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PubMed1982

Inhibition of alternative pathway factor D by factor B-related synthetic hexapeptides.

Hexapeptides mimicking the partial amino acid sequence of factor B surrounding the bond that is cleaved by factor D have been synthesized. These peptides have been assessed for their ability to inhibit factor D enzymatic activity and for their susceptibility to serine proteases. The synthetic peptides were cleaved by bovine trypsin and C1s but not by alpha-thrombin and factor D. The peptides inhibited factor B cleavage and fluid-phase or cell-bound alternative pathway C3 convertase activation by factor D. Altogether, these results suggest that peptides analogous to factor B specifically inhibit factor D enzymatic activity. Thus, they constitute an interesting tool for study of alternative pathway activation and can be of use when attempting to manipulate this pathway, since factor D is an essential component for alternative pathway initiation and amplification.

Animals↗
PubMed1997

Immunoglobulin and C3-activator profile of the microbiology laboratory worker.

The levels of immunoglobulin, C3-Activator and C3c in the sera of members of staff who have been continuously engaged for upwards of 10 years in the microbiological routine laboratory of a teachings hospital were determined. These were compared with the local normals, with those of the junior members of staff in the same laboratory but who had put in less than 5 years continuous service and with a control group. The means in all five determinations were much higher in the senior members of staff than the normal standards of the area, particularly the IgG, which was almost double the local normals. The levels for the junior staff were on the whole slightly higher than the control group. The increase in the levels of immunoglobulins with corresponding increases in C3-Activator and 3Cc suggest that immunological reactions, with the binding of complement in the alternate and classical pathways of activation are taking place in most of these senior workers. In the absence of any manifest illness in the senior staff, the findings may suggest a high degree of immunological protection.

Adult↗
PubMed1989

Secretion, cleavage and binding of complement component C3 by the human monocytic cell line U937.

Secretion of complement component C3 by U937 cells was studied. Preliminary evidence for a cell-associated proteolytic activity specific for C3 is given, as well as for a covalent-like binding of C3 fragments to the cell membranes. Secretion of C3, in the presence of 10 ng of phorbol 12-myristate 13-acetate/ml, is 120-140 ng/10(6) cells per 24 h on the third day after addition of the activator. As shown by SDS/polyacrylamide-gel electrophoresis, the intracellular pro-C3 (200 kDa) and the extracellular secreted C3 (alpha-chain 110 kDa and beta-chain 75 kDa) are identical with the forms of C3 previously characterized from human serum. Incubation of U937 cells in the presence of exogenous radiolabelled C3 shows that membrane-bound proteinase(s), not related to the classical-pathway or the alternative-pathway C3 convertases, is (are) able to cleave C3; this cleavage leads to the binding of the resulting C3 fragments to the cell membrane through reaction of membrane acceptors with the carbonyl group of C3 revealed after disruption of the intramolecular thioester bond. The proteolysis appears to be fairly specific to C3, as C4, which also possesses an intramolecular thioester bond, is not cleaved and does not bind to the cells. p-Nitrophenyl p'-guanidinobenzoate (1 mM) and di-isopropyl phosphorofluoridate (2 mM) are potent inhibitors of the proteolysis, whereas soya-bean trypsin inhibitor (1 mM), leupeptin (0.1 mg/ml) and 1,10-phenanthroline (1 mM) were ineffective. Immunological characterization of the cell-bound C3 fragments with monoclonal antibodies shows an evolution of the proteolysis of the fragments from iC3b to C3dg epitopes. Extraction of membrane-bound fragments by detergent, followed by SDS/polyacrylamide-gel electrophoresis, shows two fragments, of 43 kDa and 46 kDa, with C3dg-like characteristics.

Cell Line↗
PubMed1984

Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes.

