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.     

Characteristics of complement subcomponents C1r and C1s synthesized by Hep G2 cells.

The association and activation states of complement subcomponents C1r and C1s biosynthesized by Hep G2 cells were studied. C1r and C1s are secreted in stoichiometric amounts; in the presence of Ca2+ they are associated in a complex that sediments similarly to plasma C1r2-C1s2. Both compounds are synthesized as monomer proteins of apparent Mr 86 000. C1r is secreted as a dimer. Secreted C1r is not autoactivatable but undergoes proteolysis by exogenous C1r; secreted C1s is also proteolysed by exogenous C1r. In the presence of immune-complex-bound C1q, secreted C1r and C1s are able to reconstitute C1, but normal activation requires extrinsic C1r2-C1s2.     

Biosynthesis in vitro of complement subcomponents C1q, C1s and C1 inhibitor by resting and stimulated human monocytes.

The capacity of cultured human monocytes to synthesize and to secrete the subcomponents of C1 and C1 inhibitor was examined. Non-stimulated monocytes secreted C1q and C1s from day 5 of culture. C1s reached a plateau immediately at its maximum level, whereas C1q secretion increased progressively until the end of the second week. Between day 12 and day 25, C1q secretion remained nearly constant (1-15 fmol/day per microgram of DNA, depending on the donor), whereas C1s secretion decreased and even in some cases stopped. C1r and C1 inhibitor were not secreted in detectable amounts by these resting cells. Stimulation of monocytes by yeasts, immunoglobulin G-opsonized sheep red blood cells or latex beads did not modify consistently C1q and C1s secretion. Activation by conditioned media from mitogen-, antigen- or allogeneic-stimulated lymphocyte cultures increased C1q production from 2 to 7 times and re-activated C1s secretion. Under the same conditions of activation, C1 inhibitor was secreted (up to 300 fmol/day per microgram of DNA) and C1r became detectable in culture supernatants. Isolated human monocytes are thus able to synthesize the whole C1 subcomponents; C1, if assembled, could be protected from non-immunological activation by locally produced C1 inhibitor. Activated monocytes appear to be a good tool for studying the assembly of C1 subcomponents and the role of C1 inhibitor in this process.     

Fluid-phase interaction of C1 inhibitor (C1 Inh) and the subcomponents C1r and C1s of the first component of complement, C1.

Interactions between proenzymic or activated complement subcomponents of C1 and C1 Inh (C1 inhibitor) were analysed by sucrose-density-gradient ultracentrifugation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The interaction of C1 Inh with dimeric C1r in the presence of EDTA resulted into two bimolecular complexes accounting for a disruption of C1r. The interaction of C1 Inh with the Ca2+-dependent C1r2-C1s2 complex (8.8 S) led to an 8.5 S inhibited C1r-C1s-C1 Inh complex (1:1:2), indicating a disruption of C1r2 and of C1s2 on C1 Inh binding. The 8.5 S inhibited complex was stable in the presence of EDTA; it was also formed from a mixture of C1r, C1s and C1 Inh in the presence of EDTA or from bimolecular complexes of C1r-C1 Inh and C1s-C1 Inh. C1r II, a modified C1r molecule, deprived of a Ca2+-binding site after autoproteolysis, did not lead to an inhibited tetrameric complex on incubation with C1s and C1 Inh. These findings suggest that, when C1 Inh binds to C1r2-C1s2 complex, the intermonomer links inside C1r2 or C1s2 are weakened, whereas the non-covalent Ca2+-independent interaction between C1r2 and C1s2 is strengthened. The nature of the proteinase-C1 Inh link was investigated. Hydroxylamine (1M) was able to dissociate the complexes partially (pH 7.5) or totally (pH 9.0) when the incubation was performed in denaturing conditions. An ester link between a serine residue at the active site of C1r or C1s and C1 Inh is postulated.     

The structure and enzymic activities of the C1r and C1s subcomponents of C1, the first component of human serum complement.

