First Component Of Human Complement Research Articles

Molecular mechanisms involved in the inactivation of the first component of human complement by Trypanosoma cruzi calreticulin

Trypanosoma cruzi ( T. cruzi), the agent of Chagas’ disease, the sixth most important neglected tropical disease worldwide, causes 50,000 deaths per year in Latin America. T. cruzi calreticulin (TcCRT), a highly pleiotropic chaperone molecule, plays important roles in several host/parasite interactions. Among other functions, we have previously shown that TcCRT, translocated from the endoplasmic reticulum to the area of flagellar emergence, binds human C1q and inhibits activation of the classical pathway in vitro. Based on a series of in vitro experiments, we propose here two mechanisms to explain how TcCRT inhibits the classical pathway at the initial stages of C1 (q, r, s) activation. First, TcCRT interacts in vitro with both solid phase bound active C1s and C1, but impairment of C4 activating capacity is evident only when the serine proteases are within the structural context of the macromolecular first component. Although C1s activity, in this context, is inhibited by TcCRT, the serine protease is not displaced from the C1 complex. Second, TcCRT prevents C1 formation, by interfering with the ability of the (C1r–C1s) 2 tetramer to bind C1q. These complement inhibitory effects are better explained by direct interaction of the parasite protein with C1, rather than by the TcCRT capacity to bind calcium, an essential element for the functional integrity of C1.

F(ab')2 antibody fragments against Trypanosoma cruzi calreticulin inhibit its interaction with the first component of human complement

Trypanosoma cruzi calreticulin (TcCRT), described in our laboratory, retains several important functional features from its vertebrate homologues. We have shown that recombinant TcCRT inhibits the human complement system when it binds to the collagenous portion of C1q. The generation of classical pathway convertases and membrane attack complexes is thus strongly inhibited. In most T. cruzi-infected individuals, TcCRT is immunogenic and mediates the generation of specific antibodies. By reverting the C1q / TcCRT interaction, a parasite immune evasion strategy, these antibodies contribute to the host/parasite equilibrium. In an in vitro correlate of this situation, we show that the Clq/TcCRT interaction is inhibited by F(ab')2 polyclonal anti-TcCRT IgG fragments. It is therefore feasible that in infected humans anti-TcCRT antibodies participate in reverting an important parasite strategy aimed at inhibiting the classical complement pathway. Thus, membrane-bound TcCRT interacts with the collagenous portion Clq, and this Clq is recognized by the CD91-bound host cell CRT, thus facilitating parasite internalization. Based on our in vitro results, it could be proposed that the in vivo interaction between TcCRT and vertebrate Clq could be inhibited by F(ab')2 fragments anti-rTcCRT or against its S functional domain, thus interfering with the internalization process.

Dynamic equilibria between subcomponents of CĪ, the first component of human complement

Clr̄ and Cls̄, the serine protease components of activated Cl, form a tetramer in the presence of Ca 2+. The stability of this tetramer is sufficient that its association with the third component, Clq, has been successfully treated as a reversible bimolecular equilibrium reaction [Siegel and Schumaker, Molec. Immun. 20, 53–66 (1983)]. We have used the fluorescence anisotropy ( A) of fluorescein-labeled Cls̄ (s ∗) to monitor assembly and subcomponent exchange in 0.15 mol/1 NaCl, 0.001 mol/l Ca 2+ 0.02 mol/l Tris, pH 7.4. Addition of q to r 2s 2 ∗ causes a small but measurable Δ A of 0.01–0.02. The response is too fast to measure at 37° but can be readily followed at 4° where t 1 2 = 0.6 min when [q] = [r̄ 2s̄ 2 ∗] = 0.5 μmol/l. The increase in A can be readily reversed by dilution or by addition of unlabeled Cls̄. Slow incremental addition of q to a solution of r̄ 2s̄ 2 ∗ produces a dose-dependent Δ A from which stoichiometry and dissociation constants can be derived. Measurements of K d as a function of temperature establish an inverse temperature dependence with Δ H = − 15 kcal/mol and a value of K d = 0.031 μmol/l at 37° († G = + 11, T† S = − 26 kcal/mol). Thus, the assembly process appears to be entropy-driven presumably due to the exclusion of structured water from protein-protein interfaces in the complex.