Decay-accelerating factor (DAF), extracted from the stroma of human erythrocytes, was purified to homogeneity and incorporated into the membrane of sheep red cell complement intermediates, where its functional properties were analyzed. Incorporation of DAF into the cell membranes was temperature dependent, took place on pronase- or trypsin-treated erythrocytes, and did not depend on prior deposition of antibody, C1 or C4. Serum lipoproteins (high and low density) effectively inhibited DAF incorporation, but had no effect on the activity of DAF after its association with the cell membrane. The incorporated DAF could not be removed from the red cell surface by repeated washings in the presence of high salt concentration but was solubilized when the stroma were extracted with 0.1% Nonidet P-40. The presence of DAF in the membrane of EA did not affect the deposition of C1 and C4, but as few as 10(2) DAF molecules per cell profoundly inhibited the assembly of C3 and C5 convertases of both the classical and alternative pathways. The DAF inhibitory effect on EAC14 or EAC43 was not overcome by supplying an excess of C2 or factor B, but the alternative pathway C3 convertase could be assembled in the presence of Ni++, or nonphysiological concentrations of Mg++, which enhances the binding affinity of factor B for C3b. The DAF effect on EAC14 or EAC143 was entirely reversed by treating the cells with specific anti-DAF antibodies, showing that DAF did not alter the structure of C4b or C3b. Taken together, the experimental evidence suggests that DAF interacts directly with membrane-bound C3b or C4b and prevents subsequent uptake of C2 and factor B. DAF can function only within the cell membrane. Indeed, the decay dissociation of the C4b2a enzyme on DAF-containing sheep intermediates was not changed by varying the cell concentration. DAF-treated EA had no influence on the decay of nontreated EAC142 present in the same mixture. Moreover, the inhibitory activity of intact human erythrocytes on C4b2a was not blocked by antibodies to DAF, but was abolished by antibodies to the C3b/C4b receptor (CR1). When incorporated into the membrane of rabbit erythrocytes, human DAF inhibited their lysis by human complement. In conclusion, on the basis of these and previous results, it appears that DAF plays a central role in preventing the amplification of the complement cascade on host cell surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Effect of radiographic contrast media on complement components C3 and C4: generation of C3b-like C3 and C4b-like C4.

Various water soluble iodinated radiographic contrast media (RCM) have been studied for their effect on complement components C3 and C4, purified and in serum. Hepatotropic RCM, and at higher concentration also some nephrotropic RCM, were found to exert a direct effect on purified C3 and C4. RCM treated human C3 and C4 are characterized by (a) loss of haemolytic function, (b) retention of activity in the formation of fluid phase C3 convertases and (c) an antigenic relationship to activated C3 and C4 (C3b and C4b, respectively). This direct alteration of C3 and C4 can probably also occur in serum since loss of haemolytic function is observed at similar RCM concentrations after incubation of serum and of purified components. It is concluded that RCM treated C3 and C4 represent altered forms of these components that resemble C4b and C3b in activity and conformation (C3b-like C3 and C4b-like C4). The alteration is probably caused by binding of RCM, exerting a mild denaturing effect. C3b-like C3 is a potential activator of the alternative pathway, and both C3b-like C3 and C4b-like C4 are known to be cleaved by serum inactivators. A possible pathological significance of the generation of C3b-like C3, C4b-like C4 and their split products remains to be evaluated.

Animals↗
PubMed1998

Complementary recognition of alternative pathway activators by decay-accelerating factor and factor H.

The alternative complement pathway (ACP) functions as a surveillance mechanism by which microorganisms are opsonized with C3b in the absence of specific antibodies. The effectiveness of the ACP relies on its ability to distinguish self from non-self. This recognition function is mediated by C3 regulatory proteins including serum factor H, membrane cofactor protein (MCP), and membrane decay-accelerating factor (DAF). H activity against bound C3b can be increased by host components such as sialic acid and decreased by microbial polysaccharides. DAF and MCP may also recognize cell surface changes such as the presence of viral glycoproteins, since some virus-infected and tumor cells activate the ACP. In the present study, liposomes containing wild-type and mutant Salmonella minnesota lipopolysaccharide (LPS) were tested for ACP activation in serum. LPS-containing liposomes with bound C3b were then tested for their susceptibility to C3 convertase regulation by H and membrane DAF and for the sensitivity of their bound C3b to the cofactor activity of H. The results indicate that while the shortest mutant, Re595 LPS, did not induce ACP activation, R7 LPS containing an additional disaccharide did. This activation was poorly regulated by DAF but was inhibited by H. The regulatory activity of H for liposome-bound C3b, however, decreased when LPS of greater polysaccharide size was present in the membrane. In contrast the ACP activation induced by the phospholipid phosphatidylethanolamine was effectively inhibited by DAF but only poorly inhibited by H.

CD55 Antigens↗
PubMed1994

Expression of a hybrid complement regulatory protein, membrane cofactor protein decay accelerating factor on Chinese hamster ovary. Comparison of its regulatory effect with those of decay accelerating factor and membrane cofactor protein.

