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• W2055978589 abstract "The complement system membrane cofactor protein (MCP) CD46 serves as a C3b/C4b inactivating factor for the protection of host cells from autologous complement attack and as a receptor for measles virus (MV). MCP consists of four short consensus repeats (SCR) which are the predominant extracellular structural motif. In the present study, we determined which of the four SCR of MCP contribute to its function using Chinese hamster ovary cell clones expressing each SCR deletion mutants. The results were as follows: 1) SCR1 and SCR2 are mainly involved in MV binding and infection; 2) SCR2, SCR3, and SCR4 contribute to protect Chinese hamster ovary cells from human alternative complement pathway-mediated cytolysis; and 3) SCR2 and SCR3 are essential for protection of host cells from the classical complement pathway. These results on cell protective activity of the mutants against the human classical and the alternative complement pathways were compatible with factor I-mediated inactivation profiles of C4b and C3b, respectively, in the fluid-phase assay using solubilized mutants and factor I; the results were mostly consistent with those reported by Adams et al. (Adams, E. M., Brown, M. C., Nunge, M., Krych, M., and Atkinson, J. P.(1991) J. Immunol. 147, 3005-3011). SCR2 and SCR3 were required for C3b and C4b inactivation, and SCR4-deleted MCP showed weak cofactor activity for C4b cleavage but virtually no cofactor activity for C3b cleavage. The functional domains of MCP for the three natural ligands C3b, C4b, and MV, therefore, map to different, although partly overlapping, SCR domains. The complement system membrane cofactor protein (MCP) CD46 serves as a C3b/C4b inactivating factor for the protection of host cells from autologous complement attack and as a receptor for measles virus (MV). MCP consists of four short consensus repeats (SCR) which are the predominant extracellular structural motif. In the present study, we determined which of the four SCR of MCP contribute to its function using Chinese hamster ovary cell clones expressing each SCR deletion mutants. The results were as follows: 1) SCR1 and SCR2 are mainly involved in MV binding and infection; 2) SCR2, SCR3, and SCR4 contribute to protect Chinese hamster ovary cells from human alternative complement pathway-mediated cytolysis; and 3) SCR2 and SCR3 are essential for protection of host cells from the classical complement pathway. These results on cell protective activity of the mutants against the human classical and the alternative complement pathways were compatible with factor I-mediated inactivation profiles of C4b and C3b, respectively, in the fluid-phase assay using solubilized mutants and factor I; the results were mostly consistent with those reported by Adams et al. (Adams, E. M., Brown, M. C., Nunge, M., Krych, M., and Atkinson, J. P.(1991) J. Immunol. 147, 3005-3011). SCR2 and SCR3 were required for C3b and C4b inactivation, and SCR4-deleted MCP showed weak cofactor activity for C4b cleavage but virtually no cofactor activity for C3b cleavage. The functional domains of MCP for the three natural ligands C3b, C4b, and MV, therefore, map to different, although partly overlapping, SCR domains. INTRODUCTIONHuman membrane cofactor protein (MCP)1( 1The abbreviations used are: MCPmembrane cofactor protein (CD46)DACMN-(dimethylamino-4-methylcoumarinyl)maleimideDAFdecay-accelerating factor (CD55)mAbmonoclonal antibodyAbantibodyMVmeasles virusSCRshort consensus repeat (DSCR1 means the SCR1-deleted form)STserine/threonine-rich domainCYTcytoplasmic tailPAGEpolyacrylamide gel electrophoresisCHOChinese hamster ovaryDMEMDulbecco's modified Eagle's mediumpfuplaque-forming unit(s).) CD46 was first identified as a C3b-binding protein (1Cole J.L. Housley Jr., G.A. Dykman T.R. MacDermott R.P. Atkinson J.P. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 859-863Crossref PubMed Scopus (140) Google Scholar) distinct from other membrane complement-associated proteins such as CR1 (CD35), CR2 (CD21), and decay-accelerating factor (DAF, CD55) (reviewed in Ref. 2). MCP inactivates cell-bound C3b/C4b, acting as a cofactor for plasma protease factor I(3Seya T. Turner J.R. Atkinson J.P. J. Exp. Med. 1986; 163: 837-855Crossref PubMed Scopus (321) Google Scholar, 4Seya T. Atkinson J.P. Biochem. J. 1989; 264: 581-588Crossref PubMed Scopus (122) Google Scholar), and protects host cells from complement-mediated cell damage(5Seya T. Hara T. Matsumoto M. Sugita Y. Akedo H. J. Exp. Med. 1990; 172: 1673-1680Crossref PubMed Scopus (101) Google Scholar, 6Lublin D.M. Coyne K.E. J. Exp. Med. 1991; 174: 35-44Crossref PubMed Scopus (98) Google Scholar, 7Oglesby T.J. Allen C.J. Liszewski M.K. White D.J.G. Atkinson J.P. J. Exp. Med. 1993; 175: 1547-1551Crossref Scopus (132) Google Scholar). This molecule composed of an amino terminus of four short consensus repeating units (SCR), a Ser/Thr (ST)-rich domain, 13 amino acids of unknown significance, a transmembrane region, and a cytoplasmic tail (CYT)(8Lublin D.M. Liszewski M.K. Post T.W. Arce M.A. Le Beau M.M. Rebentisch M.B. Lemons R.S. Seya T. Atkinson J.P. J. Exp. Med. 1988; 168: 181-194Crossref PubMed Scopus (172) Google Scholar). The region responsible for complement regulation is the SCR(9Adams E.M. Brown M.C. Nunge M. Krych M. Atkinson J.P. J. Immunol. 