Convertase Formation Research Articles

Structure of Complement Component C2a: Implications for Convertase Formation and Substrate Binding

C2a provides the catalytic center to the convertase complexes of the classical and lectin-binding pathways of complement activation. We determined two crystal structures of full-length C2a, with and without a pseudo ligand bound. Both structures reveal a near-active conformation of the catalytic center of the serine protease domains, while the von Willebrand factor A-type domains display an intermediate activation state of helix alpha7 with an open, activated metal-ion-dependent adhesion site. The open adhesion site likely serves to enhance the affinity for the ligand C4b, similar to "inside-out" signaling in integrins. Surprisingly, the N-terminal residues of C2a are buried in a crevice near helix alpha7, indicative of a structural switch between C2 and C2a. Extended loops on the protease domain possibly envelop the protruding anaphylatoxin domain of the substrate C3. Together with a putative substrate-induced completion of the oxyanion hole, this may contribute to the high substrate specificity of the convertases.

CRIT peptide interacts with factor B and interferes with alternative pathway activation

Complement C2 receptor inhibitor trispanning (CRIT) inhibits the classical pathway (CP) C3 convertase formation by competing with C4b for the binding of C2. The C-terminal 11-amino-acid of the first CRIT-extracellular domain (CRIT-H17) has a strong homology with a sequence in the C4β chain, which is responsible for the binding of C2. Since the CP and alternative pathway (AP) C3 convertases have many functional and structural similarities, we further investigated the effects of CRIT-H17 on the AP. The factor D-mediated cleavage of factor B (FB) was blocked by CRIT-H17. By ELISA and immunoblot, CRIT-H17 was shown to bind FB. CRIT-H17 had no decay activity on the C3bBb complex as compared to decay-accelerating factor. Binding of CRIT-H17 to FB did not interfere with the assembly of C3bB complex. In a haemolytic assay using C2-deficient serum, CRIT-H17 interfered with AP complement activation.

Complement amplification revisited

Complement amplification in blood takes place not only on activating surfaces, but in plasma as well, where it is maintained primarily by C3b 2–IgG complexes. Regular products of C3 activation in serum, these complexes are inherently very efficient precursors of the alternative pathway C3 convertase. Moreover, they can bind properdin bivalently, thus creating preferred sites for convertase formation. C3b 2–IgG complexes have a half-life that is substantially longer than that of free C3b, since both C3b molecules are partially protected from inactivation by factor H and I. These complexes are preferentially generated on certain naturally occurring and induced antibodies that exhibit a paratope-independent affinity for C3/C3b. Such antibodies are known to stimulate alternative complement pathway activation. We have assembled the evidence for the generation and the functional potency of the C3b 2–IgG complexes, which have been studied during the last two decades. We illustrate their roles in immune complex solubilization, phagocytosis, immune response, and their ability to initiate devastating effects in ischemia/reperfusion and in aggravating inflammation.

