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PubMed2001

Structure/function of C5 convertases of complement.

C5 convertases are serine proteases that cleave both C3 and C5. Alternative pathway C3/C5 convertases formed with monomeric C3b (C3b,Bb) because of their weak interaction with C5 primarily cleave C3 thereby opsonizing the cell surface with C3b. In contrast, C3/C5 convertases formed with a high density of C3b/cell exhibit higher affinities for C5 as indicated by Km values well below the physiological concentration of C5 in blood. These C3/C5 convertases bind C5 efficiently and cleave it at a velocity approaching Vmax thereby switching the enzyme from C3 cleavage to production of the cytolytic C5b-9 complex. Studies of the structure of C3/C5 convertases have postulated that C4b-C3b and C3b-C3b dimers from high affinity C5 binding sites while indel studies have shown two binding sites in C5 for the convertase in addition to the C5 cleavage site. Together, these studies indicate that with increasing deposition of C3b on the surface, C3b complexes are formed which through multivalent attachment bind the substrate C5 with higher affinities, thereby converting the low affinity C3/C5 convertases to high affinity C5 convertases. The process underlying the formation of high affinity C5 convertases during complement activation is discussed.

Animals↗
PubMed1986

Endogenous association of decay-accelerating factor (DAF) with C4b and C3b on cell membranes.

Decay-accelerating factor (DAF) is a membrane glycoprotein found on various cells that are in contact with complement. It inhibits the formation of the C3 convertases of the complement system, both the classic (C4b2a) and alternative (C3bBb) pathways. In this investigation, we used a homobifunctional cross-linking reagent to search for a DAF ligand on the surface of cells subjected to complement attack. We found that DAF forms complexes with C4b and C3b deposited on the same erythrocytes, but not with the physiologic degradation products of these complement fragments, that is, C4d or C3dg. Taken together with prior observations that DAF action is reversible, and DAF does not affect the structure of C4b or C3b, these findings suggest that DAF functions by competitively inhibiting the uptake of C2 or factor B, and preventing the assembly of the C3 convertases.

Animals↗
PubMed1998

Mice deficient for the complement factor B develop and reproduce normally.

Factor B is an essential component of the complement cascade which forms the C3 and C5 convertase of the alternative pathway. Factor B cleavage products also function as cofactors in antibody-independent monocyte-mediated cytotoxicity, macrophage spreading, plasminogen activation and proliferation of B lymphocytes. Several healthy kindreds heterozygous for the factor B null or non-functional allele have been reported but the absence of homozygous factor B deficiency in humans or in animals has been speculated to be caused by the lethality of the phenotype. Here we report the generation of factor B-deficient mice by gene targeting in vivo. These mice were born at the expected Mendelian ratio and they both develop and breed normally in a conventional animal facility. These mice represent a model of complete alternative pathway deficiency. This model enables the dissection of the complement cascade in vivo and the elucidation of the relative contribution of this complement pathway in the various physiological and pathological phenomena ascribed to the complement system.

Animals↗
PubMed1990

The influence of membrane components on regulation of alternative pathway activation by decay-accelerating factor.

Decay-accelerating factor (DAF) is a C regulatory protein which functions in membranes to inhibit autologous C activation on cell surfaces. A liposome model was used to study the mechanism of DAF action and examine the effects of membrane-bound glycophorin and LPS on the regulatory activity of DAF. Liposomes were incubated in MgEGTA-treated human serum and activation of the alternative pathway measured by C3b binding. Liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and cholesterol activated the alternative pathway in proportion to their content of PE. Incorporation of 10(-7) mol/mol phospholipid of either human E or HeLa cell-derived DAF inhibited C activation by liposomes containing 40% phosphatidylethanolamine by 50%, an efficiency comparable to that observed in intact E. HeLa DAF that had been treated with phosphatidylinositol-specific phospholipase C to remove its glycolipid anchor had no effect on C activation by liposomes at concentrations as high as 10(-5) mol/mol phospholipid. Incorporation of DAF into liposomes prepared with bound C3b inhibited the deposition of additional C3b by C3bBbP. However, the incorporated DAF increased the amount of Bb generated from B in the presence of D indicating that accelerated decay of the convertase was the primary effect of DAF. Similarly, treatment of intact human E with anti-DAF decreased the amount of Bb generated by the alternative pathway convertase. To study the effects of other membrane components on DAF activity, liposomes were prepared with purified human glycophorin A or LPS. In glycophorin liposomes the presence of PE was required to activate the alternative pathway and DAF inhibited this activation. In contrast, LPS liposomes bound C3b independently of PE and the incorporation of DAF had no effect. These results demonstrate that within a membrane, DAF's inhibitory activity on the alternative pathway C3 convertase is mediated independently of other membrane proteins, that in this model the major activity of DAF is to accelerate convertase decay, and that the presence of other membrane molecules that may serve as C3 acceptors can circumvent DAF function.