1991; 147: 3005-3011PubMed Google Scholar, 10Matsumoto M. Seya T. Nagasawa S. Biochem. J. 1992; 281: 493-499Crossref PubMed Scopus (35) Google Scholar). The structural gene for MCP maps to 1q32(8Lublin D.M. Liszewski M.K. Post T.W. Arce M.A. Le Beau M.M. Rebentisch M.B. Lemons R.S. Seya T. Atkinson J.P. J. Exp. Med. 1988; 168: 181-194Crossref PubMed Scopus (172) Google Scholar), where the genes of complement regulatory proteins, C4b-binding protein, DAF, CR1, CR2, and factor H, are clustered(11Bora N. Lublin D.M. Kumar V. Hockett R.D. Holers V.M. Atkinson J.P. J. Exp. Med. 1989; 169: 597-602Crossref PubMed Scopus (39) Google Scholar). MCP is a member of the regulator of complement activation gene family.Naniche et al.(12Naniche D. Varior-Krishnan G. Cervoni F. Wild T.F. Rossi B. Rabourdin-Combe C. Gerlier D. J. Virol. 1993; 67: 6025-6032Crossref PubMed Google Scholar) and Dorig et al.(13Dorig R.E. Marcil A. Chopra A. Richardson D. Cell. 1993; 75: 295-305Abstract Full Text PDF PubMed Scopus (864) Google Scholar) have suggested that MCP also serves as a receptor for measles virus (MV). MV, however, has no C3b-like molecules on its envelope. H protein of MV is thought to act as a ligand for target cell receptors, whereas F protein then induces viral-cell fusion(14Richardson C.D. Scheid A. Choppin P.W. Virology. 1980; 105: 205-222Crossref PubMed Scopus (234) Google Scholar, 15Wild T.F. Malvoisin E. Buckland R. J. Gen. Virol. 1991; 72: 439-442Crossref PubMed Scopus (209) Google Scholar). Indeed, MV can infect human and various monkey species, the tropism correlating with the expression of MCP(13Dorig R.E. Marcil A. Chopra A. Richardson D. Cell. 1993; 75: 295-305Abstract Full Text PDF PubMed Scopus (864) Google Scholar, 16Nickells M.W. Atkinson J.P. J. Immunol. 1990; 144: 4262-4267PubMed Google Scholar).There are many MCP phenotypes, which are distinguishable on SDS-PAGE (17-19). This polymorphism is caused by alternative splicing of mRNA encoding the ST-rich and CYT regions(17Post T.W. Liszewski M.K. Adams E.M. Tedja I. Miller E.A. Atkinson J.P. J. Exp. Med. 1991; 174: 93-102Crossref PubMed Scopus (146) Google Scholar). CHO cell clones expressing MCP variants with a variety of ST-rich (20Iwata K. Seya T. Ueda S. Ariga H. Nagasawa S. Biochem. J. 1994; 304: 169-175Crossref PubMed Scopus (34) Google Scholar) or CYT domains (21Manchester M. Liszewski M.K. Atkinson J.P. Oldstone M.B.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2161-2165Crossref PubMed Scopus (154) Google Scholar) have been reported to become permissive to MV. Thus, the MV-binding site on MCP must be located within the SCR. The MV-binding site and its structural relationship to the complement-binding site, therefore, remain to be elucidated.In the present study, we established CHO transfectants expressing various SCR deletion mutants of MCP and mapped the functional domains for complement regulation and MV binding.MATERIALS AND METHODSCells, Antibodies, and ProteinsCHO cells were obtained from American Type Culture Collection (ATCC). Vero cells, green monkey erythrocytes, and MV, a modified Nagahata strain(22Wong T. Ayata M. Ueda S. Hirano A. J. Virol. 1991; 65: 2191-2199Crossref PubMed Google Scholar), were obtained from the Research Institute for Microbial Diseases, Osaka University. Monoclonal antibodies (mAbs) against MCP, M75, M160, and M177, were produced in our laboratory(23Seya T. Hara T. Matsumoto M. Akedo H. J. Immunol. 1990; 145: 238-245PubMed Google Scholar), E4.3 (24Sparrow R. McKenzie I.F.C. Hum. Immunol. 1983; 7: 1-6Crossref PubMed Scopus (37) Google Scholar) was from Dr. B. Loveland (Austin Institute, Melbourne, Australia), and other mAbs were reported previously(25Pesando J.M. Hoffman P. Abed M. J. Immunol. 1986; 137: 3689-3695PubMed Google Scholar, 26Stern P.L. Beresford N. Thompson S. Johnson P.M. Webb P.D. Hole N. J. Immunol. 1986; 137: 1604-1609PubMed Google Scholar). Serum from a patient with subacute sclerosing panencephalitis containing a high titer of anti-MV Ab was obtained from Dr. M. B. A. Oldstone (The Scripps Research Institute, La Jolla, CA)(27Yanagi Y. Cubitt B.A. Oldstone M.B.A. Virology. 1992; 187: 280-289Crossref PubMed Scopus (81) Google Scholar).Complement C3(28Nagasawa S. Stroud R.M. Immunochemistry. 1977; 14: 749-756Crossref PubMed Scopus (112) Google Scholar), C4(29Nagasawa S. Ichihara C. Stroud R.M. J. Immunol. 1980; 125: 578-582PubMed Google Scholar), factor H(30Seya T. Nagasawa S. J. Biochem. (Tokyo). 