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PubMed1999

Human membrane cofactor protein (MCP, CD46): multiple isoforms and functions.

Human membrane cofactor protein (MCP, CD46) is a 45-70 kDa protein with genetic and tissue-specific heterogeneity, and is expressed on all nucleated cells. MCP consists from N-terminus of 4 short consensus repeats (SCRs), 1-3 serine/threonine-rich (ST) domains, a transmembrane domain (TM) and a cytoplasmic tail (CYT). More than 8 isoforms are generated secondary to alternative splicing due to combinations of various exons encoding the ST, TM and CYT domains. It serves as a cofactor of serine protease factor I for inactivation of complement C3b and C4b. Its primary role is to protect host cells from homologous complement attack by inactivating C3b/C4b deposited on the membrane. It also acts as receptors for measles virus (MV), some kinds of bacteria and for a putative ligand on oocytes. MV infection causes temporal host immune suppression, which may appear secondary to signaling events through MCP on macrophages and dendritic cells. These functional properties of human MCP may facilitate xenotransplantation and may be useful in the generation of animal models of measles by creating human MCP-expressing animals.

Amino Acid Sequence↗
PubMed2000

Membrane cofactor protein (MCP; CD46): isoform-specific tyrosine phosphorylation.

Membrane cofactor protein (MCP; CD46) is a widely expressed type 1 transmembrane glycoprotein that inhibits complement activation on host cells. It also is a receptor for several pathogens including measles virus, Streptococcus pyogenes, Neisseria gonorrhea, and Neisseria meningitidis. That MCP may have signaling capability was suggested by its microbial interactions. That is, binding of MCP on human monocytes by measles virus hemagglutinin or cross-linking by an anti-MCP Ab resulted in IL-12 down-regulation, while binding to MCP by Neisseria on epithelial cells produced a calcium flux. Through alternative splicing, MCP is expressed on most cells with two distinct cytoplasmic tails of 16 (CYT-1) or 23 (CYT-2) amino acids. These play pivotal roles in intracellular precursor processing and basolateral localization. We investigated the putative signal transduction pathway mediated by MCP and demonstrate that CYT-2, but not CYT-1, is phosphorylated on tyrosine. We examined MCP tail peptides and performed Ab cross-linking experiments on several human cell lines and MCP isoform transfectants. We found an MCP peptide of CYT-2 was phosphorylated by a src kinase system. Western blots of the cells lines demonstrated that cells bearing CYT-2 were also phosphorylated on tyrosine. Additionally, we provide genetic and biochemical evidence that the src family of kinases is responsible for the latter phosphorylation events. In particular, the src kinase, Lck, is required for phosphorylation of MCP in the Jurkat T cell line. Taken together, these studies suggest a src family-dependent pathway for signaling through MCP.

Amino Acid Sequence↗
PubMed2006

ADAM10-mediated release of complement membrane cofactor protein during apoptosis of epithelial cells.

Membrane cofactor protein CD46 controls complement activation on cells, is a receptor for several pathogens, and modulates immune responses by affecting CD8(+) T cells. Cells can release CD46 in an intact form on membrane vesicles and in a truncated form by a metalloproteolytic cleavage. The mechanism of shedding and its relationship to cell physiology has remained unclear. We have found using RNA interference analysis that a disintegrin and metalloproteinase (ADAM) 10 is responsible for the regulated shedding of the ectodomain of CD46 in apoptotic cells. The shedding of CD46 was initiated with staurosporine and UVB. Exposure of cell cultures to either UVB or staurosporine resulted in changes of cell morphology and detachment of cells from their matrices within 8-24 h. During this process CD46 was released both in apoptotic vesicles (vCD46) and proteolytically (sCD46) into the medium. Both the metalloproteinase inhibitor GM6001 and RNA interference of ADAM10 completely prevented the release of sCD46 and increased the expression of vCD46 on HaCaT cell vesicles, suggesting that ADAM10 releases sCD46 from the apoptotic vesicles. To explore whether the release of sCD46 is associated with apoptosis we analyzed the effects of caspase inhibitors. As expected, the inhibition of caspase activity attenuated the characteristic features of apoptosis and also decreased the release of sCD46. Our results reveal ADAM10 as an important regulator of CD46 expression during apoptosis. The ADAM10-mediated release of CD46 from apoptotic vesicles may represent a form of strategy to allow restricted complement activation to deal with modified self.

