Identification and characterization of membrane cofactor protein (CD46) in the human kidneys

Membrane cofactor protein (MCP, CD46) is an integral protein that serves as a cofactor for factor I in inactivating C3b/C4b deposited on the same cell-membrane as C3bi/C4c+C4d. This C3b/C4b inactivation is closely associated with self-protection of host cells from autologous complement attack. We have studied the distribution and properties of MCP in the normal human kidney by immunohistochemical and immunoblotting methods using monoclonal antibodies against MCP. MCP was predominantly expressed on the juxtaglomerular apparatus. Glomerular capillary walls, mesangial areas, and tubulus were also MCP positive. Glomerulus MCP was composed of two major bands of 45–65 kDa, which were similar to those of lymphocyte MCP. The proportion of the high and low molecular weight components in glomerulus MCP, however, was considerably different from that of lymphocyte MCP among the individual samples tested. Glomerular epithelial cells and mesangial cells from an individual having equal amounts of high and low molecular weight components in the lymphocytes were cultured seperately and the properties of their MCP investigated. MCP in the mesangial cells and glomerular epithelial cells showed profiles in which the upper band was predominant. The results may explain the unique distribution of the high and low molecular weight forms in the glomerulus. These forms of MCP together with factor I were all capable of inactivating C3b to C3bi. Message analysis suggested that glomerular epithelial cells and mesangial cells synthesized a single species of mRNA of 4.2 kb from which the polymorphic MCP species were generated. Flow cytometric analysis suggested that MCP was minimal in mesangial cells. These results, taken together with the previous reports on the distribution of other complement regulatory proteins, infer that the distribution profile of MCP is rather similar to that of DAF but differs from those of CD59 and CR1 in the normal human kidney; this may reflect the differences between their roles or functional properties in renal tissue.     

麻疹病毒血凝素和融合蛋白基因的克隆与表达及其重组蛋白的免疫学评价

采用逆转录－多聚酶链反应（ＲＴ－ＰＣＲ）的方法从麻疹病毒Ｅｄｍｏｎｓｔｏｎ株基因组中扩增出血凝素Ｈ基因和融合蛋白Ｆ基因，并利用转移质粒将这两个基因分别重组到杆状病毒多角体蛋白启动子（ＰＨ）控制之下，获得重组病毒ｖＢＭＶＨ和ｖＢＭＶＦ。重组杆状病毒感染Ｓｆ９昆虫细胞，表达的重组蛋白分别具有血凝、血溶活性。红细胞吸附抑制试验、免疫印迹、酶联免疫试验结果显示重组蛋白在生物学、生化学特性与天然蛋白类似，并能被天然麻疹免疫血清（人、鼠、兔）所识别。动物实验表明，重组蛋白具有诱生中和抗体的能力。重组血凝素免疫血清还具有血凝抑制抗体活性。这些结果说明杆状病毒－昆虫细胞体系表达的麻疹病毒血凝素和融合蛋白具有较好的生物学活性及免疫原性和抗原性。     

人补体膜辅助调节蛋白MCP的基因克隆及其真核表达的研究

目的　克隆获得编码人补体膜辅助调节蛋白（ ＭＣＰ） 的ｃＤＮＡ,并对其在真核细胞的表达及功能进行研究。方法　应用ＲＴＰＣＲ 方法,从Ｕ９３７ 细胞总ＲＮＡ 中扩增编码人ＭＣＰ 分子的ｃＤＮＡ片段, 快速克隆于ｐＧＥＭＴＥａｓｙ 载体,测定其序列。将该片段重组于ｐＬＸＳＮ 载体,电穿孔转染ＮＩＨ３Ｔ３ 细胞,经ＦＡＣＳ 检测筛选表达ＭＣＰ 的阳性细胞克隆,用补体溶破试验鉴定其抑制人补体溶破的功能。结果　ＲＴＰＣＲ 扩增得到１ １４４ｂｐ 的编码人ＭＣＰ 分子的ｃＤＮＡ 片段,序列分析表明该ｃＤＮＡ编码的蛋白为ＳＴＣ＋ ＣＹＴ２ 亚型。细胞转染筛选获得多个表达ＭＣＰ 的ＮＩＨ３Ｔ３ 阳性细胞克隆,补体溶破试验证实其具有抑制人补体经典途径和旁路途径溶破的功能。结论　本研究为进一步探讨不同亚型结构的ＭＣＰ 分子与功能的关系及其应用奠定了基础。     

