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PubMed1993

Characterization of a cDNA clone coding for human testis membrane cofactor protein (MCP, CD46).

Membrane cofactor protein (MCP) is a complement regulatory protein that acts as a cofactor for the cleavage of C3b and C4b by the serine protease factor I. We have previously reported the characterization of a functional MCP molecule on the acrosomal membrane. This protein migrated as a single band with a molecular weight of 40,000 Da, which is 10,000-20,000 Da smaller than the known MCP molecules, and is devoid of N- and O-linked sugars. We have proposed that the difference in molecular weight resulted from the lack of sugars. To investigate if this is due to the absence of glycosylation sites, we have characterized a cDNA clone from a human testis cDNA library. This cDNA corresponds to a peculiar MCP form previously described, which is characterized by the presence of the serine/threonine/proline-rich exon C (STPC) and the cytoplasmic tail known as CYT2, and we conclude that the absence of mature oligosaccharide of the sperm MCP cannot be totally attributed to a defect of N- and O-glycosylation sequences but rather reflects an alteration of the mechanisms of glycosylation in spermatozoa. The presence of functional MCP on the acrosomal membrane, as well as the other complement regulatory protein, decay-accelerating factor, strongly suggests that these proteins may act concomitantly to protect the acrosome-reacted spermatozoa from the attack of the complement present in the female genital tract.

Amino Acid Sequence↗
PubMed1992

Membrane cofactor protein (CD46) protects cells from complement-mediated attack by an intrinsic mechanism.

The cleavage of C3 is a critical step for complement (C) activation in the classical and alternative pathways. This reaction is controlled by the regulators of C activation protein family. Membrane cofactor protein (MCP) is a cofactor for the factor I-mediated inactivation of C3b and C4b. As a widely distributed membrane protein, MCP may protect host cells from inadvertent C activation. Human MCP has recently been shown to protect transfected rodent cells from human C-mediated lysis. In this report the relationship of MCP expression to C3b deposition and cytoprotection was examined using NIH/3T3 cells transfected with human MCP and exposed to human serum as a source of C and naturally occurring anti-mouse antibody. MCP inhibited C3b deposition in a dose-dependent fashion and inhibited lysis of the mouse cells expressing it. MCP did not inhibit lysis on bystander cells. These results demonstrate the protective role of MCP, at the cellular level, by an intrinsic mechanism.

3T3 Cells↗
PubMed1994

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.

Acrosome↗
PubMed1999

Epitope mapping of 10 monoclonal antibodies against the pig analogue of human membrane cofactor protein (MCP).

Pig membrane cofactor protein (MCP; CD46) is a 50 000-60 000 MW glycoprotein that is expressed on a wide variety of cells, including erythrocytes. Pig MCP has cofactor activity for factor I-mediated cleavage of C3b and is an efficient regulator of the classical and alternative pathway of human and pig complement. A panel of 10 monoclonal antibodies (mAbs) was collected from two different laboratories; all of these mAbs were raised against pig leucocytes and all recognized the same complex banding pattern on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of erythrocyte membranes. All were shown to be reactive with pig MCP and were divided into four groups of mutually competitive antibodies based on competition studies for membrane-bound MCP and for soluble MCP, the latter by surface plasmon resonance (SPR) analysis. The antigenic properties of membrane-bound and soluble MCP were similar, although some interesting differences were revealed. None of the 10 mAbs were cross-reactive with human MCP and only one showed cross-reactivity with leucocytes from a panel of large mammals - a weak cross-reactivity with a subset of dog leucocytes. All antibodies in one of the epitope groups and some in a second epitope group were able to block the functional activity of pig MCP, as measured by inhibition of MCP-catalysed C3 degradation by factor I.

Animals↗
PubMed1989

Functional properties of membrane cofactor protein of complement.

