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PubMed1998

Solution structure of the epidermal growth factor (EGF)-like module of human complement protease C1r, an atypical member of the EGF family.

The calcium-dependent interaction between C1r and C1s, the two homologous serine proteases of the first component of human complement C1, is mediated by their N-terminal regions. The latter comprise an epidermal growth factor (EGF)-like module exhibiting the consensus sequence characteristic of Ca(2+)-binding EGF modules, surrounded by two CUB modules. Due to its Ca2+ binding ability, the C1r EGF-like module (C1r-EGF) is supposed to participate in the C1r-C1s interaction. An additional interesting feature of C1r-EGF is the unusually large loop connecting the first two conserved cysteine residues. The solution structure of synthetic C1r-EGF (residues 123-175) has been determined using nuclear magnetic resonance and combined simulated annealing-restrained molecular dynamics calculations. The resulting family of 19 structures is characterized by a well-ordered C-terminal part (residues Cys 144-Ala174) with a backbone rmsd of 0.7 A and a disordered N-terminal, including the large loop between the first two cysteines (Cys129 and Cys144). This loop is known to be surface exposed and may be expected to participate in domain-domain or protein-protein interactions. In its C-terminal part, C1r-EGF possesses the characteristic EGF fold with a major and a minor beta-sheet. The latter comprises a beta-bulge, and comparison with other EGF-like modules reveals the existence of two distinct structural and sequential motifs in the bulged part. Additional experiments in the presence of 80 mM Ca2+ did not show significant structural variation of C1r-EGF, in keeping with previous observations on blood-clotting factors IX and X.

Amino Acid Sequence↗
PubMed1990

An improved method for complement subcomponent C1R typing.

An improved method has been developed for the reliable classification of different C1R genetic variant forms from human serum or plasma. The method combines the use of neuraminidase-digested samples followed by monodimensional isoelectric focusing in the pH range 5 to 8 followed by immunoblotting. The method yields a simple pattern, with one major band in homozygote and two major bands in heterozygote cases.

Blood Grouping and Crossmatching↗
PubMed1976

Physicochemical and functional characterization of the C1r subunit of the first complement component.

C1r was isolated from serum by an improved method and found to be a glycoprotein with a sedimentation coefficient of 7.0S. Under conditions of physiologic ionic strength and pH, C1r consist of two apparently identical noncovalently linked 95,000 dalton polypeptide chains. Antisera to C1r detected a protein of gamma-mobility on electrophoresis of serum in agarose in the presence of calcium, and a Beta-mobility protein when the electrophoretic separation was carried out in EDTA. On sucrose gradient ultracentrifugation of normal human serum in the presence of calcium, C1r antigenicity was found in the 19 S region of the gradient. On the other hand, when the gradient contained EDTA, C1r antigenicity was found in the 7 S region. No reaction of anti-C1r with C1r-deficient sera was observed. C1r had a high affinity for active C1s or proenzyme C1s in the presence of calcium and was able to activate C1s and to form C1 in conjunction with C1q and C1s. Activation of C1s by C1r was inhibited by calcium, C1 inactivator, polyanethol sulfonate, and DFP. Activation of C1s by C1r occurred only after a preliminary incubation of C1r for a brief time at 37 degrees C before addition of C1s. The ability of C1r to form C1 in conjunction with C1q and C1s was, however, progressively lost on incubation at 37 degrees C. Trypsin, although potentiating the activity of crude C1r, did not modify the activity of purified C1r. Its action was on a trypsin-sensitive inhibitor separated from C1r in the final step of the isolation procedure. The binding of 125I-C1r to sensitized sheep erythrocytes required the presence of C1q and calcium but not C1s, whereas the binding of 125I-C1s required C1q, C1r and calcium. Thus, C1r functions as not only the activator of C1s, but also serves as the physical link between C1q and C1s in macromolecular C1.

Animals↗
PubMed1994

Ca(2+)-linked association of human complement C1s and C1r.

The weight-average molecular weight of Clr, an activated serine protease subcomponent of complement Cl, was measured in the presence of widely varying concentrations of Ca2+ and the other serine protease subcomponent, Cls, by utilizing the technique of tracer sedimentation equilibrium. A quantitative model for heteroassociation between the two subcomponents, which takes into account the previously observed Ca(2+)-dependent self-association of Cls, was fit to the combined data at each Ca2+ concentration. The results indicate that Clr, which exists as a dimer under all of the conditions explored in this work, can bind up to two molecules of Cls at both low and high Ca2+ concentrations, but the association constant for binding a single molecule of Cls to dimeric Clr is estimated to increase on the order of a 1000-fold as [Ca2+] increases from 1 nM to 1.0 mM. Heteroassociation of Clr and Cls is favored over self-association of Cls at all conditions. The results clearly indicate the necessity of taking into account a multiplicity of states of association when attempting to understand the equilibrium average properties of a mixture of the two subcomponents and their binding to Clq in solution.

Binding Sites↗
PubMed1991

Measurement of macromolecular interactions between complement subcomponents C1q, C1r, C1s, and immunoglobulin IgM by sedimentation analysis using the analytical ultracentrifuge.

