Chemical studies on the isolated collagen-like and globular fragment of complement component C1q. Comparative studies on bovine and human C1q

Both the collagen-like and the globular fragments of a subcomponent C1q of the first component of bovine and human complement were highly purified by enzymic digestion followed by gel filtration. Analyses by polyacrylamide gel electrophoresis showed that the former was composed of covalently linked peptide chains with an average molecular weight of 14 000, and that the latter was composed of three non-covalently linked peptide chains each having a molecular weight of approximately 15 000. Great similarities between amino acid compositions of the globular fragments and some similarities between those of the collagen-like fragments were found. Moreover, great similarities of amino acid compositions were found among three non-covalently linked chains of each globular fragment as well as between the corresponding chains of both globular fragments. These results suggested that both the collagen-like and the globular domains on the C1q molecule remained highly conserved in its evolution.     

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.     

Purification and characterization of subcomponent C1q of the first component of bovine complement

Bovine C1q, a subcomponent of the first component of complement, was purified in high yield by a combination of euglobulin precipitation, and ion-exchange and molecularsieve chromatography on CM-cellulose and Ultrogel AcA 34. Approx. 12-16mg can be isolated from 1 litre of serum, representing a yield of 13-18%. The molecular weight of undissociated subcomponent C1q, as determined by equilibrium sedimentation, is 430000. On sodium dodecyl sulphate/polyacrylamide gels under non-reducing conditions, subcomponent C1q was shown to consist of two subunits of mol.wts. 69000 and 62000 in a molar ratio of 2:1. On reduction, the 69000-mol.wt. subunit gave chains of mol.wts. 30000 and 25000 in equimolar ratio, and the 62000-mol.wt. subunit decreased to 25000. The amino acid composition, with a high value for glycine, and the presence of hydroxyproline and hydroxylysine, suggests that there is a region of collagen-like sequence in the molecule. This is supported by the loss of haemolytic activity and the degradation of the polypeptide chains of subcomponent C1q when digested by collagenase. All of these molecular characteristics support the structure of six subunits, each containing three different polypeptide chains, with globular heads connected by collagen triple helices as proposed by Reid & Porter (1976) (Biochem. J.155, 19-23) for human subcomponent C1q. Subcomponent C1q contains approx. 9% carbohydrate; analysis of the degree of substitution of the hydroxylysine residues revealed that 91% are modified by the addition of the disaccharide unit Gal-Glc. Bovine subcomponent C1q generates full C1 haemolytic activity when assayed with human subcomponents C1r and C1s.     

Stepwise dissociation of subcomponents of C1, the first component of human complement, upon activation on an affinity sorbent

An affinity sorbent comprising macroporous glass coated with the polymer with the polymer with immobilized immunoglobulin IgG was used for the isolation from human serum of the first component of the complement and for its separation into subcomponents C1r, C1s and C1q by the one-step procedure. Serum C1 was quantitatively bound to the sorbent at 0 degrees C. The unbound part of the serum can be used as a R1 reagent for determining the hemolytic activity of C1. After activation of bound C1 by heating (30 degrees C, 40 min) the activated subcomponent C1r is eluted from the sorbent. Stepwise elution with EDTA at pH 7.4 or with EDTA + 1 M NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent C1s and subcomponent C1q. Stepwise elution of C1 subcomponents from the affinity sorbent after activation reflects the process of C1 breakdown following its activation on immune complexes.     

Purification and characterization of subcomponent C1q of the first component of mouse complement.

1. Mouse C1q, a subcomponent of the first component of complement, has been purified in a highly haemolytically active form by a combination of precipitation with EGTA, ion-exchange chromatography and gel filtration. Yields ranged from 3 to 5 mg/200 ml of serum, and the activity of final preparations was in the range of 2 X 10(13)-4 X 10(13) C1q effective molecules/mg. 2. The molecular weight of mouse C1q was 439 500 +/- 1586, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. Mouse C1q was shown to be composed of non-covalently linked subunits, all being in the molecular-weight range 45 000-46 000, and three covalently linked chains each having a molecular weight of approx. 23 000 as determined on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by using non-covalently and covalently linked subunits of human C1q as markers with known molecular weights calculated theoretically previously [Porter & Reid (1978) Nature (London) 275, 699-704]. 4. Mouse C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approx. 9% carbohydrate. The absorption coefficient and nitrogen content of C1q were also determined.     

