The covalent binding reaction of complement component C3

The covalent binding of C3 to target molecules on the surfaces of pathogens is crucial in most complement-mediated activities. When C3 is activated, the acyl group is transferred from the sulfhydryl of the internal thioester to the hydroxyl group of the acceptor molecule; consequently, C3 is bound to the acceptor surface by an ester bond. It has been determined that the binding reaction of the B isotype of human C4 uses a two-step mechanism. Upon activation, a His residue first attacks the internal thioester to form an acyl-imidazole bond. The freed thiolate anion of the Cys residue of the thioester then acts as a base to catalyze the transfer of the acyl group from the imidazole to the hydroxyl group of the acceptor molecule. In this article, we present results which indicate that this two-step reaction mechanism also occurs in C3.     

Activation of the alternative pathway of human complement by apoptotic human umbilical vein endothelial cells

Complement activation generally does not occur on homologous cells. We observed C3 deposition on cultured human umbilical vein endothelial cells (HUVEC) when those which had died of apoptosis were treated with human serum. The C3 deposition on apoptotic HUVEC required Mg2+ but not Ca2+. In addition, the incubation of apoptotic HUVEC with purified C3, B, and D in the presence of Mg2+ resulted in C3 deposition. These results indicated that the C3 deposition on apoptotic HUVEC is mediated by the activation of the alternative complement pathway. C3 contains an intrachain thioester bond in the alpha chain (110 kDa) and upon activation to C3b, binds with membrane molecules by forming an ester or amide bond. Western blotting of reduced C3b-membrane molecule complexes, isolated from serum-treated apoptotic HUVEC by immunoprecipitation with anti-C3, revealed the covalent binding of C3b to several membrane molecules. Most of the C3b-membrane molecule complexes were cleaved by hydroxylamine, suggesting covalent binding via an ester bond. The molecular mass of the major alpha chain fragment released by hydroxylamine treatment was not 105 kDa but 68 kDa, which corresponds to the alpha 1 fragment of iC3b. These results indicate that most of the C3b on HUVEC was cleaved at its alpha' chain to yield iC3b, which consists of three disulfide-linked polypeptide chains and is a ligand of the complement receptor type 3 (CR3) of phagocytes. These results suggest that apoptotic HUVEC can activate the alternative pathway of the homologous complement and that the complement is related to the clearance of apoptotic cells by phagocytes.     

Opsonic complement component C3 in the solitary ascidian, Halocynthia roretzi

The recent identification of two mannose-binding lectin-associated serine protease clones from Halocynthia roretzi, an ascidian, suggested the presence of a complement system in urochordates. To elucidate the structure and function of this possibly primitive complement system, we have isolated cDNA clones for ascidian C3 (AsC3) and purified AsC3 protein from body fluid. The deduced primary structure of AsC3 shows overall similarity to mammalian C3, including a typical thioester site with the His residue required for nucleophilic activation of the thioester. AsC3 has a two-subunit chain structure, and the alpha-chain is cleaved at a specific site near to the N terminus upon activation. Ascidian body fluid contains an opsonic activity which enhances phagocytosis of yeast by ascidian blood cells, and Ab against AsC3 inhibits this opsonic activity. These results indicate that the complement system played a pivotal role in innate immunity by enhancing phagocytosis before the emergence of the vertebrates and well ahead of the establishment of adaptive immunity, which is believed to have occurred at about the time of the appearance of cartilaginous fish.     

X-ray crystal structure of C3d: a C3 fragment and ligand for complement receptor 2

Activation and covalent attachment of complement component C3 to pathogens is the key step in complement-mediated host defense. Additionally, the antigen-bound C3d fragment interacts with complement receptor 2 (CR2; also known as CD21) on B cells and thereby contributes to the initiation of an acquired humoral response. The x-ray crystal structure of human C3d solved at 2.0 angstroms resolution reveals an alpha-alpha barrel with the residues responsible for thioester formation and covalent attachment at one end and an acidic pocket at the other. The structure supports a model whereby the transition of native C3 to its functionally active state involves the disruption of a complementary domain interface and provides insight into the basis for the interaction between C3d and CR2.     

