C1 inhibitor removes the entire C1qr2s2 complex from anti-C1Q monoclonal antibodies with low binding affinities.

Evidence is presented for a new C1 Inhibitor (C1 INH) function. C1 INH was capable of dislodging the entire C1qr2s2 complex from C1-activating substances that bound weakly to the globular heads of C1q. Two different mouse IgG1 monoclonal antibodies with different affinities for C1q globular heads were compared for their complement-activating properties in the presence of normal human serum. As expected the higher affinity monoclonal antibody (Qu) was more effective in binding C1q and causing C1-mediated C4b deposition. Unexpectedly, time responses of C1 (C1q) binding to immobilized 3C7 reached a peak then gradually decreased. However, C1q remained constantly bound to immobilized Qu. These results indicated that after C1 activation in human serum, the entire C1 complex (including C1q) was dislodged from 3C7, but not from immobilized Qu. The addition of purified C1 INH to purified C1, which had bound to immobilized 3C7, resulted in removal of C1 (C1q). Removal of the entire C1qr2s2 did not occur when C1 INH preparations were first neutralized by the addition of purified activated C1s. In summary, it is suggested that C1 INH plays a prominent role in dislodging the entire C1qr2s2 from immunoglobulin preparations which have a low binding affinity for the globular heads of C1q.     

Rat polymeric IgA binds C1q, but does not activate C1

Immune complexes, prepared with monoclonal rat IgA antibodies directed against DNP, activate the alternative pathway of the complement system in rat serum. In this study, the interaction of these monoclonal IgA antibodies with the classical pathway of complement was investigated. Monoclonal polymeric IgA (p-IgA) was shown to inhibit the IgG2b-mediated classical pathway-dependent lysis of TNP-coated sheep red blood cells. In addition, the binding of C3 to solid phase IgG2b immune complexes was inhibited by p-IgA. Monoclonal monomeric IgA (m-IgA) was much less efficient in this respect. To further analyse the effect of p-IgA on the activation of the classical pathway by IgG2b immune complexes, the interaction of p-IgA with C1 was studied. It was found that p-IgA antibodies bind C1q. No species-specificity was observed, since both rat and human C1q were bound. Whereas binding of C1q in C1 to IgG2b resulted in activation of C1, binding to p-IgA did not. The binding of C1q to both p-IgA and IgG2b could be inhibited by monoclonal antibodies directed against the globular heads of C1q, but not by monoclonal antibodies directed against the collagen tail. The formation of insoluble p-IgA immune complexes was inhibited in the presence of rat serum or C1. These studies indicate that C1q binds to p-IgA by its globular heads, and thereby may modulate classical pathway-mediated reactions such as the inhibition of immune precipitate formation.     

Biochemical and functional characterization of the interaction between pentraxin 3 and C1q

Pentraxin 3 (PTX3) is a recently characterized member of the pentraxin family of acute-phase proteins produced during inflammation. Classical short pentraxins, C-reactive protein, and serum amyloid P component can bind to C1q and thereby activate the classical complement pathway. Since PTX3 can also bind C1q, the present study was designed to define the interaction between PTX3 and C1q and to examine the functional consequences of this interaction. A dose-dependent binding of both C1q and the C1 complex to PTX3 was observed. Experiments with recombinant globular head domains of human C1q A, B, and C chains indicated that C1q interacts with PTX3 via its globular head region. Binding of C1q to immobilized PTX3 induced activation of the classical complement pathway as assessed by C4 deposition. Furthermore, PTX3 enhanced C1q binding and complement activation on apoptotic cells. However, in the fluid-phase, pre-incubation of PTX3 with C1q resulted in inhibition of complement activation by blocking the interaction of C1q with immunoglobulins. These results indicate that PTX3 can both inhibit and activate the classical complement pathway by binding C1q, depending on the way it is presented. PTX3 may therefore be involved in the regulation of the innate immune response.     