Semi-flexible joint in the C1q subunit of the first component of human complement

C1q appears in electron micrographs in two different projections: lateral projections, in which the molecules resemble bunches of tulips; and top projections, in which the molecules are seen as six terminal subunits connected to a central portion. We have measured 39 particularly well-formed top views to determine the distribution of distances of terminal subunits from the central portion, from which may be calculated the distribution of the angles made by the connecting strands with an axis through the central portion. This distribution peaks sharply at a preferred angle of 50 °. A limited degree of flexibility must exist, however, for a few molecules are found with angles ranging from 20 ° to 80 °. Therefore, we suggest the existence of a semi-flexible joint at the point of interruption of the collagen-like amino acid sequence where the connecting strands join to the central portion of the C1q molecule.     

Effects of human polymorphonuclear leukocyte collagenase on sub-component C1q of the first component of human complement

The similarities in the structure and properties of C1q and collagen prompted us to examine the susceptibility of C1q to human polymorphonuclear leukocyte collagenase. Incubation of C1q with a collagenase preparation resulted in no change in (1) the binding of C1q to immunoglobulin aggregates, (2) the hemolytic function of C1q as measured by reconstitution of C1q-depleted serum in immune hemolysis, or (3) the structural properties of C1q as revealed by gel electrophorettic patterns of the whole molecule or its polypeptide chains. In contrast, rapid inactivation and degradation of C1q was caused by leukocyte elastase.The collagenase preparation was, however, capable of cleaving reduced and carboxamidomethylated C1q into discrete fragments. This activity was attributed to a gelatinase present in the enzyme preparation since (1) the cleavage reaction was inhibited by denatured collagen but not by native collagen and (2) a collagenase fraction free of gelatinolytic activity could not degrade reduced and carboxamidomethylated C1q, while a gelatinase fraction devoid of collagenase activity retained the capacity to effect reduced and carboxamidomethylated C1q. Both collagenase and gelatinase activities were activated from the latent form by trypsin, and inhibited by EDTA.Therefore, it appears that native C1q lacks the structural features present in collagen which are recognized by leukocyte collagenase for hydrolytic action even though the denatured molecule still contains that region capable of being cleaved by gelatinase.     

Subcomponents C1q of the first component of guinea pig and mouse complement: Comparative study of their asparagine-linked sugar chains

Guinea pig and mouse C1q, subcomponents of the first component of complement, contained six asparagine-linked sugar chains on the C-terminal non-collagenous globular regions of each molecule. After N-acetylation and successive NaB³H₄-reduction of asparagine-linked sugar chains liberated by hydrazinolysis, their structure was analysed by sequential exoglycosidase digestion in combination with sugar composition analyses. The sugar chains of C1q molecules of both animals were very similar and composed of the biantennary complex type sugar chains with the following outer chains in various combinations: (± NeuNAcα → )Galß1 → GlcNAcß1 → and Galß1 → Galß1 → GlcNAcß1 →. These chain moieties were found to be linked to a common core structure of Manα1 → (Manα1 → )Manß1 → GlcNAcß1 → (Fucα1 → )GlcNAc.     

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.     

Subunit composition and structure of subcomponent C1q of the first component of human complement.

1. Unreduced human subcomponent C1q was shown by electrophoresis on polyacrylamide gels run in the presence of sodium dodecyl sulphate to be composed of two types of non-covalently linked subunits of apparent mol.wts. 69 000 and 54 000. The ratio of the two subunits was markedly affected by the ionic strength of the applied sample. At a low ionic strength of applied sample, which gave the optimum value for the 54 000-apparent mol.wt. subunit, a ratio of 1.99:1.00 was obtained for the ratio of the 69 000-apparent mol.wt. subunit to the 5400-apparent-mol.wt. subunit. The amount of the 54 000-apparent-mol.wt. subunit detected in the expected position on the gel was found to be inversely proportional to increases in the ionic strength of the applled sample. 2. Human subcomponent C1q on reduction and alkylation, or oxidation, yields equimolar amounts of three chains designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. The results obtained by Yonemasu & Stroud [(1972) Immunochemistry 9, 545-554], which showed that the 69 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the A and B chains and that the 54 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the C chain, were confirmed. 3. Gel filtration on Sephadex G-200 in 6.0M-guanidinium chloride showed that both types of unreduced subunit were eluted together as a single symmetrical peak of apparent mol.wt. 49 000-50 000 when globular proteins were used as markers. The molecular weights of the oxidized or reduced A, B and C chains have been shown previously to be very similar all being in the range 23 000-24 000 [Reid et al. (1972) Biochem. J. 130, 749-763; Reid (1974) Biochem. J. 141, 189-203]. 4. It is proposed that subcomponent C1q (mol.wt. 410000) is composed of nine non-covalently linked subunits, i.e. six A-B dimers and three C-C dimers. 5. A structure for subcomponent C1q is proposed and is based on the assumption that the collagen-like regions of 78 residues in each of the A, B and C chains are combined to form a triple-helical structure of the same type as is found in collagens.     

