. Bright J.R. Fritchley S.J. Morgan B.P. Lea S.M. Smith G.P. Smith R.A. Protein Sci. 2004; 13: 2406-2415Crossref PubMed Scopus (36) Google Scholar). In brief, the purified protein was added dropwise with continuous stirring into a refolding buffer containing 0.02 m ethanolamine, 1 mm EDTA, 0.5 m l-arginine, 1 mm reduced glutathione, and 1 mm oxidized glutathione, pH 11.0. The sample was then left static for 36 h. The refolded sample was concentrated, dialyzed against phosphate-buffered saline, and subjected to SDS-PAGE, circular dichroism, and sequencing and mass analysis by mass spectrometry (17Mullick J. Bernet J. Panse Y. Hallihosur S. Singh A.K. Sahu A. J. Virol. 2005; 79: 12382-12393Crossref PubMed Scopus (40) Google Scholar). Measurement of Factor I Cofactor Activity—Analysis of factor I cofactor activities of sCCPH and the mutant was essentially performed as described (34Bernet J. Mullick J. Panse Y. Parab P.B. Sahu A. J. Virol. 2004; 78: 9446-9457Crossref PubMed Scopus (37) Google Scholar). These assays were performed in physiological ionic strength buffer (phosphate-buffered saline). Measurement of Decay-accelerating Activity—The classical pathway (CP) decay-accelerating activity of sCCPH and the R118A mutant were determined by forming EAC142 (35Pan Q. Ebanks R.O. Isenman D.E. J. Immunol. 2000; 165: 2518-2527Crossref PubMed Scopus (28) Google Scholar), and the alternative pathway (AP) C3-convertase decay-accelerating activity was measured by forming C3b,Bb on sheep (ES) as well as rabbit (ER) erythrocytes. The details of these methods have been described previously (17Mullick J. Bernet J. Panse Y. Hallihosur S. Singh A.K. Sahu A. J. Virol. 2005; 79: 12382-12393Crossref PubMed Scopus (40) Google Scholar, 36Pryzdial E.L. Isenman D.E. Mol. Immunol. 1986; 23: 87-96Crossref PubMed Scopus (23) Google Scholar). Circular Dichroism (CD)—The sCCPH and its mutant R118A were subjected to CD spectra in the far UV region (190-360 nm) using a Jasco J18 spectropolarimeter with a cylindrical quartz cell with a path length of 0.01 cm. The resolution was 1 nm, the sensitivity was 20 millidegrees, and the speed was 10 nm/min. Each presented spectrum is the measure of eight measurements. The concentration of both the proteins was 200 μg/ml in 10 mm phosphate containing 145 mm NaCl, pH 7.4. All the data were subtracted against the background data using the spectral analysis software. Flow Cytometry for Measurement of Inhibition of C3b Deposition—Inhibition of the classical and alternative pathway-mediated C3b deposition on erythrocytes by sCCPH and VCP was measured by flow cytometry (19Mullick J. Bernet J. Singh A.K. Lambris J.D. Sahu A. J. Virol. 2003; 77: 3878-3881Crossref PubMed Scopus (57) Google Scholar). For measurement of the classical pathway-mediated C3b deposition, 5 μl of EA(109/ml in GVB2+) was mixed with 2 μl of C8-deficient human serum (Calbiochem) and 2 μm sCCPH or VCP in a total volume of 44 μl and incubated for 30 min at 37 °C. The cells were washed with GVB2+, centrifuged, mixed with 100 μl of 1/100-diluted fluorescein isothiocyanate-conjugated F(ab′)2 anti-C3 goat IgG (Cappel Laboratories, Warrington, PA), and further incubated on ice for 1 h. After incubation, the cells were washed twice with 400 μl of GVB, resuspended in 1.0 ml of the same buffer, and analyzed on a FACS Vantage (BD Biosciences). For measurement of alternative pathway-mediated deposition of C3b, 5 μl of rabbit erythrocytes (109/ml in GVB) was mixed with 2 μl of 0.1 m MgEGTA, 3 μl of C8-deficient human serum (Calbiochem), and 30 μl of GVB or GVB containing 2 μm sCCPH or VCP and incubated for 30 min at 37 °C. The cells were washed with GVB, and deposition of C3b was detected as described above. Results are expressed as mean channel fluorescence of 10,000 cells. Surface Plasmon Resonance Measurements—The kinetics of sCCPH and the R118A mutant binding to C3b and C4b was determined on the surface plasmon resonance-based biosensor BIACORE 2000 (Biacore AB, Uppsala, Sweden). The experiments were performed in phosphate-buffered saline-Tween (10 mm sodium phosphate, 145 mm NaCl, pH 7.4, containing 0.05% Tween 20) at 25 °C. For proper orientation of these proteins, the free SH groups of both C3b and C4b were biotinylated and then immobilized on the streptavidin chip (Sensor Chip SA, Biacore AB) (34Bernet J. Mullick J. Panse Y. Parab P.B. Sahu A. J. Virol. 