244 GATA-1 PLAYS A CRITICAL ROLE IN ENDOTHELIAL CELL-SPECIFIC EXPRESSION OF THE 1.1-kb CD55 PROMOTER REGION

The complement system is composed of a complex group of soluble proteins that have important roles in the immune response against foreign cells such as xenografted tissue. Some of the cell surface regulators, also known as membrane complement regulatory proteins (CRPs), are the membrane cofactor protein (CD46), the decay-accelerating factor (CD55), and protectin (CD59). The CD55 is a 70 kDa glycolipid-anchored membrane-bound protein that has regulatory activity by preventing C3 convertase formation and is reported to be expressed in blood and vascular endothelial cells. Its high expression in leukocytes and endothelium is thought to safeguard against locally augmented C3 activation that can occur with inflammation. In this study, we originally cloned the 1.1-kb promoter region of the CD55 gene and constructed a luciferase reporter plasmid, pGL3-1.1CD55. The pGL3-1.1CD55 plasmid and its control plasmid, pGL3-control (SV40 promoter), were transfected into endothelial (MS-1 and BAEC) and epithelial (HaCaT and HEK293) cells, and the soluble fraction of the cell lysates was assayed for luciferase activity. Its promoter activity was compared with that of the promoters of endothelial-specific genes such as MCP, Flk-1, ICAM-2, and throbomodulin. Luciferase activity in each sample was normalized to the �-galactosidase activity of the same sample and the data were analyzed by Sigma Plot program (P < 0.01 versus control). Our results showed that the 1.1 kb CD55 promoter was the strongest among the endothelial cell-specific promoters. To define the important region for the strong expression, the deletion constructs containing 0.96, 0.86, and 0.74, CD55 promoter regions were prepared. Relative to the activity of the control SV40 promoter, decreased luciferase activity was obtained with these deletion constructs, suggesting that the about 0.2 kb 52-flanking region (between -1125 and -967) of the 1.1 kb CD55 promoter region was important for the strong gene expression. Interestingly, on the 52-end of the 0.2 kb region, we could detect two GATA-1 binding sites (from -1122 to -1118 and from -1111 to -1107); the deletion of the sequences between -1125 and -1100 (ΔGATA-1) significantly reduced the promoter activity. Furthermore, ectopic expression of GATA-1 dose-dependently induced the 1.1 kb CD55 promoter activity, but not the ΔGATA-1 activity, strongly implying that the GATA-1 binding site is important for the strong expression. Finally, we confirmed the binding of GATA-1 on the CD55 promoter region with the electrophoretic mobility shift assay (EMSA) using the 28 base pair oligonucleotide probes corresponding to the GATA-1 binding site and chromatin immunoprecipitation (Ch-IP) using anti-GATA-1 antibody. Taken together, these results strongly suggested that GATA-1 plays a critical role in endothelial cell-specific expression of the 1.1 kb CD55 promoter region. This work was supported by the Research Project on the Production of Bio-organs, Ministry of Agriculture and Forestry, Republic of Korea, and by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005-070-C00095).

Complement C2 Receptor Inhibitor Trispanning: A Novel Human Complement Inhibitory Receptor

The complement system presents a powerful defense against infection and is tightly regulated to prevent damage to self by functionally equivalent soluble and membrane regulators. We describe complement C2 receptor inhibitor trispanning (CRIT), a novel human complement regulatory receptor, expressed on hemopoietic cells and a wide range of tissues throughout the body. CRIT is present in human parasites through horizontal transmission. Serum complement component C2 binds to the N-terminal extracellular domain 1 of CRIT, which, in peptide form, blocks C3 convertase formation and complement-mediated inflammation. Unlike C1 inhibitor, which inhibits the cleavage of C4 and C2, CRIT only blocks C2 cleavage but, in so doing, shares with C1 inhibitor the same functional effect, of preventing classical pathway C3 convertase formation. Ab blockage of cellular CRIT reduces inhibition of cytolysis, indicating that CRIT is a novel complement regulator protecting autologous cells.

Investigation of the mechanisms of anti-complement activity in Ixodes ricinus ticks

The feeding success of a tick upon a host depends on its ability to suppress host anti-tick responses which include activation of the complement system. We investigated the mechanism of inhibition of the alternative pathway of complement by salivary gland extract (SGE) of the ixodid tick species, Ixodes ricinus. SGE treatment strongly inhibited C3a generation and factor B cleavage in serum when rabbit erythrocytes were used as complement activator, but not when cobra venom factor (CVF) was used as an activator. SGE treatment strongly inhibited C3b deposition on rabbit erythrocytes, and the turnover of C3 (to C3b/iC3b) in serum. However, there was no significant effect upon the formation, stability or activity of C3 convertase (C3bBb) when formed from purified C3b, factor B and factor D. SGE treatment of isolated C3 resulted in a shift in mobility of the α-chain (by about 5 kDa). N-terminal sequencing of this species suggests that cleavage occurs at the C-terminus of the α-chain of C3. Consistent with this hypothesis, the modified α-chain was still a substrate for pre-formed convertase. The activity was specific for the α-chain of C3 but not of C3(H 2O) nor the α′-chain of C3b. It is proposed that SGE-modified C3 does not participate in convertase formation, probably having a reduced affinity for factor B.

Inhibitor of complement, Compstatin, prevents polymer-mediated Mac-1 up-regulation of human neutrophils independent of biomaterial type tested.