Blocking measles virus infection with a recombinant soluble form of, or monoclonal antibodies against, membrane cofactor protein of complement (CD46).

Human membrane cofactor protein (MCP, CD46) functions as an inhibitor of the complement (C) cascade to protect host cells from C attack, and as a receptor for measles virus (MV). Normal human sera contains 10-60 ng/ml of naturally produced soluble forms of MCP, which is also a cofactor for the factor I-mediated inactivation of C3b. We produced monoclonal antibodies (mAb) against MCP and a recombinant soluble form of MCP similar to the natural soluble forms, and tested their ability to block MV infection. Vero cells and CHO cells expressing human MCP were the targets. Of the antibodies tested, M75 and M177, which blocked the C regulatory activity of MCP, efficiently blocked MV infection. More than 50 micrograms/ml of the soluble form moderately blocked MV infection of CHO cells expressing MCP, but barely blocked that of Vero cells. The two mAb and the soluble form also inhibited MV H protein-mediated green monkey erythrocyte rosette formation. A quantitative analysis suggested that 30 micrograms/ml of the soluble form functionally corresponded to 0.2 microgram/ml of M177 or M75. These data established that the C regulatory function and the MV receptor function of MCP were blocked simultaneously by the individual mAb, and that soluble forms of MCP could inhibit MV infection in cells expressing human MCP, although doses far higher than the natural concentration of soluble MCP were required.     

Distribution of membrane cofactor protein (MCP/CD46) on pig tissues. Relevance To xenotransplantation.

Membrane cofactor protein (MCP; CD46) is a 50-60 000 MW glycoprotein, expressed on a wide variety of cells and tissues in man, which plays an important role in regulating complement activation. Human MCP has also been shown to be the receptor for measles virus. We have recently identified the pig analogue of MCP and demonstrated that pig MCP has cofactor activity for factor I-mediated cleavage of C3b when these components are derived either from pig or human. As a consequence, pig MCP is an efficient regulator of the classic and alternative pathways of human and pig complement. In order to define the potential importance of MCP in protecting against complement activation in the pig, we have conducted a comprehensive survey of its distribution in pig cells and organs. As in humans, MCP in the pig is broadly and abundantly distributed. Pig MCP is highly expressed on all circulating cells, including erythrocytes, in contrast to its absence on human erythrocytes. Multiple isoforms of MCP are found on cells and in tissues, probably representing products of alternative splicing analogous to those found in man. MCP is abundantly expressed throughout all tissues examined with particularly strong staining on the vascular endothelium. Connective tissue elements within liver and testis are also strongly stained by anti-pig MCP antibodies. Pig MCP is expressed only weakly on skeletal muscle cells and expression is absent from smooth muscle cells in the lung and vessel walls, sites at which human MCP is expressed. Of particular note, MCP is not expressed in B-cell areas of the germinal centres of lymph nodes.     

Molecular cloning of a pig homologue of membrane cofactor protein (CD46)

