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PubMed1992

Membrane cofactor protein.

MCP serves to down-regulate the activation of complement on host tissue. It performs this function by serving as a cofactor for the factor I-mediated cleavage of C3b and C4b. MCP is most likely an intrinsic regulator, i.e., it primarily protects its home cell. The wide tissue distribution of MCP mirrors this critical function of host cell protection. With the exception of erythrocytes, every cell and tissue examined expresses this protein. MCP is represented as two broad heterogeneous bands on SDS-PAGE with M(r)s of 51,000-58,000 and 59,000-68,000. The quantity of each form expressed is inherited in an autosomal codominant fashion. In most cells and cell lines, four isoforms of MCP predominate and arise by alternative splicing of a single MCP gene. All forms possess four repeating modules of--60 aminoacids, an area enriched in serines, threonines, and prolines [(STP), probable site of O-linked glycosylation], a short area of unknown function, a transmembrane domain, and a cytoplasmic tail. The isoforms differ, however, in the length and composition of the STP region and in the cytoplasmic tail. Alternative splicing of a single exon within the STP region determines the protein phenotype. Alternative splicing at the COOH_terminus gives rise to two distinct cytoplasmic tails. The biological significance of these structural variations in the STP and cytoplasmic tail regions is being investigated.

Animals↗
PubMed1992

Effects of complement activation on platelet-activating factor and eicosanoid synthesis in rat mesangial cells.

The effects of in vitro complement activation and of isolated complement components (C3a, C5a, C3b) on the activity of the microsomal enzyme acetyl coenzyme A: 1-0-alkyl-glycero-3-phosphocholine acetyl transferase (AcTr) in cultured mesangial cells and on the synthesis of prostaglandin E2 (PGE2) were assessed. In vitro complement activation induced by the introduction of purified cobra venom factor (CVF) in culture media containing human serum enhanced mesangial cell PGE2 synthesis and had no effect on microsomal AcTr activity. When media containing C6-deficient serum were used, the stimulatory effect of CVF on PGE2 synthesis was abolished. Introduction of CVF in these media enhanced mesangial cell AcTr activity. These effects were partially reversed when the C6-deficient serum was supplemented with C7-deficient serum to allow formation of the C5b-9 complex. Isolated C3a, C5a, and C3b had opposite effects on PGE2 synthesis and on AcTr activity. Specifically, all components enhanced mesangial cell PGE2 synthesis. In contrast, AcTr activity was enhanced by C3a alone; C5a had no effect, whereas C3b had an inhibitory effect. The observations indicate that in response to complement activation and specific anaphylatoxin agonists, mesangial cell eicosanoid synthesis is not coupled with changes in the activity of AcTr and PAF synthesis.

Acetyltransferases↗
PubMed2002

Complement C3b/C3d and cell surface polyanions are recognized by overlapping binding sites on the most carboxyl-terminal domain of complement factor H.

Factor H (FH) is a potent suppressor of the alternative pathway of C in plasma and when bound to sialic acid- or glycosaminoglycan-rich surfaces. Of the three interaction sites on FH for C3b, one interacts with the C3d part of C3b. In this study, we generated recombinant constructs of FH and FH-related proteins (FHR) to define the sites required for binding to C3d. In FH, the C3d-binding site was localized by surface plasmon resonance analysis to the most C-terminal short consensus repeat domain (SCR) 20. To identify amino acids of FH involved in binding to C3d and heparin, we compared the sequences of FH and FHRs and constructed a homology-based molecular model of SCR19-20 of FH. Subsequently, we created an SCR15-20 mutant with substitutions in five amino acids that were predicted to be involved in the binding interactions. These mutations reduced binding of the SCR15-20 construct to both C3b/C3d and heparin. Binding of the wild-type SCR15-20, but not the residual binding of the mutated SCR15-20, to C3d was inhibited by heparin. This indicates that the heparin- and C3d-binding sites are overlapping. Our results suggest that a region in the most C-terminal domain of FH is involved in target recognition by binding to C3b and surface polyanions. Mutations in this region, as recently reported in patients with familial hemolytic uremic syndrome, may lead to indiscriminatory C attack against self cells.

Amino Acid Sequence↗
PubMed1994

The susceptibility of S-layer-positive and S-layer-negative Aeromonas strains to complement-mediated lysis.