The interactions between the complement components and with immunoglobulins are greatly enhanced by lowering the ionic strength and become readily measurable by physical techniques. Thus, the binding between C1q and IgM was previously shown to be appreciable (k = 1 x 10(6) M-1) at 0.084 M ionic strength (Poon, P.H., Phillips, M.L., and Schumaker, V.N. (1985) J. Biol. Chem. 260, 9357-9365). We have now found that, at 0.128 M ionic strength, the binding between human C1- (the activated first component of complement) and IgM was strong at physiological concentrations (k = 1 x 10(7) M-1), while under the same conditions binding between C1q and IgM was not observed. To explore the nature of the interactions responsible for this enhanced binding by C1- over C1q, mixtures of the various subcomponents of C1- were studied alone and with IgM. C1r2 did not bind to C1q, even when the ionic strength was reduced to 0.098 M, nor did the presence of C1r2 enhance the binding of C1q to IgM. In contrast, two C1s2 independently bound to C1q (k = 1 x 10(6) M-1), and caused a marked increase in its association with IgM (k = 5 x 10(6) M-1) at 0.098 M ionic strength. No detectable interaction was found between C1s2 and/or C1r2 and IgM in the absence of C1q. Moreover, there was no detectable interaction between the C1(-)-like complex formed between C1r2C1s2 and the collagenous C1q stalks (pepsin-digested C1q) and IgM. These data suggest that the binding of C1s2 to C1q, either alone or together with C1r2, induces a conformational change in C1q which results in additional C1q heads binding to complementary sites on IgM.

Complement C1q↗
PubMed1990

Neutron scattering study of the (gamma-B) catalytic domains of complement proteases activated C1r and C1s.

The catalytic domains of activated C1r and C1s, comprising the C-terminal region of the A chain (gamma), disulphide-linked to the B chain, were obtained by limited proteolysis of the native proteases with chymotrypsin and plasmin, respectively, and studied by small angle neutron scattering. For activated C1s (gamma-B), a molar mass of 45,000 +/- 5000 g/mol, and a relatively large radius of gyration (Rg) of 28 +/- 1 A were determined, excluding a single globular domain. The corresponding values for activated C1r (gamma-B)2 (90,000 g/mol, Rg = 34 +/- 1 A) are consistent with a dimer involving the loose packing of two (gamma-B) subunits. Various models of the dimer are discussed in the light of neutron scattering and other data.

Chymotrypsin↗
PubMed1997

Baculovirus-mediated trans-epithelial transport of proteins in infected caterpillars.

Baculovirus-mediated abnormal protein trafficking was studied in infected caterpillars by using heterologous proteins. The gene for human complement C1r was expressed in larvae of Mamestra brassicae by a recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) vector. By following the time-course of recombinant C1r distribution among various tissues, cell types and cell organelles, we concluded that the dominant site of recombinant protein synthesis was the fat body, although some production in the haemocytes and midgut was also observed. Only about 4% of the cells of the infected organs expressed recombinant C1r, which was then secreted into the haemolymph. The tracheal and integumental cuticle was rich in recombinant protein from the fourth day after infection although epidermal cells did not synthesize recombinant C1r. The morphological picture suggested that the accumulation was a consequence of a trans-epithelial transport. This transport process was checked by following the fate of the 49 kDa haemolymph protein and injected ovalbumin in AcMNPV-infected Mamestra brassicae and in Lymantria dispar nuclear polyhedrosis virus-infected Lymantria dispar larvae. Both proteins were able to pass the basal membrane of the epidermis and accumulated in the cuticle, while in control larvae neither was transported. The observed trans-epithelial transport points to the role of baculoviruses in directing recombinant, endo- and exo-genous proteins to cuticulated tissues. Based on these results we conclude that the permeability of basal membranes undergoes a characteristic change during the course of baculovirus infection.

Animals↗
PubMed1984

Lack of activation of C1, despite circulating immune complexes detected by two C1q methods, in patients with rheumatoid arthritis.

The activation of C1 by circulating immune complexes in patients with rheumatoid arthritis was investigated. C1rC1s(C1-In)2 complexes in EDTA-plasma, reflecting C1 activation in vivo, were slightly raised in 35 of 57 patients with rheumatoid arthritis, though most patients had elevated levels of circulating immune complexes as measured with either the 125I-C1q binding test or the C1q solid phase assay. The activation of C1 by circulating immune complexes in vitro was investigated by measuring the generation of C1rC1s(C1-In)2 complexes during 60 minutes at 37 degrees C in diluted recalcified EDTA-plasma. In 16 of the 57 patients, a slightly increased C1 activation in vitro was observed. These patients tended to have high levels of circulating immune complexes. However, the majority of the patients with high levels of circulating immune complexes showed a normal C1 activation in vitro. Therefore, it was concluded that measurement of circulating immune complexes by either of the two C1q methods in patients with rheumatoid arthritis does not imply that these circulating immune complexes are able to activate C1.