Rapid activation of the complement system by cuprophane depends on complement component C4

Hemodialysis with cuprophane dialyzer membranes promotes rapid activation of the complement system, which is thought to be mediated by the alternative pathway. Complete hereditary deficiency of complement C4, a classical pathway component, in two hemodialysis patients provided the opportunity to investigate a possible role of the classical pathway. In two hemodialysis patients with both C4 isotypes, C4A and C4B, and in one patient with C4B deficiency complement activation occurred immediately after the onset of hemodialysis, with peak levels of C3a and terminal complement complex (TCC) after ten to fifteen minutes. In patients with complete C4 deficiency, C3a and TCC remained unchanged for fifteen minutes and increased thereafter, reaching the highest level after thirty minutes. The leukocyte nadir was also delayed from fifteen to thirty minutes. In vitro incubation of normal, C4A- or C4B-deficient serum with cuprophane caused complement activation after fifteen minutes. In contrast, no activation was observed in sera of four C4-deficient patients. The addition of normal serum or purified human C4 restored the capacity for rapid complement activation. In one patient with severe immunoglobulin deficiency, C3a and TCC levels increased only moderately after 25 minutes of cuprophane dialysis. This patient's serum also exhibited delayed complement activation in vitro, which was normalized after pretreatment of cuprophane with immunoglobulins. Preincubation of normal serum with MgEGTA, a blocker of the classical pathway, inhibited rapid complement activation through cuprophane. As basal levels of C4a are markedly increased in hemodialysis patients (3450 +/- 850 ng/ml) compared to healthy controls (224 +/- 81 ng/ml), no further elevation of C4a was detectable during cuprophane hemodialysis. Incubation of normal serum with cuprophane, however, caused a slight increase in C4a after five minutes. These results indicate that the initial deposition of complement C3b on the cuprophane membrane, necessary for activation of the amplification loop of the alternative pathway, is mediated by the classical pathway C3-convertase C4b2a. We propose an extended concept of complement activation through cuprophane, which is based on four steps: (a) binding of anti-polysaccharide antibodies, (b) classical pathway activation, (c) alternative pathway activation and (d) terminal pathway activation.     

Multiple forms of complement C3 in trout that differ in binding to complement activators

In all other species analyzed to date, the functionally active form of complement component C3 exists as the product of a single gene. We have now identified and characterized three functional C3 proteins (C3-1, C3-3, and C3-4) in trout that are the products of at least two distinct C3 genes. All three proteins are composed of an alpha-and a beta-chain and contain a thioester bond in the alpha-chain. However, they differ in their electrophoretic mobility, glycosylation, reactivity with monospecific C3 antibodies, and relative ability to bind to various surfaces (zymosan, Escherichia coli, erythrocytes). A comparison of the partial amino acid sequences of the three proteins showed that the amino acid sequence identity/similarity of C3-3 to C3-4 is 87/91%, while that of C3-3 and C3-4 to C3-1 is 51.5/65.5% and 60/73% respectively. Thus, trout possess multiple forms of functional C3 that represent the products of several distinct genes and differ in their ability to bind covalently to various complement activators.     

Modulation of antigen processing and presentation by covalently linked complement C3b fragment

Ligands such as complement fragments (C3, C4), IgG or alpha 2-macroglobulin, which bind antigen (Ag) before their uptake by antigen-presenting cells (APC), are likely to modulate the different steps of Ag processing and presentation. These ligands contribute to internalization and endosomal targeting of Ag; they also influence its processing and, consequently, the binding of resulting peptides to major histocompatibility complex (MHC) class II molecules before presentation to T cells. Complement protein C3 contains, like other members of the alpha 2-macroglobulin family, an intrachain thiolester bond. Conformational alteration or limited proteolysis of C3 into C3b leads to breaking of the thiolester with transient capacity of the revealed carbonyl group to esterify hydroxyl groups of Ag. Ester-linked complexes including tetanus toxin (TT) and C3b were prepared to analyse the influence of bound C3b on TT processing and presentation by APC. Covalent binding of C3b to TT resulted in increased and prolonged stimulation of specific T-cell proliferation. This effect was observed with non-specific B cells, as well as with a TT-specific B-cell clone, as APC. On the other hand, SDS-PAGE analysis of proteolysates of TT or C3b-TT, obtained with endosome/lysosome-enriched subcellular fractions prepared from human Epstein-Barr virus (EBV)-transformed B cells, indicated a delay of TT proteolysis when TT was associated to C3b. Treatment of APC with protease inhibitors, before and during exposure of the cells to Ag, resulted in differences in the inhibition of TT and C3b-TT proteolysis. Using purified cathepsins B and D, we demonstrated that covalent binding of C3b to TT totally abolished TT proteolysis by cathepsin D, while proteolysis by cathepsin B was preserved. This finding and the absence of cathepsin B in endosomes may explain a delay in TT processing when it is associated to C3b. Confirming these data, presentation by formaldehyde-fixed cells of C3b-TT proteolysates showed higher stimulation of specific T-cell clones than formaldehyde-fixed TT proteolysates.     