2004; 78: 9446-9457Crossref PubMed Scopus (37) Google Scholar). FC-2 was immobilized with C3b (1592 RU), FC-3 was immobilized with C4b (1197 RUs), and FC-1 (blank flow cell) served as the control flow cell. Because sCCPH showed very little binding to C3b, more C3b molecules were deposited onto FC-2 by forming C3-convertase (37Jokiranta T.S. Hellwage J. Koistinen V. Zipfel P.F. Meri S. J. Biol. Chem. 2000; 275: 27657-27662Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 38Harris C.L. Abbott R.J. Smith R.A. Morgan B.P. Lea S.M. J. Biol. Chem. 2005; 280: 2569-2578Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In brief, ∼6000 RUs of C3b were deposited using three cycles of C3b deposition. In each cycle, the C3-convertase was formed by injecting a mixture of factors B and D (5 μg of B and 0.35 μg of D) and then 45 μg of native C3 was injected using the co-inject option. Deposition of C3b onto the chip was performed in veronal-buffered saline containing 1 mm NiCl2. For measurement of binding of sCCPH and the mutant protein to C3b and C4b, various concentrations of these proteins were injected for 120 s at 50 μl/min. Dissociation was measured for 180 s. The sensor chips were regenerated with 30-s pulses of 0.2 m sodium carbonate, pH 9.5. Sensograms obtained for the control flow cell (FC-1) were subtracted from the data for the flow cell immobilized with C3b or C4b, and the surface plasmon resonance data obtained were evaluated by BIAevaluation software version 4.1 using global fitting. Expression and Characterization of sCCPH—Because a large quantity of protein was required for conducting multiple assays, we chose to express HVS sCCPH in E. coli using the pET expression system. The soluble form of HVS CCPH was amplified from the HVS clone pCEX-1 and cloned into the expression vector pET29. The expressed protein was purified to homogeneity using histidine affinity (Fig. 1), and the identity of the expressed protein was confirmed by sequencing using mass spectrometry. The amino acid sequence of the expressed protein was consistent with the predicted sequence confirming the identity; the sequence coverage obtained was 86%. The expressed protein was refolded according to the method described by R. A. Smith and co-workers (33White J. Lukacik P. Esser D. Steward M. Giddings N. Bright J.R. Fritchley S.J. Morgan B.P. Lea S.M. Smith G.P. Smith R.A. Protein Sci. 2004; 13: 2406-2415Crossref PubMed Scopus (36) Google Scholar). This method typically provided a 20% yield. Analysis of the refolded protein by circular dichroism yielded a peak around 230 nm, which is a characteristic of CCP domains (39Kirkitadze M.D. Krych M. Uhrin D. Dryden D.T. Smith B.O. Cooper A. Wang X. Hauhart R. Atkinson J.P. Barlow P.N. Biochemistry. 1999; 38: 7019-7031Crossref PubMed Scopus (44) Google Scholar) (Fig. 1). These data confirmed proper folding of the protein. The expressed protein was >95% pure as judged by SDS-PAGE analysis, and it migrated as a single band of 32,000 Da on the gel. Further mass analysis by mass spectrometry confirmed that its molecular mass was similar to its calculated mass (within error <1%) (Fig. 1). Previously it has been shown that the membrane form of CCPH inhibits C3d deposition on the target cells (30Fodor W.L. Rollins S.A. Biancocaron S. Rother R.P. Guilmette E.R. Burton W.V. Albrecht J.C. Fleckenstein B. Squinto S.P. J. Virol. 