The inflammatory reaction after cell contact with polymer materials is primarily mediated by activated neutrophils and may, in some cases, lead to exhaustion of neutrophil cell function. A direct consequence of this can be impairment of local or even systemic host defense mechanisms, which in turn can result in foreign body infections. Neutrophil activation, as indicated by the up-regulation of the Mac-1 adhesion receptor, is a reliable parameter for estimating the inflammatory risk due to implanted biomaterials. Because at blood contact, biomaterials immediately acquire a material-specific layer of blood proteins on their surface, including fibrinogen, complement, and immunoglobulin G, it is generally believed that after biomaterial contact, neutrophil activation primarily occurs by interaction with this protein layer. In this study, using our recently established polymer bead in vitro assay, we investigated whether complement inhibition alone can reduce biomaterial-mediated neutrophil activation, independent of the type of polymer and, hence, also its surface chemistry. Complement inhibition was achieved by using Compstatin, a recently developed complement inhibitor that binds to the complement component C3 preventing C3 convertase formation. We revealed significantly reduced (p < or = 0.025) Mac-1 receptor expression levels after 45 min of blood contact with the following polymers (without and with Compstatin): 1. polyurethane, 98.3%, 13.6%; 2. polymethylmetacrylate, 88.5%, 11.0%; and poly-D,L-lactide, 71.8%, 8.4%. Although these three polymer types acquire material-specific protein layers because of their different surface chemistry, complement inhibition by Compstatin alone proved to be sufficient to reduce neutrophil activation after surface contact, thus reducing the risk of biomaterial-mediated inflammatory reaction.

Development of a Novel Cytomedical Treatment that can Protect Entrapped Cells from Host Humoral Immunity.

Cell therapy is expected to relieve the shortage of donors needed for organ transplantation. When patients are treated with allogeneic or xenogeneic cells, it is necessary to develop a means by which to isolate administered cells from an immune attack by the host. We have developed "cytomedicine, " which consists of functional cells entrapped in semipermeable polymer, and previously reported that alginate-poly-l-lysine-alginate microcapsules and agarose microbeads could protect the entrapped cells from injury by cellular immunity. However, their ability to isolate from humoral immunity was insufficient. It is well known that the complement system plays an essential role in rejection of transplanted cells by host humoral immunity. Therefore, the goal of the present study was to develop a novel cytomedical device containing a polymer capable of inactivating complement. In the screening of various polymers, polyvinyl sulfate (PVS) exhibited high anticomplement activity and low cytotoxicity. Murine pancreatic β-cell line (MIN6 cell) entrapped in agarose microbeads containing PVS maintained viability and physiological insulin secretion, replying in response to glucose concentration, and resisted rabbit antisera in vitro. PVS inhibited hemolysis of sensitized sheep erythrocytes (EAs) and rabbit erythrocytes by the complement system. This result suggests that PVS inhibits both the classical and alternative complement pathways of the complement system. Next, the manner in which PVS exerts its effects on complement components was examined. PVS was found to inhibit generation of C4a and Ba generation in activation of the classical and alternative pathways, respectively. Moreover, when the EAC1 cells, which were carrying C1 on the EAs, treated with PVS were exposed to C1-deficient serum, hemolysis decreased in a PVS dose-dependent manner. These results suggest that PVS inhibits C1 in the classical pathway and C3 convertase formation in the alternative pathway. Therefore, PVS may be a useful polymer for developing an anticomplement device for cytomedical therapy.

Complement C2 receptor inhibitor trispanning and the beta-chain of C4 share a binding site for complement C2.

Complement C2 receptor inhibitor trispanning (CRIT) of the Schistosoma parasite binds human C2 via the C2a segment. The receptor in vivo functions as C2 decoy receptor by directly competing with C4b for binding to C2. As a result, CRIT is able to limit the extent of classical pathway (CP) C3 convertase formation. We report that the CRIT-extracellular domain 1 (ed1) peptide inhibits CP-mediated complement activation with an ICH(50) of approximately 0.1 microM, the C-terminal 11 aa of CRIT-ed1, named H17, even more effectively. The beta-chain region F222-Y232 of C4 shares 55% identity and 73% similarity with H17. Peptides based on this region also inhibit CP in a dose-dependent manner. As further evidence of C2 binding we showed CRIT-ed1 peptides and homologous C4 beta-chain peptides to inhibit complement in C2 hemolytic assays. We have predicted C4 beta-c F222-Y232 as a C2 binding site which we have termed the CRIT-ed1 domain, and the sequence [F/H]EVKX(4/5)P as a consensus C2-binding sequence. Anti-CRIT-ed1 cross-reacts with the C4 beta-chain and F222EVKITPGKPY232 appears to be the key epitope recognized by this Ab. Furthermore, anti-CRIT-ed1 was found to inhibit CP activation in a total hemolytic assay. We believe that Schistosoma CRIT-ed1, as well as C4 beta-chain peptides based on the CRIT-ed1 domain, function as interface peptides. These peptides, based on C2-binding sequences in CRIT, or C4, competitively inhibit the binding of C2 to C4b and thus limit the activation of C. The C4 peptides, unlike CRIT-ed1, did not inhibit the cleavage of C2 by C1s.