The subcomponents C1r and C1s and their activated forms C-1r and C-1s were each found to have mol.wts. in dissociating solvents of about 83000. The amino acid compositions of each were similar, but there were significant differences in the monosaccharide analyses of subcomponents C1r and C1s, whether activated or not. Subcomponents C1r and C1s have only one polypeptide chain, but subcomponents C-1r and C-1s each contain two peptide chains of approx. mol.wts. 56000 ("a" chain) and 27000 ("b" chain). The amino acid analyses of the "a" chains from each activated subcomponent are similar, as are those of the "b" chains. The N-terminal amino acid sequence of 29 residues of the C-1s "a" chain was determined, but the C-1r "a" chain has blocked N-terminal amino acid. The 20 N-terminal residues of both "b" chains are similar, but not identical, and both show obvious homology with other serine proteinases. The difference in polysaccharide content of the subcomponents C-1r and C-1s is most marked in the 'b' chains. When tested on synthetic amino acid esters, subcomponent C-1r hydrolysed both lysine and tyrosine ester bonds, but subcomponent C-1r did not hydrolyse any amino acid esters tested nor any protein substrate except subcomponent C1s. The lysine esterase activity of subcomponent C1s provides a rapid and sensitive assay of the subcomponent.     

Conformational changes of the subunits C1q, C1r and C1s of human complement component C1 demonstrated by 125I labeling

C1s and C1r proenzymes and enzymes (C1s, C1r) and C1q were labeled with 125I. The distribution of the 125I label between H- and L-chain of C1s was only slightly dependent on the state of activation of C1s, and approx. 90% of the label was found in the H-chain. In the C1r proenzyme molecules 50% of the label was incorporated into the H-chain. The C1r H-chain label was reduced to 10% on activation of C1r to C1r, while the L-chain label increased to 90% of the total label. The presence of either C1s, C1q or C1qs during labeling reduced the C1r H-chain level, although C1r remained in the proenzyme form. The presence of C1s or C1rs enhanced the 125I uptake of C1q in Ca2+ or EDTA medium. This was unexpected because one would have anticipated a diminution of the C1q label due to the apposition of C1r and C1s, similarly as it occurs during C1rs complex and C1s dimer formation for the H-chain label of C1s. The results show that C1r and C1q alter their conformation during activation and C1 complex formation.     

Characterization of complement 1q binding protein of tiger shrimp,Penaeus monodon,and its C1q binding activity

The receptor for the globular heads of C1q, C1qBP/gC1qR/p33, is a multicompartmental, multifunctional cellular protein with an important role in infection and in inflammation. In the present study, we identified and characterized the complement component 1q subcomponent binding protein (C1qBP) from the tiger shrimp Penaeus monodon (designated as PmC1qBP). The open reading frame of PmC1qBP encodes 262 amino acid residues with a conserved MAM33 domain, an arginine-glycine-aspartate cell adhesion motif, and a mitochondrial targeting sequence in the first 53 amino acids. PmC1qBP shares 32%–81% similarity with known C1qBPs and clusters with lobster gC1qR under phylogenetic analysis. The temporal PmC1qBP mRNA expression in the hepatopancreas was significantly enhanced at 9 h after Vibrio vulnificus challenge. The native PmC1qBP was expressed in the gills, hepatopancreas, ovaries, and intestines as a precursor (38 kDa) and the active peptide (35 kDa). The recombinant PmC1qBP protein was expressed in Escherichia coli BL21, and was purified using nickel–nitrilotriacetic acid agarose. A complement 1q binding assay indicated that the rC1qBP protein competitively binds to C1q in mouse serum. The data reveal that PmC1qBP is not only involved in shrimp immune responses to pathogenic infections, but also cross-binding to the mouse C1q.     

Molecular modelling of human complement subcomponent C1q and its complex with C1r2C1s2 derived from neutron-scattering curves and hydrodynamic properties.

Models for the structures of subcomponent C1q of first component C1 of human complement and its complex with subunit C1r2C1s2 are compared with experimental neutron-scattering curves. The length of the C1q collagenous arm is closer to 14.5 nm than to 11.5 nm proposed from electron microscopy, and this is consistent with the primary sequence of C1q. The mean C1q base-arm angle is 40-45 degrees and C1q is found to be flexible: the base-arm angle can vary up to 30 degrees from equilibrium at any moment. The complex of C1r2C1s2 and C1q requires a large shape change in C1r2C1s2. Ring-like models for C1r2C1s2 are not as successful at rationalizing the scattering data as are models that involve C1r2C1s2 binding to one side of C1q. Hydrodynamic calculations of the sedimentation coefficients for C1q and C1 are generally consistent with these neutron models.     