Membrane cofactor protein (MCP or gp45-70) of the complement system is a cofactor for factor I-mediated cleavage of fluid-phase C3b and C3b-like C3, which opens the thioester bond. In the present study the activity of MCP was further characterized. Unexpectedly, in the absence of factor I, MCP stabilized the alternative- and, to a lesser extent, the classical-pathway cell-bound C3 convertases and thereby enhanced C3b deposition. Soluble MCP, if added exogenously, hardly functioned as cofactor for the cleavage of erythrocyte-bound C3b to iC3b; i.e. its activity, compared with the cofactor activity of factor H, was inefficient, since less than 10% of the bound C3b was MCP-sensitive. Further, exogenously added soluble MCP was also a weak cofactor for the cleavage of C3b bound to zymosan. Likewise, factor I, in the presence of cells bearing MCP, cleaved fluid-phase C3b inefficiently. These results imply that MCP has very little extrinsic cofactor activity for factor I. In contrast, exogenously added MCP and factor I mediated efficient cleavage of erythrocyte-bound C3b if the concentration of Nonidet P40 was sufficient to solubilize the cells. Interestingly, soluble MCP and factor I degraded C3b attached to certain solubilized acceptor membrane molecules more readily than others. The cleavage reaction of fluid-phase and cell-bound C3b by soluble MCP and factor I produced iC3b, but no C3c and C3dg. These and prior data indicate that soluble MCP has potent cofactor activity for fluid-phase C3b or C3b bound to solubilized molecules, but acts inefficiently towards C3b on other cells. This functional profile is unique for a C3b/C4b binding protein and, taken together with its wide tissue distribution, suggests an important role for MCP in the regulation of the complement system.

Animals↗
PubMed2000

Dissecting sites important for complement regulatory activity in membrane cofactor protein (MCP; CD46).

Membrane cofactor protein (MCP; CD46), a widely distributed regulator of complement activation, is a cofactor for the factor I-mediated degradation of C3b and C4b deposited on host cells. MCP possesses four extracellular, contiguous complement control protein modules (CCPs) important for this inhibitory activity. The goal of the present study was to delineate functional sites within these modules. We employed multiple approaches including mutagenesis, epitope mapping, and comparisons to primate MCP to make the following observations. First, functional sites were located to each of the four CCPs. Second, some residues were important for both C3b and C4b interactions while others were specific for one or the other. Third, while a reduction in ligand binding was invariably accompanied by a parallel reduction in cofactor activity (CA), other mutants lost or had reduced CA but retained ligand binding. Fourth, two C4b-regulatory domains overlapped measles virus interactive regions, indicating that the hemagglutinin docks to a site important for complement inhibition. Fifth, several MCP regulatory areas corresponded to functionally critical, homologous positions in other CCP-bearing C3b/C4b-binding proteins. Based on these data and the recently derived crystal structure of repeats one and two, computer modeling was employed to predict MCP structure and examine active sites.

Amino Acid Sequence↗
PubMed1992

Identification and characterization of membrane cofactor protein of human spermatozoa.

Membrane cofactor protein (MCP) regulates C activation by serving as a cofactor for the cleavage of C3b and C4b by the serine protease factor I. An MCP-like molecule on the inner acrosomal membrane of human spermatozoa has been characterized. Three mAb and a rabbit polyclonal antibody against MCP recognized the sperm protein. On SDS-PAGE, it migrated as a single band with a molecular mass of 38,000 and 44,000 Da under nonreducing or reducing conditions, respectively. The molecular mass was 10,000 to 20,000 Da less than the two forms of MCP expressed on others cells. The electrophoretic pattern, by one- and two-dimensional gel analysis, and the isoelectric point profile (4.5 to 5.0) of the sperm protein were similar among multiple individuals. In contrast to MCP of other cells, digestion with endoglycosidases did not alter either the m.w. or the pI of the protein, suggesting that it is a poorly or nonglycosylated form of MCP. The solubilized sperm protein bound C3 with broken thioester bond to Sepharose and possessed cofactor activity for factor I-mediated cleavage of C3 with the broken bond. A mAb that blocks the regulatory function of MCP inhibited the cofactor activity of the sperm lysate. Thus, the sperm protein is an antigenic and functional homologue of MCP but has the distinct structural features of a lower m.w. and an apparent lack of glycosylation. MCP may play an essential role in the survival of the acrosome-reacted spermatozoa by modulating C activation in the female genital tract.