CD55 Antigens↗
PubMed1979

Recurrence of dense deposits in transplanted kidney: II. Serum complement and nephritic factor profiles.

Dense deposit disease of the kidney is a rare form of chronic glomerulonephritis frequently associated with serum complement abnormalities (low C3 levels) and a circulating C3 convertase activator of the alternative pathway, the C3 nephritic factor (NF). Eleven patients with end-stage dense deposit disease underwent kidney transplantation. Of the 11, 7 had pretransplant low C3 and NF. In the posttransplant period, persisting low C3 levels were associated with persisting NF, although not quantitatively so. The original glomerular lesion recurred in the graft within 6 months in 9 of 11. Of these 9, 2 had no complement abnormalities either prior to or after transplantation. Pretransplant complement abnormalities were rapidly corrected in 4 of 7 patients whether or not recurrence of the original lesion occurred. Thus, serum complement profiles before and after transplantation are neither predictive nor indicative of recurrence.

Complement C3↗
PubMed1998

Active sites in complement component C3 mapped by mutations at indels.

Engineered mutants of human complement component C3 were used to test the idea that sites of length polymorphisms in protein families (indels) can guide a search for protein:protein interaction sites. Sequence changes were introduced at each of the 27 indels in the C3/4/5 protein family, and mutants at 26 indels were expressed by transiently transfected COS cells. Expressed proteins were assayed 1) for concentration, by ELISA and by autoradiography of radiolabeled protein; 2) for classical pathway hemolytic activity; 3) for susceptibility to proteolytic activation by the alternative pathway and cobra venom factor C3 convertases; and 4) for susceptibility to complement factor I in the presence of factor H. Most of the mutations did not appreciably alter expression or activity relative to wild-type C3, consistent with the idea that most indels occur at the protein surface. Mutations at four indels severely damaged C3 functional activity, but did not affect the stability or structure of the protein, as assessed by their effects on expression by COS cells and on susceptibility to cleavage by C3 convertases and factor I. These indels are therefore near functionally important amino acid residues; they represent good candidates for sites of protein:protein interactions. Mutation of the sequence at a fifth indel altered the equilibrium between the latent and reacted C3 conformations, and mutations at 4 other indels substantially decreased both protein activity and expression. The mutants provided an overview of the structural and functional roles played by different parts of C3.

Amino Acid Sequence↗
PubMed1996

Mouse complement component C4 is devoid of classical pathway C5 convertase subunit activity.