An amphioxus gC1q protein binds human IgG and initiates the classical pathway: Implications for a C1q‐mediated complement system in the basal chordate

The origin of the classical complement pathway remains open during chordate evolution. A C1q‐like member, BjC1q, was identified in the basal chordate amphioxus. It is predominantly expressed in the hepatic caecum, hindgut, and notochord, and is significantly upregulated following challenge with bacteria or lipoteichoic acid and LPS. Recombinant BjC1q and its globular head domain specifically interact with lipoteichoic acid and LPS, but BjC1q displays little lectin activity. Moreover, rBjC1q can assemble to form the high molecular weight oligomers necessary for binding to proteases C1r/C1s and for complement activation, and binds human C1r/C1s/mannan‐binding lectin‐associated serine protease‐2 as well as amphioxus serine proteases involved in the cleavage of C4/C2, and C3 activation. Importantly, rBjC1q binds with human IgG as well as an amphioxus Ig domain containing protein, resulting in the activation of the classical complement pathway. This is the first report showing that a C1q‐like protein in invertebrates is able to initiate classical pathway, raising the possibility that amphioxus possesses a C1q‐mediated complement system. It also suggests a new scenario for the emergence of the classical complement pathway, in contrast to the proposal that the lectin pathway evolved into the classical pathway.     

The interaction of 1-anilino-8-naphthalene sulphonate with human C1q

C1q has 12 binding sites for 1-anilino-8-naphthalene sulphonate (ANS), two per peripheral subunit. This number increases to 18 upon weak-acid-induced conformational transition in the globular heads. One ANS binding site is present in each C gamma 2 domain of human IgG. ANS is bound by C1q with a higher affinity (Ka = 2.07 X 10(6) M-1) than by the Fc fragment (Ka = 9.07 X 10(4) M-1) of human IgGl. Hence the inhibitory capacity of C1q binding to IgG immune complexes of ANS probably reflects its preferential binding to the globular heads of C1q. The characteristics of ANS-C1q binding may in part explain the hydrophobic component of the C1q-IgG interaction. It is suggested that an ionic-hydrophobic two-step process is involved in the contact between C1q and IgG.     

Recent progress in the understanding of the structure-function relationships of the globular head regions of C1q

The first step in the activation of the classical pathway of complement cascade by immune complexes involves the binding of the C-terminal globular head regions of C1q to the Fc regions of IgG or IgM, each globular head being composed of the C-terminal halves of one A-, one B- and one C-chain. Recent studies using recombinant forms of globular region appear to suggest that each globular head of C1q may be composed of three, structurally and functionally, independent domains/modules. The heterotrimeric organisation thus could offer functional flexibility and versatility to the whole C1q molecule. The crystal structure of an adipocyte-specific serum protein, Acrp-30, has revealed the existence of a structural fold shared by members of a new C1q/tumor necrosis factor (TNF) superfamily, characterized by a distinctive globular domain. The protein members seem to be active as self-assembling noncovalent trimers, whose individual chains fold as compact ‘jellyroll' b sandwiches. The recognition of a C1q/TNF superfamily, which has wide-ranging functions, highlights the possibility that the globular regions of C1q may fulfill more binding functions than previously envisaged.     

The distortive mechanism for the activation of complement component C1 supported by studies with a monoclonal antibody against the "arms" of C1q

A mouse monoclonal antibody (IgG1 isotype) against human C1q (MAb 130) is presented that activates C1 in serum through its antigen-binding sites at an optimal molar ratio of 3 MAbs:1 C1q. The antibody does not inhibit binding of C1q to IgG. Experiments with pepsin- and collagenase-digested C1q showed that MAb 130 binds to the fibril-like strands (arms) of C1q, close to the globular heads. Bivalency of MAb 130 was a requirement for C1-activation, but not for binding to C1q. Increasing the segmental flexibility of the intact antibody by reduction and alkylation destroyed its capacity to activate C1. A MAb against the globular heads of C1q completely inhibited C1-activation by aggregated IgG (AHG), but did not prevent activation by MAb 130. C1, reconstituted by adding C1q-stalks that lack the globular heads to C1q-depleted serum was not activated by AHG, whereas activation by MAb 130 was not affected. Activation of serum-C1 by AHG and MAb 130 was inhibited by addition of excess purified C1-inhibitor in a comparable and dose-dependent manner. Sucrose-gradient analysis indicated a predominance of stable complexes of a single C1q-molecule with three MAbs at the optimal activating ratio. When isolated and added to C1q-depleted serum, these complexes activated C1 efficiently. A mechanism for activation by MAb 130 is proposed that supports the "distortive" model of C1-activation.     