1985; 97: 373-382Crossref PubMed Scopus (33) Google Scholar), and factor I (29Nagasawa S. Ichihara C. Stroud R.M. J. Immunol. 1980; 125: 578-582PubMed Google Scholar) were purified from human plasma as described previously. C3b and C4b were prepared (29Nagasawa S. Ichihara C. Stroud R.M. J. Immunol. 1980; 125: 578-582PubMed Google Scholar, 30Seya T. Nagasawa S. J. Biochem. (Tokyo). 1985; 97: 373-382Crossref PubMed Scopus (33) Google Scholar) and labeled with an SH reagent, N-(dimethylamino-4-methylcoumarinyl)maleimide (DACM)(31Seya T. Okada M. Nishino H. Atkinson J.P. J. Biochem. (Tokyo). 1990; 107: 310-315Crossref PubMed Scopus (21) Google Scholar). The DACM-labeled C3b and C4b have been characterized as substrates for factor I and MCP(31Seya T. Okada M. Nishino H. Atkinson J.P. J. Biochem. (Tokyo). 1990; 107: 310-315Crossref PubMed Scopus (21) Google Scholar, 32Masaki T. Matsumoto M. Nakanishi I. Yasuda R. Seya T. J. Biochem. (Tokyo). 1992; 111: 573-578Crossref PubMed Scopus (29) Google Scholar). Glycosidases were obtained as follows: N-glycanase F (Boehringer Mannheim, GmbH, Mannheim, Germany), neuraminidase (Sigma), and O-glycanase (Genzyme, Cambridge, MA).cDNA Construction and Expression of SCR Deletion Mutants of MCP in CHO CellsMCP cDNA of STC/CYT2 phenotype (8Lublin D.M. Liszewski M.K. Post T.W. Arce M.A. Le Beau M.M. Rebentisch M.B. Lemons R.S. Seya T. Atkinson J.P. J. Exp. Med. 1988; 168: 181-194Crossref PubMed Scopus (172) Google Scholar) was mutated using a T7-GEN In Vitro Mutagenesis Kit (U. S. Biochemical Corp.). Briefly, oligonucleotides looping out the sequences encoding a single SCR (corresponding to amino acids 1-61 of MCP, in reference to its amino acid sequence(8Lublin D.M. Liszewski M.K. Post T.W. Arce M.A. Le Beau M.M. Rebentisch M.B. Lemons R.S. Seya T. Atkinson J.P. J. Exp. Med. 1988; 168: 181-194Crossref PubMed Scopus (172) Google Scholar)) were synthesized. For example, the oligonucleotide, 5′-TGGACATGTTTCTCTGGCATCGGAGAAGGA-3′ was synthesized to delete SCR1 (ΔSCR1) of MCP. Similarly, oligonucleotide sequences were determined to generate a mutant ΔSCR2 (lacking the 62-124 amino acid sequence), ΔSCR3 (lacking the 125-190 amino acid sequence), and ΔSCR4 (lacking the 192-251 amino acid sequence). The nucleotide sequences of the resulting cDNAs were all confirmed on a nucleotide sequencer (ABI 373A). Intact and mutated MCP cDNAs were subcloned into the EcoRI/PstI-digested expression vector pME18S(33Kojima A. Iwata K. Seya T. Matsumoto M. Ariga H. Atkinson J.P. Nagasawa S. J. Immunol. 1993; 151: 1519-1527PubMed Google Scholar). The mutated cDNAs were again confirmed on the nucleotide sequencer.CHO cells were cotransfected with vectors containing MCP cDNA (20 μg) and 1 μg of pSV2hph (a hygromycin resistance gene vector) (34Blochlinger K. Diggelmann H. Mol. Cell. Biol. 1984; 4: 2929-2937Crossref PubMed Scopus (186) Google Scholar) by calcium phosphate precipitation(35Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Vol. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 16.32-16.37Google Scholar). The transfected CHO cells were maintained for 24 h in Ham's F-12 medium, 10% fetal calf serum, 0.06% kanamycin, in an atmosphere of humidified 5%CO2, 95% air at 37°C. The cells were transferred to the same medium containing 0.7 mg/ml of hygromycin B (Sigma) for selection. The hygromycin-resistant colonies were isolated with cloning cylinders and expanded in tissue culture plates. The cDNAs incorporated into CHO cells were confirmed by Southern blotting(35Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Vol. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 16.32-16.37Google Scholar). The expression of these mutants was confirmed by flow cytometry using M160 (not shown), M177, and E4.3.Flow Cytometry and ImmunoblottingThe protein levels expressed on the transfected cells (1 ´ 106) were assessed by flow cytometry (FACScan and/or Profile II) as described previously (33) using M177 and E4.3, and fluorescein isothiocyanate-labeled second Ab. CHO cells, transfected with vectors only, pME18S and pSV2hph, were used as controls.Immunoblotting was performed as described previously(36Towbin H. Staehlin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44708) Google Scholar). MCP and its mutants were solubilized from transfectants (about 2 ´ 107 cells) as reported previously(3Seya T. Turner J.R. Atkinson J.P. J. Exp. Med. 1986; 163: 837-855Crossref PubMed Scopus (321) Google Scholar). The proteins were resolved by SDS-PAGE (10-12.5% acrylamide), blotted onto membranes, and detected with a mAb against MCP. The conditions for blotting analyses were described in detail previously(10Matsumoto M. Seya T. Nagasawa S. Biochem. J. 1992; 281: 493-499Crossref PubMed Scopus (35) Google Scholar).Glycosidase TreatmentSolubilized ΔSCR4 mutant was mixed with 0.10 volume of 1% SDS and incubated with 1 unit of N-glycanase F. After incubation for 12 h at 37°C, 1 unit of N-glycanase F was further added and incubated