©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Diversity of Sites for Measles Virus Binding and for Inactivation of Complement C3b and C4b on Membrane Cofactor Protein CD46 (*)

Kazunori Iwata (1), Tsukasa Seya (8), Yusuke Yanagi (3), John M. Pesando (4), Peter M. Johnson (5), Masaru Okabe (7), Shigeharu Ueda (6), Hiroyoshi Ariga (2), Shigeharu Nagasawa (1)(§)

From the (1)Division of Hygienic Chemistry and (2)Division of Molecular Biology, Department of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060, Japan, the (3)Department of Bacteriology, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, (4)Clinical Immunology, Oncomembrane, Inc., Seattle, Washington 98102, the (5)Department of Immunology, Royal Liverpool University Hospital, P. O. Box 147, Liverpool L69 3BX, United Kingdom, the (6)Department of Neurovirology and (7)Department of Experimental Animals, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan, and the (8)Department of Immunology, Center for Adult Diseases Osaka, Higashinari-ku, Osaka 537, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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.


INTRODUCTION

Human membrane cofactor protein (MCP)()CD46 was first identified as a C3b-binding protein (1) 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(3, 4) , and protects host cells from complement-mediated cell damage(5, 6, 7) . 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)(8) . The region responsible for complement regulation is the SCR(9, 10) . The structural gene for MCP maps to 1q32(8) , where the genes of complement regulatory proteins, C4b-binding protein, DAF, CR1, CR2, and factor H, are clustered(11) . MCP is a member of the regulator of complement activation gene family.

Naniche et al.(12) and Dorig et al.(13) 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(14, 15) . Indeed, MV can infect human and various monkey species, the tropism correlating with the expression of MCP(13, 16) .

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(17) . CHO cell clones expressing MCP variants with a variety of ST-rich (20) or CYT domains (21) 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 METHODS

Cells, Antibodies, and Proteins

CHO cells were obtained from American Type Culture Collection (ATCC). Vero cells, green monkey erythrocytes, and MV, a modified Nagahata strain(22) , 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(23) , E4.3 (24) was from Dr. B. Loveland (Austin Institute, Melbourne, Australia), and other mAbs were reported previously(25, 26) . 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)(27) .

Complement C3(28) , C4(29) , factor H(30) , and factor I (29) were purified from human plasma as described previously. C3b and C4b were prepared (29, 30) and labeled with an SH reagent, N-(dimethylamino-4-methylcoumarinyl)maleimide (DACM)(31) . The DACM-labeled C3b and C4b have been characterized as substrates for factor I and MCP(31, 32) . 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 Cells

MCP cDNA of ST/CYT2 phenotype (8) 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(8) ) were synthesized. For example, the oligonucleotide, 5`-TGGACATGTTTCTCTGGCATCGGAGAAGGA-3` was synthesized to delete SCR1 (SCR1) of MCP. Similarly, oligonucleotide sequences were determined to generate a mutant SCR2 (lacking the 62-124 amino acid sequence), SCR3 (lacking the 125-190 amino acid sequence), and SCR4 (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(33) . 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) (34) by calcium phosphate precipitation(35) . 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%CO, 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(35) . The expression of these mutants was confirmed by flow cytometry using M160 (not shown), M177, and E4.3.

Flow Cytometry and Immunoblotting

The protein levels expressed on the transfected cells (1 10) 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(36) . MCP and its mutants were solubilized from transfectants (about 2 10 cells) as reported previously(3) . 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(10) .

Glycosidase Treatment

Solubilized SCR4 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 SCR4 mutant was mixed with an equal volume of incubation buffer (40 mM Tris maleate, 20 mMD-galactono--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 Activity

A 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 10 cells were prepared by acid precipitation(3) , and the amounts of wild-type and mutant MCP were measured by sandwich enzyme-linked immunosorbent assay(37) . 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(31, 32) . Cofactor activity was mostly abrogated by the addition of M177(5) , suggesting that the expressed MCP was a major cofactor in these CHO cells (not shown).

Complement-dependent Cytolysis of CHO Transfectants

Anti-CHO cell Ab was used as a sensitizer(33) , 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 MgCl and 10 mM EGTA (Mg-EGTA), respectively. Briefly, the transfected cells (2 10/well) were seeded on 96-well plates. Fifteen h later, the cells were incubated with Cr (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 -counter. Cytotoxicity was calculated as described previously (38). All determinations were performed in triplicate.

MV Binding Assay

Cells 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 10 cells were incubated for 2 h at 4 °C with 2 ml of concentrated MV (10 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(27) . 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 Infectivity

CHO 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 (27) 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 (13) 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).


RESULTS

Levels and Properties of SCR Deletion Mutants Expressed on CHO Cells

The expression levels of the MCP mutants SCR1, SCR2, SCR3, and SCR4 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 SCR1 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(9) . 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 .