Development of a humanized C1q A chain knock-in mouse: assessment of antibody independent beta-amyloid induced complement activation

Evidence has been accumulating for a role of inflammation in the development of Alzheimer's disease (AD), a progressive neurodegenerative disorder causing a common form of dementia in the elderly. C1q, part of the initiation component of the classical complement pathway (CCP), is associated with beta-sheet, fibrillar amyloid plaques in AD brain. In vitro, beta-amyloid peptide in fibrillar beta-sheet conformation (fAbeta) can activate CCP via interaction of specific negatively charged amino acids of the beta-amyloid fibril with human C1q. Previous results using peptide inhibitors led to the hypothesis that a highly positively charged domain consisting of three arginine residues, such as that present in the N-terminal collagen-like region of the human C1q A chain, may be critical for the activation event. However, mouse C1q A chain lacks two of the three arginines in the corresponding C1q A chain collagen-like region. To test the hypothesis that this divergent activation domain results in a weaker C' activation and thus may contribute to the lower neuronal loss observed in transgenic mouse models of AD, a partially humanized C1q A chain knock-in mouse was generated. The mouse C1q A chain gene was modified by homologous recombination to replace 4 residues in the 13-20 amino acid region to mimic the corresponding sequence from human A chain. No significant differences in the expression of C1q were found in sera from mice homozygous for the humanized C1q A chain compared to littermate wild type mice. Two distinct C1 activation assays demonstrated that activation by fAbeta was not significantly different in the homozygous humanized C1q A chain mice. Activation of C1 by DNA, previously hypothesized to interact with this C1q A chain arginine-rich sequence was also not significantly different in the knock-in mouse. Molecular modeling based on the published crystal structure of human C1q B chain globular head and a beta-sheet model for fibrillar amyloid suggests an alternative arginine ladder in the globular head domain may provide the functional C1 activating interaction domains. The humanized C1q mouse generated here should provide a better animal model for assessing the mechanisms of C1 activation and the contribution of C1q to human health and disease.     

Direct binding of C1q to apoptotic cells and cell blebs induces complement activation

Deficiency of early components of the classical pathway of complement, particularly C1q, predisposes to the development of systemic lupus erythematosus. Several studies have suggested an association between the classical complement pathway and the clearance of apoptotic cells. Mice with a targeted deletion of the C1q gene develop a lupus-like renal disease, which is associated with the presence of multiple apoptotic bodies in the kidney. In the present study we demonstrate that highly purified C1q binds to apoptotic cells and isolated blebs derived from these apoptotic cells. Binding of C1q to apoptotic cells occurs via the globular heads of C1q and induces activation of the classical complement pathway, as shown by the deposition of C4 and C3 on the surface of these cells and on cell-derived blebs. In addition, for the first time, we demonstrate that surface-bound C1q is present on a subpopulation of microparticles isolated from human plasma. Taken together, these observations demonstrate that C1q binds directly to apoptotic cells and blebs derived therefrom and support a role for C1q, possibly in concert with C4 and C3, in the clearance of apoptotic cells and blebs by the phagocytic system.     