Complement (C3, C4) and C-reactive protein responses to cardiopulmonary bypass and protamine administration

Complement activation has been deemed responsible for the damaging effects of cardiopulmonary bypass (CPB) in patients undergoing open heart surgery. We studied C3, C4 and C-reactive protein (CRP) in 22 patients undergoing CPB. In Group 1 (11 patients), protamine was given intravenously and in Group 2 (11 patients), via the aortic root after CPB. Significant decreases were observed in C3 and C4 during CPB in both groups indicating complement activation primarily by the classic pathway. Protamine did not lead to further activation of the complement system. In both groups, C3 levels gradually returned toward baseline within 24 hours but C4 levels were still lower than baseline 24 hours postoperatively. CPB and protamine administration did not cause any significant changes in CRP levels, but CRP increased abruptly 24 hours after operation. Although activation of complement system during CPB is expected to invoke an acute phase response, we conclude that this period is not long enough to induce an increased production of CRP in response to tissue injury or inflammation.     

Alkylation of cellulosic membranes results in reduced complement activation

4-Vinyl pyridine was grafted to the surface of the cellulosic membrane Cuprophan, and subsequently alkylated with both C10 and C16 aliphatic chains. Complement activation of heparinized human blood, corrected for anaphylatoxin adhesion, was measured by radioimmunoassay. The surface treatments both yielded substantial reductions in C5a activity, with a lessor reduction in C3a and C4a activity. Alkylation with 10 and 16 carbon chains resulted both in enhancements of albumin adsorption and stability. These enhancements as well as the reductions in complement activation were statistically indistinguishable between the two treatments. The reduction in complement activation was influenced more by adsorption of endogenous albumin and possibly by the vinyl pyridine graft, than the removal of surface active hydroxyl groups from Cuprophan.     

Application of flow injection analysis in trace element determination of body fluids

Nucleophiles activated the catalytic actions of beta-galactosidases with neutral or positively charged substitutions for Glu-461. Aliphatic carboxylic acids increased the rate of hydrolysis of o-nitrophenyl beta-D-galactopyranoside if the pKa values of the carboxyl groups were > approximately 3.5. Amino compounds activated if their pKa values were < approximately 8.5. Imidazole, azide, and 2-mercaptoethanol also activated. Nucleophiles with high pKa values were able to activate the catalysis if the pH was high, and this showed that the lack of activation at pH 7.0 was because of protonation. Kinetic analysis showed that most of the nucleophiles that activated were bound to the active site, since the activation followed Michaelis-Menten type saturation kinetics. The binding seemed to be dependent upon the hydrophobicity; the longer the aliphatic chain, the stronger the binding. Gas-liquid chromatographic analysis showed that adducts of some type were formed during the reactions in the presence of many of the nucleophiles. Three of these adducts were purified and the nucleophiles were found beta-linked to D-galactose. This indicates that if an intermediate covalent bond is formed in the mechanism of beta-galactosidase action and if the nucleophile reacts to displace it, the intermediate covalent bond must have the alpha configuration and involve a group other than Glu-461.     

Domain-switched mouse IgM/IgG2b hybrids indicate individual roles for C mu 2, C mu 3, and C mu 4 domains in the regulation of the interaction of IgM with complement C1q

Although polymeric IgM and monomeric IgG are potent activators of the classical complement pathway, previous studies have indicated that monomeric IgM is inactive. To understand this and to examine the roles of the individual mu domains in complement activation, we created a set of IgM/IgG2b mouse chimeric Abs in which homologous domains of both Abs have been interchanged, either singly or together with adjacent domains. The monomer subunits (H2L2) of the resulting chimeras were analyzed for their capacities to bind C1q and to initiate complement-mediated lysis (CML) of haptenated erythrocytes. When C gamma 2 was flanked by C mu 4, the inherent C1q-binding activity of the C gamma 2 domain was lost. This demonstrates that C mu 4 can suppress the C1q-binding activity of the adjacent C gamma 2 domain, and suggests that C mu 4 may exert a similar effect on the C mu 3 domain in the IgM monomer subunit. When C mu 3 was located in an IgG2b background and potentially freed from the constraints imposed by the IgM background, the monomer was not able to bind C1q or initiate CML. This suggests that these activities are not expressed inherently in the C mu 3 domain. The transplantation of C mu 3 together with C mu 4 into the IgG background permitted polymer formation. This polymer was able to bind C1q, although neither the monomer nor the polymer forms were active in CML; conversely, all IgM polymers with a transplanted C gamma 2 domain were active in both C1q binding and CML, and demonstrated apparent Kd values similar to that of wild-type IgM.     