for an additional 12 h. Another ΔSCR4 mutant was mixed with an equal volume of incubation buffer (40 mM Tris maleate, 20 mMD-galactono-g-lactone, 2 mM calcium acetate, 0.2% Nonidet P-40, pH 6.0) and incubated with 100 microunits of neuraminidase for 1 h at 37°C. Then, 3 milliunits of O-glycanase and 1 unit of N-glycanase F were added and incubated for 12 h. The sample was mixed with an additional 1 unit of N-glycanase F and incubated for an additional 12 h at 37°C. The glycanase-treated samples were resolved by SDS-PAGE and Western blotted with M177 mAb, which recognizes SCR2.Factor I-Cofactor ActivityA fluid-phase assay was used (31, 32). Briefly, DACM-labeled C3b or C4b (10 μg) and factor I (0.5 μg) were incubated for 3-12 h at 37°C with various amounts of MCP or its SCR deletion mutants, which were prepared as follows. The samples solubilized from 2 ´ 107 cells were prepared by acid precipitation(3Seya T. Turner J.R. Atkinson J.P. J. Exp. Med. 1986; 163: 837-855Crossref PubMed Scopus (321) Google Scholar), and the amounts of wild-type and mutant MCP were measured by sandwich enzyme-linked immunosorbent assay(37Hara T. Kuriyama S. Kiyohara H. Nagase Y. Matsumoto M. Seya T. Clin. Exp. Immunol. 1992; 89: 490-494Crossref PubMed Scopus (55) Google Scholar). SCR2 and SCR3 deletion mutants could not be detected in this enzyme-linked immunosorbent assay, so the concentrations of the mutants were estimated from the copy numbers of the mutants on the CHO cells assuming the solubilization efficiencies to be the same as that of the wild type. We obtained MCP samples of 7 μg/ml, and 1-10 μl was used as an MCP source. At timed intervals, 10 μl of 10% SDS and 3 μl of 2-mercaptoethanol were added to terminate the reaction and to reduce the substrates. The samples were analyzed by SDS-PAGE (8-10% acrylamide), and the percentage conversions of C3b to C3bi, and of C4b to C4d, were determined by fluorescence spectrophotometry as described previously(31Seya T. Okada M. Nishino H. Atkinson J.P. J. Biochem. (Tokyo). 1990; 107: 310-315Crossref PubMed Scopus (21) Google Scholar, 32Masaki T. Matsumoto M. Nakanishi I. Yasuda R. Seya T. J. Biochem. (Tokyo). 1992; 111: 573-578Crossref PubMed Scopus (29) Google Scholar). Cofactor activity was mostly abrogated by the addition of M177(5Seya T. Hara T. Matsumoto M. Sugita Y. Akedo H. J. Exp. Med. 1990; 172: 1673-1680Crossref PubMed Scopus (101) Google Scholar), suggesting that the expressed MCP was a major cofactor in these CHO cells (not shown).Complement-dependent Cytolysis of CHO TransfectantsAnti-CHO cell Ab was used as a sensitizer(33Kojima A. Iwata K. Seya T. Matsumoto M. Ariga H. Atkinson J.P. Nagasawa S. J. Immunol. 1993; 151: 1519-1527PubMed Google Scholar), and the complement sources for the classical and the alternative pathways were normal human serum diluted with gelatin veronal buffer and normal human serum diluted with gelatin veronal buffer containing 1 mM MgCl2 and 10 mM EGTA (Mg2+>-EGTA), respectively. Briefly, the transfected cells (2 ´ 104/well) were seeded on 96-well plates. Fifteen h later, the cells were incubated with 51Cr (1 μCi/well) in complete medium for 3 h at 37°C. After three washes with gelatin veronal buffer, the labeled cells were incubated with anti-CHO Ab for 30 min at 4°C, then 100 μl of 2-fold-diluted complement sources were added. The plates were incubated for 60 min at 37°C, and the released radioactivities were measured in a g-counter. Cytotoxicity was calculated as described previously (38). All determinations were performed in triplicate.MV Binding AssayCells to be assayed for MV binding were detached from flasks at 80% confluence by the addition of 2 ml of phosphate-buffered saline containing 5 mM EDTA. After two washes, aliquots containing 1 g 106 cells were incubated for 2 h at 4°C with 2 ml of concentrated MV (107 pfu/ml) in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum. After three washes with DMEM, 5% fetal calf serum, cells were incubated at 4°C for 45 min with 5 μg of anti-H mAb(27Yanagi Y. Cubitt B.A. Oldstone M.B.A. Virology. 1992; 187: 280-289Crossref PubMed Scopus (81) Google Scholar). After three washes with 10 ml of DMEM, cells were incubated with 5 μg of fluorescein isothiocyanate-labeled goat anti-mouse IgG. The levels of the MV H protein in each CHO cell strain were assessed by flow cytometry.Determination of MV InfectivityCHO cell clones with or without a variety of MCP mutants were cultured at 70% confluence in 24-well plates (Corning) for 15 h and infected with MV at 0.001-0.5 pfu/cell. Simultaneously, we performed plaque-forming assays (27Yanagi Y. Cubitt B.A. Oldstone M.B.A. Virology. 