Acrosome↗
PubMed2002

Identification of the streptococcal M protein binding site on membrane cofactor protein (CD46).

Adherence of group A streptococcus (GAS) to keratinocytes is mediated by an interaction between human CD46 (membrane cofactor protein) with streptococcal cell surface M protein. CD46 belongs to a family of proteins that contain structurally related short consensus repeat (SCR) domains and regulate the activation of the complement components C3b and/or C4b. CD46 possesses four SCR domains and the aim of this study was to characterize their interaction with M protein. Following confirmation of the M6 protein-dependent interaction between GAS and human keratinocytes, we demonstrated that M6 protein binds soluble recombinant CD46 protein and to a CD46 construct containing only SCRs 3 and 4. M6 protein did not bind to soluble recombinant CD46 chimeric proteins that had the third and/or fourth SCR domains replaced with the corresponding domains from another complement regulator, CD55 (decay-accelerating factor). Homology-based molecular modeling of CD46 SCRs 3 and 4 revealed a cluster of positively charged residues between the interface of these SCR domains similar to the verified M protein binding sites on the plasma complement regulators factor H and C4b-binding protein. The presence of excess M6 protein did not inhibit the cofactor activity of CD46 and the presence of excess C3b did not inhibit the ability of CD46 to bind M6 protein by ELISA. In conclusion, 1) adherence of M6 GAS to keratinocytes is M protein dependent and 2) a major M protein binding site is located within SCRs 3 and 4, probably at the interface of these two domains, at a site distinct from the C3b-binding and cofactor site of CD46.

Amino Acid Sequence↗
PubMed1994

Modulation of complement regulatory function and measles virus receptor function by the serine-threonine-rich domains of membrane cofactor protein (CD46).

Three major membrane cofactor protein (MCP) phenotypes with different serine-threonine (ST)-rich regions, namely STc (L-phenotype), STBC (H or U phenotype) and STABC, and the MCP without the ST domain (delta ST) were expressed in Chinese hamster ovary (CHO) cells by transfecting the respective cDNAs. The expressed molecules migrated with a larger molecular mass on SDS/PAGE than those expected from their amino acid sequences. O-Glycanase digestion showed that this was due to O-linked sugar chains. The apparent sugar contents in each ST segment were compatible with their serine and threonine contents in the ST regions. The functional properties of these phenotypes as inhibitors of human complement (C) and receptors of measles virus (MV) were compared. The classical pathway-dependent CHO cell lysis by human C was more effectively suppressed by the expressed delta ST and STC than by the STABC and STBC phenotypes, although the difference was not so prominent. In contrast, alternative C pathway-dependent CHO-cell lysis was most effectively suppressed by the STABC phenotype and was only slightly blocked by the ST-deleted mutant. MV infection occurred with all of the phenotypes, but the infectious dose required to cause the same level of syncytium formation was 100-times higher in large ST (STABC and STBC) than in small ST (STC and delta ST) phenotypes. Thus, the ST domain serves as a functional modulator in MCP: MCP with a large ST domain having high O-linked sugar contents is favourable to the effective suppression of both the alternative C pathway-mediated cytolysis and MV infection, whereas MCP with a small ST domain is favourable to the suppression of the classical C pathway.

Animals↗
PubMed1993

Characterization of the promoter region of the membrane cofactor protein (CD46) gene of the human complement system and comparison to a membrane cofactor protein-like genetic element.