CR2‐mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes

Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane-bound C3 convertase. We have investigated whether this might lead to the generation of a C5 convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4.4 mM MgCl2/20 mM EGTA, which abrogates the classical pathway of complement without affecting the alternative pathway. Blockade of the CR2 ligand-binding site with the monoclonal antibody FE8 resulted in 56 +/- 13% and 71 +/- 9% inhibition of the C3-fragment and MAC deposition, respectively, whereas the monoclonal antibody HB135, directed against an irrelevant CR2 epitope, had no effect. Blockade of the CR1 binding site with the monoclonal antibody 3D9 also resulted in a minor reduction in MAC deposition, while FE8 and 3D9, in combination, markedly reduced deposition of both C3 fragments (91 +/- 5%) and C9 (95 +/- 3%). The kinetics of C3-fragment and MAC deposition, as well as the dependence of both processes on CR2, indicate that MAC formation is a consequence of alternative pathway activation.

CD59 homologue regulates complement-dependent cytolysis of rat Schwann cells

<h2>Abstract</h2> Antibody (Ab) sensitized sciatic nerve Schwann cells (SchC) of 2-day-old rats (SchC/2d) were significantly more susceptible to cytolysis by both heterologous, guinea pig (GP), and homologous rat serum complement (40 ± 3.8% and 21.2 ± 3.1%, respectively) than SchC of 6-day-old rats (SchC/6d) (7.9 ± 5.9% and 2.6 ± 3.1%, respectively). To determine if resistance to complement (C)-mediated cytolysis correlated with expression of membrane proteins which regulate C activation, we used Western blot and FACS analysis. Binding of specific polyclonal Ab demonstrated similar concentrations of Crry, a regulator of C3 convertase formation, on plasma membranes of SchC 2d and 6d. During C activation, both C3b deposition and iC3b formation were greater on SchC/6d than on SchC/2d and the C3b deposition did not correlate with enhanced cytolysis. In contrast, 2.1-fold more rat CD59, a regulator of C8 and C9 incorporation into C5b-9, detected with Western blot on SchC/6d compared with SchC/2d was confirmed by FACS. Further, both rat and GP C8/C9 lysed SchC/2d expressing human C5b-7 (20.1 ± 3.7 and 21.6 ± 4.7%, respectively), while only GP C8/C9 caused cytolysis of 10.7 ± 4.3% SchC/6d expressing hu C5b-7 and rat C8/C9 did not (0.5 ± 0.5%). Preincubation of SchC/6d with an F(ab)₂ fragment of an mAb to rCD59 with blocking capacity, increased cytolysis mediated by rat serum C more than 6-fold to 16.7 ± 3.0% but only 1.7-fold (maximum cytolysis 37.4 ± 11.2%) in SchC/2d. Our data suggest that expression of rat CD59 on SchC increased almost two-fold between postnatal days 2 and 6, and this increased expression on more terminally differentiated SchC is a significant factor in regulating terminal complement complex formation and limiting cytolysis of rat SchC by homologous serum complement.