Forty strains of Aeromonas hydrophila and Aeromonas veronii recovered from invasive and non-invasive infections were tested for their susceptibility to complement-mediated lysis by 65% pooled human serum (PHS). Based upon the results of this assay, two major populations could be defined. The first group (n = 20) consisted of serogroup O:11 strains, all of which possessed a paracrystalline surface layer (S layer); all of these strains were refractory to the bactericidal activity of 65% PHS with the exception of A. hydrophila strain AH-121, which was composed of mixed subpopulations of serum-susceptible and serum-resistant clones. A second collection of isolates (n = 20), all of which were S-layer-negative, contained a subgroup of strains (n = 7) that were highly susceptible to complement-mediated lysis, showing a greater than 100-fold reduction of viable progeny within 30 min of exposure to 65% PHS. Serum-resistant strains from both groups could not be lysed by exposure of bacterial cells to polyclonal somatic or whole cell antisera or to 30 micrograms ml-1 of polymyxin B nonapeptide prior to challenge with 65% PHS. Analysis of selected serum-resistant and serum-susceptible strains from both groups showed that all isolates activated the complement pathway and most bound C3b to the cell surface, indicating that the inability of complement to lyse serum-resistant strains was related to a defect in the terminal portions of the complement pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Aeromonas↗
PubMed1979

The septic burned patient: a model for studying the role of complement and immunoglobulins in opsonization of opportunist micro-organisms.

Studies were performed to determine the effects of septicemia on complement levels and activities and opsonic function in septic and nonseptic burned patients. None of the nonseptic burned patients had consumption of classical pathway activity during their clinical course. Patients who did not survive septicemia had consumption of all of the classical complement components (C1-C5) prior to and during their septic episodes. Patients who survived septicemia had multiple patterns of classical complement pathway consumption. In these patients, classical pathway activity was restored to normal following the last positive blood culture. Alternative complement pathway consumption was demonstrated in only one of the septic burned patients, as evidenced by decreased factor B and C3b INA levels and decreased C3 and C5 conversion in sera treated with 10 mM ethylene glycol tetraacetic acid and 10 mM MgCl(2) (MgEGTA) and in untreated sera. In all of the other septic patients and in the nonseptic patients, reduction in C3 and C5 conversion in MgEGTA sera and untreated sera was not associated with decrease in factor B or C3b INA. Reduction in complement levels and activities did not reduce the ability of the patients' sera to promote phagocytosis and intracellular killing of their infecting micro-organisms by normal human peripheral polymorphonuclear leukocytes. The results indicate that measurement of classical pathway activity in burned patients can be used as a diagnostic tool for predicting the severity of septic episodes and for monitoring recovery. In addition, the observation that complement consumption did not reduce the opsonic capacity of the patients' sera for their infecting micro-organisms suggests that current concepts regarding the role of immunoglobulins and complement in opsonization of opportunist micro-organisms require re-evaluation.

Adolescent↗
PubMed1989

Elevated NK-mediated lysis of Raji and Daudi cells carrying fixed iC3b fragments.

Raji and Daudi cells were opsonized with C3b, iC3b, and C3d fragments by using purified complement components. The sensitivity of C3-opsonized cells to lysis mediated by low density blood lymphocytes was studied. Raji and Daudi cells carrying C3b or C3d fragments were lysed with similar efficiencies as the nonopsonized cells. The presence of iC3b on the target surface imposed elevated NK sensitivity. The iC3b-mediated enhancement of NK lysis was inhibited when iC3b fragments or rabbit anti-human C3 antibodies were included into the lytic assays. These results indicate that the iC3b fragments fixed on the targets bind to the CR3 on the lymphocytes. Results obtained in immobilized conjugate-lytic assays showed that iC3b-opsonized targets interact more readily with the lymphocytes. This was reflected by the elevated proportion of lymphocytes that were bound to the iC3b-carrying targets. The proportions of conjugates in which target damage occurred were similar with the control and with the iC3b-carrying cells. It seems therefore that opsonization of targets with iC3b leads to recruitment of effector lymphocytes due to contact with their CR3. However, once the effector-target contact is established, the triggering of lytic function does not seem to be influenced by the iC3b/CR3 bridge.

Burkitt Lymphoma↗
PubMed1993

Extracorporeal circulation: in vivo and in vitro analysis of complement activation by heparin-bonded surfaces.

Complement activation was analyzed during extracorporeal CO2 removal to compare heparin-coated with standard surfaces where systemic heparinization was required. In vivo studies were performed in adult sheep for up to 5 days under standardized conditions using a capillary membrane oxygenator. Applying assays for hemolytic complement function (CH50, APH50) and C3-derived split products, we found that complement activation was markedly reduced in sheep connected to an extracorporeal circuit where heparin was covalently bound by end-point attachment. In addition, incubation of human serum in a miniaturized circulation system revealed less complement activation by heparin-bonded surfaces, as evaluated by enzyme-linked immunosorbent assays for C3a and the activation-specific protein-protein complexes, C1rsC1 inhibitor (classical pathway) and C3b(Bb)P (alternative pathway). Our results provide further evidence that biocompatibility can be improved by end-point attachment of heparin to the surfaces of the extracorporeal circuit.