Studies on the mechanisms of allergen-induced activation of the classical and lectin pathways of complement

Allergen extracts are efficient activators of the complement system trough the classical pathway. Involvement of the lectin pathway was not previously studied. To further examine the mechanism of complement activation by allergens, in vitro experiments, which covered early steps both of classical and lectin pathways, were performed. Two types of allergens used in these studies: parietaria (PA) and house dust (HD) mite extracts. These allergen extracts bound to the globular head of C1q and interacted with purified mannan-binding lectin (MBL) as measured by solid-phase ELISA. None of the allergen extracts was able to activate human C1 in vitro, as measured by the determination of the split products of C1s in a reconstituted precursor C1 preparation.Neither the HD nor the PA extracts induced C4d generation above background in the serum of three subjects with hypogammaglobulinaemia but normal complement haemolytic activity. After reconstitution to normal level with purified human IgG, allergen extracts induced C4d formation above control at a level comparable to that measured in normal serum incubated with the same amounts of the extracts. HD-induced C4d generation was about the same comparable in MBL-depleted serum and in normal sera. In contrast PA induced no C4d formation in the MBL-depleted serum, whereas reconstitution with purified MBL restored C4d generation.These in vitro findings indicate that although the allergen extracts can bind purified C1q and MBL, they require IgG for efficient complement activation. Depending on the allergens, this activation may be initiated through C1, MBL, or both.     

Immunoblot analysis of IgG subclasses of circulating antibodies in bullous pemphigoid.

Sera from 20 patients with bullous pemphigoid (BP) were analyzed for the subclass distribution of IgG autoantibodies by Western immunoblotting of normal human heat-separated epidermal extracts. Strips were sequentially probed with patient sera, monoclonal antibodies (Mab) anti-human IgG1-4, and a labeled anti-mouse Ig. The relative dilutions of each subclass-specific Mab were determined in dot immunobinding experiments in order to give uniform reactivity to the corresponding human IgG subclass. Circulating BP autoantibodies reacting with the major BP antigen of 220 kDa and/or a 165-kDa band showed a stricking predominance of IgG4. Overall, IgG4 was the major subclass reactive with the 220-kDa band, whereas, in addition to IgG4, IgG1 and IgG2 were frequently represented in the response to the 165-kDa band. Complement fixation of circulating BP antibodies was studied by complement indirect immunofluorescence using human serum as source of complement and FITC goat anti-human C3, C1q, and C4, on normal human skin substrate. When circulating BP antibodies contained IgG1 or IgG2 in addition to IgG4, they fixed C3 and, in some cases, C1q and C4, suggesting complement activation by either the classical or the alternate pathway.     

The complement subcomponent C1q mediates binding of immune complexes and aggregates to endothelial cells in vitro

The present studies were initiated to investigate whether soluble immune complexes, upon interaction with complement, can bind to endothelial cells. Human umbilical vein endothelial cells (HUVE) were incubated with purified human 125I-labeled C1q at 4 degrees C in RPMI-0.5% bovine serum albumin and assayed for binding. Optimal binding of 125I-labeled C1q to HUVE was reached within 2 h, and saturation of binding was found at concentrations of 5 micrograms/well input. The binding of 125I-labeled C1q was inhibitable with unlabeled C1q and by the collagenous region of pepsin-cleaved C1q. No inhibition was observed with the globular heads of C1q, suggesting that C1q binds to HUVE via the collagenous region of C1q. When HUVE were first reacted with various concentrations of C1q, washed and subsequently incubated with 125I-labeled aggregated human IgM (AIgM), binding of 125I-labeled AIgM to HUVE occurred depending on the dose of C1q. Only those aggregates of IgM which react with C1q in a solid-phase C1q binding assay were able to bind to HUVE presensitized with C1q. In addition it was shown that C1q mediated binding of aggregated IgG to HUVE. Furthermore, immune complexes (IC), that were prepared with bovine thyroglobulin (BTg) and rabbit anti-BTg, bound to C1q-preincubated HUVE. These studies suggest that localization of IC on endothelium can be enhanced following interaction of the IC with complement.     