CD59 homologue regulates complement-dependent cytolysis of rat Schwann cells

Antibody (Ab) sensitized sciatic nerve Schwann cells (SchC) of 2-day-old rats (SchC/2d) were significantly more susceptible to cytolysis by both heterologous, guinea pig (GP), and homologous rat serum complement (40 ± 3.8% and 21.2 ± 3.1%, respectively) than SchC of 6-day-old rats (SchC/6d) (7.9 ± 5.9% and 2.6 ± 3.1%, respectively). To determine if resistance to complement (C)-mediated cytolysis correlated with expression of membrane proteins which regulate C activation, we used Western blot and FACS analysis. Binding of specific polyclonal Ab demonstrated similar concentrations of Crry, a regulator of C3 convertase formation, on plasma membranes of SchC 2d and 6d. During C activation, both C3b deposition and iC3b formation were greater on SchC/6d than on SchC/2d and the C3b deposition did not correlate with enhanced cytolysis. In contrast, 2.1-fold more rat CD59, a regulator of C8 and C9 incorporation into C5b-9, detected with Western blot on SchC/6d compared with SchC/2d was confirmed by FACS. Further, both rat and GP C8/C9 lysed SchC/2d expressing human C5b-7 (20.1 ± 3.7 and 21.6 ± 4.7%, respectively), while only GP C8/C9 caused cytolysis of 10.7 ± 4.3% SchC/6d expressing hu C5b-7 and rat C8/C9 did not (0.5 ± 0.5%). Preincubation of SchC/6d with an F(ab) 2 fragment of an mAb to rCD59 with blocking capacity, increased cytolysis mediated by rat serum C more than 6-fold to 16.7 ± 3.0% but only 1.7-fold (maximum cytolysis 37.4 ± 11.2%) in SchC/2d. Our data suggest that expression of rat CD59 on SchC increased almost two-fold between postnatal days 2 and 6, and this increased expression on more terminally differentiated SchC is a significant factor in regulating terminal complement complex formation and limiting cytolysis of rat SchC by homologous serum complement.

Control of the Complement System

The complement system has developed a remarkably simple but elegant manner of regulating itself. It has faced and successfully dealt with how to facilitate activation on a microbe while preventing the same on host tissue. It solved this problem primarily by creating a series of secreted and membrane-regulatory proteins that prevent two highly undesirable events: activation in the fluid phase (no target) and on host tissue (inappropriate target). Also, if not checked, even on an appropriate target, the system would go to exhaustion and have nothing left for the next microbe. Therefore, the complement enzymes have an intrinsic instability and the fluid-phase control proteins play a major role in limiting activation in time. The symmetry of the regulatory process between fluid phase and membrane inhibitors at the C4/C3 step of amplification and convertase formation as well as at the MAC steps are particularly striking features of the self/nonself discrimination system. The use of glycolipid anchored proteins on membranes to decay enzymes and block membrane insertion events is unlikely to be by chance. Finally, it is economical for the cofactor regulatory activity to produce derivatives of C3b that now specifically engage additional receptors. Likewise, C1-Inh leads to C1q remaining on the immune complex to interact with the C1q receptor. Thus the complement system is designed to allow rapid, efficient, unimpeded activation on an appropriate foreign target while regulatory proteins intervene to prevent three undesirable consequences of complement activation: excessive activation on a single target, fluid phase activation, and activation on self.

A novel bifunctional chimeric complement inhibitor that regulates C3 convertase and formation of the membrane attack complex.

Abstract Human cells express cell surface complement regulatory molecules that inhibit the activity of the C3/C5 convertases (DAF, MCP, CR1) or inhibit the membrane attack complex (CD59). A single molecule that inhibits both the convertase activity and formation of the membrane attack complex has never been characterized. To this end, we have developed two reciprocal chimeric complement inhibitors (CD, NH2-CD59-DAF-GPI; and DC, NH2-DAF-CD59-GPI) that contain the functional domains of decay accelerating factor (DAF; CD55) and CD59. Cell surface expression of the CD and DC chimeric proteins was detected with DAF- and CD59-specific antisera. Cell surface C3d deposition was inhibited on cells expressing the chimeric molecules, thereby indicating that the DAF moiety was functional in both molecules. Conversely, Ab-blocking experiments demonstrated that only the DC molecule retained CD59 function. Therefore, the DC molecule represents a novel potent chimeric bifunctional complement inhibitor that retains the functional domains of two distinct complement regulatory molecules.

In vitro cleavage of serum complement protein C3: a comparison between patients with adult periodontitis and periodontally healthy persons.

