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PubMed1979

The role of C4-binding protein and beta 1H in proteolysis of C4b and C3b.

Two forms of C4-binding protein (C4-bp) (C4-bp low, C4-bp high), which differ slightly in net charge and apparent molecular weight, as determined by SDS- PAGE, were separated by ion-exchange chromatography and contaminants removed with specific antisera. Both forms of C4-bp served as cofactors for the cleavage of C4b in solution by C3b inactivator, and the resulting fragments of the a'-chain of C4b had identical molecular weights. In addition, similarly to beta1H, C4-bp low or high served as cofactors for the cleavage of fluid phase C3b by C3bINA. However, important quantitative differences between the activities of C4-bp and beta1H were observed. With regard to C3b in solution, the cofactor activity of beta1H was {approximately equal to}20 times greater than that of C4-bp on a weight basis. In relation to cell-bound C3b, the differences in activity were even more marked. Whereas beta1H enhanced the effects of C3bINA on the erythrocyte intermediate EC3b, inhibiting the assembly of EC3bBb, C4-bp was without effect even at concentrations {approximately equal to}300 times greater than beta1H. Therefore, under physiological conditions, it is likely that beta1H is the key protein which controls the function of C3b, and that C4-bp activity is directed mainly toward the cleavage of C4b. We also examined the relation between C4-bp and the C3b-C4bINA cofactor described by Stroud and collaborators (3, 4). By functional, physico-chemical and immunological criteria, they are the same protein.

Complement C3↗
PubMedDate not supplied

Reproductive tract secretions and bull spermatozoa contain different clusterin isoforms that cluster cells and inhibit complement-induced cytolysis.

Clusterin from bull rete testis fluid (RTF), cauda epididymal fluid (CEF), and octyl-beta-D-glucopyranoside extract of cauda epididymal sperm (CES) was identified and characterized using monoclonal and polyclonal antibodies (Abs) developed against ram clusterin and a beta-subunit-specific oligopeptide of porcine clusterin. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting showed that bovine RTF clusterin had dimeric and monomeric molecular weights (M(r)s) of approximately 94 kDa and of 42 and 43 kDa, respectively. Clusterin in CEF and CES had similar dimeric M(r)s (74 kDa). Reduced CEF clusterin appeared as three monomers (M(r)=40, 39, and 38 kDa), whereas reduced CES clusterin appeared only at M(r)40 kDa. Enzymatic deglycosylation resulted in similar M(r)s of clusterin from RTF, CEF, and CES. The M(r) of RTF clusterin decreased from 94 kDa to 51 kDa, indicating a carbohydrate content of 45%. After deglycosylation, the M(r) of the CEF clusterin decreased from 74 kDa to two distinct bands at 51 and 50 kDa (with carbohydrate contents of 31 and 32%, respectively), suggesting that two isoforms of the heterodimeric protein are present because of the two isoforms of the alpha-subunit. Under nonreduced conditions, a beta-subunit-specific Ab reacted with M(r) of 36-38 kDa, indicating the existence of free clusterin beta-subunits in CES. RTF, CEF, and CES extracts all caused mouse fibroblastic L-cell aggregation. CEF cell aggregation was inhibited by Hyb-17 Ab but not by other Abs. Both RTF and CEF caused a dose-dependent inhibition of complement-induced cytolysis, although RTF clusterin was more potent than CEF clusterin. We conclude that several isoforms of clusterin occur in the bull reproductive tract and that the variation in carbohydrate content among these isoforms may affect the biological or functional activity of the protein.

Animals↗
PubMed2003

Complement: structure, functions, evolution, and viral molecular mimicry.

The complement (C') system has long been recognized as an important mediator of innate immune defense and inflammation. In recent years there is increasing evidence suggesting that complement components may also participate in non-inflammatory and developmental processes. Here we review our current work on the structural-functional aspects of C3-ligand interactions and the rational design of small-sized complement inhibitors. We present a novel, proteomics-based, approach to studying protein-protein interactions within the C' system and discuss our progress in the study of viral immune evasion strategies. Furthermore we discuss the involvement of complement proteins in organ regeneration and hematopoietic development.

Animals↗
PubMed1995

One-step sandwich enzyme immunoassays for human C4b-binding protein (C4BP) and protein S-C4BP complex using monoclonal antibodies.