Degradation of type I and II collagen by human C1̄s

The activated first component of human complement, C1̄s, was shown to cleave type I and II collagen and gelatin. The proteolytic activity was heat labile and was inhibited by a monoclonal antibody (M241) which recognized light chain of active human C1s or by a serine protease inhibitor, DFP, but not by a chelating agent.

Calorimetric investigation of the domain structure of human complement C1.lovin.s: reversible unfolding of the short consensus repeat units

Cl-s is a multidomain serine protease that participates in Ca2+-dependent protein-protein interactions with other subcomponents of Cl, the first component of human complement. Proteolytically derived fragments that retain some of the functional properties of the parent protein have been isolated, and their thermal stability has been investigated by differential scanning calorimetry. Three endothermic transitions are observed in whole Cl-s near 37, 49, and 60 degrees C in 0.05 M Tris-HCl, pH 7.2, containing 0.22 M NaCl and 0.1 mM EDTA. The first (37 degrees C) and third (60 degrees C) transitions are also seen in Cl-s-A, a derivative comprised mainly of the intact nonenzymatic A chain. The second (49 degrees C) and third transitions are seen in Cl-s-gamma B, a fragment comprised of the intact B chain, disulfide linked to the C-terminal gamma region of the A chain. Thus, the first transition, which is alone stabilized by Ca2+, corresponds to the melting of the N-terminal alpha beta region of the A chain, the second to the melting of the catalytic B chain domain, and the third to the gamma region. The gamma region is comprised of two homologous short consensus repeat (SCR) motifs that are also found in several other complement and coagulation proteins. A new 24-kDa fragment, Cl-s-gamma, which contains these two SCRs, was isolated from plasmic and chymotryptic digests of Cl-s-A. Cl-s-gamma exhibits a reversible transition near 60 degrees C corresponding to the highest temperature peak in whole Cl-s and Cl-s-A.(ABSTRACT TRUNCATED AT 250 WORDS)

Restriction fragment length polymorphism of the C1 inhibitor gene in hereditary angioneurotic edema.

Hereditary angioneurotic edema (HANE) results from the deficiency of the inhibitor of the first component of human complement (C1-INH). It is inherited as an autosomal dominant trait. Heterogeneity of this defect has been shown at the protein and mRNA level. Southern blot analysis of genomic DNA was performed after digestion with six different restriction endonucleases in 24 families affected with type 1 HANE (low antigenic and functional C1-INH levels) and five with type 2 (low functional C1-INH levels and normal or elevated levels of dysmorphic C1-INH). Blots were hybridized with a C1-INH cDNA probe of 1,227 bp. With one enzyme (Pst I), two different patterns of restriction fragment length polymorphism (RFLP) were detected. One was present in one kindred with type 1 HANE and the other appeared the same in one type 1 and in one type 2 family, thus indicating that each RFLP resulted from a different mutation. Analysis of a total of 34 members of these three families suggested that the polymorphisms are tightly linked to the mutation responsible for the disease. Using a 170-bp probe we showed that the three different mutations leading to these polymorphisms are located in the same region of the C1-INH gene. These data suggest that different mutations in the same region of the C1-INH gene are responsible for C1-INH deficiency in these families. Most of these mutations are probably point mutations or other "minor" defects and do not appear to be due to major deletions or rearrangements.

Activation of human C1: analysis with Western blotting reveals slow self-activation.