This study tested the hypothesis that in vitro cleavage of C3 could be triggered with similar case in serum samples from patients with adult periodontitis (n = 26) as in samples from periodontally healthy subjects (n = 13). A lipoteichoic acid, a lipopolysaccharide and an aggregated IgG served as activators of complement. On the average, the periodontitis group generated significantly (p < 0.01) more C3d activation fragments than did the healthy group, as judged from rocket immunoelectrophoresis measurements. Cleavage of C4 and factor B were then assayed through immunoblotting, without prior purification of the sera. C4c fragments were seen in all activated samples, the healthy group causing significantly (p < 0.05) more C4 conversion than did the periodontitis group. Cleavage of factor B, taken as a measure of soluble amplification convertase formation, was about equal between the groups. We inferred therefore that the 2 groups produced comparable amounts of C3b. The results suggested, however, that periodontitis sera favour breakdown of the opsonin C3b, most likely by activating the regulatory proteins factor H and I. Lipoteichoic acid, causing moderate depletion of C4 and factor B, produced significantly (p < 0.01) more C3d fragments than the other two activators examined. It may be that complement activation is down-regulated in periodontitis sera, perhaps at the expense of adequate local opsonic function.

Tear lactoferrin levels and ocular bacterial flora in HIV positive patients.

Keratoconjunctivitis Sicca(4) has recently been reported to occur at a greater rate in HIV-positive symptomatic patients. We looked at HIV positive asymptomatic patients, compared to age matched HIV negative patients to study external ocular resistant factors, namely lactoferrin levels in tears, bacterial flora in lid margins, conjunctiva and tears, and evidence of dry eyes using a Schirmer test and tear osmolarity. Eighteen eyes of nine HIV positive patients and eighteen eyes of HIV negative controls were studied. Results showed markedly decreased lactoferrin levels in HIV positive asymptomatic patients with a mean of 85.8 mgs/dcl compared to HIV negative patients with a mean 156 mgs/dcl (P < 0.01). There were increased numbers of colonies of bacterial flora on the lids of HIV positive asymptomatic patients with an average colony count 4.1 colonies/patient compared to 1.5 colonies/patients in the control group (P < 0.025). Seventy eight percent of the study group had bacterial growth compared to 33% in the control group. The tear osmolarity in both groups had no significant difference; mean in HIV positive being 312 mosml/litre; mean in control 306 mosml/litre. The Schirmer test also showed no significant difference, with the mean in HIV positive patients being 11 mm wetting, and in control patients being 12.7 mm wetting. Therefore, despite no symptomatic or clinical evidence of dry eyes, asymptomatic HIV-positive patients had markedly decreased levels of lactoferrin in tears and increased colony counts of bacterial flora in the lids.

Membrane defence against complement lysis: the structure and biological properties of CD59.

The complement system is an important branch of the innate immune response, constituting a first line of defence against invading microorganisms which activate complement via both antibody-dependent and -independent mechanisms. Activation of complement leads to (a) a direct attack upon the activating cell surface by assembly of the pore-forming membrane attack complex (MAC), and (b) the generation of inflammatory mediators which target and recruit other branches of the immune system. However, uncontrolled complement activation can lead to widespread tissue damage in the host, since certain of the activation products, notably the fragment C3b and the C5b-7 complex, can bind nonspecifically to any nearby cell membranes. Therefore it is important that complement activation is tightly regulated. Our own cells express a number of membrane-bound control proteins which limit complement activation at the cell surface and prevent accidental complement-mediated damage. These include decay-accelerating factor, complement receptor 1 and membrane cofactor protein, all of which are active at the level of C3/C5 convertase formation. Until recently, cell surface control of MAC assembly had been attributed to a single 65-kD membrane protein called homologous restriction factor (alternatively named C8-binding protein and MAC-inhibiting protein). However a second MAC-inhibiting protein has since been discovered and it is now clear that this protein plays a major role in the control of membrane attack. This review charts the rapid progress made in elucidating the protein and gene structure, and the mechanism of action of this most recently discovered complement inhibitor, CD59.