Animals↗
PubMed1977

Studies on C3 convertases. Inhibition of C5 convertase formation by peptides containing aromatic amino acids.

The influence of various peptides containing the aromatic amino acids phenylalanine and tyrosine on the formation of the enzyme EAC1423 of the complement system from component C3 and enzyme EAC142 was investigated. Kinetic analysis of enzyme EAC1423 formation and studies on the binding of the C3b fragment of 125I-labelled component C3 to enzyme EAC142 both showed that binding of the C3b fragment of component C3 was decreased by the peptides. Kinetic studies on component-C3 turnover in the fluid phase of enzyme EAC142 failed to reveal effects of the peptides. However, an initial lag in component-C3 turnover occurred that at constant component-C3 concentration was inversely proportional to enzyme EAC142 concentration. This lag in enzyme EAC142 activity is considered as an indication that the interaction of enzyme EAC142 with component C3 possibly does not follow simple Michaelis-Menten kinetics, as was previously assumed. It is shown that the stages after enzyme EAC1423 formation are not influenced by the peptides, suggesting a high degree of specificity of the peptides for the inhibition of enzyme EAC1423 formation.

Complement Activating Enzymes↗
PubMed1998

The requirement of localized, CR2-mediated, alternative pathway activation of complement for covalent deposition of C3 fragments on normal B cells.

We have shown previously that normal B cells share, with Epstein-Barr virus-transformed and malignant B cells, the ability to activate the alternative pathway (AP) of complement in vitro, resulting in the deposition of C3 fragments on the cell surface. Complement receptor type 2 (CR2, CD21) has been implicated directly as the site for formation of an AP convertase, which provides nascent C3b for deposition at secondary sites on the B-cell surface. In the present study, we have examined C3 fragment deposition in vitro in more detail by (1) assessing the importance of locally generated C3b for the deposition process, (2) investigating whether CR2 is the sole requirement for conferring AP activation capacity on a cell, and (3) determining whether CR2's function, as an AP activator, has different structural requirements from ligand binding. Increasing the availability of native C3, by increasing the serum (NHS) concentration, resulted in enhanced C3 fragment deposition on the B cells, whereas use of factor 1-depleted NHS, which showed massive fluid phase C3 conversion during the incubation, diminished the deposition. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting of untreated and hydroxylamine-treated lysates from B cells, after in vitro activation, revealed that the majority of C3 fragments (primarily iC3b and C3dg) had been covalently bound to the cell surface. Transfection of COS cells with wild-type CR2 or a deletion mutant lacking 11 of the molecule's 15 homologous domains, but retaining the ligand-binding site, revealed that expression of intact CR2 conferred a 12-fold increase in AP-activating capacity on these cells, while no increase in AP activity was apparent on cells transfected with the mutant CR2.

B-Lymphocytes↗
PubMed2001

Formation of high-affinity C5 convertases of the alternative pathway of complement.

Cleavage of C5 by C5 convertase is the last enzymatic step in the complement activation cascade leading to the formation of the cytolytic proteolytically activated form of C5 (C5b)-9 complex. In the present study, we examined the effect of the density of C3b (the proteolytically activated form of C3) on the function of the noncatalytic subunit of natural surface-bound forms of the enzyme. A comparison of the kinetic parameters of C5 convertases assembled on three surfaces (zymosan, rabbit erythrocytes, and sheep erythrocytes) were similar and revealed that the average K:(m) decreased approximately 28-fold (5.2-0.18 microM) when the density of C3b was increased from approximately 18,000 to 400,000 C3b/cell. Very-high-affinity C5 convertases were generated when preformed C3 convertases were allowed to self amplify by giving them excess C3. These convertases exhibited K(m) from 0.016 to 0.074 microM, well below the normal plasma concentration of C5 in blood (0.37 microM). The results suggest that in serum convertases formed with monomeric C3b will be relatively inefficient in capturing C5 but will continue to cleave C3 opsonizing the cell surface for phagocytosis, whereas convertases formed with C3b-C3b complexes in areas of high C3b density will primarily cleave C5. The catalytic rate of these convertases approaches maximum velocity, thereby switching the enzyme from cleavage of C3 to cleavage of C5, and production of the cytolytic C5b-9 complex.