C activation on the cell surface is supposedly regulated by membrane cofactor protein (MCP) and decay accelerating factor (DAF). These are complementary in function: MCP acts as a cofactor in factor I-mediated C3b and C4b inactivation, thus preventing the assembly of C3 convertases, whereas DAF accelerates spontaneous decay of the assembled C3 convertase. In this report, a hybrid MCP-DAF was expressed on Chinese hamster ovary cells by transfecting cDNA, and its regulatory activity was compared with those of MCP and DAF transfectants and with a transfectant expressing both MCP and DAF (MCP + DAF). The C3 deposition on sensitized CHO cells through activation of the classical pathway was blocked to a different degree with these transfectants, the order being MCP + DAF > DAF > hybrid MCP-DAF > MCP. Likewise, the C3 deposition via the alternative pathway was blocked efficiently in the order hybrid > MCP + DAF > MCP. The C-mediated cytolysis of CHO cells virtually reflected the degree of C3 fragment deposition. The MCP-DAF transfectant acquired additive protective activity against alternative pathway-mediated C3 deposition and cytolysis but was less potent in circumventing classical pathway attack than cells that expressed DAF alone or DAF + MCP. Hybrid MCP-DAF may be useful for alleviating C-mediated cell damage, especially via the alternative pathway.

Animals↗
PubMed2004

Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli.

Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 A, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.

Amino Acid Sequence↗
PubMed1999

Regulation of complement activation by C-reactive protein.

C-reactive protein (CRP) is an acute-phase serum protein and a mediator of innate immunity. CRP binds to microbial polysaccharides and to ligands exposed on damaged cells. Binding of CRP to these substrates activates the classical complement pathway leading to their uptake by phagocytic cells. Complement activation by CRP is restricted to C1, C4, C2 and C3 with little consumption of C5-9. Surface bound CRP reduces deposition of and generation of C5b-9 by the alternative pathway and deposition of C3b and lysis by the lectin pathway. These activities of CRP are the result of recruitment of factor H resulting in regulation of C3b on bacteria or erythrocytes. Evidence is presented for direct binding of H to CRP. H binding to CRP or C3b immobilized on microtiter wells was demonstrated by ELISA. Attachment of CRP to a surface was required for H binding. H binding to CRP was not inhibited by EDTA or phosphocholine, which inhibit ligand binding, but was inhibited by a 13 amino acid CRP peptide. The peptide sequence was identical to the region of CRP that showed the best alignment to H binding peptides from Streptococcus pyogenes (M6) and Neisseria gonorrhoeae (Por1A). The results suggest that CRP bound to a surface provides secondary binding sites for H resulting in greater regulation of alternative pathway amplification and C5 convertases. Complement activation by CRP may help limit the inflammatory response by providing opsonization with minimal generation of C5a and C5b-9.

Amino Acid Sequence↗
PubMed1980

Numerical changes in T cell subsets (T gamma and T mu) in leprosy patients.

Eighty-six leprosy patients (49 active lepromatous, 24 inactive lepromatous 7 borderline, and 6 tuberculoid) and nine healthy controls were examined for numerical changes in T cell subsets (T gamma and T mu), and complement levels in peripheral blood to determine the roles of T cell subsets and complement in the etiology of leprosy. The percentage and number of T gamma and T mu showed no significant differences among the different clinical groups, but 4 out of 49 active lepromatous, 3 out of 24 inactive lepromatous and 3 out of 7 borderline cases showed a high prcentage of T gamma cells. Serum concentrations of C4, C3c, and C3 activator, an important factor in the alternative pathway of complement activation, were not significantly different among the groups. However, C3 activator and C3c concentrations were significantly high in active lepromatous patients complicated by an immune complex disease called "erythema nodosum leprosum" (ENL) compared with ENL-free active lepromatous leprosy.

Adult↗
PubMed1990

Proteolytic elimination of decay-accelerating factor (DAF): lytic abnormality coincides with removal of DAF in papain-treated human erythrocytes.