Organs of transgenic pigs that express human complement regulatory proteins are under assessment as an alternative to transplantation. A major barrier to the transplantation of pig organs is the hyperacute rejection caused by pre-existing antibodies and complement. Pig cells are very susceptible to human complement, presumably because pig cell- surface complement regulatory proteins are inefficient against it. Expression of human complement regulatory proteins, such as decay- accelerating factor and membrane cofactor proteins (MCP or CD46), by means of transgenes would confer resistance to human complement upon pig cells, thereby preventing hyperacute rejection. To express sufficient levels of human complement regulatory proteins at appropriate sites, regulatory elements of genes of pig membrane-bound complement regulatory proteins would be useful. To obtain their cDNAs, we transfected human cells with a pig cDNA library, selected cells by incubation with pig complement and rescued the plasmids. We cloned a cDNA for the pig homologue of MCP, pMCP. The cDNA encoded a predicted protein of 363 amino acids with 42% amino acid identity with human MCP. The pMCP consisted of four short consensus repeats, a Ser/Thr/Pro-rich domain, and transmembrane and cytoplasmic domains. Recombinant soluble pMCP that lacked transmembrane and cytoplasmic domains had factor I cofactor activity in C3b cleavage, indicating that it is functionally, as well as structurally homologous to MCP. FACS analysis with anti-pMCP mAb demonstrated that pMCP is expressed on all blood leukocytes, erythrocytes, and on endothelial and epithelial cell lines.      

CD46 (membrane cofactor protein) associates with multiple beta1 integrins and tetraspans

The tetraspans associate with a large number of surface molecules, including a subset of beta1 integrins and, indirectly through CD19, with the complement receptor CD21. To further characterize the tetraspan complexes we have raised and selected monoclonal antibodies (mAb) for their ability to immunoprecipitate a molecule associated with CD9. A unique mAb was identified which recognizes the complement regulator CD46 (membrane cofactor protein). CD46 associated in part with several tetranspans and with all beta1 integrins that were tested (CD29/CD49a, CD29/CD49b, CD29/CD49c, CD29/CD49e, CD29/CD49f) but not with beta4 integrins. These data, together with cross-linking experiments showing the existence in living cells of CD46/integrin complexes, suggest that CD46 associates directly with beta1 integrins and indirectly with tetraspans. CD46 also acts as a receptor for measles virus; however, mAb to various integrins and tetraspans did not modify the virus fusion entry step.     

Complement inhibitor membrane cofactor protein (MCP; CD46) is constitutively shed from cancer cell membranes in vesicles and converted by a metalloproteinase to a functionally active soluble form

Human cell-surface protein CD46 protects cells from complement damage, regulates immune functions through signaling and acts as a receptor for certain pathogenic microbes. Multiple molecular weight isoforms of membrane bound CD46 are produced by alternative splicing of the CD46 mRNA in an area coding for the serine/threonine/proline-rich region or for the cytoplasmic tail. We demonstrate that CD46 becomes proteolytically modified on cell membranes. We observed that tumor cells liberated intact 60-65 kDa forms of CD46 into the cell culture medium on the surface of vesicles with a diameter of 200 nm. Furthermore, soluble CD46 (55-60 kDa) containing the glycosylated STP-region but lacking the hydrophobic transmembrane sequence and cytoplasmic domains was released from tumor cell membranes. The use of selective inhibitors indicated that CD46 release is due to specific cleavage by a metalloproteinase. Exposure of the cells to hydrogen peroxide (H2O2) or their detachment from the pericellular matrix increased the shedding of soluble CD46. Both vesicular and soluble forms of CD46 remained functional and promoted C3b cleavage by factor I. The results show that the functional activity of CD46 is not restricted to the tumor cell membranes but can be liberated in vesicles and by a metalloproteinase.     

Membrane cofactor protein (CD46) in seminal plasma is a prostasome-bound form with complement regulatory activity and measles virus neutralizing activity.