It has long been known that mouse C4 has unusually low hemolytic activity relative to the C4 of other mammalian species (e.g. human and guinea pig), the measurements being done in most cases using a C4-deficient guinea pig serum reagent in a one-step assay with EA. This low activity for mouse C4 previously had been attributed to "technical" difficulties such as lability of the protein during blood collection and partial species incompatibilities with guinea pig components. Recently, we presented evidence for the involvement of human C4 beta-chain residues 455-469, a putatively exposed hydrophilic segment, in contributing to a C5 binding site in the C4b subunit of the classical pathway C5 convertase, C4b3b2a. Given that there were five sequence differences between the human and mouse protein within this segment, we hypothesized that these substitutions may have compromised the C5 convertase subunit activity of mouse C4, thereby resulting in its low hemolytic activity. Using a multi-step hemolytic assay which was totally dependent upon C5 cleavage by the classical pathway, we found that mouse C4 was completely devoid of classical pathway C5 convertase subunit activity. We have been able to rule out the most obvious potential species incompatibilities (e.g. between C4mo and C5gp) as being responsible for this lack of activity. Moreover, we found that the low level of hemolytic activity of mouse C4 measured in the one-step assay can be ascribed totally to C5 cleavage, and subsequent terminal component assembly, by the alternative pathway C5 convertase, (C3b)2Bb. However, the assembly of the latter enzyme complex is dependent upon the presence of C3b molecules deposited initially via the classical pathway C3 convertase in which mouse C4b is a subunit. Finally, whereas conversion of human residues 458RP to the mouse-like sequence PL was sufficient to abrogate classical pathway C5 convertase subunit activity in human C4, the five substitutions which "humanized" the 452-466 segment of mouse C4 (corresponding to human residues 455-469) were on their own insufficient to impart this activity to mouse C4. This implies that, in addition to the 455-469 beta-chain segment of human C4, there are other regions of the molecule contributing to C5 binding which are also non-conserved between human and mouse C4.

Amino Acid Sequence↗
PubMed1998

Expression of factor I-resistant mutants of the human complement component C3 in heterologous systems.

Complement plays a major role in hyperacute rejection of xenografts. In order to overcome this, we are developing, by minimal mutagenesis, a modified C3 molecule that, like cobra venom factor (CVF), escapes normal complement regulatory processes and inhibits complement-mediated responses by systemic depletion of C3. Unlike CVF, this protein should have little or no immunogenicity and be suitable for repeat administrations. As an initial step in this process, we have modified human C3 to make it resistant to inactivation by factor I. The factor I resistant C3 is capable of forming an active C3 convertase. Preincubation with normal human serum abrogated subsequent complement-mediated cytolysis by both the classical and alternative pathways, while wild-type (wt) C3 was inactive. The modified human C3 also blocked complement activity of guinea-pig serum. For economical and rapid production, we have developed expression of recombinant C3 wt and mutant proteins in the Baculovirus system. Large quantities are also being produced from stably transfected CHO cell lines. In addition, we have developed a fast C3 purification method by engineering a 6XHIS tag into the C3a portion of the molecule, thereby avoiding the need for subsequent separation of the tag from active C3b molecules.

Animals↗
PubMed1979

[Alternative complement pathway (author's transl)].

The dual role of the alternative complement pathway in recognition of foreign substances by a non-immune host and in the intrinsic regulation of the complement sequence is now well recognized. Activation of this pathway occurs through escape from its regulatory mechanisms induced by the activating principle; its functional expression depends on the respective levels of the component proteins C3, factor B, factor D and properdin, and on the control proteins beta 1H and C3bINA. This article presents recently acquired knowlege on the molecular mechanisms of activation, regulation and behaviour under pathological conditions of the alternative complement pathway.

Complement Activation↗
PubMed1998

Interaction of vaccinia virus complement control protein with human complement proteins: factor I-mediated degradation of C3b to iC3b1 inactivates the alternative complement pathway.

Vaccinia virus complement control protein (VCP) is a virulence determinant of vaccinia virus that helps protect the virus from the complement attack of the host. To characterize the interaction of VCP with C3 and C4 and understand the mechanism by which VCP inactivates complement, we have expressed VCP in a yeast expression system and compared the biologic activity of the purified protein to that of human factor H and complement receptor 1 (CR1). Recombinant VCP bound to C3 and the proteolytically cleaved form of C3 (C3b), but not to the 135,300-m.w. fragment of C3 generated using elastase (C3c) and the 35,000-m.w. fragment of C3 generated using elastase (C3d) and inhibited both the classical and alternative pathways of complement activation. Although rVCP was less effective at inhibiting the alternative pathway than factor H or CR1, it was more effective than factor H at inhibiting the classical pathway. Unlike factor H, rVCP was unable discriminate between alternative pathway-mediated lysis of rabbit and sheep E. A comparison of the cofactor activity in factor I-mediated cleavage of C3b suggested that in contrast to factor H and CR1, which displayed cofactor activity for the three sites, rVCP displayed cofactor activity primarily for the first site, leading to generation of C3b cleaved by factor I between Arg1281-Ser1282 (iC3b1). Its cofactor activity for C4b cleavages was similar to that of soluble complement receptor type 1. Purification and functional analysis of iC3b1 showed that it was unable to interact with factor B to form the alternative pathway C3 convertase, C3b,Bb. These results suggest that the interaction of VCP with C3 is different from that of factor H and CR1 and that VCP-supported first cleavage of C3b by factor I is sufficient to render C3b nonfunctional.