A complement inhibitory fragment of human vttronectin

Antibody (Ab) sensitized sciatic nerve Schwann cells (SchC) of 2-day-old rats (SchC/2d) were significantly more susceptible to cytolysis by both heterologous, guinea pig (GP), and homologous rat serum complement (40 ± 3.8% and 21.2 ± 3.1%, respectively) than SchC of 6-day-old rats (SchC/6d) (7.9 ± 5.9% and 2.6 ± 3.1%, respectively). To determine if resistance to complement (C)-mediated cytolysis correlated with expression of membrane proteins which regulate C activation, we used Western blot and FACS analysis. Binding of specific polyclonal Ab demonstrated similar concentrations of Crry, a regulator of C3 convertase formation, on plasma membranes of SchC 2d and 6d. During C activation, both C3b deposition and iC3b formation were greater on SchC/6d than on SchC/2d and the C3b deposition did not correlate with enhanced cytolysis. In contrast, 2.1-fold more rat CD59, a regulator of C8 and C9 incorporation into C5b-9, detected with Western blot on SchC/6d compared with SchC/2d was confirmed by FACS. Further, both rat and GP C8/C9 lysed SchC/2d expressing human C5b-7 (20.1 ± 3.7 and 21.6 ± 4.7%, respectively), while only GP C8/C9 caused cytolysis of 10.7 ± 4.3% SchC/6d expressing hu C5b-7 and rat C8/C9 did not (0.5 ± 0.5%). Preincubation of SchC/6d with an F(ab)2 fragment of an mAb to rCD59 with blocking capacity, increased cytolysis mediated by rat serum C more than 6-fold to 16.7 ± 3.0% but only 1.7-fold (maximum cytolysis 37.4 ± 11.2%) in SchC/2d. Our data suggest that expression of rat CD59 on SchC increased almost two-fold between postnatal days 2 and 6, and this increased expression on more terminally differentiated SchC is a significant factor in regulating terminal complement complex formation and limiting cytolysis of rat SchC by homologous serum complement.

Analysis of complement C3 activation products in human atherosclerotic lesions

Cleavage of the complement C3 protein is essential for complement activation. Saline extracts of human atherosclerotic lesions were examined by various techniques for the presence of C3 cleavage fragments. Crossed intermediate gel immunoelectrophoresis revealed that native C3 was the predominate C3 protein in extracts and that the C3dg fragment was also detected. SDS-PAGE/Western blot analyses of lesion extracts employing monoclonal antibodies directed at C3c and C3dg fragment determinants demonstrated molecular weight bands corresponding to the known molecular weights of all the physiologic C3 cleavage fragments, except C3b which is known to have a short half-life. After C3, the two most common fragments observed were C3c and C3dg. No bands other than those corresponding to known C3 cleavage fragments were observed and control antibody stains were always negative. In some blots bands with a greater molecular mass than C3 were evident, indicating that some of the C3 in lesions may be covalently bound to an activator. We have previously identified a large (100-500 nm) nonapoprotein containing lipid particle (LCA) as a major complement activating structure in human atherosclerotic lesions. Fractionation of lesion extracts by molecular sieve chromatography and sucrose density gradient centrifugation failed to reveal a concordance between LCA and C3 antigens. The results indicate that complement activation, i.e. C3 convertase formation, takes place in human atherosclerotic lesions and that activated C3 is degraded according to normal complement regulatory mechanisms.

Characterization of a Trypanosoma cruzi C3 binding protein with functional and genetic similarities to the human complement regulatory protein, decay-accelerating factor.

Evasion of the complement system by microorganisms is an essential event in the establishment of infection. In the case of Trypanosoma cruzi, the causative agent of Chagas disease, resistance to complement-mediated lysis is a developmentally regulated characteristic. Infectious trypomastigotes are resistant to complement-mediated lysis in the absence of immune antibodies, whereas the insect forms (epimastigotes) are sensitive to lysis via the alternative complement pathway. We have purified a developmentally regulated, trypomastigote glycoprotein, gp160, and shown that it has complement regulatory activity. The T. cruzi gp160 restricts complement activation by binding the complement component C3b and inhibiting C3 convertase formation. The protein is anchored in the parasite membrane via a glycosyl phosphatidylinositol linkage, similar to the human complement regulatory protein, decay-accelerating factor. Using anti-gp160 antibodies we have isolated a bacteriophage lgt11 clone expressing a portion of the gp160 gene that shares significant DNA sequence homology with the human DAF gene. These results provide functional, biochemical, and genetic evidence that the T. cruzi gp160 is a member of the C3/C4 binding family of complement regulatory proteins, and that gp160 may provide the infectious trypomastigotes with a means of evading the destructive effects of complement.