Human seminal plasma contains 0.55 microgram/ml of membrane cofactor protein (MCP; CD46) of 60,000 MW. By ultracentrifugation, gel filtration and immunoelectron microscope methods, we found that the MCP in seminal plasma was associated with prostasomes. The functional properties of the prostasome-bound MCP were assessed in comparison with a recombinant soluble form, gamma MCP1, which is composed of four short consensus repeats (SCR), type C of the serine/threonine-rich domain (STC), and unknown significance (UK). The MCP in seminal plasma, although demonstrably bound to prostasomes, behaved more like the soluble form of MCP. In the absence of detergent it, together with factor I, degraded the fluid-phase ligand, methylamine-treated C3 [C3(MA)], which is insensitive under no-detergent conditions to the membrane form of MCP and factor I. Moreover, C3dg fragment was generated as a final product instead of C3bi during the incubation, indicating that the prostasomal MCP and proteases may be responsible for the C3dg generation. The prostasomes neutralized measles virus (MV) infectivity, while gamma MCP1, for the most part, did not. These results, taken together with the CD59 concentration on the prostasomes, suggest that the prostasomes are potential immunomodulators for complement activation, providing the C3- and C9-step inhibitors. The present report also reinforces the idea that there are two different forms of MCP in semen. One is located in the inner acrosomal membrane of spermatozoa, which appears through acrosomal reaction and spermatoon-egg interaction. The other is a prostasome-bound form maintaining activities sufficient to regulate complement activation and, probably, MV infection.     

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.     

Differential expression of complement regulatory proteins decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 during human spermatogenesis.

We have examined the distribution of the complement (C) regulatory proteins CD59, membrane cofactor protein (MCP) and decay-accelerating factor (DAF) on mature sperm and compared expression of these proteins in parallel both during spermatogenesis and in the prostate. Enhanced immunoperoxidase staining and radioimmunoassay confirmed that C regulators are differentially expressed on sperm; CD59 was strongly expressed on the surface of acrosome intact sperm while MCP and DAF appear to be located primarily on the inner acrosomal membrane. While the MW of CD59 on sperm is typical of other systems, we confirm that in addition to a novel 40,000-46,000 MW MCP protein, sperm also express a novel 55,000 MW DAF product. Examination of normal testis by immunostaining revealed that although C regulators are differentially expressed within the germinal epithelium, all three proteins were present on the acrosomal region of condensing spermatids. We show that novel, low MW forms of MCP and DAF are expressed in normal testis membranes but are absent from testis membranes obtained from patients undergoing gender reassignment surgery in whom the germinal epithelium is diminished. Novel MW C3 convertase regulators are therefore associated with differentiating germinal epithelium. Typical CD59 components were also present on normal testis membranes confirming that CD59 is acquired during spermatogenesis. We demonstrate that the prostatic epithelium, in addition to MCP, expresses CD59 but not DAF. By comparison with CD59, therefore, our studies suggest that DAF may be acquired only in the testis. Overall, our data suggest that, on leaving the testis, sperm express the repertoire of C regulators required for protection from C during their transit through the male and female reproductive tracts.     

Enhanced MHC class II-restricted presentation of measles virus (MV) hemagglutinin in transgenic mice expressing human MV receptor CD46

This study analyzes the role of the measles virus (MV) receptor, i.e. the human CD46 molecule, in the MHC class II-restricted presentation of MV hemagglutinin (H). We generated transgenic mice ubiquitously expressing CD46, with a similar level of transgene expression on the surface of antigen-presenting cells (APC), i.e. B cells, dendritic cells (DC) and macrophages. APC isolated from transgenic mice and nontransgenic controls were tested for their ability to present MV H to H-specific CD4⁺ I-E ^d -restricted T cell hybridomas. All three populations of APC were capable of presenting MV to T cell hybridomas, DC being the most efficient. Expression of CD46 on B lymphocytes increased MHC class II-dependent presentation of MV H up to 100-fold, while CD46-transgenic DC stimulated H-specific T cell hybridomas up to 10-fold better than nontransgenic DC. Interestingly, expression of CD46 did not change the presentation efficiency of transgenic macrophages, indicating that CD46-dependent enhancement of antigen presentation depends on the nature of the APC. Furthermore, a single injection of UV-inactivated MV particles into CD46-transgenic mice, but not nontransgenic controls, induced generation of MV-specific T lymphocytes and production of anti-H antibodies, suggesting a role for CD46 in the efficient capture of MV in vivo. These results show for the first time that one ubiquitously expressed cell surface receptor, like CD46, could function in receptor-mediated antigen presentation both in vitro and in vivo and its performance depends on the type of APC which expresses it.     