Erythrocytes (E) from patients with paroxysmal nocturnal hemoglobinuria (PNH) lack decay-accelerating factor (DAF) and this partly causes increasing susceptibility of the E to complement. Several reagents have been used to convert normal E to the complement-sensitive (PNH-like) cells. The relationship between DAF amounts and complement susceptibility of these PNH-like cels has been examined. Of the reported reagents for preparation of PNH-like cells, 2-amino-ethylisothiouronium bromide (AET), papain, and periodate efficiently converted normal E to the complement-sensitive cells, but only papain reduced the quantity of DAF on the cells. Further, of the proteases we tested only papain cleaved DAF to liberate its major fragment from the cells. The papain-treated cells lysed in a similar fashion to PNH cells as the serum concentration increased. The major papain-digested product of DAF had Mr, 55,000, lacked hydrophobicity, and retained the ability to inhibit the C3 convertases. These findings suggest that papain allows liberation from cells of functional domains as well as most of the antigenic epitopes of DAF to generate a PNH-like cell.

Blood Proteins↗
PubMed1980

The Croonian Lecture, 1980. The complex proteases of the complement system.

The assembly and activation of the early components of complement, after their interaction with antibody-antigen complexes, are described in terms of the structures of the different proteins taking part. C1q, a molecule of unique half collagen--half globular structure, binds to the second constant domain of the antibody molecules through its six globular heads. A tetrameric complex of C1r2-C1s2 binds to the collagenous tails and leads to formation of the serine-type proteases C1r and C1s. C1s activates C4, which forms a covalent bond between its alpha' chain and the Fab section of the antibody. C2 is also activated by C1s and associates with the bound C4 molecule to form C42, a labile protease that activates C3, but which loses activity as the C2 peptide chains dissociate from C4. C2, by analogy with factor B, the equivalent component of the alternative pathway of activation, appears to be a novel type of serine protease with a similar catalytic site but different activation mechanism to the serine proteases that have been described previously.

Amino Acid Sequence↗
PubMed2002

The effect of pH and nucleophiles on complement activation by human proximal tubular epithelial cells.

BACKGROUND: Activation of urinary complement proteins in situ by proximal tubular epithelial cells (PTEC) may contribute to the mediation of tubulointerstitial injury in patients with significant proteinuria. However, the mechanism involved is unclear, and the role of changes in urinary pH and in the concentrations of urea or ammonia requires further clarification. METHODS: The protein fraction of urine samples from nine patients with proteinuria >1.5 g/day was purified. A cell ELISA involving cultured HK-2 PTEC was used to investigate the capacity of urinary protein to promote the deposition of both C3 and C9 on the cell surface. The effect of variations in pH (5.5-8.0) and in the concentration of urea and ammonia was also examined. C3 was purified and used to further investigate the mechanism of complement deposition. RESULTS: Urine samples from the majority of patients induced deposition of C3 and C9 on the surface of HK-2 cells via the alternative pathway. This process was maximal at acidic pH values. Preincubation of urinary complement or serum with urea or ammonia inhibited C3 deposition. Purified C3 incubated with HK-2 cells showed no evidence of activation in the absence of other complement components. CONCLUSIONS: These data suggest that bicarbonate protects against complement-mediated damage in the lumen by increasing the local pH, rather than by inhibiting the generation of ammonia. PTEC appear to activate complement through provision of a 'protected site' on their surface, rather than by the activation of C3 by convertase-like protease(s).

Ammonia↗
PubMed1996

Inhibition of human complement by a C3-binding peptide isolated from a phage-displayed random peptide library.

We have screened a phage-displayed random peptide library for binding to C3b, the proteolytically activated form of complement component C3, and have identified a novel peptide that suppresses complement activation. This phage-displayed peptide bound to C3, C3b, and C3c, but not to C3d, indicating that it binds to the C3c region of C3. A synthetic 27-amino acid peptide corresponding to the phage-displayed peptide also bound to C3 and C3 fragments and inhibited both the classical and alternative pathways of complement activation. The inhibition of complement activation was reversible. Studies with overlapping peptides indicated that the functional activity was located in the cyclic 13-amino acid N-terminal region (ICVVQDWGHHRCT) of the parent peptide. Reduction and alkylation of this 13-residue synthetic peptide destroyed its inhibitory activity. Analysis of the mechanism of inhibition revealed that the peptide inhibited C3 cleavage in normal human serum as well as when the alternative pathway was reconstituted with purified complement components, and the observed inhibition was not due to sterically hindered access to the C3a/C3b cleavage site. Further, the peptide did not inhibit the cleavage of factor B, indicating that it did not affect the interaction of CA with factor B or the formation of C3b,Bb. The peptide also had no effect on the binding of properdin to C3, demonstrating that the observed inhibition of C3 cleavage in normal human serum was not due in part to its effect on the properdin-stabilized C3 convertase, C3b,Bb,P. These results indicate that the peptide we have identified interacts with C3 to inhibit its activation.