1992; 187: 280-289Crossref PubMed Scopus (81) Google Scholar) and confirmed the correlation between CHO cell syncytium formation and plaque formation. The syncytia formed were counted, and the cytopathic characteristics of the CHO cell transfectants (13Dorig R.E. Marcil A. Chopra A. Richardson D. Cell. 1993; 75: 295-305Abstract Full Text PDF PubMed Scopus (864) Google Scholar) were observed 3 days post-infection. Virtually, no background infection was observed at our doses of MV within 3 days. Cells were photographed under a Nikon inverted microscope (not shown).The supernatants of the infected cells were harvested after sonication, and the MV titer was determined using Vero cells by the standard method (12, 13).RESULTSLevels and Properties of SCR Deletion Mutants Expressed on CHO CellsThe expression levels of the MCP mutants ΔSCR1, ΔSCR2, ΔSCR3, and ΔSCR4 were examined by flow cytometry. All SCR deletion mutants were translated into proteins and expressed on the CHO cells (Fig. 1). The expression levels of MCP mutants were similar on all clones used except for the ΔSCR1 transfectant, the density of which was elevated 3-4-fold. Based on the reaction profile, the epitope of E4.3 was mapped in SCR1 (right panel of Fig. 1), consistent with the results of a previous report(9Adams E.M. Brown M.C. Nunge M. Krych M. Atkinson J.P. J. Immunol. 1991; 147: 3005-3011PubMed Google Scholar). Likewise, the epitope of M177 was mapped in SCR2 (left panel of Fig. 1). Epitope mapping was performed with 10 mAbs against MCP, and the results are summarized in. Table ITable I:Epitopes and effects on MCP functions of mAbs Open table in a new tab Immunoblotting suggested that the wild-type MCP has an molecular mass of 57 kDa, consistent with that of the previously reported STC/CYT2 form(6Lublin D.M. Coyne K.E. J. Exp. Med. 1991; 174: 35-44Crossref PubMed Scopus (98) Google Scholar, 33Kojima A. Iwata K. Seya T. Matsumoto M. Ariga H. Atkinson J.P. Nagasawa S. J. Immunol. 1993; 151: 1519-1527PubMed Google Scholar). The molecular masses of ΔSCR1, ΔSCR2, and ΔSCR3 mutants were 42, 44, and 54 kDa, respectively (Fig. 1A). The present results supported those of previous reports that SCR1 and SCR2 are N-glycosylated (9, 39). The ΔSCR4 mutant yielded two bands on SDS-PAGE of 44 and 36 kDa, resulting from the single expected nucleotide sequence. The two bands were accumulated into a single 33-kDa band on SDS-PAGE after N-glycanase treatment, and this band was further decreased by 3 kDa by O-glycanase treatment (Fig. 1B). Thus, on CHO cells the ΔSCR4 mutant protein consists of two forms, one heavily glycosylated with a molecular mass of 43 kDa and a lightly glycosylated 34-kDa form. All mutants possessed O-linked sugars estimated on SDS-PAGE to be 3 kDa.Complement Regulatory Function of the MCP MutantsComplement regulatory activities of the mutants were determined by fluid-phase factor I-cofactor assay(31Seya T. Okada M. Nishino H. Atkinson J.P. J. Biochem. (Tokyo). 1990; 107: 310-315Crossref PubMed Scopus (21) Google Scholar, 32Masaki T. Matsumoto M. Nakanishi I. Yasuda R. Seya T. J. Biochem. (Tokyo). 1992; 111: 573-578Crossref PubMed Scopus (29) Google Scholar). CHO(-) cells possessed minimal C3b-C3bi converting activity (percent conversion by CHO(-) cells was 20-22% under the conditions shown in). With DACM-labeled C3b as a substrate, SCR2-, SCR3-, and SCR4-deletion mutants showed no cofactor activity relevant to the expressed MCP, whereas ΔSCR1 retained its activity (). Similar results were obtained with another substrate, DACM-labeled C4b, except that the cofactor activity for C4b cleavage remained minimal in the ΔSCR4 mutant (). M177 completely blocked this ΔSCR4-mediated C4b inactivation (data not shown), excluding the possibility of involvement of other cofactors in the C4b cleavage.The cell protection assay from human complement was performed with 50Cr-labeled CHO cells with the mutants and the complement sources for the classical and the alternative pathways (Table II). Two CHO cell clones expressing wild-type MCP were used as controls: one clone, whose expression level of MCP was as high as that of ΔSCR1, and the other clone, whose expression level of MCP was similar to those of ΔSCR2, ΔSCR3, and ΔSCR4. The ΔSCR1 mutant expressed as potent protective activity from complement attack as a wild-type. Judging from the MCP copy numbers expressed on the CHO cells, ΔSCR1 and wild-type MCP protected cells from the two pathways with similar potencies. The ΔSCR2 and ΔSCR3 mutants showed no protective activity from the two pathways, and the ΔSCR4 had virtually no effect on alternative pathway-mediated cell damage, while retaining marginal protective activity against the classical pathway. Thus, the degrees of cell protection in the mutants were essentially consistent with their factor I-cofactor activities. The results are summarized in.Tabled 1 Open table in a new tab The Domain of MCP Responsible for MV InfectionMV binding assay was performed by flow cytometry (). Binding was observed in CHO cells expressing ΔSCR3 and ΔSCR4 mutants, but not in those expressing ΔSCR1 or ΔSCR2. The degrees of MV binding were in the order: wild type = ΔSCR3 mutant>ΔSCR4 mutant.Syncytium formation is a representative