Molecular remodelling of human CD46 for xenotransplantation: designing a potent complement regulator without measles virus receptor activity

In pig-to-human discordant xenotransplantation, human complement (C) is a major barrier to long survival of xenografts. The current idea on how to cope with this barrier is that human complement regulatory proteins are forcibly expressed on xenografts to serve as safeguards against host C-induced hyperacute rejection of xenografts. Co-expression of decay-accelerating factor (DAF) (CD55) and membrane cofactor protein (MCP) (CD46) would be the first choice for this trial, because most of the human cells are protected from C-mediated damage by two different modes with these two kinds of C-regulators. Many problems have arisen, however, for MCP expression on grafts. (i) MCP acts as a measles virus receptor, which may function to render donor pigs measles virus (MV) sensitive. (ii) MCP signals immune suppression which causes devastation of the recipient's immune responses. (iii) MCP exerts relatively low self-protective activity against C compared with other cofactors; development of more efficient forms is desirable. (iv) Grafts with a high expression level of MCP are difficult to produce. In this study, we made a number of cDNA constructs of MCP, expressed them on swine endothelial cell lines, and tested cell-protective potency and MV susceptibility. The short consensus repeat 1 (SCR1)-deleted MCP with glycosyl phosphatidylinositol (GPI)-anchored form (Delta1MCP-PI) of MCP was found to be most suitable for the purpose of overcoming these problems. However, it was also found that MV induces two modes of cytopathic effect (CPE) on swine endothelial cells, either MCP-dependent or -independent. Here, we discuss these two points which will be raised through study of MCP-transgenic animals.     

Urine levels of CD46 (membrane cofactor protein) are increased in patients with glomerular diseases

Soluble membrane cofactor protein (MCP, CD46) has not been detected by conventional ELISA in human urine. Here, we established a highly sensitive assay method for determination of urinary MCP (uMCP) using monoclonal antibody-coated paramagnetic beads. This method enabled us to detect less than 0.05 ng/ml of purified membrane and recombinant soluble MCP, a sensitivity 10-fold higher than that of conventional ELISA. In normal subjects, the levels of uMCP were <0. 05 ng/ml. The levels of uMCP were elevated in patients with IgA nephropathy and more prominently in patients with rapidly progressive glomerulonephritis. The levels of uMCP were correlated significantly with those of serum MCP (sMCP) and N-acetyl-beta-glucosaminidase and nonsignificantly with those of beta(2)-microglobulin, total urine protein, or serum creatinine. The properties of uMCP were inconsistent with those of the reported sMCP, since uMCP showed three bands on SDS-PAGE/immunoblotting with molecular mass profiles different from those of sMCP. uMCP exhibited factor I cofactor activity for cleavage of C3b comparable to that of sMCP. The origin of uMCP, however, remains to be determined. These results, taken together with the parameter correlation profiles, suggested that uMCP is secreted or produced secondary to tubular or glomerular damage. The physiological role and clinical significance of uMCP are now within the scope of our investigation by establishment of this assay.     

Membrane cofactor protein (MCP; CD46): deficiency states and pathogen connections

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Inhibition of human spermatozoon--oocyte interaction in vitro by monoclonal antibodies to CD46 (membrane cofactor protein)