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.     

Biosynthesis of the complement component C1q in mouse peritoneal macrophage cultures

Biosynthesis of C1q complement component by resident peritoneal macrophages from (CBA X C57BL/6)F1 mice has been studied in in vitro experiments. Using anti-mouse C1q antibodies immobilized on CNBr Sepharose it has been demonstrated that 14C glycine incorporates both into intracellular C1q and C1q secreted into the medium. The maximum radioactivity of intracellular C1q was observed 48 h after cultivation, with it dropping drastically between hours 72-96. Kinetics of radiolabelled C1q was similar, but 24 hours delayed. Cell viability during 96 h of cultivation remained unchanged. These data can be considered as the indication of feedback regulation of C1q biosynthesis at the cellular level.     

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.     

Isolation and characterization of C1q, a subcomponent of the first component of complement, from human and rabbit sera

1. C1q, a subcomponent of the first component of complement, has been isolated, in a haemolytically active and soluble form, by ion-exchange chromatography and gel filtration, from human and rabbit sera. Yields ranged from 10 to 25mg/litre of serum and the activity of final preparations was consistently in the range 5x10(3)-15x10(3) C1qH(50) units/mg. 2. The molecular weights of human and rabbit subcomponent C1q were 409600 and 417600, as determined by sedimentation equilibrium studies. 3. Subcomponent C1q from both species was shown to be composed of non-covalently linked subunits of approximately 57000 molecular weight as determined by gel-filtration or sedimentation equilibrium studies in 5.3m-guanidinium chloride. Reduction or oxidation of human and rabbit subcomponent C1q yielded three chains each having a molecular weight of approximately 23000 and which differed slightly in amino acid composition but markedly in carbohydrate content. The oxidized chains were separated, on a preparative scale, by ion-exchange chromatography in 8m-urea on DEAE-cellulose. 4. Both human and rabbit subcomponent C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approximately 8% carbohydrate. Glutamic acid and aspartic acid were the free N-terminal amino acids of human subcomponent C1q whereas only serine was found in rabbit subcomponent C1q. 5. Collagenase digestion of human or rabbit subcomponent C1q caused a rapid loss of haemolytic activity which correlated with the breakdown of collagenous regions in the molecule.     

The unactivated form of the first component of human complement, C1.

The first component of complement, C1, was isolated unactivated from human serum by repeated additions of di-isopropyl phosphorofluoridate during isolation. The unactivated subcomponents were also isolated, and evidence is given that the three subcomponents C1q, C1r and C1s account wholly for the activity of component C1 in serum. No evidence could be found for a fourth subcomponent, C1t. The approximate molar proportions of the subcomponents in serum are C1q/C1r/C1s = 1:2:2. Optimum activity by haemolytic assay was found at approximate molar proportions C1q/C1r/C1s of 1:4:4. No activity was found when subcomponents were assayed singly or in pairs, except for subcomponents C1q and C1s, which in molar ratio 1:4 gave 15-20% of the activity of the mixture C1q + C1r + C1s. The proteolytic activity of the isolated subcomponent C1s varied according to the method of activation used. Subcomponents C1q + C1r + C1s and C1q + C1s in the presence of antibody-antigen aggregates were activated and inactivated simultaneously, showing a peak of activity and subsequent loss of activity. Both reactions are probably due to proteolysis, and analysis of the peptide bonds split will be necessary to distinguish these two phenomena.