Deposition of complement activation products on plastic-adsorbed immunoglobulins. A simple ELISA technique for the detection of defined complement deficiencies

The activation of complement components in human serum has been studied using immunoglobulins adsorbed to microtiter plates. The sequential deposition of complement fragments was detected by a series of mono- and polyclonal antibodies in an indirect enzyme-linked immunosorbent assay (ELISA). Antibodies against C1q, C1s, C4b/d, C3b/d, factor B, C5b-9 membrane attack complex (MAC), the regulatory complement proteins C4 binding protein (C4bp) and properdin were reactive. Several lines of evidence suggest that complement activation was via the classical pathway: (1) complement activation was highly isotype-restricted with regard to the adsorbed Igs (human IgG1 and IgG3 as well as mouse IgM, IgG2a and IgG2b isotypes are strong activators in contrast to human IgG2, IgG4, IgA and mouse IgG1); (2) Ca2+ depletion, heat treatment (56 degrees C for 45 min), incubation with 0.5 M KSCN or heat-aggregated immunoglobulins (aggIgG) abrogated serum activity; (3) complement deficient sera (C1q def', C2 def', C6 def' human sera; C2 def', C4 def' guinea pig sera) showed impaired deposition of the complement components that follow the missing component in the cascade of activation. In a clinical study sera from patients with systemic lupus erythematosus (SLE) were investigated in order to measure the effect of hypocomplementemia due to complement consumption. The results obtained suggest that this new and simple assay is well suited for (1) the detection of various inherited complement deficiencies, (2) the semiquantitative evaluation of sera with decreased complement levels, (3) a more detailed study of complement components bound to a solid phase.     

Interaction of the envelope glycoprotein of human immunodeficiency virus with C1q and fibronectin under conditions present in human saliva

Human saliva has been shown to reduce the infectivity of human immunodeficiency virus (HIV) particles in vitro. The factors in human saliva involved in this inhibition of HIV infectivity are unknown, although the salivary sediment of normal individuals has the major HIV neutralizing activity. Interestingly, the first complement component (C1) has been detected on the surface of the salivary sediment in the whole saliva of normal individuals. At the relatively low ionic strength of saliva, we determined that purified human C1q bound with high affinity to the envelope glycoprotein of HIV. Normally, the interaction of the C1q globular heads with immune complexes causes C1 activation. However, direct interactions between C1 and rgp120 (or rgp160) did not lead to C1 fixation, as determined by hemolytic studies with rate-limiting levels of C1, nor did rgp120 cause C1 activation as determined by activated C1s-mediated C4 conversion in normal human serum. Using ELISA, it was observed that intact C1, with the C1r2C1s2 tetramer associated with the collagen-like stem of C1q, did not bind to immobilized rgp120, whereas free C1q did bind. In addition, digestion of the C1q stem portion with collagenase completely eliminated its binding to rgp120. These findings suggest that the collagen-like stem region of C1q, rather than the globular heads, may participate in the binding to the envelope glycoprotein of HIV. Fibronectin, which is present in submandibular saliva, appeared to bind to rgp120 and to enhance the interaction of C1q with rgp120. It is conceivable that C1q and fibronectin, in binding and sequestering HIV particles (i.e. to the salivary sediment), may play an important role in the reduction of HIV transmission via saliva. Further studies will be needed to test the latter speculation.     

Activation of the Classical Pathway of Complement by Non-Immune Complexes of Immunoglobulins with Human Protein Fv (Fv Fragment-Binding Protein)

Protein Ev, a human sialoprotein recently described in the stools of patients suffering from liver diseases, binds the variable domain of the heavy chains of immunoglobulins. We show here that preincubation of this protein with monoclonal human IgG1 and IgM activates the complement cascade by forming nonimmune complexes, as evidenced by haemolysis inhibition of antibody-coated sheep erythrocytes. As negative controls, no inhibition was observed after incubation either with immunoglobulins or with protein Fv alone, and with the protein Ev-depleted medium. Activation was due to the binding of immunoglobulins with protein Fv, as shown by inhibition of protein Fv-induced agglutination of the sensitized erythrocytes in the absence of complement. Activation of the classical pathway was demonstrated both by using a buman IgG4 or F(ab)₂ fragments unable to activate C1q, and by Western blot analysis of the cleavage of C4 in human serum. These results confirm that protein Fv-binding mimicks antigen-antibody reactions, and suggest its involvement in hepatitis-associated vasculitis and in local lesions of some infiammatory gut diseases.     