Amyloid Precursor Protein Secretases↗
PubMed1996

Soluble CD46 (membrane cofactor protein, MCP) in human reproductive tract fluids.

CD46 (membrane cofactor protein, MCP) is a cell surface complement regulatory protein which may have an additional role in human sperm-egg interaction. A soluble form (sCD46) has also been detected in a number of biological fluids, most notably seminal plasma. The present study has employed a monoclonal antibody-based ELISA to assay sCD46 in reproductive tract fluids in normal and pathological conditions. Large amounts of sCD46 were detected in seminal plasma of both fertile and infertile men (combined mean, 4859 ng/ml). Vasectomized men had lower levels (mean, 2421 ng/ml), indicating contributory sources both before and after the vas deferens ligation site. Pre-colostrum also contained relatively high quantities (mean, 445 ng/ml), whereas breast milk (mean, 117 ng/ml), peritoneal fluid (mean, 154 ng/ml) and follicular fluid (mean, 107 ng/ml), as well as uterine (mean, 208 ng/ml), umbilical (mean, 166 ng/ml) and peripheral (mean, 206 ng/ml) blood plasma, had sCD46 levels within a comparable range. Amniotic fluid had low sCD46 concentrations (mean, 22 ng/ml). In endometriosis, peritoneal fluid levels of sCD46 were significantly raised (mean, 199 mg/ml). These results indicate distinctive fluid compartmentalisation of sCD46 consistent with a biological function in human reproductive tract fluids.

Amniotic Fluid↗
PubMed1998

Molecular cloning of a murine homologue of membrane cofactor protein (CD46): preferential expression in testicular germ cells.

Human membrane cofactor protein (MCP, CD46) has been suggested, although no convincing evidence has been proposed, to be a fertilization-associated protein, in addition to its primary functions as a complement regulator and a measles virus receptor. We have cloned a cDNA encoding the murine homologue of MCP. This cDNA showed 45% identity in deduced protein sequence and 62% identity in nucleotide sequence with human MCP. Its ectodomains were four short consensus repeats and a serine/threonine-rich domain, and it appeared to be a type 1 membrane protein with a 23-amino acid transmembrane domain and a short cytoplasmic tail. The protein expressed on Chinese hamster ovary cell transfectants was 47 kDa on SDS/PAGE immunoblotting, approximately 6 kDa larger than the murine testis MCP. It served as a cofactor for factor I-mediated inactivation of the complement protein C3b in a homologous system and, to a lesser extent, in a human system. Strikingly, the major message of murine MCP was 1.5 kb and was expressed predominantly in the testis. It was not detected in mice defective in spermatogenesis or with immature germ cells (until 23 days old). Thus, murine MCP may be a sperm-dominant protein the message of which is expressed selectively in spermatids during germ-cell differentiation.

Amino Acid Sequence↗
PubMed2001

Membrane cofactor protein (MCP; CD46) expression in transgenic mice.

Human membrane cofactor protein (MCP; CD46) is a widely distributed complement regulator. In the mouse, expression of MCP is largely restricted to the testis while a related, widely expressed protein (Crry) appears to perform MCP's (CD46) regulatory activity. We have developed two mouse strains transgenic for human MCP (CD46) utilizing an approximately 400 kb YAC clone carrying the complete gene. A third mouse strain was generated using an overlapping YAC clone isolated from a second library. The expression of human MCP (CD46) in these mouse strains was characterized by immunohistochemistry, FACS, Western blotting and RT-PCR. No differences were detected in the isoform pattern or distribution among the three strains, although the expression level varied according to how many copies of the gene were integrated. The expression profile closely mimicked that observed in humans, including the same pattern of isoform expression as the donor. In addition, tissue-specific isoform expression in the kidney, salivary gland and brain paralleled that observed in man. The transgenic mice expressed low levels of MCP (CD46) on their E, in contrast to humans but in line with most other primates. These mice should be a useful tool to analyse tissue-specific expression, to establish animal models of infections and to characterize the role of MCP (CD46) in reproduction.