Animals↗
PubMed1989

Activation of C3 and binding to Aspergillus fumigatus conidia and hyphae.

Complement activation by Aspergillus fumigatus may play a crucial role in stimulating binding and killing of this organism by phagocytes. We examined the amount and type of C3 deposited on resting conidia, swollen conidia, and hyphae of A. fumigatus after incubation in pooled human serum. All three life forms of A. fumigatus were potent activators of the complement cascade, with deposition on the organisms of similar amounts of C3 per unit of surface area. The rate of deposition was slowest for resting conidia, although maximal deposition was still achieved within 40 min. The roles of the alternative and classical pathways were assessed by use of serum chelated with magnesium EGTA [magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and with an alternative pathway reconstituted from the six purified alternative-pathway proteins. Complement activation by resting conidia was mediated by the alternative pathway. In contrast, there was a progressive dependence on the classical pathway as the fungal particles matured into swollen conidia and then hyphae. Treatment with hydroxylamine, which disrupts ester linkages, removed 89 to 95% of the C3 bound to all three forms of A. fumigatus. This released C3 contained a mixture of C3b and iC3b, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. These data demonstrate that although all three forms of A. fumigatus are potent activators of the complement system, the transition from resting conidia to swollen conidia to hyphae results in progressive changes in the manner in which the fungal particles interact with the complement system. The lack of participation of the classical pathway in complement activation by resting conidia may have important implications regarding their ability to effectively stimulate phagocytes.

Aspergillus fumigatus↗
PubMed1983

Inhibition of phagocytosis of complement C3- or immunoglobulin G-coated particles and of C3bi binding by monoclonal antibodies to a monocyte-granulocyte membrane glycoprotein (Mol).

Events that lead to phagocytosis of complement (C3)- or IgG-coated particles after their interaction with specific cell surface receptors are poorly understood. Two mouse monoclonal antibodies (an IgM and an IgG2a) to a human granulocyte-monocyte surface membrane differentiation antigen (Mol) inhibited ingestion by granulocytes both of oil Red O particles opsonized with normal human serum or with IgG and of sheep erythrocytes sensitized with IgG. In addition, they specifically inhibited rosetting between phagocytes and sheep erythrocytes coated with C3bi, a fragment of the complement component C3, generated by cleaving C3b with C3b inactivator and beta IH protein. These monoclonal anti-Mol antibodies did not inhibit IgG Fc, C3b or C3d receptor-mediated binding of erythrocytes coated with the respective proteins. The Fab fragment of the IgG2a monoclonal antibody inhibited noncytotoxic enzyme release from granulocytes when these cells were stimulated with zymosan coated with C3bi. Electrophoretic transfer of polymorphonuclear leukocyte detergent lysates to nitrocellulose, followed by immunofixation with monoclonal antibody, showed that these antibodies were directed to a 155,000-mol wt glycoprotein. This surface membrane structure appears to be involved in Fc and C3 receptor-dependent phagocytosis and closely associated with the C3bi receptor.

Antibodies, Monoclonal↗
PubMed1998

The complement cofactor protein (SBP1) from the barred sand bass (Paralabrax nebulifer) mediates overlapping regulatory activities of both human C4b binding protein and factor H.

We have previously shown that serum of the teleost fish barred sand bass (Paralabrax nebulifer) cleaves the alpha'-chain of human C4b and C3b. The proteins that participate in these reactions were purified, and a specific protease and a single cofactor protein were identified. Functional characterization of the recombinantly expressed sand bass cofactor protein (SBP1) and truncated forms containing short consensus repeats (SCRs) 1-2, 1-3, 1-4, 1-5, and 12-17 revealed that SBP1 and SCRs 1-4 mediate the functional activities of the human plasma regulatory protein C4bp and factor H. They form a complex with C4b, inhibit the formation, and accelerate the decay of the classical pathway C3 convertase and display cofactor activity for the cleavage of C4b. In contrast, the interaction of SBP1 and SCRs 1-4 with human C3b in all these activities was limited. This difference is due to species-specific incompatibilities between the cofactor protein and human C3b. SBP1 and SCRs 1-5 displayed full binding and cofactor activity for methylamine-treated C3 from trout, a species closely related to the sand bass. The presence of only one cofactor in the fish plasma that combines the functional activities of C4bp and factor H demonstrates that the sand bass cofactor protein is the ancestral precursor to the two complement regulatory proteins in human plasma.

Animals↗
PubMed1999

Complement processing and immunoglobulin binding to Neisseria gonorrhoeae determined in vitro simulates in vivo effects.