1995; 69: 3889-3892Crossref PubMed Google Scholar). To verify if the refolded protein is biologically active, we tested its ability to inhibit C3b deposition on erythrocytes during complement activation. As depicted in Fig. 2, sCCPH inhibited both the classical as well as alternative pathway-mediated deposition of C3b on erythrocytes. Importantly, the data indicated that sCCPH was more active than VCP in inhibiting the alternative pathway-mediated deposition of C3b. Kinetic Analysis of Interaction of sCCPH with Complement Proteins C3b and C4b—The human complement control proteins inactivate complement by targeting C3b and/or C4b. Because sCCPH inhibited C3b deposition mediated by both the classical and alternative pathways, we sought to analyze its interaction with C3b and C4b by surface plasmon resonance technology. In this assay, C3b and C4b were immobilized in their physiological orientation on a streptavidin chip by labeling their free SH groups with biotin (34Bernet J. Mullick J. Panse Y. Parab P.B. Sahu A. J. Virol. 2004; 78: 9446-9457Crossref PubMed Scopus (37) Google Scholar), and sCCPH was injected over the chip to measure binding. The sCCPH showed good binding to C4b but very weak binding to C3b (Fig. 3, upper left panel). Binding data obtained by injecting various concentrations of sCCPH fitted well to 1:1 binding model (ka = 158; kd = 5.32 × 10−3; KD = 3.35 × 10−5 ; χ2 = 0.273). Because the binding response was very low, we further deposited C3b on the sensor chip to increase the response and reevaluate affinity. More C3b was deposited by forming AP C3-convertase on the chip and flowing native C3 (37Jokiranta T.S. Hellwage J. Koistinen V. Zipfel P.F. Meri S. J. Biol. Chem. 2000; 275: 27657-27662Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 38Harris C.L. Abbott R.J. Smith R.A. Morgan B.P. Lea S.M. J. Biol. Chem. 2005; 280: 2569-2578Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Three cycles of AP amplification resulted in deposition of ∼6000 RUs of C3b (Fig. 3, upper right panel); non-covalently associated Bb and C3b were removed by injection of brief pulses of 0.2 m sodium carbonate, pH 9.5. As expected, deposition of C3b using this approach resulted in a decaying surface, suggesting that C3b was attached to the surface by forming ester linkages (40Sahu A. Pangburn M.K. J. Biol. Chem. 1994; 269: 28997-29002Abstract Full Text PDF PubMed Google Scholar). When sCCPH was flown over the enzyme-coupled C3b, it showed good binding response (Fig. 3, lower left panel). To calculate affinities for C3b and C4b, various concentrations of sCCPH were injected over the chip. The sCCPH bound to both C3b and C4b in a dose-dependent manner (Fig. 3). Global fitting analysis of the sensograms showed a good fit of sCCPH-C3b data to the 1:1 binding model with a drifting base line (χ2 = 1.83), but sCCPH-C4b data could not be fitted to 1:1 model, and therefore, it was evaluated by steady-state analysis. These data indicated that sCCPH-C3b interaction follows a simple 1:1 binding model, whereas sCCPH-C4b interaction is complex. A comparison of the affinities showed that sCCPH has a 24-fold higher affinity for C4b than C3b (Table 1).TABLE 1Kinetic and affinity data for the interactions of sCCPH, R118A mutant, and VCP with human complement proteins C3b and C4bLigandAnalytekd/kaS.E. (kd/ka)KDX2s−1/M−1MC3bsCCPH2.63 × 10−3/1372.05 × 10−5/3.391.92 × 10−51.83aData were calculated by global fitting to a 1:1 Langmuir binding model (BIA evaluation 4.1).C4bsCCPHNANA8.04 × 10−72.87bData were calculated by steady-state analysis (BIA evaluation 4.1).C3bR118A4.06 × 10−3/18.74.75 × 10−5/0.732.17 × 10−49.44aData were calculated by global fitting to a 1:1 Langmuir binding model (BIA evaluation 4.1).C4bR118ANANA9.64 × 10−71.23bData were calculated by steady-state analysis (BIA evaluation 4.1).C3bVCPNANA2.0 × 10−61.47bDa