Therapy with monoclonal antibodies. II. The contribution of Fc gamma receptor binding and the influence of C(H)1 and C(H)3 domains on in vivo effector function

An in vivo model is used to define Fc motifs engaged by mAbs to deplete target cells. Human IgG1 and human IgG4 were very potent, and mutations within a motif critical for Fc gammaR binding (glutamate 233 to proline, leucine/phenylalanine 234 to valine, and leucine 235 to alanine) completely prevented depletion. Mouse IgG2b was also potent, and mutations to prevent complement activation did not impair depletion with this isotype, as previously shown for human IgG1. In contrast, a mutation that impaired binding to mouse Fc gammaRII (glutamate 318 to alanine) eliminated effector function of mouse IgG2b and also reduced the potency of human IgG4. To reveal potential contributions of domains other than C(H)2, domain switch mutants were created between human IgG1 and rat IgG2a. Two hybrid mAbs were generated with potencies exceeding anything previously seen in this model. While their mechanism of depletion was not defined, their activity appeared dependent upon interdomain interactions in the Fc region.     

A second serine protease associated with mannan-binding lectin that activates complement

The complement system comprises a complex array of enzymes and non-enzymatic proteins that is essential for the operation of the innate as well as the adaptive immune defence. The complement system can be activated in three ways: by the classical pathway which is initiated by antibody-antigen complexes, by the alternative pathway initiated by certain structures on microbial surfaces, and by an antibody-independent pathway that is initiated by the binding of mannan-binding lectin (MBL; first described as mannan-binding protein) to carbohydrates. MBL is structurally related to the complement C1 subcomponent, C1q, and seems to activate the complement system through an associated serine protease known as MASP (ref. 4) or p100 (ref. 5), which is similar to C1r and C1s of the classical pathway. MBL binds to specific carbohydrate structures found on the surface of a range of microorganisms, including bacteria, yeasts, parasitic protozoa and viruses, and exhibits antibacterial activity through killing mediated by the terminal, lytic complement components or by promoting phagocytosis. The level of MBL in plasma is genetically determined, and deficiency is associated with frequent infections in childhood, and possibly also in adults (for review, see ref. 6). We have now identified a new MBL-associated serine protease (MASP-2) which shows a striking homology with the previously reported MASP (MASP-1) and the two C1q-associated serine proteases C1r and C1s. Thus complement activation through MBL, like the classical pathway, involves two serine proteases and may antedate the development of the specific immune system of vertebrates.     

Pertussis toxin modification of PC12 cells lowers cytoskeletal F-actin and enhances norepinephrine secretion: involvement of protein kinase C and protein phosphatases

In order to understand the modifications of proteins produced by aldehydes of lipid peroxidation, [1-13C]-2(E)-hexenal, [1-13C]-4-oxopentanal, and a mixture of [1-13C]- and [2-13C]-4-hydroxynon-2(E)-enal were synthesized and the reaction of each of the labeled aldehydes with bovine serum albumin was analyzed by 13C NMR spectroscopy. Protein nucleophiles add to the 3-position of hexenal, and the resulting propanal moieties appear to undergo aldol condensation, form imine cross-links with lysyl residues, or lead to pyridinium rings. During the reaction of 4-oxopentanal with the lysyl residues of bovine serum albumin, only 1-alkyl-2-methylpyrrole and a possible intermediate leading to the pyrrole were observed. Hydroxypyrrolidine cross-links such as 25 could not be detected, leaving the pyrrole as the mediator of protein cross-linking. The Michael adducts are the major products in the reaction between 4-hydroxynon-2-enal and proteins. They exist almost exclusively in the cyclic hemiacetal form and do not appear to cross-link through imine formation with lysyl residues. A minor pathway involves the reaction of 4-hydroxynon-2-enal with the lysyl amino groups of protein resulting in 2-pentylpyrrole adducts that may mediate protein cross-linking. The Michael adducts appear not to be the direct source of the pyrrole, but the imine 32 and the enamine 35 are likely intermediates toward the five-membered ring.     