The first component of human complement was separated from C1-INH by sucrose linear gradient ultracentrifugation. Activation of C1 was studied in the absence and presence of immune complexes; activation was monitored by SDS-PAGE and Western blot. When the partially purified native C1 preparation was incubated at 37 degrees C without immune complexes, activated C1s appeared after 30 min in the case of eightfold dilution with respect to the original serum, and after 45 min with 32-fold dilution. Kinetics of appearance of activated C1r was the same as that of activated C1s. From the following results, we concluded that spontaneous activation may be partially due to proteolytic enzymes contaminating the preparation: 1) a nonspecific protease inhibitor, PMSF, completely inhibited spontaneous activation but did not inhibit the activation of C1 by immune complexes; 2) alpha 2-macroglobulin partially inhibited spontaneous activation, and 3) although spontaneous activation in the absence of PMSF was relatively slow, activated C1 accelerated spontaneous activation that was completely blocked by C1-INH. In contrast to spontaneous activation, the partially purified native C1 was rapidly activated by immune complexes: within 5 min almost all C1 was activated by rabbit IgG anti-human IgM-human IgM complexes. These results support conclusions derived from activation studies when using native C1 and hemolytic assays, and do not support those derived from the activation studies with reconstituted C1 and SDS-PAGE analysis. We suggest that the contradictions can be resolved if one assumes that C1 activation can be both an intra- and intermolecular process; which process dominates is determined by the state of C1 and by experimental conditions.

Functional analysis of activated C1s, a subcomponent of the first component of human complement, by monoclonal antibodies.

Three mouse monoclonal antibodies (M365, M81, and M241) directed against human C1s were used to analyze the structure of C1s related to the enzymatic activity. M365 and M81 recognized different epitopes on the heavy chain of C1s and could bind to C1s, as well as to C1s. The C4 cleaving activity of C1s was completely blocked by M81 and was partially blocked by M365. Although the C2 cleaving activity of C1s was partially inhibited by M81, no blocking was observed with M365. Both antibodies had no effect on the esterolytic activity of C1s. These results indicate that the C4 and C2 binding sites on C1s reside in the heavy chain, and they are distinct from each other. M241 could bind only to C1s, an active form of C1s. After reduction of C1s, M241 could not react with either heavy or light chain of C1s. The esterolytic activity of C1s was markedly reduced by M241. Furthermore, M241 blocked not only the cleavage of C4 and C2 by C1s but also the complex formation of C1s and C1 inactivator. From these observations, we suggest that M241 reacts with the active site of C1s, and both heavy and light chains of C1s participate in the composition of the active site.

Activation of the first component of human complement, C1, by monoclonal antibodies directed against different domains of subcomponent C1q.

Two monoclonal antibodies directed against C1q, and their (Fab)2 and Fab fragments, were used to study the mechanism of C1 activation. Monoclonal antibody 2A10, an IgG2a, was digested by pepsin to yield fully immunoreactive (Fab')2. Monoclonal antibody 1H11, an IgG1, was digested by papain to yield fully immunoreactive, bivalent (Fab)2. Previously 1H11 had been shown to bind to the C1q "heads," whereas 2A10 bound to stalks. Activation of C1 was followed by the cleavage of 125I-C1s in the presence of C1 inhibitor (C1-Inh) at 37 degrees C. Spontaneous activation was minimal at inhibitor concentrations above 0.4 micron (1.3 X physiologic inhibitor concentration); all results were corrected for the spontaneous activation background. Heat-aggregated IgG activated completely in this system and was taken as 100% activation. Monoclonal antibody 2A10 caused precipitation of C1 and slow activation; neither the (Fab')2 nor the Fab' derived from 2A10-caused activation. Probably, aggregates of intact 2A10 and C1 were serving as immune complexes to activate other molecules of C1. In contrast, both 1H11 and its (Fab)2 activated completely and stoichiometrically; that is, maximal activation was achieved at a ratio of one C1q head to one antibody combining site. The monovalent Fab derived from 1H11 bound well to C1q, but no activation of C1 was observed. Thus, bivalent binding of this head-binding monoclonal is required for C1 activation, but not the presence of the antibody Fc portion. Neither 1H11 nor its (Fab)2 fragments caused C1 precipitation; however, the 1H11 did form complexes composed of two C1q cross-linked by multiple 1H11, which were visualized by electron microscopy. The presence of these dimeric complexes correlated well with activation. A model for C1 activation is proposed in which two C1q subcomponents are held together by multiple (Fab)2 bridging C1q heads. The model is roughly analogous to touching opposing pairs of fingers and thumb tips, the two hands representing the two C1q, forming a cage. C1-Inh, which probably binds to C1r through the open end of the C1 cone, is too long asymmetric to be included within the cage. Thus, according to this model, the dimers of C1 are released from the inhibitory action of C1-Inh, and activation proceeds spontaneously and rapidly at 37 degrees C.

