[Show abstract][Hide abstract] ABSTRACT: Factor B is a zymogen that carries the catalytic site of the complement alternative pathway convertases. During C3 convertase assembly, factor B associates with C3b and is cleaved at a single site by factor D. The Ba fragment is released, leaving the active complex, C3bBb. During the course of this process, the protease domain becomes activated. The type A domain of factor B, also part of Bb, is similar in structure to the type A domain of the complement receptor and integrin, CR3. Previously, mutations in the factor B type A domain were described that impair C3b-binding. This report describes "gain of function" mutations obtained by substituting factor B type A domain amino acids with homologous ones derived from the type A domain of CR3. Replacement of the betaA-alpha1 Mg2+ binding loop residue D254 with smaller amino acids, especially glycine, increased hemolytic activity and C3bBb stability. The removal of the oligosaccharide at position 260, near the Mg2+ binding cleft, when combined with the D254G substitution, resulted in increased affinity for C3b and iC3b, a C3b derivative. These findings offer strong evidence for the direct involvement of the type A domain in C3b binding, and are suggestive that steric effects of the D254 sidechain and the N260-linked oligosaccharide may contribute to the regulation of ligand binding.
The Journal of Immunology 04/1999; 162(5):2906-11. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Factor B and C2 are serine proteases that carry the catalytic sites of the complement C3 and C5 convertases. Their protease domains are activated by conformational changes that occur during convertase assembly and are deactivated upon convertase dissociation. Factor B and C2 share an 8-amino acid conserved sequence near their serine protease termini that is not seen in other serine proteases. To determine its importance, 24 factor B mutants were generated, each with a single amino acid substitution in this region. Whereas most mutants were functionally neutral, all five different substitutions of aspartic acid 715 and one phenylalanine 716 substitution severely reduced hemolytic activity. Several aspartic acid 715 mutants permitted the steps of convertase assembly including C3b-dependent factor D-mediated cleavage and activation of the high affinity C3b-binding site, but the resulting complexes did not cleave C3. Given that factor B and C2 share the same biological substrates and that part of the trypsin-like substrate specificity region is not apparent in either protein, we propose that the conserved region plays a critical role in the conformational regulation of the catalytic site and could offer a highly specific target for the therapeutic inhibition of complement.
Journal of Biological Chemistry 11/1998; 273(40):25996-6000. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The membrane-associated proteins that regulate human complement activation are ubiquitously expressed and function cooperatively to protect cells from autologous complement damage. For classical and alternative pathways, the primary regulators at the stage of C3 proteolysis and deposition are membrane cofactor protein (MCP; CD46) and decay-accelerating factor (DAF;CD55), whereas protectin or CD59 regulates terminal component assembly. There is increasing awareness in reproductive, tumor, and transplantation immunology of the conventional and non-complement roles of these proteins. The human reproductive system may serve as a model of the non-complement functions.
We performed immunohistochemical analyses of multiple normal ovaries, fallopian tubes, cervices, and uterine corpi by using well-characterized monoclonal antibodies to provide a detailed, direct comparison of complement regulator expression.
Membrane cofactor protein was diffusely and strongly expressed on all epithelia and vascular endothelium and was the predominant regulator on oocytes. In contrast, decay-accelerating factor had variable expression in intensity and distribution on epithelia and was notably absent on certain epithelia and oocytes. It was the only regulator present on the connective tissue between muscle bundles in the myometrium and the cervix and was found on most stroma. CD59, although staining intensity varied, was present on virtually all epithelia, vascular tissue, and stroma.
Distinct reproducible patterns of complement regulator expression are found throughout the female reproductive tract. Differential expression on certain epithelia and oocytes may suggest non-complement activities. This comprehensive study should provide a basis for further characterization of pathological tissues and mechanisms of cellular localization.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have suggested that the residues 727-768 of human (Hu) C3 contain the binding sites for CR1, factor H, and factor B. Here, we have (1) characterized further some of the C3 structural requirements for its binding to CR1, H, and B, (2) investigated the functions associated with these C3-ligand interactions, and (3) studied the relationship of MCP-binding sites in C3 with those for CR1, H, and B. Hu C3 molecules in which residues 727-768 were deleted (designated C3delta727-768) or substituted with the corresponding segment of cobra venom factor, Xenopus, or trout C3 (chimeric C3s) were expressed in the baculovirus system and analyzed for their reactivity with C3-binding proteins. In contrast to wild-type iC3 which, in the presence of CR1, is cleaved by factor I to iC3b-a and C3c-a and C3dg, all chimeric C3s were cleaved only to iC3b-a. In addition, the cleavage of deleted (C3delta727-768) iC3 to iC3b-a by factor I in the presence of CR1 was significantly reduced, whereas it remained unaltered in the presence of MCP. Cleavage of iC3 to iC3b-a by factor I and H was similar in all expressed C3s except C3delta727-768, whose cleavage was significantly reduced. All of the expressed molecules except C3delta727-768 were capable of forming the fluid-phase alternative pathway C3 convertase, and all reacted with properdin. These results suggest that during cleavage of iC3 by factor I and CR1, or H, CR1 and H bind to at least two sites on C3 and that the MCP binding site(s) on C3b are different from those for CR1. They also indicate that some or all of the C3 residues that are directly involved in, or contribute to, the structure of one of the CR1 and H binding sites are located within residues 727-768. These studies also demonstrate that, although this segment of C3 may be involved in C3-factor B interaction, other residues in addition to 736EE (previously implicated in B binding) must also contribute significantly to this interaction.