Arginine↗
PubMed1993

Hemolysis of normal human erythrocytes by autologous serum complement.

Unsensitized normal human erythrocytes (E) were shown to be lysed when incubated with autologous serum in the presence of zymosan (Zy). The hemolysis proceeded slowly with a relatively constant rate for at least 24 h at 37 degrees C. It was shown that the hemolytic reaction is antibody independent and mediated by complement activation through the alternative pathway and that hemolysis is not due to the decay or inactivation of complement regulators present on the E membrane. The mechanism of the phenomenon was studied by use of several kinds of sera genetically deficient in C3, C5, C7 or C9. The reaction was found to be divided into two stages: in the first step, neither E, C5, C7 nor C9 but Zy, serum factors containing C3 and metal ions are necessary, and in the second step, neither C3 nor metal ions but E, C5, C7 and C9 are necessary. Thus, E seem to be lysed by reactive lysis induced by C5 convertase formed on Zy through alternative complement pathway activation.

Complement Activation↗
PubMed1987

[Alternative complement pathway activation by anti-band 3 antibodies].

Naturally occurring antibodies against the anion exchange protein of red cells (band 3 protein) can elicit in whole serum a strong C3b deposition to red cells under conditions which favor alternative complement pathway activation. Such a mode of opsonization calls for generation of an alternative C3 convertase nucleated by C3b covalently bound to anti-band 3. Senescent, but not young, red cells should also carry "C3b-anti-band 3" complexes, if clearance of in vivo aged red cells occurred by the same mechanism. We succeeded in isolating covalently linked complexes of C3b and IgG primarily from membranes of senescent red cells.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1998

C5 convertase of the alternative pathway of complement. Kinetic analysis of the free and surface-bound forms of the enzyme.

Although proteolytic activation of the complement protein C5 initiates important defensive and occasionally pathological inflammatory reactions, the enzymatic properties of the enzymes responsible for this cleavage have never been examined. We have studied the kinetic parameters of the C5 convertase of the alternative pathway of complement, either bound to a zymosan surface or in its monomeric soluble form. C5 convertase enzymatic activity was measured as a function of C5 concentration by quantitating production of C5b,6 under physiological conditions of temperature, pH, and ionic strength. The C5 convertases appeared to follow Michaelis-Menten kinetics and exhibited similar catalytic rate constants (kcat). However, the surface-bound enzyme, ZymC3b,Bb had a Km (1.4 microM) that was 17 times lower than that of the soluble monomeric form of the enzyme, C3b,Bb (Km = 24 microM). The kcat for the cell-bound enzyme, ZymC3b,Bb was 0.0048 s-1 and that for soluble C3b,Bb was 0.0110 s-1. Both forms of the enzyme had a low turnover number at Vmax (0.23 to 0.68 C5/min/enzyme). Substituting Mg2+ for Ni2+ did not alter the kinetic parameters but lowered the half-life of the enzyme by 5-7-fold. The kinetic data presented demonstrate that the fluid phase C5 convertase, C3b,Bb, can cleave C5 without the aid of a second C3b molecule. The results also show that the greater enzymatic activity previously observed for the surface-bound C5 convertases is not due to higher catalytic efficiency but is solely due to higher affinity for the substrate C5. In blood, C5 concentrations are 3-4-fold below the Km determined for the surface-bound C5 convertase suggesting a direct correlation between the local C5 concentration and production of the anaphylatoxin C5a and the cytolytic C5b-9 complex.