Membrane cofactor protein (MCP; CD46) is a widely expressed C regulatory protein that inhibits C activation on self-tissue. MCP binds C3b and C4b deposited on autologous cells and then serves as a cofactor for their inactivation by limited proteolytic cleavage. To characterize the DNA sequence elements responsible for controlling MCP expression, the 5' flanking region of the human MCP gene was cloned. Sequencing of 1350 nucleotides upstream from the ATG codon revealed a GC-rich region in the initial 500 nucleotides that is especially rich in the CpG dinucleotide. A CAAT box in reverse orientation, surrounded by four putative SP1 binding sites but lacking a typical TATA element, was within the first 200 nucleotides of this GC-rich region. The major transcriptional initiation site for HeLa cells, determined by primer extension and S1 nuclease protection analyses, was located 105 nucleotides from the translational start site. This overall orientation of the promoter region is characteristic of "housekeeping" genes. The MCP promoter region was further examined in HEp-2 cells by the chloramphenicol acetyltransferase (CAT) reporter gene assay, using various constructs derived from the 5' region of the MCP gene. The MCP promoter activity was confined to the GC-rich region from -624 to +96 (start site of transcription being +1). Inclusion of an AT-rich sequence from -624 to -1204 resulted in a 42% reduction in CAT activity suggesting that an inhibitor is present among the AT-rich sequences. The 5' flanking region of a highly homologous partial duplication of the MCP gene was also cloned and sequenced, and various constructs were assessed in the CAT reporter system. Many of the functionally relevant sequences seen in MCP are also found in the MCP-like 5' UT region, which is 85% homologous to MCP. The most striking difference was a 224 nucleotide deletion that was upstream from the corresponding MCP region harboring most of the promoter activity. Although expression of an MCP-like protein has not been reported, the MCP-like promoter region produced promoter activity comparable with that of MCP. These results serve as a basis for subsequent analyses of the expression of MCP in various cells and tissues and for understanding the mechanism of its modulation in inflammatory conditions. Also, through a comparison of the 5' region of MCP with other genes in the regulators of C activation gene cluster (at 1 q32), we propose a model for the evolution of the promoters in this tight linkage group.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗
PubMed1992

Expression of human decay accelerating factor or membrane cofactor protein genes on mouse cells inhibits lysis by human complement.

Mouse cells expressing the human complement regulatory proteins decay accelerating factor (DAF) or membrane cofactor protein (MCP) were produced both by hybridoma technology and by transfection with the appropriate cDNAs. The expression of either or both of these products protected the mouse cell from lysis by human (though not rabbit) complement in the presence of naturally occurring human anti-mouse antibody. This effect could be abrogated by the addition of monoclonal antibody against DAF or MCP. These data suggested that the production of animals transgenic for human complement regulatory proteins should in principle be similarly protected from hyperacute xenograft rejection.

Animals↗
PubMed1993

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 homologous 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/immunoblotting 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.

Antigens, CD↗
PubMed1995

Mouse complement regulatory protein Crry/p65 uses the specific mechanisms of both human decay-accelerating factor and membrane cofactor protein.

Normal host cells are protected from the destructive action of complement by cell surface complement regulatory proteins. In humans, decay-accelerating factor (DAF) and membrane cofactor protein (MCP) play such a biologic role by inhibiting C3 and C5 convertases. DAF and MCP accomplish this task by specific mechanisms designated decay-accelerating activity and factor I cofactor activity, respectively. In other species, including mice, structural and/or functional homologues of these proteins are not yet well characterized. Previous studies have shown that the mouse protein Crry/p65 has certain characteristics of self-protecting complement regulatory proteins. For example, Crry/p65 is expressed on a wide variety of murine cells, and when expressed on human K562 erythroleukemic cells, it prevents deposition of mouse C3 fragments on the cell surface during activation of either the classical or alternative complement pathway. We have now studied factor I cofactor and decay-accelerating activities of Crry/p65. Recombinant Crry/p65 demonstrates cofactor activity for factor I-mediated cleavage of both mouse C3b and C4b. Surprisingly, Crry/p65 also exhibits decay-accelerating activity for the classical pathway C3 convertase strongly and for the alternative pathway C3 convertase weakly. Therefore, mouse Crry/p65 uses the specific mechanisms of both human MCP and DAF. Although Crry/p65, like MCP and DAF, contains tandem short consensus repeats (SCR) characteristic of C3/C4 binding proteins, Crry/p65 is not considered to be a genetic homologue of either MCP or DAF. Thus, Crry/p65 is an example of evolutionary conservation of two specific activities in a single unique protein in one species that are dispersed to individual proteins in another. We propose that the repeating SCR motif in this family has allowed this unusual process of evolution to occur, perhaps driven by the use of MCP and DAF as receptors by human pathogens such as the measles virus.