Local inflammation elicited by Neisseria gonorrhoeae correlates closely with sensitivity to killing by normal human serum. Serum-sensitive (SS) isolates are rendered resistant in vitro by lipooligosaccharide sialylation. Differences in C3b processing on N. gonorrhoeae in vitro were found to match findings at the cervical level in vivo. Nonsialylated SS gonococci bound 5-fold more C3b than did stably serum-resistant (SR) gonococci; most was processed to iC3b, yet significant C3b persisted. Sialylated SS gonococci bound 4-fold less total C3 antigen than did SR gonococci, which was promptly converted to iC3b. C3b bound later on stably SR gonococci but again was processed swiftly to iC3b. In vivo, the iC3b/C3 ratio of SS isolates more closely resembled nonsialylated SS isolates in vitro, implying heterogeneous sialylation or desialylation in vivo. In vitro, total IgM bound was unchanged by sialylation of SS isolates, but total C4 bound decreased by 75%, suggesting that sialylation may indirectly regulate the classical complement pathway.

Animals↗
PubMed1993

Studies on the mechanism of complement-mediated inhibition of antibody binding to HIV gp41.

We have previously demonstrated that HIV envelope gp41 binding to specific antibodies decreases after preincubation of fluid-phase gp41 in normal human serum. This inhibition is proven to be mediated by the classical complement pathway. In this study recombinant gp41 (rgp41) and/or synthetic peptides were preadsorbed to solid phase, and then complement (normal human serum/heated human serum/purified Clq/heated Clq) and anti-gp41 antibodies were added either after each other or simultaneously, and the amounts of bound antibody, and deposited C3b, C4b and Clq were measured. Complement-dependent inhibition of antibody binding to solid-phase rgp41 was found, and Clq seems to be at least partially responsible for this phenomenon. Heating of Clq did not affect this process. Higher amounts of anti-gp41 antibodies significantly and dose-dependently enhanced C4b and C3b fixation to solid-phase rgp41. In the case of synthetic peptides corresponding to the immunodominant region of gp41, significant antibody binding to the solid-phase peptides was also detected, and pretreatment of peptides preadsorbed to solid phase with normal human serum almost totally abolished the antibody binding.

Amino Acid Sequence↗
PubMed1984

The biochemistry of opsonization: central role of the reactive thiolester of the third component of complement.

In these studies, we have defined the mechanism by which the opsonic fragment of the third component of complement (C3) binds to pathogenic bacteria. With use of purified human C3 to reconstitute the alternative pathway in human serum in which both C3 and C4 had been chemically inactivated, we showed that opsonization of pathogenic serotypes of Streptococcus pneumoniae (serotypes 3, 4, 6A, 14, and 18C) requires the reactive thiolester of native C3. When purified human C3 (thiolester intact) is added to serum deficient in C3 and C4, phagocytic uptake of 3H-labeled pneumococci by polymorphonuclear leukocytes from normal adults is fully reconstituted. However, hydrolysis of the thiolester or reaction of the thiolester with the inhibitor methylamine abolishes opsonization and phagocytosis. Finally, by characterizing those C3 fragments released from pneumococcal surfaces after treatment with 1.0 M hydroxylamine, we have defined a role for covalent-bond formation in the opsonic interaction. Therefore, the presence of the reactive thiolester of C3 is an absolute requirement for the opsonic and covalent binding of the C3b molecule to pathogenic bacteria.

Chemical Phenomena↗
PubMed1990

The mechanism of activation of the alternative pathway of complement by cell-bound C4b.

Investigations into the mechanism of alternative pathway-dependent lysis of C4b-coated cells are reported. Test cells (EAC1q4b) were formed by reaction of sheep erythrocytes with antibody, C1 and C4. In C5-deficient serum, more C3b was deposited onto EAC1qC4b than onto control cells (EAC1q). The possibility that the C4bBb enzyme could form was considered, but no C3 convertase activity was generated when magnesium, properdin and factors B and D were added to EAC1qC4b. Binding studies employing radiolabeled components provided evidence that C4b bound the C3 convertase, C3bBbP, through a weak interaction with C3b. These data implied C3 conversion would be localized to the cell surface, thereby amplifying C3b deposition. This could be demonstrated in vitro. C3b, properdin, factor B and factor D were all required and the amplified C3b deposition was not due to deposition onto C4b itself. In serum, C5 convertase activity would be consequently expressed and cell lysis would result. This could be the mechanism by which the sera of C2-deficient patients mediate lysis of antibody coated sheep erythrocytes.

Animals↗

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