Binding and activation of human precursor C1 by soluble aggregates of human and rabbit IgG

The capacities of soluble human and rabbit IgG aggregates to bind and to activate human C1 were compared. Aggregates prepared by incubation of purified IgG at 63 degrees C were fractionated by gel filtration and hemolytic assays were used to measure the binding and activation of isolated human precursor C1. The C1 binding and activation capacities of both human and rabbit IgG aggregates were highly dependent on their size. Human IgG aggregates had a slightly higher binding avidity for human C1 than rabbit IgG aggregates of comparable size, but no clear differences were found between their capacities to activate C1. Experiments with nonaggregated IgG also indicated that although human IgG binds human C1 somewhat more avidly, human and rabbit IgG do not differ in their capacities to initiate fluid-phase activation of the human classical complement pathway.     

The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation

Complement activation with formation of biologically potent mediators like C5a and the terminal C5b-9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway-induced C5 activation than previously recognized and used pathway-selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that selective blockade of the alternative pathway by neutralizing factor D in human serum diluted 1 : 2 with mAb 166-32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid- and fluid-phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation.     

Complement-coated antibody-transfer (CCAT); serum IgA1 antibodies intercept and transport C4 and C3 fragments and preserve IgG1 deployment (PGD)

In periodontal disease, IgG1 and IgA1 antibodies produced in situ deposit on antigens in the affected tissues. Thus, there is an interest in the effect of co-deposited IgA1 antibodies on complement activation by IgG1-immune complexes. In the present study, we first analyzed the effect of IgA1-immune complexes on complement using human IgA1 antibodies to dansyl (with dansylated human serum albumin serving as the immobilized antigen). It was observed that these IgA1-immune complexes when incubated for prolonged times with 33% human serum as a source of complement received C4b and C3b deposition. As C4b and C3b deposited on the IgA1 antibodies and on the antigenic surface, the complement-coated IgA1 antibodies departed. These fluid-phase complement-coated IgA1 antibodies were transferred to antigen-coated microtiter-ELISA plates, where they became bound to the antigens. Thus, the complement-coated IgA1 antibodies retained their antigen-binding function, especially as a proportion of their covalently bound C3b progressively degraded to iC3b and C3d. Genetically engineered carbohydrate-deficient mutant human IgA1 antibodies were used to assess the role of carbohydrate in accepting the C4b and C3b depositions, and these studies indicated that the carbohydrate on the Fc-region of IgA1 played a positive role. Another interesting finding generated by this study was that when IgA1 was co-deposited with IgG1 antibodies, and serum complement was added, the IgG1 antibodies tended to remain on the antigenic surface. The co-deposited IgA1 antibodies not only controlled (reduced) the rate of the consumption of the first component of complement (C1) and of classical complement pathway activation by IgG1-immune complexes (and therein reduced the rate of complement-mediated dissolution of the IgG1-immune complexes), but also the co-deposited IgA1 antibodies simultaneously intercepted/accepted C4b and C3b, then departed, as complement began to cover the antigenic surfaces. The process in which complement-coated IgA1 antibodies transferred to non-complement-coated antigens is termed complement-coated antibody-transfer/transport (CCAT). In this way, IgA1 antibodies extended the efficiency of the complement system by insuring the specific IgA1 antibody-mediated transport of the captured biologically active complement fragments to those antigens stimulating the IgA1 antibody response but not yet neutralized (completely coated) with complement. Simultaneously by impeding the rate of C1 consumption and by intercepting C4b and C3b, IgA1 antibodies slowed C4b and C3b deposition on the antigenic surface and on the co-deposited IgG1 antibodies. Thus, in the presence of ongoing complement activation, the deposition of serum IgA1 antibodies enabled the co-deposited IgG1 antibodies to better maintain their ability to interact with antigens. We termed this latter phenomenon, preservation of IgG antibody deployment (PGD). In summary, co-deposited IgA1 antibodies maximized the efficiency of the complement system, transported their covalently bound complement fragments to specific antigens and sustained the effective deployment of IgG1 antibodies directed to those same antigens.     