Interaction of C1q and mannan-binding lectin (MBL) with C1r, C1s, MBL-associated serine proteases 1 and 2, and the MBL-associated protein MAp19

Mannan-binding lectin (MBL) and C1q activate the complement cascade via attached serine proteases. The proteases C1r and C1s were initially discovered in a complex with C1q, whereas the MBL-associated serine proteases 1 and 2 (MASP-1 and -2) were discovered in a complex with MBL. There is controversy as to whether MBL can utilize C1r and C1s or, inversely, whether C1q can utilize MASP-1 and 2. Serum deficient in C1r produced no complement activation in IgG-coated microwells, whereas activation was seen in mannan-coated microwells. In serum, C1r and C1s were found to be associated only with C1q, whereas MASP-1, MASP-2, and a third protein, MAp19 (19-kDa MBL-associated protein), were found to be associated only with MBL. The bulk of MASP-1 and MAp19 was found in association with each other and was not bound to MBL or MASP-2. The interactions of MASP-1, MASP-2, and MAp19 with MBL differ from those of C1r and C1s with C1q in that both high salt concentrations and calcium chelation (EDTA) are required to fully dissociate the MASPs or MAp19 from MBL. In the presence of calcium, most of the MASP-1, MASP-2, and MAp19 emerged on gel-permeation chromatography as large complexes that were not associated with MBL, whereas in the presence of EDTA most of these components formed smaller complexes. Over 95% of the total MASPs and MAp19 found in serum are not complexed with MBL.     

Structure and activity of C1r and C1s

During activation of the first component of the classical complement pathway the two zymogen subcomponents, C1r and C1s are converted to active proteolytic enzymes. Activated C1r cleaves C1s which then becomes the activator of C4 and C2. Amino acid sequence studies of the proteolytic chains of C1r and C1s, carried out in Oxford and Aberdeen respectively, have shown that they belong to the serine proteinase family. Modelling of these sequences to the three-dimensional coordinates of chymotrypsin (Birktoft & Blow 1972) reveals that both molecules have a conserved structural core, and that most of the differences lie in the external loops. Catalytically functional residues (Ile-16, His-57, Asp-102, Ser-195) are conserved, and residue 189 is aspartic acid, consistent with the known trypsin-like specificity of cleavage. Examination of the amino acid sequences of C4a, and comparison with those of the homologous molecules C3a and C5a, shows that there is a marked difference in the distribution of basic residues near the C-terminal arginine residue which is the site of action of C1s. When these amino acid sequences are modelled to the coordinates of C3a (Huber et al. 1980) and docked to the active site of C1s, the basic residues of C4a appear to interact with two glutamate residues peculiar to C1s, suggesting that this interaction may contribute to the ability of C1s to discriminate C4 from C3 and C5.     

A monoclonal antibody to C1q which appears to interact with C1r2C1s2-binding site

A monoclonal antibody (SB-4) to human C1q was prepared. The equilibrium constant of the antibody for C1q was found to be greater than 10(10) M-1. It has been shown that the antibody binds to the A-B chain dimer, probably via the B chain of C1q. Pepsin digestion of C1q at pH 4.5, which fragments the globular regions but leaves the collagenous region intact, allowed the demonstration that the antigenic site is located in the collagenous region of the molecule. The effect of the antibody on haemolytic activity has shown that it is capable of inhibiting the formation of EAC1 cells from EAC1q cells plus C1r and C1s but is incapable of inhibiting the C1 activity of performed EAC1 cells. This indicates that the binding of the antibody to the collagenous portion of the B chain of C1q probably prevents interaction between C1q and the C1r2-C1s2 complex.     

Comparative studies on biological activities of subcomponents C1q of the first component of human, bovine, mouse and guinea-pig complement

Both the haemolytic activity and the binding ability to immunoglobulin G(IgG) (Fc-binding ability) were comparatively assayed among human, bovine, mouse and guinea-pig C1q. The haemolytic activity was measured by using the sensitized sheep erythrocytes with rabbit immunoglobulin M(IgM)- or IgG-haemolysin. The Fc-binding ability was assayed by using immune complexes made of rabbit IgG-antibody against human serum albumin as well as agglutination of latex particles coated with human, bovine or rabbit IgG (IgG-latex). The specific haemolytic activity was comparable with between bovine and mouse C1q, while those of guinea pig and human C1q were significantly lower than those of the others. Only the human and mouse C1q showed significantly positive agglutinating activity of human or bovine IgG-latex. In the case of the use of rabbit IgG-latex, each of these C1q gave much weaker agglutination. On the other hand, the ability of all these C1q to bind to Fc of immune complexes specifically was almost comparable. The discrepancy in specific activities between the haemolysis and the Fc-binding ability may suggest that these two biological activities are not always correlative and that these are independent biological phenomena.     