C4b-binding protein (C4BP), a regulatory component in the complement system, binds to an anticoagulant vitamin K-dependent plasma protein S (PS) which acts as a cofactor of activated protein C. We raised monoclonal antibodies against C4BP and PS, and developed two different one-step sandwich enzyme immunoassay (EIA) systems for human total C4BP (assay A) and PS-C4BP complex (assay B) by using a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody (Fab'). The reaction time of the assay was 45 min in both EIA systems: 30 min for the immunoreaction and 15 min for the color reaction. The sensitivities were 12 and 20 mg/l in assays A and B, respectively. Linearity was obtained between 31 and 500 mg/l in both EIA systems. Assay A could detect both uncomplexed C4BP and PS-C4BP complex with equal efficiency so that total C4BP level was not affected by PS. The levels of total C4BP and PS-C4BP complex were found to significantly increase in sera from patients with membranous nephropathy and decrease with liver cirrhosis in comparison with the levels in normal subjects. On the other hand, a difference in the total C4BP and PS-C4BP complex levels was not shown between IgA nephropathy and normal subjects. Affinity column analysis and difference of total C4BP and PS-C4BP complex levels showed that most of C4BP in sera exists as PS-C4BP complex.

Animals↗
PubMed1998

The expression of complement regulatory proteins by adult human oligodendrocytes.

In multiple sclerosis, infiltrating T lymphocytes and perivascular microglia may initiate demyelinating lesions, but a role for antibody and complement in the ensuing inflammatory damage to myelin and oligodendrocytes is likely. In most tissues, ubiquitously expressed complement regulatory proteins prevent autologous destruction, protecting host cells from the powerful cytolytic activity of activated complement. We have studied the surface expression of a comprehensive range of complement regulatory proteins by live adult human oligodendrocytes in vitro. Only DAF of the activation pathway regulators was expressed, not CR1 or MCP. Of the membrane attack pathway regulatory proteins, HRF was not expressed, while substantial heterogeneity of CD59 expression by oligodendrocytes was found. Clusterin expression was not found. A relative deficiency of protective complement regulatory proteins on human oligodendrocytes may contribute to their selective damage in multiple sclerosis.

CD59 Antigens↗
PubMed1997

Human immunodeficiency virus type 1 incorporates both glycosyl phosphatidylinositol-anchored CD55 and CD59 and integral membrane CD46 at levels that protect from complement-mediated destruction.

Human immunodeficiency virus type 1 (HIV-1) can be either resistant or sensitive to complement-mediated destruction depending on the host cells. Incorporation of different levels of host cell CD46, CD55 and CD59 may account for this differential sensitivity to complement. However, it has not been determined whether CD46, CD55 and CD59 can all be incorporated at levels which protect virions. To determine whether each of these proteins can protect HIV-1, virions were derived from CHO cells expressing either human CD46, CD55 or CD59. Virions were shown to incorporate both glycosyl phosphatidylinositol (GPI)-anchored CD55 and CD59 as well as transmembrane CD46. Importantly, all three virus preparations were significantly more resistant to complement lysis than control virus. This study demonstrates that HIV-1 incorporates both transmembrane and GPI-anchored complement control proteins from host cells and that both types of protein increase complement resistance of virus.

Animals↗
PubMed1988

Complement inhibitor S protein is associated with membranes of red blood cells from patients with paroxysmal nocturnal haemoglobinuria.