Functional model of subcomponent C1 of human complement

The domain organization of the zymogen subunits of the first component of human complement † † The nomenclature of complement is that recommended by the World Health Organization (1968); activated components are indicated by a bar, e.g. CIs. Cls, Clr 2 and the complex Cls-Clr 2-Cls was studied by electron microscopy. In the absence of Ca 2+, monomeric Cls was visualized as a dumb-bell-shaped molecule consisting of two globular domains (center-to-center distance 11 nm) connected by a rod. One of the globular domains is assigned to the light chain (B-chain) of the activated molecule, which is homologous to trypsin and other serine proteases. The second globular domain and the rod are assigned to the heavy chain (A-chain) of C 1 ̄ s. The subunit Clr is a stable dimer in the presence or absence of Ca 2+. This dimer Clr 2 was visualized as composed of two dumb-bells of dimensions similar to those observed for Cls. These are connected near the junctions between the rod and one of the globular domains. This leads to the structure of an asymmetrical X with two inner closely spaced globules (center-to-center distance 7 nm) and two outer globules at a larger distance (14 nm). By comparison with fragment ClrII 2, in which part of the A-chain is removed, the inner globular domains were assigned to the catalytic B-chains. This characteristic structure of Clr 2 is readily recognized in the central portion of the thread-like 54 nm long Cls-Clr 2-Cls complex formed in the presence of Ca 2+. By affinity-labeling of Cls with biotin and visualization of avidin-ferritin conjugates in the reconstituted complex, it was demonstrated that Cls forms the outer portion of the complex. A detailed model of Cls-Clr 2-Cls is proposed, according to which two Cls monomers bind to the outer globes of Clr 2 by contacts between their heavy chains and those of Clr. According to this model the catalytic domains of Clr are located in the center and those of Cls at the very tips of the Cls-Clr 2-Cls complex. On the basis of the structure of Cls-Clr 2-Cls, we derived a detailed model of the C1 complex (composed of C1q and the tetrameric complex) and we discuss this model with a view to finding a possible activation mechanism of C1. By analogy with mechanisms proposed for the activation of trypsinogen and plasminogen, we propose that activation of Clr 2 in C1 may be mediated by interaction of the Cls light chain with the Clr light chain domain, thus inducing enzymatic activity in the Clr zymogen, which in turn may lead to a cleavage and final activation of the closely adjacent catalytic domain in the Clr 2 dimer.

Antibody-independent interaction between the first component of human complement, C1, and the outer membrane of Escherichia coli D31 m4.