Animals↗
PubMed2004

Do poor-prognosis breast tumours express membrane cofactor proteins (CD46)?

UNLABELLED: CD46 or membrane cofactor protein (MCP) is a complement regulatory protein that has been identified on all nucleated cells and which protects them from attack by autologous complement. Breast carcinomas are reported to consistently express CD46. AIM AND METHODS: Our previous immunohistochemical study showed that in breast carcinomas, loss of CD59 and CD55 correlated with poor survival. This study aimed to investigate the prognostic significance of CD46 on breast tumours using a rabbit polyclonal anti-CD46 antibody with a standard immunohistochemistry method. A total of 510 breast tissues from patients with primary operable breast cancer diagnosed between 1987 and 1992 had previously been included in tissue microarrays. They included patients 70 years of age or less (mean = 54 years) with a long-term follow-up (median = 82 months). RESULTS: Immunohistochemical study revealed that 507/510 (99.4%) of breast tumours expressed CD46. Strong immunoreactivity was exhibited by 136/510 (27%) tumours, while moderate and weak staining was observed in 43% and 29% of tumours, respectively. Intensity of CD46 expression was significantly associated with tumour grade (p < 0.05), histological type of tumour (p < 0.001) and tumour recurrence (p < 0.05). There was no correlation with lymph node stage or the presence of vascular invasion, nor with patient age or menopausal status. Interestingly, as most tumours expressed CD46, it would appear that poor-prognosis tumours that lose CD55 and CD59 still express CD46. CONCLUSION: Breast tumours express high levels of CD46 that correlates with tumour grade and recurrence. It is therefore likely that loss of CD55 and CD59 could be compensated by expression of CD46. However, loss of CD55 and CD59, even for tumours that still express CD46, is still associated with a poor prognosis. This may suggest that CD46 alone can protect from complement lysis but that loss of CD55 and CD59 are associated with other roles in immune regulation.

Adult↗
PubMed1988

Biochemical characterization of membrane cofactor protein of the complement system.

Membrane cofactor protein (MCP; formerly termed glycoprotein 45-70 to indicate its Mr) of complement is a widely distributed iC3/C3b binding protein with co-factor activity. On human mononuclear cells and cell lines and platelets, MCP is a doublet. The two forms differ in Mr by approximately 5 k and the upper species is predominant in most individuals. To further characterize these two forms, limited proteolytic digestions were performed. Of the four peptides produced, three have identical Mr indicating that the molecules are similar proteins. Both forms also have acidic isoelectric points and shift to a less acidic isoelectric point after treatment with neuraminidase. Glycosidase digestions indicate that both species contain N- and O-linked oligosaccharides but that the quantity of sialic acid is greater on the larger one. Pulse-chase experiments demonstrate approximately equal quantities of two precursor forms with Mr of 41 and 43 k. These two precursors possess N-linked high-mannose type of oligosaccharides and chase into the mature molecules which have complex sugars. The smaller precursor chases at a slower rate, possibly accounting for the reduced quantity of the smaller form of the mature form of MCP. These experiments indicate that the two forms of MCP are structurally similar and are derived from two distinct precursors. They also suggest that variations in the rate of processing of two intracellular precursors may account for the different quantities of the mature forms of this membrane protein.