S protein is a plasma glycoprotein (Mr = 78,000) which binds to nascent C5b-7 complexes upon complement activation in the fluid phase in whole serum. It thereby protects innocent bystander cells from complement mediated lysis. It is unknown whether S protein also functions as complement inhibitor on cell surfaces. We here report that S protein is recognized on red blood cells (RBC) from patients with paroxysmal nocturnal haemoglobinuria (PNH), but not on normal RBC. RBC from eight PNH patients showed 12-48% haemolysis subsequent to complement activation in the fluid phase, while normal RBC did not respond. Preincubation of the PNH cells with affinity-purified antibodies against human S protein resulted in a three- to five-fold increase of haemolysis, while preincubation of these cells with S protein decreased haemolysis by 40%. In contrast, haemolysis remained unaffected by other unrelated antibodies, i.e. IgG anti-Rh(D) and anti-A. If PNH RBC, normal RBC pretreated with 2-amino-ethylisouronium bromide (AET), or untreated normal RBC, respectively, were incubated with purified S protein in vitro, the uptake of antibodies against S protein was significantly enhanced with PNH and with AET-treated, but not with untreated normal RBC. Additionally, while normal RBC did not respond to reactive lysis initiated by purified C5b-6 and C7, PNH as well as AET-RBC showed significant haemolysis that could be inhibited by S protein in a dose-dependent fashion. These findings strengthen the assumption that the increased sensitivity of PNH cells towards reactive complement lysis is either due to the lack of an inhibitor of the terminal complement sequence and/or enhanced insertion of the membrane attack complex. These defects of PNH RBC may partly be overcome by the fluid phase complement inhibitor S protein which binds to PNH RBC and may thereby suppress homologous cytolysis.

Complement Inactivator Proteins↗
PubMed2004

Complement inactivation by recombinant human C3 derivatives.

From the implications of the complement system in a large number of diseases, an urgent need for therapeutics effecting reduced complement activity in vivo has emerged. In this study we report the design of a novel class of enzymes of human origin that obliterate functional complement by a noninhibitory, catalytic mechanism. Combining the framework of human C3 and the enzymatic mechanism of cobra venom factor, a nontoxic snake venom protein, we established molecules capable of forming stable C3 convertase complexes. Although the half-life of naturally occurring C3 convertase complexes ranges between 1 and 2 min, these complexes exhibit a half-life of up to several hours. Because the overall identity to human C3 could be extended to >90%, the novel C3 derivatives can be assumed to exhibit low immunogenicity and, therefore, represent promising candidates for therapeutic reduction of complement activity in vivo.

Animals↗
PubMed1977

[Action of hyperoxia on hemolytic activity of the complement system].

Normobarie oxygen exposures momentarily raise by about 20% the haemolytic activity of the guinea pig complement likely by a rise of protein synthesis. About the end of this treatment the complement activity quickly decreases back to its initial value. Hyperbaric oxygen exposure immediately decreases the haemolytic activity. This shift might be result of a release in the serum of cytoplasmic elements bearing an anticomplementary activity or of an inactivation of the complement components involved in the stress reaction appearing in the treated animals. 2 days after the first exposure, during the resting phase, the complement rate increases by about 25% then decreases slowly back to normal.

Animals↗
PubMed1989

C3 nephritic factor and C4 nephritic factor in the serum of two patients with hypocomplementaemic membranoproliferative glomerulonephritis.

Both C3 nephritic factor (C3NeF) and C4 nephritic factor (C4NeF) were found in the serum of two patients with hypocomplementaemic membranoproliferative glomerulonephritis type 1 (H-MPGN type 1). Both stabilizing activities were associated with the IgG fraction. The serum levels of C3 and C5 were quite low, whereas the levels of early components and control proteins were in the normal range.

Adolescent↗
PubMed2001

Complement and its implications in cardiac ischemia/reperfusion: strategies to inhibit complement.

Although reperfusion of the ischemic myocardium is an absolute necessity to salvage tissue from eventual death, it is also associated with pathologic changes that represent either an acceleration of processes initiated during ischemia or new pathophysiological changes that were initiated after reperfusion. This so-called "reperfusion injury" is accompanied by a marked inflammatory reaction, which contributes to tissue injury. In addition to the well known role of oxygen free radicals and white blood cells, activation of the complement system probably represents one of the major contributors of the inflammatory reaction upon reperfusion. The complement may be activated through three different pathways: the classical, the alternative, and the lectin pathway. During reperfusion, complement may be activated by exposure to intracellular components such as mitochondrial membranes or intermediate filaments. Two elements of the activated complement contribute directly or indirectly to damages: anaphylatoxins (C3a and C5a) and the membrane attack complex (MAC). C5a, the most potent chemotactic anaphylatoxin, may attract neutrophils to the site of inflammation, leading to superoxide production, while MAC is deposited over endothelial cells and smooth vessel cells, leading to cell injury. Experimental evidence suggests that tissue salvage may be achieved by inhibition of the complement pathway. As the complement is composed of a cascade of proteins, it provides numerous sites for pharmacological interventions during acute myocardial infarction. Although various strategies aimed at modulating the complement system have been tested, the ideal approach probably consists of maintaining the activity of C3 (a central protein of the complement cascade) and inhibiting the later events implicated in ischemia/reperfusion and also in targeting inhibition in a tissue-specific manner.