The heptoseless mutant of Escherichia coli, E. coli D31 m4, binds C1q and C1 at 0 degrees C and at low ionic strength (I0.07). Under these conditions, the maximum C1q binding averages 3.0 X 10(5) molecules per bacterium, with a Ka of 1.4 X 10(8) M-1. Binding involves the collagen-like region of C1q, as shown by the capacity of C1q pepsin-digest fragments to bind to E. coli D31 m4, and to compete with native C1q. Proenzyme and activated forms of C1 subcomponents C1r and C1s and their Ca2+-dependent association (C1r-C1s)2 do not bind to E. coli D31 m4. In contrast, the C1 complex binds very effectively, with an average fixation of 3.5 X 10(5) molecules per bacterium, and a Ka of 0.25 X 10(8) M-1, both comparable with the values obtained for C1q binding. C1 bound to E. coli D31 m4 undergoes rapid activation at 0 degrees C. The activation process is not affected by C1-inhibitor, and only slightly inhibited by p-nitrophenyl p'-guanidinobenzoate. No turnover of the (C1r-C1s)2 subunit is observed. Once activated, C1 is only partially dissociated by C1-inhibitor. Our observations are in favour of a strong association between C1 and the outer membrane of E. coli D31 m4, involving mainly the collagen-like moiety of C1.

Immunoglobulin M possesses two binding sites for complement subcomponent C1q, and soluble 1:1 and 2:1 complexes are formed in solution at reduced ionic strength.

Soluble complexes were formed between C1q, a subunit of the first component of human complement, and four different Waldenström IgM proteins at reduced ionic strengths. The equilibria between these complexes and the free proteins were studied in the ultracentrifuge. Complex formation was found to be a very sensitive function of the salt concentration, and at physiological ionic strength complex formation was negligible. The complexes were cross-linked with a water-soluble carbodiimide and separated by sucrose gradient centrifugation. Both 22 S 1:1 and 26 S 2:1 C1q X IgM complexes were formed; stoichiometry was established by cross-linking 125I-C1q with 131I-IgM and determining the ratios of the specific activities of the gradient-purified materials. The association process was studied as a function of protein concentration and was analyzed by Scatchard and Hill plots to yield stoichiometry, association constant, and degree of cooperativity. The results indicated that IgM has two identical and independent binding sites for C1q. The intrinsic association constant was found to vary between 10(6) M-1 at 0.084 M ionic strength to 10(4) M-1 at physiological ionic strength; the slope of the log-log plot gave a value of -6.0. The cross-linked complexes were examined by electron microscopy, and the C1q appeared to be attached to the IgM through the C1q heads, implying that the biologically significant binding sites were involved in this interaction. For the 2:1 complexes, the two C1q appeared to attach to opposite surfaces of the IgM, suggesting the presence of a pseudo-2-fold axis lying in the plane of the IgM disk.

Domain structure and associated functions of subcomponents C1r and C1s of the first component of human complement.

The serine protease subcomponents of the activated form of the first component of human complement (C1), C1r and C1s, were observed by electron microscopy after the native proteins and their limited proteolysis products, obtained from autolytic cleavage (C1r) or from incubation with plasmin (C1s) were rotary shadowed. At the monomeric level, both C1r and C1s comprised two globular domains, a smaller interaction domain (corresponding to the NH2-terminal half of the A chain, alpha, and responsible for calcium binding and C1r-C1s interaction) and a larger catalytic domain (corresponding to the COOH-terminal part of the A chain, gamma, disulfide-linked to the B chain and bearing the serine protease active site). The two globular domains are linked by a connecting strand, beta. The (C1r)2 dimer appeared as a "croissant"-like association, where the two monomers interact through their catalytic domains. On the basis of the domain structure of C1r and C1s, a model of the calcium-dependent C1s dimer is proposed, in which the two monomers interact through their NH2-terminal interaction domains; in the same way, a model of the C1s-(C1r)2-C1s catalytic subunit of C1 is presented, in which (C1r)2 forms a core, its distal interaction domains interacting with the corresponding domains of C1s.