marker for MV infection of CHO cells expressing MCP(13Dorig R.E. Marcil A. Chopra A. Richardson D. Cell. 1993; 75: 295-305Abstract Full Text PDF PubMed Scopus (864) Google Scholar). In this study, syncytia were visualized under the light microscope and the number of syncytia formed at each dose of MV was counted to determine the titer of infectivity. The minimum doses of MV for syncytium formation are shown in. The results reflected those of the MV binding assay. These results suggested that SCR1 and SCR2 are the domains essential for MV binding/infection.Importance of the SCR1 and SCR2 domains for MV infection was also supported by the inhibition studies with anti-MCP mAb (). SCR2 was particularly important, since SCR2-recognizing mAbs M75 and M177 blocked MV infection in both CHO transfectants and Vero cells. An SCR3-recognizing mAb MH61 blocked MV infection in CHO transfectants, but not Vero cells. This blocking effect may be due to steric hindrance or a conformational change in SCR2 secondary to the binding of this mAb to SCR3.Replication of MV in MCP Mutant-expressing CHO CellsMV replicated in CHO transfectants expressing ΔSCR3 and ΔSCR4 mutants, as well as wild-type, whereas no replication occurred in CHO transfectants expressing ΔSCR1 or ΔSCR2 mutants. The results were confirmed by determination of MV H protein synthesis by immunostaining using subacute sclerosing panencephalitis serum and mAb against MV H. The results again reinforce the notion that the SCR1 and SCR2 domains are essential for MV infection and that MV replication is permitted regardless of deletion of SCR3 or SCR4.DISCUSSIONWe studied which of the four SCR domains of MCP contribute to the cofactor activity for factor I-mediated cleavage of C3b and C4b, the protection of host cells from complement-mediated cytolysis, and MV binding activity, using deletion mutants. In summary: 1) SCR1 and SCR2 are mainly involved in MV binding and infection; 2) SCR2, SCR3, and SCR4 sustain sufficient C3b inactivation by factor I; and 3) SCR2 and SCR3 are essential for factor I-mediated inactivation of C4b. The last two points are essentially similar to those reported by Adams et al.(9Adams E.M. Brown M.C. Nunge M. Krych M. Atkinson J.P. J. Immunol. 1991; 147: 3005-3011PubMed Google Scholar) and Oglesby et al.(40Oglesby T.J. Allen C.J. Liszewski M.K. White D.J.G. Atkinson J.P. Mol. Immunol. 1993; 30 (abstr.): 40Crossref Google Scholar), except that the ΔSCR4 mutant retained weak cofactor activity for factor I-mediated C4b cleavage. They also determined the domains responsible for C3b and C4b binding(9Adams E.M. Brown M.C. Nunge M. Krych M. Atkinson J.P. J. Immunol. 1991; 147: 3005-3011PubMed Google Scholar). Our results taken together with their findings indicate that the three ligands of MCP, C3b, C4b, and MV, bind to different sets of SCR and confer distinct functions of MCP. Hence, MCP is a multifunctional receptor with both classical and alternative complement regulatory and MV binding activities.In our permanent expression system using CHO cells, N-glycosylation diverged in ΔSCR4 resulting in the two forms of the ΔSCR4 protein. This is not the case in a transient COS cell expression system reported by Adams et al.(9Adams E.M. Brown M.C. Nunge M. Krych M. Atkinson J.P. J. Immunol. 1991; 147: 3005-3011PubMed Google Scholar), which may explain the differences between previous results (9Adams E.M. Brown M.C. Nunge M. Krych M. Atkinson J.P. J. Immunol. 1991; 147: 3005-3011PubMed Google Scholar, 40Oglesby T.J. Allen C.J. Liszewski M.K. White D.J.G. Atkinson J.P. Mol. Immunol. 1993; 30 (abstr.): 40Crossref Google Scholar) and our present findings. That is, the presence of the two forms of the ΔSCR4 protein may explain the findings that our ΔSCR4 mutant retain, albeit weak, cofactor activity for C4b and that CHO cells expressing ΔSCR4 are less sensitive to MV than those expressing ΔSCR3 or wild-type MCP. It is not surprising that MCP molecules are differently glycosylated in different cell lines. In fact, a variety of MCP size variants secondary to cell type- and organ-specific glycosylation have been reported(17Post T.W. Liszewski M.K. Adams E.M. Tedja I. Miller E.A. Atkinson J.P. J. Exp. Med. 1991; 174: 93-102Crossref PubMed Scopus (146) Google Scholar, 18Russell S.M. Sparrow R.I. McKenzie I.F.C. Purcell D.F.J. Eur. J. Immunol. 1992; 22: 1513-1518Crossref PubMed Scopus (98) Google Scholar, 19Johnstone R.W. Russell S.M. Loveland B. McKenzie I.F.C. Mol. Immunol. 1993; 30: 1231-1242Crossref PubMed Scopus (80) Google Scholar).Some viruses and bacteria such as HIV (reviewed in Ref. 41), Listeria (42), and Mycobacteria (43Schlesinger L.S. Horwitz M.A. J. Exp. Med. 1991; 174: 1031-1038Crossref PubMed Scopus (67) Google Scholar) activate the host complement system to allow the deposition of C3b/C3bi on their own membranes, and this deposited C3b/C3bi facilitates cellular invasion by the microorganisms, even into their specific receptor-negative cells via complement receptors. Naniche et al.