C1q augments platelet activation in response to aggregated Ig

Immune complexes and aggregated IgG (agg-IgG) induce platelet aggregation and the release reaction. Immune complexes also activate the complement system and interact with the complement component C1q. Since platelets possess both Fc and C1q receptors capable of signal transduction, the present study focused on the interaction between these binding sites and platelet activation. Subaggregating doses of agg-IgG (20-400 microg/ml) were identified for washed platelets from each of 11 healthy donors, and platelet aggregation was monitored in the presence or the absence of increasing concentrations of C1q (5-100 microg/ml). C1q produced a dose-dependent potentiation of platelet alphaIIb/beta3 integrin activation, platelet aggregation, and granule secretion when combined with low doses of agg-IgG. C1q alone was without effect. Maximal enhancement of agg-IgG-induced platelet activation was noted at C1q concentrations ranging from 50 to 100 microg/ml. The observed C1q-induced potentiation of platelet aggregation in response to agg-IgG was blocked by polyclonal antibody F(ab')2 directed against platelet binding sites recognizing the collagen-like domain of C1q (cC1qR) or by mAb Fab (IV.3) directed against platelet FcgammaRII receptors. These data suggest a cooperative interaction between platelet FcgammaRII and cC1q receptors and support a potential role for platelet cC1q receptors in pathologic platelet activation by circulating immune complexes often associated with in vivo thrombosis and thrombocytopenia.     

Immunoglobulins and complement in pleural effusions associated with bronchogenic carcinoma

Levels of IgG, IgA, IgM, the total haemolytic complement (CH50), and the individual components C1q, C3, C4, C6, and C7 were measured in 29 pleural effusions. Of these, 18 were associated with carcinoma of the bronchus and 11 were non-malignant effusions including empyemas. The level of IgG was significantly lower in the malignant group when compared with non-malignant effusions. The usefulness of measurements of IgG with respect to malignant effusions associated with carcinoma of the bronchus requires an expanded study to show whether it has any real diagnostic value. There were no significant differences in other immunoglobulins, the CH50, and individual complement components between the two groups. The identification of total haemolytic activity in the majority of effusions in both groups indicates that all nine components of the classical pathway of complement, including macromolecules such as C1, can be present in pleural fluids.     

His-357 of beta-galactosidase (Escherichia coli) interacts with the C3 hydroxyl in the transition state and helps to mediate catalysis

The His at position 357 of beta-galactosidase (Escherichia coli) was substituted by an Asp, an Asn, a Leu, and a Phe, and studies done with the substituted enzymes showed that the main role of His-357 is to stabilize the transition state by interacting with the C3 hydroxyl. The substituted enzymes were less stable to heat than was wild-type enzyme (40-90% of the activity was lost in 10 min at 52 degreesC compared to wild-type beta-galactosidase which lost no activity), but the gross physical properties of the substituted enzymes at normal temperatures were not changed. There were also no differences in the ability to bind or to be activated by Mg2+. The substitutions (except Asp) did not affect the pKa for binding substrate in the ground state, but the pKa of the kcat was altered as would be expected for a residue important for binding the transition state. Substitution by Asp may cause a conformational change at high pH values. Activation energy differences (Delta DeltaGS), as determined by differences in kcat/Km values, indicated that substitutions for His-357 caused significant destabilizations of the first transition state (for the step in which the galactoside bond is cleaved and the covalent reaction intermediate is formed). This resulted in decreases of up to 900-fold in k2 for the mononitrophenyl substrates. In contrast, the k3 values (which depend on the energy level of the second transition state) were not decreased as much (<90-fold). In some cases, the k3 values even increased (when Asn was substituted for His-357). The importance of His-357 for stabilization of the transition state was confirmed by studies with transition state analogue inhibitors that showed that His-357 forms strong specific interactions with the C3 hydroxyl of the galactose moiety of the transition state. Studies with substrate analogue inhibitors indicated that His-357 is probably not important for the binding of the substrates themselves.     

Double resonance probes for close frequencies

Halorhodopsin, isolated from Halobacterium salinarium cells incubated with tritiated palmitic acid, co-elutes with labeled palmitate in phenylsepharose CL-4B chromatography. Halorhodopsin-bound 3H-palmitate is not readily displaced by prolonged exposure to a large excess of detergents and by re-chromatography of radiolabeled halorhodopsin on phenylsepharose. On other hand, the association of labeled palmitate with purified halorhodopsin is not resistant to denaturation induced either by isopropanol/hexane or by SDS gel electrophoresis. We have tested the hypothesis that tightly associated palmitate is bound to halorhodopsin through a thioester bond, which is unstable in denaturing conditions. Using GC/MS, we have analysed the reaction products of native halorhodopsin with specific thioester reagents, thiols and NaBH4, which are inactive on free fatty acids. The results of this analytical approach indicate that there is no thioester bond between halorhodopsin and palmitic acid and that palmitic acid is associated with halorhodopsin as a free fatty acid.