Nature of the interaction between the C1q and C1r 2s 2 subunits of the first component of human complement

The strength of interaction between the C1q and C1r 2s 2 subunits of C1 was studied as a function of temp. During centrifugation through sucrose density gradients at 4°C, macromolecular C1 readily dissociated as it sedimented away from its free subunits. In contrast, at 20°C, C1 remained associated as the 16S complex throughout centrifugation, thus indicating a stronger interaction between C1q and C1r 2s 2 at the higher temp. C1-inhibitor (C1-In) or nitrophenylguanidinobenzoate was present during centrifugation to prevent C1 activation. That native C1 was in fact the species being studied was confirmed by SDS-PAGE analysis. To investigate this temp dependence without using inhibitors, an alternative approach was used. Trace amounts of 125I-C1q were centrifuged through numerous sucrose density gradients, each of which contained a different concn of native C1r 2s 2 throughout the gradient. The s-rate of 125I-C1q increased with increasing C1r 2s 2 input. An association constant of 4.9 × 10 7 M −1 was calculated for this reversible interaction at 4°C. However, at 20°C, the data indicated a much higher affinity reaction since the addition of far less C1r 2s 2 was required for the s-rate of 125I-C1q to reach the 16S plateau. The presence of C1-In did not affect these results. We have demonstrated that the association of C1q with C1r 2s 2 increases with increasing temp, a finding suggestive of a hydrophobic interaction. However, since we also show that C1 readily dissociates with increasing NaCI concn, the C1q-C1r 2s 2 interaction must, in fact, be ionic in nature. We therefore conclude that the temp dependence of the inter-subunit interaction is the result of a conformational change(s) within one of the subunits, and propose that this change may be similar to that occurring during C1 activation.

A simple two-step procedure for the preparation of the first component of human complement (C1) in its native form

Native human C1 was purified from fresh human serum by affinity chromatography on protein A-bound Sepharose in the presence of 4-nitrophenyl-4-guanidinobenzoate hydrochloride (NPGB) taking advantage of the successive binding of IgG to protein A followed by C1 binding to IgG. After elution the C1 preparation contained IgG as a major contaminant as shown by SDS-PAGE. C1 was further purified by gel filtration. The yield of C1 was 12% and less than 4% of this C1 was activated during purification as assessed by a C4 consumption assay.

Autocatalytic activation of C1r subcomponent of the first component of human complement.

Autoactivation of the proenzyme form of a subunit of the first component (C1r) was performed in the presence and absence of diisopropyl fluorophosphate (DFP). The time-course of autoactivation of zymogen C1r followed a sigmoidal curve and was accelerated by addition of the enzyme C1r and by increasing the concentration of C1r, suggesting that autoactivation of C1r consists of two intermolecular reactions, i.e. zymogen(C1r)- and enzyme(C1r)-catalyzed reactions. In the presence of 10 mM DFP, the enzyme-catalyzed autoactivation of C1r was completely inhibited, while the zymogen-catalyzed autoactivation still proceeded depending upon C1r concentration. These results suggested that the zymogen-catalyzed autoactivation of C1r is a DFP-insensitive second-order reaction and is mediated by an active site generated in a single chain C1r through a conformational change (Kassahara et al. (1982) FEBS lett. 141, 128-131). Based on these results, a possible reaction process of autoactivation of C1r was proposed, as follows: (formula; see text) where C1r represents a conformational isomer which catalyzes the autoactivation of C1r, and the rate constants, k2 and k3, are of second-order. Utilizing a computer, we simulated the autoactivation of C1r and found the above scheme to be a reasonable model of C1r autoactivation. Evidence which supports the formation of a conformational isomer of C1r, C1r, as an intermediate in its autoactivation was also obtained by a surface radiolabeling method.

Activation of C1 by monoclonal antibodies directed against C1q

Eleven monoclonal antibodies directed against the subcomponent C1q of the first component of human complement, C1, were prepared and tested for binding to intact C1q and to the collagenous portion, the C1q stalks. All of the monoclonals bound well to the intact C1q. Eight out of the eleven exhibited strong binding to the collagenous stalks, while three bound very weakly, if at all, to the stalks and, thus, were presumed to bind to the pepsin-sensitive region which includes the C1q heads. For one of the latter monoclonals, this was confirmed by electron microscopy. Five of the monoclonals were purified by C1q affinity chromatography. When tested with C1 reassembled from its subunits, two of these purified monoclonal antibodies markedly enhanced the rate of spontaneous activation.