Complement Activation↗
PubMed1985

Characterization of human S protein, an inhibitor of the membrane attack complex of complement. Demonstration of a free reactive thiol group.

S protein, an inhibitor to the membrane attack complex of complement, was purified from human plasma. The procedure involved barium citrate adsorption and fractionation by poly(ethylene glycol) 4000 precipitation, followed by chromatography on DEAE-Sephacel, Blue Sepharose, Sephacryl S-200, and finally anti-albumin-Sepharose. Reduced glutathione was added throughout to inhibit spontaneous formation of disulfide-linked S-protein dimers. The recovery was 7%, resulting in approximately 10 mg of pure S protein from 1 L of starting plasma. S protein is a single-chain molecule; sedimentation equilibrium ultracentrifugation yielded a molecular weight of 83 000; the s020,W value was estimated to be 4.0 S. The purified protein contained a free, reactive thiol group causing spontaneous formation of disulfide-linked S-protein dimers. Alkylated and nonalkylated S proteins were equally active in inhibiting C9 polymerization, catalyzed by the C5b-8 complex. In parallel with the inhibition of C9 polymerization, nonalkylated S protein catalyzed the formation of disulfide-linked C9 dimers, presumably through disulfide interchanges.

Amino Acid Sequence↗
PubMed1998

X-linked vacuolated myopathy: membrane attack complex deposition on the surface membrane of injured muscle fibers is not accompanied by S-protein.

We have studied the expression of S-protein on the muscle from patients with X-linked vacuolated myopathy [characterized by the deposition of the complement C5b-9 membrane attack complex (MAC) over abnormal muscle fibers] and controls by immunocytochemistry and immunoblotting. No expression was detected on muscle from controls and patients with X-linked vacuolated myopathy. These findings suggest that S-protein does not render the MAC inactive in X-linked vacuolated myopathy. This situation may be due to the fact that the pathways of MAC activation and the expression of S-protein in X-linked vacuolated myopathy are different from the ones observed in ischemic and/or necrotic, or immune diseases. These results emphasize the role of the membrane complement regulatory proteins (i.e., CD59) in X-linked vacuolated myopathy.

Adult↗
PubMed2000

Laminar shear stress upregulates the complement-inhibitory protein clusterin : a novel potent defense mechanism against complement-induced endothelial cell activation.

BACKGROUND: The complement system is implicated in the pathogenesis of atherosclerosis. Complement has been shown to activate endothelial cells (ECs) by inducing a proinflammatory response. Physiological levels of shear stress exert potent antiatherosclerotic effects. Therefore, we investigated whether shear stress antagonizes the effects of complement on ECs. METHODS AND RESULTS: Incubation of ECs with nonlytic concentrations of complement serum (CS: 0.2 U/mL for 6 hours) resulted in an upregulation of interleukin-8 (IL-8) (165+/-12%) and monocyte chemoattractant protein-1 (MCP-1) mRNA expression (267+/-34%). Preexposure of ECs for 18 hours with laminar shear stress (15 dyne/cm(2)) abrogated CS-induced IL-8 release to 106+/-10% (P<0.001) and reduced CS-induced MCP-1 expression (170+/-31%; P<0.05). To examine the mechanism of the protective effect of shear stress, expression of the complement-inhibitory protein clusterin was analyzed under shear exposure. Shear stress increased clusterin mRNA (225+/-76%, 6 hours) and protein expression (164+/-22%, 18 hours). Specific inhibition of clusterin by transfection with antisense oligonucleotides reversed the protective effect of shear stress on CS-induced MCP-1 and IL-8 upregulation (P<0.05 versus sense-transfected cells). Moreover, clusterin overexpression inhibited CS-induced EC activation. CONCLUSIONS: Shear stress abrogates the complement-induced proinflammatory response of ECs by upregulation of the complement-inhibitory protein clusterin. Upregulation of clusterin may contribute to the potent antiatherosclerotic effects of shear stress by preventing endothelial activation through the complement cascade.

Cells, Cultured↗