Figure 1: Immunoblotting and flow cytometric analyses for wild-type MCP and four MCP mutants. A, MCP transfectants and control CHO cells were solubilized with Nonidet P-40, and the insoluble materials were removed by centrifugation after acid precipitation (see ``Materials and Methods''). The samples were resolved by SDS-PAGE (10% acrylamide) (52) under nonreducing conditions. Immunoblotting was performed with M177 or E4.3 (see also Ref. 9) as primary Abs, and the membranes were subsequently stained with alkaline phosphatase-conjugated goat anti-mouse IgG and color reagents. The mean fluorescent shifts in flow cytometric analysis of the cells are indicated under the figure. B, the SCR4 mutant was incubated with N- and/or O-glycanases, resolved by SDS-PAGE, and Western-blotted using M177. Nontreated SCR4 mutant (lane 1), SCR4 incubated with N- and O-glycanases (lane 2), and SCR4 mutant incubated with O-glycanase were resolved by SDS-PAGE (12.5% acrylamide) and Western-blotted using M177.



Immunoblotting suggested that the wild-type MCP has an molecular mass of 57 kDa, consistent with that of the previously reported ST/CYT2 form(6, 33) . The molecular masses of SCR1, SCR2, and SCR3 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 SCR4 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 SCR4 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 Mutants

Complement regulatory activities of the mutants were determined by fluid-phase factor I-cofactor assay(31, 32) . 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 SCR1 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 SCR4 mutant (). M177 completely blocked this SCR4-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 Cr-labeled CHO cells with the mutants and the complement sources for the classical and the alternative pathways (). 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 SCR1, and the other clone, whose expression level of MCP was similar to those of SCR2, SCR3, and SCR4. The SCR1 mutant expressed as potent protective activity from complement attack as a wild-type. Judging from the MCP copy numbers expressed on the CHO cells, SCR1 and wild-type MCP protected cells from the two pathways with similar potencies. The SCR2 and SCR3 mutants showed no protective activity from the two pathways, and the SCR4 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 .

The Domain of MCP Responsible for MV Infection

MV binding assay was performed by flow cytometry (). Binding was observed in CHO cells expressing SCR3 and SCR4 mutants, but not in those expressing SCR1 or SCR2. The degrees of MV binding were in the order: wild type = SCR3 mutant>SCR4 mutant.

Syncytium formation is a representative marker for MV infection of CHO cells expressing MCP(13) . 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 Cells

MV replicated in CHO transfectants expressing SCR3 and SCR4 mutants, as well as wild-type, whereas no replication occurred in CHO transfectants expressing SCR1 or SCR2 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.


DISCUSSION

We 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.(9) and Oglesby et al.(40) , except that the SCR4 mutant retained weak cofactor activity for factor I-mediated C4b cleavage. They also determined the domains responsible for C3b and C4b binding(9) . 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 SCR4 resulting in the two forms of the SCR4 protein. This is not the case in a transient COS cell expression system reported by Adams et al.(9) , which may explain the differences between previous results (9, 40) and our present findings. That is, the presence of the two forms of the SCR4 protein may explain the findings that our SCR4 mutant retain, albeit weak, cofactor activity for C4b and that CHO cells expressing SCR4 are less sensitive to MV than those expressing SCR3 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(17, 18, 19) .

Some viruses and bacteria such as HIV (reviewed in Ref. 41), Listeria (42), and Mycobacteria (43) 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.(12) 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.(44) indeed block MV infection but have not been characterized with regard to the blocking of complement regulatory activity. One mAb, GB24(45) , which recognizes the SCR3 or SCR4, has been reported to block C3b and C4b binding to MCP (9) without suppressing MV infection(13) . 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 (23) 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 (46, 47) and some strains of the Echo virus(48) , respectively. The heads of these molecules appear to be important for virus binding, similar to other virus receptors(49) . There are many SCR proteins, which enhance cell adhesion (reviewed in Refs. 50 and 51) mediated by integrin family receptors and others(50) . 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.

  
Table: Epitopes and effects on MCP functions of mAbs

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 chain and 1 fragment by spectrofluorometry (Hitachi F2000) (31). Percent inhibition by each mAb was calculated assuming that the percent conversion of the chain to the 1 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 10 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.


  
Table: 0p4in The assay was performed as in the classical pathway, except that 40-fold-diluted anti-CHO Ab was used to potentiate activation of the alternative pathway, and human serum was substituted with Mg-EGTA human serum. Values of CHO cell clones expressing 3-fold more MCP or SCR1 than other CHO cells expressing SCR2, SCR3, and SCR4 are indicated in parentheses.(119)


FOOTNOTES

*
This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare of Japan and by grants from the Mochida Memorial Foundation, the Naito Memorial Foundation, the Nagase Science and Technology Foundation, and the Ryoichi Naito Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Hygienic Chemistry, Dept. of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060, Japan. Tel.: and Fax: 81-011-706-4990.

The abbreviations used are: MCP, membrane cofactor protein (CD46); DACM, N-(dimethylamino-4-methylcoumarinyl)maleimide; DAF, decay-accelerating factor (CD55); mAb, monoclonal antibody; Ab, antibody; MV, measles virus; SCR, short consensus repeat (DSCR1 means the SCR1-deleted form); ST, serine/threonine-rich domain; CYT, cytoplasmic tail; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; pfu, plaque-forming unit(s).


ACKNOWLEDGEMENTS

We are grateful to Drs. H. Akedo, M. Matsumoto, and M. Hatanaka (Center for Adult Diseases Osaka) for valuable discussions and to Dr. B. Loveland for providing us with mAb E4.3.


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