Binding of the human complement subcomponent C1q to hybrid mouse monoclonal antibodies

Activation of the classical pathway of the complement system is initiated by the binding of C1q to antibody complexes. Here we evaluated the C1q binding capacity of series of monospecific and bispecific hybrid mouse monoclonal antibodies (mAb) and compared them with parental (conventional) mAb. The hierarchy in C1q binding capacity of the bispecific anti-HuIgA1/HRP mAb with homologous H-H chain combinations (IgG2a-2a, IgG2b-2b and IgG1-1) and the parental anti-HuIgA1 or anti-HRP mAb was identical; IgG2a greater than IgG2b much greater than IgG1. Hybrid IgG1-2a mAb bind intermediate amounts of C1q when compared with the IgG1 and IgG2a parental antibodies. IgG1-2b and IgG1-1 hybrid mAb did not bind any C1q, like the IgG1 mAb. We could not observe any difference in C1q binding efficiency between monovalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 HRP mAb and the bivalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 mAb, respectively. Furthermore, these hybrid ms anti-HuIgA1 and bs anti-HRP/HuIgA1 mAb were able to lyse HuIgA1-coated erythrocytes, in the presence of 50% human serum, as efficiently as their parental counterparts. These data indicate that a simultaneous binding of both F(ab') fragment to antigen is not a necessary prerequisite for binding and activation of C1q.     

Lysine 322 in the human IgG3 C(H)2 domain is crucial for antibody dependent complement activation

The classical complement activation cascade of the immune system is initiated by multivalent binding of its first component, C1q, to the Fc region of immunoglobulins in immune complexes. The C1q binding site on mouse IgG2b has been shown to contain the amino acids Glu 318, Lys 320 and Lys 322 in the C(H)2 domain (Duncan, A.R., Winter, G.,1988. The binding site for C1q on IgG. Nature 322 738-740). Identical or closely related motifs are found on all IgGs in all species, and the binding site has therefore been thought to be universal. However, the results from another study indicate that the site is different in human IgG1 molecules (Morgan, A., Jones, N.D., Nesbitt, A.M., et al., 1995. The N-terminal end of the C(H)2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding. Immunology 86 319-324). To determine the site(s) responsible for complement activation in anti-NIP-mouse/human IgG3 antibodies, we have mutated amino acids Lys 276, Tyr 278, Asp 280, Glu 318, Lys 320 and Lys 322 in two beta-strands in the C(H)2 domains of human IgG3. In addition, we mutated the Glu 333, which resides in close proximity to the postulated C1q-binding site of mouse IgG2b, as well as Leu 235 in the lower hinge region. All mutants were tested in Antibody Dependent Complement Mediated Lysis (ADCML)(4) assays, where the antigen concentration on target cells was varied and human serum was complement source. Only the mutants that lacked the positively charged side chain of lysine in position 322 showed strong reduction in ADCML, particularly at low antigen density on target cells. Alanine scanning of positions 318 and 320 did not affect ADCML, contrary to what was observed for mouse IgG2b. Neither did a leucine to glutamic acid mutation in position 235 have the effect that has been reported for human IgG1. These results suggest that the complement binding site on human IgG3 molecules is different from that found on mouse IgG2b, and possibly on human IgG1 as well. Thus the contact site may not be conserved.