CD46 (membrane cofactor protein) is a cell surface complementregulatory glycoprotein that facilitates enzymatic cleavageof complement component C3b; it is expressed by both human oocytesand acrosome-reacted spermatozoa. Murine anti-CD46 monoclonalantibody (mAb) has been reported to decrease significantly theability of human spermatozoa to penetrate hamster oocytes. Wehave investigated the effect of purified anti-CD46 mAbs on spermatozoon-oocyteinteraction in an autologous zona-free oocyte penetration test.Oocytes and/or spermatozoa were preincubated with either oftwo anti-CD46 murine mAbs, TRA.2.10 (a non-blocking mAb) andMH61 (a mAb that functionally blocks C3b-ligand binding activity),or a control isotype-matched mAb, in medium supplemented withhuman serum albumin. Preincubation of both spermatozoa and zona-freeoocytes with TRA.2.10, but not MH61, caused a significant decreasein the number of oocytes showing sperm binding and pronuclearformation (9/23) compared with controls (21/26) in this complementcomponent-depleted medium. This effect was not observed if oocytesor spermatozoa alone were preincubated. These data suggest thatCD46 has a role in human spermatozoon-oocyte interaction atthe level of the ooocyte plasma membrane, and indicate thata novel function other than direct C3b binding could be involved.     

Membrane cofactor protein (MCP,CD46) in seminal plasma and on spermatozoa in normal and “sterile” subjects

A sperm protein of molecular mass 43 kDa (the spermatozoa membrane cofactor protein, smMCP) and a seminal plasma protein of 60 kDa (ssMCP) were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with four monoclonal antibodies (mAb) against membrane cofactor protein (MCP, CD46).These proteins served as factor I cofactors for the cleavage of methylamine-treated C3 (C3ma), the activity of which was blocked by M75, an MCP cofactor-activity-blocking mAb. Thus, these semen proteins are antigenic and functional homologoues of MCP. On SDS-PAGE analysis these MCP migrated as single-band proteins which differed from the two-band forms of MCP expressed on other cells. smMCP was N-glycosylated but not O-glycosylated, while ssMCP was O-glycosylated: after deglycosylation of these proteins bands were detected at 38-40 kDa and 43 kDa on SDS-PAGE, respectively. These semen MCP are therefore, structurally different from the conventional MCP. ssMCP in both normal and “sterile” subject groups was determined by sandwich enzyme-linked immunosorbent assay. Seminal plasma in the two groups contained 250-700 ng/ml ssMCP. The difference between the two groups was marginal, although samples from normal subjects tended to show higher concentrations of ssMCP than samples from “sterile” subjects. No molecular difference was observed with ssMCP and smMCP in the two groups by SDS-PAGE/immunobloblotting analysis. Immunohistochemical analysis suggested that MCP was positive in glandular epithelial cells and the lumen of the prostate, and in most intra-lumen cells of the testis. Using antibody M177, solubilized prostate and testis were analyzed by immunoblotting and compared with other cell MCP The major band of MCP in the testis, but not in the prostate, was of 60 kDa, which aligned with ssMCP. No band of testis or prostate MCP, however, aligned with smMCP. ssMCP may be produced in the testis, while the origin of smMCP remains unknown. We hypothesize that ssMCP is important in the survival of spermatozoa, protecting them against local secretion of immunoglobulin and complement in the female genital tract, and that smMCP, which is expressed on acrosome-reacted spermatozoa, plays an essential role in the interaction of spermatozoa with oocytes.     

Transforming growth factor-β isoforms regulate the surface expression on membrane cofactor protein (CD46) and CD59 on human keratinocytes

We studied the regulation of the expression of complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF) and CD59, on human keratinocytes by supernatant of activated mononuclear cells and by some individual cytokines present therein. Cultured keratinocytes expressed MCP, DAF and CD59. Supernatant of activated mononuclear cells and recombinant forms of transforming growth factor (TGF)-β variants (β1, β2 and β3) up-regulated MCP and CD59 but not DAF. Recombinant IL-1α, IL-2, IL-6, TNF-α and IFN-γ had no influence. TGF-β present in the supernatant was likely responsible for up-regulation of MCP and CD59. A monoclonal anti-TGF-β antibody, which neutralized TGF-β1, -β2 and -β3, did not inhibit the up-regulation of MCP and CD59 by the supernatant. These results indicated that TGF-β and an additional factor(s) present in the supernatant may be responsible for up-regulating the expression of MCP and CD59 on keratinocytes; both may be acting non-synergistically.