Cell Membrane↗
PubMed1986

Synthesis of complement components C5, C6, C7, C8 and C9 in vitro by human monocytes and assembly of the terminal complement complex.

Monocytes cultured under serum-free conditions secreted protein which bound covalently and non-covalently to agarose beads, an activator of the alternative pathway of complement. There was a significantly binding of monoclonal anti-C3c antibodies, polyclonal anti-C5, anti-C6, anti-C7, anti-C8, and anti-C9 antibodies, and of a monoclonal antibody against a neoantigen of polymerized C9 to agarose beads incubated with the monocytes for 24, 48, 72 or 96 h. From these results, we conclude that monocytes produce C5, C6, C7, C8 and C9 that assemble as the terminal complement complex on the surface of the agarose beads. Activation by agarose of the alternative pathway with generation of particle bound C3 and C5 convertases is a prerequisite for the subsequent formation of the terminal complement complex. Whether SC5b-9 or the membrane attack of complement (C5b-9) is formed on the beads will be examined.

Antibodies, Monoclonal↗
PubMed2000

Molecular mechanisms of target recognition in an innate immune system: interactions among factor H, C3b, and target in the alternative pathway of human complement.

In the alternative pathway of complement (APC) factor H is the primary control factor involved in discrimination between potential pathogens. The APC deposits C3b on possible Ags, and the interaction with factor H determines whether the initial C3b activates the APC. Factor H is composed of a linear array of 20 homologous short consensus repeats (SCR) domains with many functional sites. Three of these sites are involved in binding C3b and regulating complement activation; others bind to sialic acid and/or heparin and are responsible for host recognition. Using site-directed mutations we have examined the contributions of each of these sites to target discrimination and to functional activities of factor H. Decay acceleration by SCR1-4 of C3/C5 convertases bound to nonactivators was strongly dependent on SCR domains 11-15 and 16-20. Loss of these regions caused a 97% loss of activity, with SCR16-20 being the most critical (>90% loss). On APC activators the pattern of site usage was different and unique on each. On yeast, deletion of the 10 C-terminal domains (SCR11-20) had no effect on specific activity. On rabbit erythrocytes, this deletion caused loss of 75% of the specific activity. An examination of binding affinity to C3b on the four cell types demonstrated that factor H exhibits a unique pattern of SCR involvement on each cell. The results reveal a complex molecular mechanism of discrimination between microbes and host in this ancient innate defense system and help explain the different rates and intensities of APC activation on different biological particles.

Animals↗
PubMed1983

Effect of cycloheximide and of anti-C3 Fab' on the intrinsic synthesis and secretion of lysosomal enzyme and of complement components by guinea-pig peritoneal macrophages.

Without any additional stimulus, the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (beta-GLU) is secreted immediately from starch gel induced guinea-pig peritoneal macrophages. A more than three-fold increase of the total enzyme activity in the course of 3 days and the reversible inhibition of this enzyme increase by cycloheximide prove the synthesis of beta-GLU. The synthesis rate of beta-GLU remains rather constant after the second day, despite rapid cell death in the same culture period which could be explained by heterogeneity of the macrophage population. Cycloheximide immediately inhibits secretion of C3 whereas inhibition of beta-GLU secretion is only observed after a lag phase of 24 h. Secretion of factor D and of beta-GLU is not altered if secreted C3 is totally neutralized by the addition of anti-C3 Fab' to macrophage cultures. Thus, endogenous C3 as well as its endogenously generated fragments do not influence these macrophage functions. In the same cultures with anti-C3 Fab', conversion of secreted factor B into its fragments is inhibited as indicated by the detection of functional B activity. These results indicate that the secretion of factor D and of beta-GLU is also independent of endogenous B-derived fragments such as Bb. Finally, the detection of functional B in cultures with anti-C3 Fab' proves that C3b is required for factor B activation. The apparent interaction of secreted C3 and factors D and B in regular macrophage cultures suggests the constant formation of the labile C3 convertase C3bBb of the alternative pathway.

Acetylglucosaminidase↗