The Journal of Immunology 07/1996; 156(12):4821-32. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human factor B is required for the initiation and propagation of the complement alternative pathway. It also participates in the amplification of the complement classical pathway. Alone, factor B is a zymogen with little known biochemical activity, but in the context of the alternative pathway convertases, the factor B serine protease is activated in a process that first involves the association with C3b and subsequently the cleavage of factor B into two fragments, Ba and Bb. Ba, the NH2-terminal fragment, is composed mainly of three tandem short consensus repeats, globular domains found in other complement proteins. It dissociates from the convertase during assembly, leaving the active C3 convertase, C3bBb. Previous reports suggest that the Ba region may be instrumental in convertase assembly. This hypothesis was tested using site-directed mutagenesis of recombinant factor B and monoclonal antibody epitope mapping to evaluate the relative importance of specific short consensus repeat amino acid residues. Three sites of interest were identified. Site 1 is a stretch of 19 contiguous amino acids in short consensus repeat 1 that form the epitope of a monoclonal antibody that effectively blocks factor B function. Site 2, composed of 6 contiguous amino acids in short consensus repeat 2, and site 3, consisting of 7 contiguous amino acids in short consensus repeat 3, were defined by mutations that reduce factor B hemolytic activity to 3% or less. Further analyses indicated that sites 2 and 3 contribute to factor B-C3b interactions.
Journal of Biological Chemistry 09/1995; 270(34):19716-22. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of endogenous complement is inhibited both in the soluble phase and at the membrane surface by a group of structurally similar proteins. A possible solution to hyperacute rejection is to produce donor animals transgenic for human complement regulators. 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. Hyperacute rejection of discordant organ xenografts is mediated by human complement. A 6.5 kilobase minigene for DAF has been microinjected into porcine fertilised ova. Forty-five pigs transgenic for human DAF have been produced. Of these, 65% transcribe message. The amount of message produced varied substantially from animal to animal and was independent of copy number integrated. Expression of human DAF on the porcine lymphocyte surface could be detected and this was able to downregulate human complement activation. Amounts of protein expressed on different tissues varied both from pig to pig and within animals from tissue to tissue. The pigs grow and develop normally with no evidence of ill effects due to possession of the transgene.
[Show abstract][Hide abstract] ABSTRACT: Human spermatozoa were analyzed for their expression of decay-accelerating factor (DAF, CD55), a glycolipid-anchored regulatory protein of the C casade. Morphologic data showed that DAF was localized at the acrosomal region of the sperm head. Analysis with anti-DAF antibody-immunoprecipitated proteins of acrosome-reached spermatozoa revealed a 44- to 54-kDa protein. Carbohydrate analysis of sperm DAF indicated that it contains nonsialated N- and O-linked sugars. The absence of mature oligosaccharides on this protein appears to account for the difference in molecular mass between sperm DAF and the 70-kDa DAF expressed on other human tissues. Sperm DAF reinserted into sheep E and inhibited C-mediated lysis. This effect was reversed by mAb, which block DAF function. These results indicate that sperm DAF also possesses a glycolipid anchor. The expression of DAF on acrosome-reacted spermatozoa suggests that it may act concomitantly with other C regulators such as membrane cofactor protein to modulate the activation of C in the immunocompetent female genital tract and protect acrosome-reacted spermatozoa from C-mediated attack.
The Journal of Immunology 08/1993; 151(2):939-48. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The behaviour of the complement system during human reproduction is now the focus of much scientific attention. The presence of antisperm antibodies in the reproductive tracts of some infertile individuals, and of complement in cervical and ovarian follicular fluid, suggests that complement-mediated damage of spermatozoa is involved in some cases of infertility. Further, deposition of maternal IgG and of complement in the extrafetal tissues indicates that complement activation occurs within the fetoplacental unit. Recently, three complement-regulatory proteins--decay-accelerating factor, membrane cofactor protein and CD59--have been detected on spermatozoa and in the extrafetal tissues. It is likely that these inhibitors are essential for normal reproductive function. This article reviews current understanding of the interaction of the complement system with cells and tissues involved in reproduction, with emphasis on the nature and function of the controlling proteins.