(12Naniche D. Varior-Krishnan G. Cervoni F. Wild T.F. Rossi B. Rabourdin-Combe C. Gerlier D. J. Virol. 1993; 67: 6025-6032Crossref PubMed Google Scholar) suggested this possibility for MV, but no conclusive evidence supporting this infective mechanism has been reported. The observation that the SCR1 domain is responsible for MV binding but not for the C3b/C4b inactivation negates the involvement of C3b in MV-MCP interaction.There are six mAbs that specifically recognize the SCR1 of MCP (). None of these mAbs, except for an mAb 4-23SB, however, blocked MV infection in Vero cells and CHO transfectants. It may be that the epitopes for these mAbs in SCR1 are not concerned in MV infection. An mAb 4-23SB inhibited weakly MV infection in CHO transfectants. The mAbs reported by Naniche et al.(44Naniche D. Wild T.F. Rabourdin-Combe C. Gerlier D. J. Gen. Virol. 1992; 73: 2617-2624Crossref PubMed Scopus (49) Google Scholar) indeed block MV infection but have not been characterized with regard to the blocking of complement regulatory activity. One mAb, GB24(45Hsi B.-L. Yeh C.-J.G. Fenichel P. Samson M. Grivaux C. Am. J. Reprod. Immunol. Microbiol. 1988; 18: 21-26Crossref PubMed Scopus (41) Google Scholar), which recognizes the SCR3 or SCR4, has been reported to block C3b and C4b binding to MCP (9) without suppressing MV infection(13Dorig R.E. Marcil A. Chopra A. Richardson D. Cell. 1993; 75: 295-305Abstract Full Text PDF PubMed Scopus (864) Google Scholar). Although the binding sites of C3b/C4b and MV in MCP map in nearby regions, no mAb reported to date can completely blocked both MV binding and complement regulatory activities of MCP. Hence, M177 and M75 (23Seya T. Hara T. Matsumoto M. Akedo H. J. Immunol. 1990; 145: 238-245PubMed Google Scholar) are the first mAbs to simultaneously block both activities of MCP. Their epitopes were mapped in the SCR2 domain, which is shared for both MV binding and C3b/C4b inactivation. Evidence that mAbs 4-23SB and MH61 can block MV infection in CHO transfectants but not in Vero cells may suggest structural difference in the SCR domains of MCP between human and monkey.Besides MCP, CR2 and DAF have been reported to be receptors for the Epstein-Barr virus (46Carel J.-C. Myones B.L. Frazier B. Holers V.M. J. Biol. Chem. 1990; 265: 12293-12299Abstract Full Text PDF PubMed Google Scholar, 47Moore M.D. Cannon M.J. Sewall A. Finlayson M. Okimoto M. Nemerow G.R. J. Virol. 1991; 65: 3559-3565Crossref PubMed Google Scholar) and some strains of the Echo virus(48Bergelson J.M. Chan M. Solomon K.R. St. John N.F. Lin H. Finberg R.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6245-6248Crossref PubMed Scopus (269) Google Scholar), respectively. The heads of these molecules appear to be important for virus binding, similar to other virus receptors(49Koike S. Ise I. Nomoto A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4104-4108Crossref PubMed Scopus (85) Google Scholar). There are many SCR proteins, which enhance cell adhesion (reviewed in Refs. 50 and 51) mediated by integrin family receptors and others(50Springer T.A. Nature. 1990; 346: 425-434Crossref PubMed Scopus (5830) Google Scholar). Cell adhesion generally provides a convenient environment for viral fusion/infection. It may be favorable for microorganisms to adopt SCR proteins as a receptor, which facilitate efficient infection by promoting cell-to-cell attachment. However, further studies are required to substantiate this hypothesis.The purified membrane form of MCP (10 ng) (3) was preincubated with each mAb (10 μg). The mixtures were then incubated with DACM-labeled C3b (10 μg) and factor I (0.5 μg) for 3 h at 37°C in phosphate-buffered saline containing 0.02% Nonidet P-40, and samples were resolved by SDS-PAGE (8% gels) under reducing conditions. Cofactor activity was evaluated from the fluorescence intensity of the α chain and α1 fragment by spectrofluorometry (Hitachi F2000) (31). Percent inhibition by each mAb was calculated assuming that the percent conversion of the α chain to the α1 fragment in the absence of mAb was 100%. Monolayers of Vero or CHO cells in 24-well plates were incubated at 37°C for 60 min with each anti-MCP mAb (25 μg/ml) in Ham's F-12, 10% fetal calf serum, then infected with MV at a multiplicity of infection of 1 g 104~ 0.1 plaque-forming units (as determined on Vero cell monolayers) per well for 2 h at 37°C. The cells were washed three times and cultured for 3 days. At timed intervals, the cytopathic effect was evaluated under a Nikon inverted microscope. INTRODUCTIONHuman membrane cofactor protein (MCP)1( 1The abbreviations used are: MCPmembrane cofactor protein (CD46)DACMN-(dimethylamino-4-methylcoumarinyl)maleimideDAFdecay-accelerating factor (CD55)mAbmonoclonal antibodyAbantibodyMVmeasles virusSCRshort consensus repeat (DSCR1 means the SCR1-deleted form)STserine/threonine-rich domainCYTcytoplasmic tailPAGEpolyacrylamide gel electrophoresisCHOChinese hamster ovaryDMEMDulbecco's modified Eagle's mediumpfuplaque-forming unit(s).) CD46 was first identified as a C3b-binding protein (1Cole J.L. Housley Jr., G.A. Dykman T.R. MacDermott R.P. Atkinson J.P. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 859-863Crossref PubMed Scopus (140) Google Scholar) distinct from other membrane complement-associated proteins such as CR1 (CD35), CR2 (CD21), and decay-accelerating factor (DAF, CD55) (reviewed in Ref. 2). MCP inactivates cell-bound C3b/C4b, acting as a cofactor for plasma protease factor I(3Seya T. Turner J.R. Atkinson J.P. J. Exp. Med. 1986; 163: 837-855Crossref PubMed Scopus (321) Google Scholar, 4Seya T. Atkinson J.P. Biochem. J. 1989; 264: 581-588Crossref PubMed Scopus (122) Google Scholar), and protects host cells from complement-mediated cell damage(5Seya T. Hara T. Matsumoto M. Sugita Y. Akedo H. J. Exp. Med. 1990; 172: 1673-1680Crossref PubMed Scopus (101) Google Scholar, 6Lublin D.M. Coyne K.E. J. Exp. Med. 1991; 174: 35-44Crossref PubMed Scopus (98) Google Scholar, 7Oglesby T.J. Allen C.J. Liszewski M.K. White D.J.G. Atkinson J.P. J. Exp. Med. 1993; 175: 1547-1551Crossref Scopus (132) Google Scholar). This molecule composed of an amino terminus of four short consensus repeating units (SCR), a Ser/Thr (ST)-rich domain, 13 amino acids of unknown significance, a transmembrane region, and a cytoplasmic tail (CYT)(8Lublin D.M. Liszewski M.K. Post T.W. Arce M.A. Le Beau M.M. Rebentisch M.B. Lemons R.S. Seya T. Atkinson J.P. J. Exp. Med. 1988; 168: 181-194Crossref PubMed Scopus (172) Google Scholar). The region responsible for complement regulation is the SCR(9Adams E.M. Brown M.C. Nunge M. Krych M. Atkinson J.P. J. Immunol. 1991; 147: 3005-3011PubMed Google Scholar, 10Matsumoto M. Seya T. Nagasawa S. Biochem. J. 1992; 281: 493-499Crossref PubMed Scopus (35) Google Scholar). The structural gene for MCP maps to 1q32(8Lublin D.M. Liszewski M.K. Post T.W. Arce M.A. Le Beau M.M. Rebentisch M.B. Lemons R.S. Seya T. Atkinson J.P. J. Exp. Med. 1988; 168: 181-194Crossref PubMed Scopus (172) Google Scholar), where the genes of complement regulatory proteins, C4b-binding protein, DAF, CR1, CR2, and factor H, are clustered(11Bora N. Lublin D.M. Kumar V. Hockett R.D. Holers V.M. Atkinson J.P. J. Exp. Med. 1989; 169: 597-602Crossref PubMed Scopus (39) Google Scholar). MCP is a member of the regulator of complement activation gene family.Naniche et al.(12Naniche D. Varior-Krishnan G. Cervoni F. Wild T.F. Rossi B. Rabourdin-Combe C. Gerlier D. J. Virol. 1993; 67: 6025-6032Crossref PubMed Google Scholar) and Dorig et al.(13Dorig R.E. Marcil A. Chopra A. Richardson D. Cell. 1993; 75: 295-305Abstract Full Text PDF PubMed Scopus (864) Google Scholar) have suggested that MCP also serves as a receptor for measles virus (MV). MV, however, has no C3b-like molecules on its envelope. H protein of MV is thought to act as a ligand for target cell receptors, whereas F protein then induces viral-cell fusion(14Richardson C.D. Scheid A. Choppin P.W. Virology. 1980; 105: 205-222Crossref PubMed Scopus (234) Google Scholar, 15Wild T.F. Malvoisin E. Buckland R. J. Gen. Virol. 1991; 72: 439-442Crossref PubMed Scopus (209) Google Scholar). Indeed, MV can infect human and various monkey species, the tropism correlating with the expression of MCP(13Dorig R.E. Marcil A. Chopra A. Richardson D. Cell. 1993; 75: 295-305Abstract Full Text PDF PubMed Scopus (864) Google Scholar, 16Nickells M.W. Atkinson J.P. J. Immunol. 1990; 144: 4262-4267PubMed Google Scholar).There are many MCP phenotypes, which are distinguishable on SDS-PAGE (17-19). This polymorphism is caused by alternative splicing of mRNA encoding the ST-rich and CYT regions(17Post T.W. Liszewski M.K. Adams E.M. Tedja I. Miller E.A. Atkinson J.P. J. Exp. Med. 1991; 174: 93-102Crossref PubMed Scopus (146) Google Scholar). CHO cell clones expressing MCP variants with a variety of ST-rich (20Iwata K. Seya T. Ueda S. Ariga H. Nagasawa S. Biochem. J. 1994; 304: 169-175Crossref PubMed Scopus (34) Google Scholar) or CYT domains (21Manchester M. Liszewski M.K. Atkinson J.P. Oldstone M.B.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2161-2165Crossref PubMed Scopus (154) Google Scholar) have been reported to become permissive to MV. Thus, the MV-binding site on MCP must be located within the SCR. The MV-binding site and its structural relationship to the complement-binding site, therefore, remain to be elucidated.In the present study, we established CHO transfectants expressing various SCR deletion mutants of MCP and mapped the functional domains for complement regulation and MV binding." @default.
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• W2055978589 title "Diversity of Sites for Measles Virus Binding and for Inactivation of Complement C3b and C4b on Membrane Cofactor Protein CD46" @default.
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