Animals↗
PubMed1996

Characterisation of the complement-regulatory proteins decay-accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46) on a human colonic adenocarcinoma cell line.

To avoid destruction by complement, normal and malignant cells express membrane glycoproteins that restrict complement activity. These include decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and protectin (CD59), which are all expressed on colonic adenocarcinoma cells in situ. In this study we have characterised the C3/C5 convertase regulators DAF and MCP on the human colonic adenocarcinoma cell line HT29. DAF was found to be a glycosyl-phosphatidylinositol-anchored 70-kDa glycoprotein. Blocking experiments with F(ab')2 fragments of the anti-DAF monoclonal antibody BRIC 216 showed that DAF modulates the degree of C3 deposition and mediates resistance to complement-mediated killing of the cells. The expression and function of DAF were enhanced by tumour necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1 beta). Cells incubated with interferon gamma (IFN gamma) did not alter their DAF expression. Two MCP forms were expressed, with molecular masses of approximately 58 kDa and 68 kDa, the lower form predominating. MCP expression was up-regulated by IL-1 beta, but not by TNF alpha or INF gamma. Expression of DAF and MCP promotes resistance of colonic adenocarcinoma cells to complement-mediated damage, and represents a possible mechanism of tumour escape.

Adenocarcinoma↗
PubMed2002

Production and characterization of a pig line transgenic for human membrane cofactor protein.

A pig line transgenic for human membrane cofactor protein (hMCP) has been established. Offspring from the founder were produced by crossing the founder with pigs heterozygous for the human decay accelerating factor (hDAF) transgene. As a result, pigs transgenic for both hMCP and hDAF have been produced. Ribonuclease protection assay (RPA) indicated that hMCP was expressed in all the tissues analysed. In addition, immunohistochemical results indicated a high level of expression of hMCP on neural tissues and islets where hDAF was absent or weakly expressed. C3 fragment deposition and cytotoxicity assays indicated that hMCP expression alone on pig endothelial cells and peripheral blood lymphocytes (PBLs) provided protection against human complement mediated damage. However, we did not find that porcine endothelial cells expressing both hDAF and hMCP were better protected than those expressing hDAF alone. The expression of hMCP on tissues where hDAF is not expressed could provide these tissues with protection against human complement mediated lysis.

Animals↗
PubMed1992

Complement regulatory proteins at the feto-maternal interface during human placental development: distribution of CD59 by comparison with membrane cofactor protein (CD46) and decay accelerating factor (CD55).

The complement (C) regulatory proteins decay-accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), which control C3 convertases, together with CD59, an inhibitor of the membrane attack complex (MAC), were found to be present in the developing human placenta from at least 6 weeks of gestation until term. Immunostaining revealed differences in the distribution of these proteins on the fetally derived trophoblast epithelium, especially in early placentae which contain trophoblast populations of diverse proliferative potential and differentiation status. Expression of all three proteins occurred on the terminally differentiated syncytiotrophoblast epithelium covering chorionic villi and which is in direct contact with maternal blood. CD59 was also expressed on the underlying villous cytotrophoblast cells and on their extra-villous derivatives. These two populations showed differential expression of the C3 convertase regulators. Villous cytotrophoblast cells expressed MCP but were largely devoid of DAF. Proliferation of this population to generate extra-villous cytotrophoblast cell columns was associated with both an increase in DAF expression and a decrease in MCP expression. Throughout placental development, expression of DAF appeared to be lower than that of MCP and CD59 as assessed by solid-phase binding assays on isolated trophoblast membranes. Early placentae were also found to contain both DAF+ and DAF- chorionic villi. Conversely, expression of CD59 appeared comparatively high and transcripts for CD59 were found to be much more abundant than those for DAF in purified trophoblast cells. C regulatory proteins appear to play an important role throughout gestation in protecting the fetally derived human conceptus from maternal C. The differential expression patterns of the proteins on trophoblast may reflect differences in requirement for specific functional activities at different locations within the placenta.