Aged↗
PubMed1985

Antibody density on rat red cells determines the rate of activation of the complement component C1.

It is a common observation that there is variability in the rate of activation of C1, the first component of complement, when bound to immune complexes. The cause of this variation has been investigated with experiments designed to assess separately the effect of antibody, antigen and C1 density. Using 125I-labeled C1 and a rat monoclonal antibody specific for the class I antigen, it has been found that the rate of activation is primarily dependent on antibody density on the cell surface and not on antigen or C1 density. This finding supports the suggestion that direct contact between the C1r2C1s2 subcomponent of C1 and antibody may be required for potentiation of C1 activation.

Animals↗
PubMed1995

Improved method for measuring C1-r-C1-s-(C1 inh)2 complexes by an enzyme-linked immunosorbent assay.

Measurement of C1-r-C1-s-(C1 inh)2 complexes in serum or plasma by enzyme-linked immunosorbent assay (ELISA) has been proposed as a relatively convenient and sensitive means for assessing C1 activation. However, interference by unactivated C1q (r-s)2 at low serum or plasma dilutions has resulted in estimates that vary widely with the degree of serum or plasma dilution. Precipitating the interfering C1q (r-s)2 with 6% polyethylene glycol has been proposed to resolve this problem, but here it is shown that this procedure also precipitates or coprecipitates some of the C1-r-C1-s-(C1 inh)2 complexes. Satisfactory results have been achieved without PEG precipitation by testing high plasma dilutions under conditions where there is a sufficient excess of anti-C1s coating the microtitration plate wells that removal of C1q (r-s)2 is not necessary. Optimizing conditions for quantitating these complexes at high dilution have been investigated. The mean normal EDTA plasma C1-r-C1-s-(C1 inh)2 complex measurement was 36.6 +/- 7.0 (S.D.) ELISA units with a 95% confidence interval of 19.5-47.6u. Besides providing a sensitive assay for C1 activation, measuring C1-r-C1-s-(C1 inh)2 complexes may help to clarify the pathophysiologic mechanisms resulting from C1 inh deficiency under various conditions.

Adult↗
PubMed1998

A newly discovered function for C1 inhibitor, removal of the entire C1qr2s2 complex from immobilized human IgG subclasses.

A new function for C1 inhibitor (C1 INH) is reported. C1 inhibitor dislodged the entire activated C1 complex (C1qr2s2) from immobilized human IgG. C1 binding to doses of immobilized human IgG3, IgG1, or IgG2 was quantified as a function of time. When human serum, as a source of C1qr2s2, was added to relatively low doses of immobilized IgG, C1q binding peaked at 1.0 min then gradually decreased. However when purified C1q was applied to immobilized IgG, C1q binding did not diminish with time. The removal of C1q was duplicated by adding purified C1 INH to C1qr2s2 which had been bound to immobilized IgG. The dislodgement of C1q from immobilized IgG required the presence of intact C1qr2s2 and of C1 INH. This removal of C1q by purified C1 INH was prevented when activated C1s was used to neutralize C1 INH function or when relatively high levels of IgG were immobilized.

Binding, Competitive↗
PubMed1983

The first component of human complement (C1): activation and control.

The first component of human complement (C1) is a 750 000 dalton glycoprotein that requires calcium or other specific metal ions to maintain its native structure and function. Under physiologic conditions, C1 comprises two weakly interacting subunits, C1q and C1r2s2, with C1q containing the binding site(s) for activators and C1r2s2 possessing enzymatic potential. C1 circulates in a precursor state and only after "activation" does it acquire functional activity, manifested as enzymatic activity specific for its natural substrates C2 and C4. C1 activation, which is accompanied by limited proteolysis and conformational changes, can be induced by immune complexes or certain nonimmune substances. With C1 binding to an immune complex, the strength of interaction between C1q and C1r2s2 increases. C1 also spontaneously activates at 37 degrees C by an intramolecular autocatalytic mechanism although at a slower rate than that induced by activators. C1 functions are controlled by the serum glycoprotein C1-inhibitor (C1-In) which blocks the enzymatic activities of activated C1 (C1). Under physiologic conditions, C1 has a half-life of only 13 seconds in the presence of C1-In. C1 is efficiently disassembled by C1-In, thereby releasing two inactive C1rC1s(C1-In)2 complexes per C1 molecule, leaving C1q activator-bound with biologically reactive sites uncovered that are not expressed in macromolecular C1. The most recently recognized function of C1-In is that of controlling the C1 activation process itself. While having only limited effect on immune complex-induced C1 activation, C1-In effectively controls certain nonimmune-induced as well as spontaneous C1 activation. Thus C1-In plays an important role in regulating nonspecific complement activation. The latter observation is relevant for the understanding of the human disease hereditary angioedema. An overabundance of spontaneous C1 autoactivation, due to low C1-In levels, might underlie the abnormal activation of complement via the classical pathway detected in the sera of these patients. Finally, recent studies indicate that C1 may have other important biologic functions in addition to initiating the complement cascade.

Angioedema↗