Immunologic Research 02/1993; 12(3):276-94. · 2.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Experimental Medicine 07/1992; 175(6):1547-51. · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Immunology 04/1992; 148(5):1431-7. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Transplant International 02/1992; 5 Suppl 1:S648-50. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MCP is a widely distributed regulatory glycoprotein of the complement system which binds C3b and C4b and has factor I-dependent co-factor activity. Monoclonal antibodies raised to lymphocytes (E4.3), chorionic microvilli (GB24) and an embryonal carcinoma cell line (TRA-2-10) recognize MCP (CD46). GB24 inhibited both the binding of MCP to its ligand iC3 and co-factor activity; E4.3 and TRA-2-10 did not. The binding of GB24 to cells bearing MCP was not cross-inhibited by E4.3 or TRA-2.10, but TRA-2-10 blocked binding and displaced pre-bound E4.3. Using these antibodies, we developed a radioassay for quantifying the number of MCP molecules/cells. Human peripheral blood mononuclear (PBMC) and polymorphonuclear cells (PMN) had about 10,000 MCP cell; platelets had about 600/cell, and no MCP was found on erythrocytes. Neoplastic hematopoietic cell lines, of myelocytic and T lymphocytic origin, had several-fold more (20-60,000) molecules cell than peripheral blood cells or B cell lines (about 12,000). Malignant epithelial cell lines. HeLa (about 100,000/cell) and HEp-2 (about 250,000 cell) had the highest MCP expression of any cells examined. These monoclonal antibodies--especially GB24, which blocks MCP function--and the direct binding assay will facilitate the further analysis of the biology of this complement regulatory protein.
[Show abstract][Hide abstract] ABSTRACT: The non-lineage restricted human CD46 antigen, with two glycoproteins of 56,000 molecular weight (MW) and 66,000 MW, was defined using a panel of monoclonal antibodies (mAb) that included the E4.3 mAb to the HuLy-m5 antigen. Here the E4.3 mAb is used to show that two other human cell-surface molecules, membrane co-factor protein (MCP) of the complement system and trophoblast leucocyte-common antigen (TLX), are the same as HuLy-m5; thus, these three independently identified molecules are equivalently CD46. A mouse mAb to TLX (H316) and a specific rabbit antiserum to purified MCP (RA-MCP) blocked the binding of FITC-labelled E4.3 to the surface of human peripheral blood leucocytes (PBL). In sequential immunoprecipitation studies, E4.3 cleared all molecules detected by H316 and the RA-MCP antiserum. Immunoprecipitation from Chinese hamster ovary cells expressing transfected MCP cDNA showed that E4.3 detects both the mature 66,000 higher MW form of MCP and its 48,000 MW pro-MCP precursor, which lacks O-linked carbohydrate and bears only simple high-mannose-type N-linked carbohydrate. The IgG fraction of a polyclonal antiserum to purified MCP blocked factor I-mediated cleavage of C3b, whereas the E4.3 mAb did not. These data establish that three independently identified antigen systems are indeed the same: HuLy-m5, which shares a cross-reactive epitope with some primate retroviral gp 70 molecules and can be physically associated with class I major histocompatibility complex (MHC) chains in the cell membrane; MCP, of interest as a member of the regulators of complement activation gene family thought to protect autologous cells from complement activation; and TLX, a polymorphic molecule of interest for its potential role at the foeto-maternal tissue interface during pregnancy. Thus, the human CD46 antigen amalgamates the HuLy-m5, MCP and TLX cell-membrane glycoproteins.
[Show abstract][Hide abstract] ABSTRACT: Analysis of C-dependent lysis of sensitized SRBC by C2-deficient sera (C2D) led to the characterization of a C2 bypass pathway. Lysis in the total hemolytic C assay by C2D sera was Ca2+-dependent and required a high concentration of hemolysin to sensitize E. Selective component depletion indicated a requirement for C1 and C4 of the classical pathway (CP) and proteins B, P, and probably D of the alternative pathway (AP). Total hemolytic C could be restored to normal in these C2D sera by utilizing heavily sensitized E or by the addition of a supranormal concentration of B. This system most closely resembles a pathway described by J. E. May and M. M. Frank which requires antibody, C1, and the AP but not C4 or C2. It differs in its requirement for C4. We hypothesize that this pathway represents vestiges of a more primitive C pathway. It becomes evident and possibly clinically important in the setting of C2 deficiency, by allowing C activation, other than the AP, and perhaps in normal individuals, by damaging microorganisms that have evolved means to inhibit early components of the CP.
The Journal of Immunology 11/1989; 143(7):2256-61. · 5.52 Impact Factor