Amino Acid Sequence↗
PubMed1984

The influence of classical pathway components during alternative pathway--modulated immune complex aggregation: the role of C1 INH.

Alternative pathway (AP)-triggered reactions as well as classical pathway (CP)-mediated ones, were investigated turbidimetrically and/or immune electrophoretically, either in the presence or in the absence of in situ-generated immune complexes (ICs; tetanus toxoid/human anti-tetanus toxoid-IgG; ICs of equivalence) during the early stages of reaction. Monospecific Fab'- or Fab-fragments (rabbit) were used to block the complement function in normal human serum (NHS). C1q, functionally available following the addition of ethylene-glycol-bis-(beta-aminoethyl ether), N,N'-tetraacetic acid to NHS (EGTA-NHS), was found to increase the IC aggregation, thereby producing a biological surface upon which AP-dependent proteins were deposited. The functional inhibition of C1INH caused a C1s-mediated C3 conversion irrespective of the fact whether C1s was incorporated within macromolecular C1 (NHS) or dissociated from it (EGTA-NHS), thus, in the latter case inhibiting the AP-dependent portion of turbidity. It seemed probable that C3 conversion was effected by a fluid-phase CP C3 convertase. This process, normally counteracted by C1INH, worked more efficiently in EGTA-NHS than in NHS, indicating that the C1s-mediated reactions, initiated by presently unknown mechanisms, were less extensively regulated outside of the Ca2+-dependent C1 complex. The study demonstrates that in EGTA-NHS, too, where AP-triggered reactions have usually been investigated, sections of CP activation may play an important role, especially in situations where the function of C1INH is restricted.

Antigen-Antibody Complex↗
PubMed1997

A novel mechanism of alternative pathway complement activation accounts for the deposition of C3 fragments on CR2-expressing homologous cells.

Complement receptor type 2 (CD21, CR2), the receptor for the C3 fragment C3dg, activates complement via the alternative pathway and also serves as a preferential acceptor site for C3 fragments. The molecular basis for this phenomenon, which has recently been demonstrated for B lymphocytes in vivo, is currently not understood. Here we present a model for this CR2-dependent complement activation. The inactive C3 (iC3), which forms spontaneously in serum in low amounts by reaction of native C3 with H2O, binds noncovalently to the N-terminal part of CR2. Subsequent association of properdin and factor B, and cleavage of factor B by factor D lead to formation of a C3 convertase associated with CR2, thus focussing covalent C3 deposition to CR2 itself. This model is supported by the following experimental findings. 1) By FACS analysis and radioreceptor assays we showed that iC3, properdin, and factor B bound to CR2 on Raji B cells, MT2 T cells, and peripheral blood B cells. 2) Both binding of these proteins and complement activation by CR2-expressing cells were reduced in parallel by Abs against CR2. 3) 125I-labeled C3b was covalently deposited on CR2, when hemolytically active 125I-labeled C3 was added to Raji cells preincubated with iC3, factor B, properdin, and factor D, thus proving functionality of CR2-bound C3 convertase. This model of C3 convertase activity formed on CR2 domains inaccessible for decay-accelerating factor offers an explanation for the deposition of C3 found on CR2-expressing cells.

B-Lymphocytes↗
PubMed1994

Hypocomplementaemia of poststreptococcal acute glomerulonephritis is associated with C3 nephritic factor (C3NeF) IgG autoantibody activity.

We have observed that decreased plasma levels of C3 in the serum of three children with poststreptococcal acute glomerulonephritis (PSAGN) at the time of presentation were associated with the presence of C3NeF activity in purified serum IgG from the patients. C3NeF activity was determined using a sensitive assay measuring the ability of patients' IgG to stabilize a preformed cell-bound alternative pathway convertase complex. C3NeF activity of patients' IgG decreased within weeks as plasma levels of C3 progressively returned to normal values. C3NeF activity became undetectable within 1-4 months following normalization of plasma C3 levels. Our observations suggest that early alternative pathway-dependent hypocomplementaemia, a cardinal feature of PSAGN, is mediated by the transient expression of C3NeF autoantibody activity by patients' IgG.

Acute Disease↗
PubMed1996

Dissection of CR1, factor H, membrane cofactor protein, and factor B binding and functional sites in the third complement component.

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.

Amino Acid Sequence↗