Diamine-induced dissociation of the first component of human complement, C1.

Lysine has been shown to inhibit spontaneous and antibody-dependent C1 activation. This paper demonstrates that lysine does not prevent autoactivation of purified C1r. 20 mM lysine, 1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane or 1,5-diaminopentane are able to dissociate C1 into its two entities, C1q and the calcium-dependent C1r2-C1s2 complex. Ig-ovalbumin insoluble complexes bearing C1 are also dissociated by lysine and the above-mentioned diamines used at the same concentration: C1q remains bound to the complexes whereas the C1r2-C1s2 complex is partially solubilized. The effect of lysine or diamines is not due to a competition with calcium for calcium-binding sites, as increasing concentrations of calcium even slightly increase the dissociation due to the amines. The dissociative effect is dependent on the carbon chain length of the diamines, with an optimum for 1,3-diaminopropane. It is also dependent on the relative 'cis-position' of the amino groups in the diamines. Polyamines such as spermine and spermidine are also able to dissociate C1 with even a higher efficiency than lysine and putrescine. Thus, a diamine-induced 'structural inhibition' of C1 is demonstrated, of potential interest for a pharmacological control of complement activation.

The role of immune complexes in the activation of the first component of human complement.

Immune complex-induced C1 activation and fluid phase C1 autoactivation have been compared in order to elucidate the immune complex role in the C1 activation process. Kinetic analyses revealed that immune complex-bound C1 activates seven times faster than fluid phase C1 spontaneously activates. The rate of spontaneous C1 activation increased after decreasing the solution ionic strength. In fact at one-half physiologic ionic strength (i.e., 0.08 M), the kinetics of spontaneous C1 activation were indistinguishable from the kinetics of activation of immune complex-bound C1 at physiologic ionic strength. The enhanced fluid phase C1 activation at low ionic strength resulted neither from C1 nor C1q aggregation, nor from selective effects on the C1r2S2 subunit; however, at the reduced ionic strength, the C1 association constant (defined for C1q + C1r2S2 in equilibrium C1qr2S2) did increase to 2.3 X 10(8) M-1, which is equal to that for C1 bound to an immune complex at physiologic ionic strength. Therefore, C1 can spontaneously activate in the fluid phase as rapidly as C1 on an immune complex when the strength of interaction between C1q and C1r2S2 is the same in both systems. In conclusion, under physiologic conditions, C1q and C1r2S2 are two weakly interacting proteins. Immune complexes provide a site for the assembly of a stable C1 complex, in which C1q and C1r2S2 remain associated long enough for C1q to activate C1r2S2. Thus, immune complexes enhance the intrinsic C1 autoactivation process by strengthening the association of C1q with C1r2S2.

Measurement of antibody-dependent binding, proteolysis, and turnover of C1s on liposomal antigens localizes the fluidity-dependent step in C1 activation

The antibody-dependent binding and activation of the first component of human complement (C1) by liposomes containing nitroxide spin-label lipid haptens have been simultaneously measured. The liposomes were either fluid (dimyristoylphosphatidylcholine) or solid (dipalmitoylphosphatidylcholine) at the temperature of the experiments (32°C). In 10 minutes fluid liposomes activate 40% of the C1 whereas solid liposomes only activate 10% of the C1. The fraction of C1 bound at the end of the activation incubation is approx. 2% for fluid liposomes and approx. 4% for solid liposomes. This binding is consistent with the relative amounts of antibody which bind to these two types of liposomes. These results demonstrate turnover of C1 or C1r 2s 2 on the liposome surface. It is concluded that the differential activation of C1 is due to a difference in the rate of activation of C1 after it is bound to the liposome surface. Lower limits for the activation rate constant for C1 bound to fluid and solid liposomes are estimated to be 8·10 −2 s −1 and 1·10 −2 s −1, respectively.