Antigens, CD↗
PubMed1992

Human astrocytes express membrane cofactor protein (CD46), a regulator of complement activation.

Expression of membrane cofactor protein (CD46) on cultured human astrocytes was demonstrated by indirect immunofluorescence microscopy and flow cytometry following staining with a monoclonal antibody specific for CD46. Western transfer and immunoblotting detected a doublet of Mr 66,000 and 56,000. Analysis of astrocyte mRNA revealed the presence of multiple alternatively spliced transcripts encoding different extracellular regions or cytoplasmic tails of CD46. Astrocytes were also shown to express decay accelerating factor, but not the type 1 complement receptor. Upregulation of astrocyte CD46 occurred following cytomegalovirus infection. These results indicate that astrocytes express proteins involved in regulation of complement activation and protection against autologous complement.

Antigens, CD↗
PubMed1996

The N-glycan of the SCR 2 region is essential for membrane cofactor protein (CD46) to function as a measles virus receptor.

Membrane cofactor protein (MCP) (CD46), a complement-regulatory protein, serves as a cellular receptor for measles virus. Its amino-terminal portion is composed of four short consensus repeats (SCR), three of which (SCR1, SCR2, and SCR4) carry an N-linked oligosaccharide. In order to determine the importance of the three N-glycans for the function of MCP as a measles virus receptor, we established Chinese hamster ovary (CHO) cell lines that stably express mutant MCPs lacking one of the three motifs for N glycosylation (NQ1, NQ2, and NQ4). In an additional mutant (NQ1-2), two glycosylation motifs were altered, allowing the addition of an N-linked oligosaccharide only in SCR4. The abilities of the mutant MCPs to function as measles virus receptors were analyzed with three different assays: (i) binding of measles virus hemagglutinin to MCP immobilized on nitrocellulose; (ii) binding of measles virus to CHO cells expressing wild-type or mutant MCP; and (iii) infection of the transfected CHO cells by measles virus. In all three assays, the abilities of the NQ2 and NQ1-2 mutants to serve as measles virus receptors were drastically impaired. The NQ1 and NQ4 mutants were recognized by measles virus almost as efficiently as the wild-type protein. These results indicate that the N-glycan attached to SCR2 is essential for MCP to serve as a measles virus receptor, while the oligosaccharides attached to SCR1 and SCR4 are of only minor importance.

Animals↗
PubMed1999

Control of C3b and C5b deposition by CD46 (membrane cofactor protein) after alternative but not classical complement activation.

C3b and C5b deposition following complement activation, and its regulation by CD46 were studied using xenogenic Chinese hamster ovary (CHO) cells as targets and cytofluorometry. Following activation of the alternative pathway, an initial low level of C3b deposition was observed on CHO cell surfaces after a lag time of approximately 4 min. This was followed by a secondary high level of C3b deposition with a slower rate. C3b deposition was maximal within 15 min. When CD46 was expressed (B2 isoform), the kinetics of C3b deposition were essentially unchanged, but the onset of the secondary high C3b deposition was fully prevented. C5b deposition was also observed on CHO but not on CHO.CD46 cells following activation of the alternative pathway. Activation of the classical pathway on CHO and CHO.CD46 cells, using factor B-depleted human serum and anti-CHO antibodies, resulted in almost identical single-peak C3b deposition profiles. Accordingly, no regulation of C5b deposition by CD46 was evident following activation of the classical pathway. These data indicate that CD46 prevents the C3b deposition amplification loop mediated by the alternative C3 convertase and, consequently, inhibits the formation of the alternative C5 convertase. But CD46 prevents neither the spontaneous tick-over C3b deposition leading to the formation of the alternative C3 convertase nor the formation of the functional classical C3 and C5 convertases.