Antigens, CD↗
PubMed1997

Membrane cofactor protein (CD46) is a basolateral protein that is not endocytosed. Importance of the tetrapeptide FTSL at the carboxyl terminus.

Membrane cofactor protein (MCP) is a widely distributed complement regulatory protein that is expressed on the basolateral surface of polarized epithelial cells. The basolateral targeting of the BC1 isoform of MCP was analyzed by generating deletion mutants and point mutants within the cytoplasmic tail of 16 amino acids. A sequence of four amino acids, FTSL, was found to be indispensable for the basolateral transport of MCP. This tetrapeptide has two unique features compared with the targeting motifs of other basolateral proteins: (i) it contains a phenylalanine rather than a tyrosine at position 1; (ii) it is located at the very COOH-terminal end. Replacement of the phenylalanine or the leucine by an alanine resulted in a nonpolarized delivery to the cell surface. On the other hand, substitution of a tyrosine for the phenylalanine did not affect the basolateral transport of MCP. The latter mutant, however, was efficiently internalized, whereas the wild type protein was not subject to endocytosis. Our results indicate that the targeting signal YXX-large aliphatic that is involved in various sorting events has been modulated in MCP in such a way that it allows basolateral transport but not endocytosis.

Amino Acid Sequence↗
PubMed2005

Transgenic pigs expressing human CD59, in combination with human membrane cofactor protein and human decay-accelerating factor.

BACKGROUND: The expression of human complement regulators has been proved as an effective strategy to overcome hyperacute rejection in discordant xenogeneic organ transplantation. In this study, we tested the hypotheses that expression of triple transgenes for human complement regulators and provide more effective protection to the transplanted pig tissues. METHODS: Pigs transgenic for human complement regulatory proteins, human CD59 (hCD59) and human membrane cofactor protein (hMCP), have been generated using large genomic constructs. Heterozygous human decay-accelerating factor (hDAF) transgenic pigs, from a previously established line, were bred with hCD59 or hCD59 plus hMCP pigs to produce animals that expressed both hCD59 and hDAF, or expressed triple transgenes hCD59, hDAF and hMCP. RESULTS: All three transgenes were widely expressed in most of the tissues analyzed, but the expression of hMCP was at low levels. In cytotoxicity assays on porcine peripheral blood mononuclear cells, the expression of a single transgenic protein, hCD59, or hCD59 in combination with hMCP provided similar protection against human complement-mediated damage as the single expression of hDAF. However, the expression of triple transgenic proteins or double hCD59 and hDAF transgenic proteins provided greater protection than either hCD59 or hDAF alone. CONCLUSIONS: Thus, pigs transgenic for multiple transgenes provide a greater degree of human complement regulation and hence might be more suitable for xenotransplantation.

Animals↗
PubMed1993

Expression of decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma cell line, D54-MG, and primary rat astrocytes.

In this report, we have shown the expression of the complement regulatory proteins decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 on human D54-MG astroglioma cells by several methods, including immunofluorescence, flow cytometry and Western blotting and Northern blot analysis. These studies demonstrate that all three proteins are structurally and antigenically similar to their counterparts expressed on HepG2 and SW480 cells (hepatocyte and epithelial cell lines, respectively). D54-MG cells express mRNA for all three proteins of the appropriate size(s). The phosphatidylinositol-specific enzyme, PIPLC, cleaved DAF from the surface of D54-MG cells, demonstrating that DAF is linked by a glycophospholipid anchor as has been shown for other cell types. Flow cytometry demonstrates that primary rat astrocytes also constitutively express all three regulatory proteins. These data are the first to demonstrate the expression of CD59 on astrocytes, and the presence of all three regulatory proteins on astrocytes suggests that regulation of complement activation in the central nervous system is important in neural host defense mechanisms.

Animals↗