C1q binding and C1 activation by various isolated cellular membranes

Cellular and subcellular membranes obtained from heart, liver, and brain tissue from human, baboon, bovine, rabbit, and rat bound highly purified, radioiodinated human C1q with a high affinity (Ka = 10(8) to 10(10) M-1). The majority of these membrane preparations were able to activate fully assembled C1 as evidenced by the conversion of 125I-C1s, incorporated into C1 complexes, to 125I-C1s. C1 activation by baboon heart mitochondrial membranes required an intact C1 complex and appeared to be mediated by the binding of the C1q subcomponent in that excess C1q completely blocked C1 activation. Several experiments suggested that the heart mitochondrial membrane binding site for C1q is an integral component of the mitochondrial membrane and that C1q interacted with the membrane binding site through its globular head regions. It is suggested that the binding of C1q and the activation of C1 by cellular and subcellular membranes may be involved in the initiation and/or enhancement of the inflammatory process after acute tissue damage.     

Recombinant human complement subcomponent C1s lacking beta-hydroxyasparagine, sialic acid, and one of its two carbohydrate chains still reassembles with C1q and C1r to form a functional C1 complex.

In contrast to the human serum protein which is approximately one-half erythro-beta-hydroxyasparagine at asparagine 134 [Theilens et al. (1990) Biochemistry 29, 3570-3578], recombinant C1s expressed by insect cells after infection with recombinant baculovirus entirely lacks posttranslational modification at asparagine 134. It is also incompletely glycosylated, lacking, at least, sialic acid. Site-directed mutagenesis of one of the two sites of carbohydrate attachment (Asn 159 to Gln 159) yields a faster migrating recombinant C1s still abundantly secreted. Furthermore, the mutated protein displays good hemolytic activity when reassembled with C1q and either human serum or recombinant C1r, demonstrating that these posttranslational modifications are not critical for any of the multiple interactions between C1s and C1q, C1r, C2, and C4 required for reassembly of the C1 complex, activation, and initiation of the classical complement pathway. The 4.0S recombinant C1s dimerizes to yield 5.6S C1s2 in the presence of Ca2+ and forms the 9.1S C1s-C1r-C1r-C1s tetramer upon the addition of human serum C1r and the 15.6S C1 complex upon the addition of C1q to the tetramer. The recombinant C1s and human serum C1s have identical N-terminal amino acid sequences, indicating proper recognition by the insect signal peptidase. The recombinant C1s is secreted and isolated as the unactivated zymogen, and it may be activated by human serum C1r which cleaves at Arg422-Ile423 to yield the characteristic heavy and light chains. A very tight complex is formed between C1-inhibitor and the light chain of recombinant C1s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of the pentamer/hexamer forms of mannan-binding protein to zymosan activates the proenzyme C1r2C1s2 complex, of the classical pathway of complement, without involvement of C1q

The serum lectin, mannan binding protein (MBP), was isolated in a yield of 40 micrograms/liter from pooled normal human serum by affinity chromatography on mannan-Sepharose, followed by gel-filtration and ion-exchange chromatography and finally by passage down an anti-IgM Sepharose column. A rabbit antiserum was prepared against the purified MBP and an enzyme-linked immunoassay developed that used both the specificity of the polyclonal antibody and the Ca+(+)-dependent carbohydrate binding property of MBP. Assay of the sera from 103 blood-donors showed a wide range of MBP levels, ranging from 0 to 870 micrograms/liter. MBP, after interaction with zymosan, caused efficient activation of a C1r2 125I-C1s2 complex that was prepared by incubation of 125I-C1s2 with serum, from a patient with a complete genetic deficiency of C1q, followed by gel-filtration on Sepharose 6B. The purified MBP is composed of a mixture of trimers, tetramers, pentamers, and hexamers of an approximate 90-kDa structural unit as judged by chromatography, SDS-PAGE and electron microscopy studies. Only the molecules in the pentamer/hexamer fraction, which have a similar overall structure to that of C1q, appeared to cause efficient, zymosan-dependent, activation of C1s within the C1r2C1s2 complex. The pentamer/hexamer form of MBP may therefore play an important role in antibody-independent activation of the C system during the early stages of certain infections.