Animals↗
PubMed1997

Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection.

CD46 (membrane cofactor protein) is a human cell-surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA. A high level of CD46 expression in homozygotic transgenic mice was obtained with spleen cells having approximately 75% of the level found on human peripheral blood mononuclear cells. CD46 was detected in all tissues examined by immunohistochemistry, radioimmunoassay and Western blotting, showing that these mice were suitable for transplantation and measles virus infection studies. It also indicated that the transgene included the important regulatory elements of the CD46 promoter. Transgenic spleen cells were significantly protected in vitro from human complement activated by either the classical or alternative pathways and from alternative pathway rat complement. Furthermore, transgenic mouse hearts transplanted to rats regulated complement deposition in an in vivo model of antibody-dependent hyperacute xenograft rejection. Similar to human lymphocytes, transgenic lymphoblasts could be infected in vitro with measles virus; infected cells expressed viral proteins and produced infectious viral particles. The data demonstrate the suitability of this minigene for obtaining high-level CD46 expression sufficient for enhanced resistance of transgenic cells to complement attack and for obtaining wide tissue distribution of CD46, analogous to human tissues and, therefore, useful for comparative studies.

Acute Disease↗
PubMed2005

The distal short consensus repeats 1 and 2 of the membrane cofactor protein CD46 and their distance from the cell membrane determine productive entry of species B adenovirus serotype 35.

The human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single amino acids within SCR domains I and II were tested in binding and in Ad35-mediated luciferase transduction assays. In addition, anti-CD46 antibodies and soluble polypeptides constituting various CD46 domains were used in binding inhibition studies. Our data indicate that (i) CD46 SCR I or SCR II alone confers low but significant Ad35 binding; (ii) the presence of SCR I and II is required for optimal binding and transgene expression; (iii) transduction efficiencies equivalent to that of full-length CD46 are obtained if SCR I and II are at an appropriate distance from the cell membrane; (iv) ablation of the N-glycan attached to SCR I has no influence on receptor function, whereas ablation of the SCR II N-glycan results in about a two- to threefold reduction of binding and transgene expression; (v) most putative Ad35 binding residues are located on the same solvent-exposed face of the SCR I or SCR II domain, which are twisted by about 90 degrees ; and (vi) the putative Ad35 binding sites partly overlap with the measles virus binding surface.

Adenoviruses, Human↗
PubMed1990

Complement-mediated tumor cell damage induced by antibodies against membrane cofactor protein (MCP, CD46).

We have developed polyclonal and monoclonal antibodies against human membrane cofactor protein (MCP) to use as tools to investigate the functions of MCP on intact nucleated cells. Two human T cell lines, CEM and TALL, are CR1- and DAF-. Pretreatment of these cell lines with M177 and polyclonal anti-MCP, which inhibit cofactor activity almost completely, resulted in effective C3 deposition immediately following addition of these cells to Mg2+/EGTA/human sera. The deposited C3 remained expressed partly on the cell surface and most of them were gradually converted to C3bi. Some of the deposited C3 were complexed with membrane proteins, since 140- and 250-kD bands became significantly accumulated on SDS-PAGE by treatment with the antibodies. We next tested whether these C3-coated cells were damaged by complement-mediated cytolysis. p18, an inhibitor of membrane attack complex (MAC) formation, was negative in TALL but positive in CEM. TALL was lysed efficiently only by treatment with the polyclonal anti-MCP, while CEM showed only slight lysis with the same treatment. Monoclonal antibodies to MCP, including M177, caused only minimal cell destruction. Based on these results, together with the fact that decay-accelerating factor (DAF) serves as a factor for preventing C3 attack on human cells, we conclude that MCP and DAF cooperatively protect host cells from C3 targeting and, in these T cell lines, MCP is sufficient for preventing C3 deposition even without DAF. After all, human cells undergo almost no autologous complement-mediated cytolysis if they express at least one of the functionally active inhibitors, MCP, DAF, or p18.

Animals↗