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 binding of human complement component C4 to antibody-antigen aggregates.

The binding of human complement component C4 to antibody-antigen aggregates and the nature of the interaction have been investigated. When antibody-antigen aggregates with optimal C1 bound are incubated with C4, the C4 is rapidly cleaved to C4b, but only a small fraction (1-2%) is bound to the aggregates, the rest remaining in the fluid phase as inactive C4b. It has been found that C4b and th antibody form a very stable complex, due probably to the formation of a covalent bond. On reduction of the C4b-immunoglobulin G (IgG) complex, the beta and gamma chains, but not the alpha' chain, of C4b are released together with all the light chain, but only about half of the heavy chain of IgG. The reduced aggregates contain two main higher-molecular-weight complexes, one shown by the use of radioactive components to contain both IgG and C4b and probably therefore the alpha' chain of C4b and the heavy chain of IgG, and the other only C4b and probably an alpha' chain dimer. The aggregates with bound C1 and C4b show maximal C3 convertase activity, in the presence of excess C2, when the alpha'-H chain component is in relatively highest amounts. When C4 is incubated with C1s in the absence of aggregates, up to 15% of a C4b dimer is formed, which on reduction gives an alpha' chain complex, probably a dimer. The apparent covalent interaction between C4b and IgG and between C4b and other C4b molecules cannot be inhibited by iodoacetamide and hence cannot be catalysed by transglutaminase (factor XIII). The reaction is, however, inhibited by cadaverine and putrescine and 14C-labelled putrescine is incorporated into C4, again by a strong, probably covalent, bond. It is suggested that a reactive group, possibly an acyl group, is generated when C4 is activated by C1 and that this reactive group can react with IgG, with another C4 molecule, or with water.     

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.     

Polymorphism of human complement component C4

An assessment has been made of the polymorphism of human complement component C4 by comparing derived amino acid sequences of cDNA and genomic DNA with limited amino acid sequences. In all, one complete and six partial sequences have been obtained from material from three individuals and include two C4A and two C4B alleles. Differences were found between the 4 alleles from 2 loci in only 15 of the 1722 amino acid residues, and 12 lie within one section of 230 residues, which in 1 allele also contains a 3-residue deletion. In three variable positions, an allelic difference in one C4 type was common to the other types. Three nucleotide differences were found in four introns. In spite of marked differences in their chemical reactivity, the many allelic forms appear to differ in less than 1% of their amino acid residue positions. This unusual pattern of polymorphism may be due to recent duplication of the C4 gene, or may have arisen by selection as a result of the biological role of C4, which interacts in the complement sequence with nine other proteins necessitating conservation of much of the surface structure.     

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.     

A synthetic complement C1q-like peptide selectively interacts with immune complexes

A water-soluble peptide possessing an immune complex selective affinity was synthesized and its primary structure established as: Leu-Glu-Gln-Gly-Glu-Asn-Val-Phe-Leu-Gln-Ala-Thr-Ser-Asp-Asp-Cys. This peptide, designated as C1q-like peptide (CLP), represents a possible immune complex binding epitope of complement C1q. CLP has a hydrophilicity value of 0.21. At 0.5 M, it inhibited by 50% natural human C1q from binding to horseradish peroxidase-rabbit anti-peroxidase immune complex. CLP failed to inhibit Staphylococcus aureus protein A from binding monomeric IgG. When coated to a microplate, CLP showed selective binding to the immune complex, and could be used for application in immunochemical detection of immune complex. © Rapid Science Ltd. 1998     

Functional complement C1q abnormality leads to impaired immune complexes and apoptotic cell clearance

C1q plays a key role in apoptotic cell and immune complex removal. Its absence contributes to the loss of tolerance toward self structures and development of autoimmunity. C1q deficiencies are extremely rare and are associated with complete lack of C1q or with secretion of surrogate C1q fragments. To our knowledge, we report the first case of a functional C1q abnormality, associated with the presence of a normal C1q molecule. Homozygous GlyB63Ser mutation was found in a patient suffering from lupus with neurologic manifestations and multiple infections. The GlyB63Ser C1q bound to Igs, pentraxins, LPSs, and apoptotic cells, similarly to C1q from healthy donors. However, the interaction of C1r(2)C1s(2) and C1 complex formation was abolished, preventing further complement activation and opsonization by C3. The mutation is located between LysB(61) and LysB(65) of C1q, suggested to form the C1r binding site. Our data infer that the binding of C1q to apoptotic cells in humans is insufficient to assure self-tolerance. The opsonization capacity of C4 and C3 fragments has to be intact to fight infections and to prevent autoimmunity.     

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.     

The sequence and topology of human complement component C9.

A partial nucleotide sequence of human complement component C9 cDNA representing 94% of the coding region of the mature protein is presented. The amino acid sequence predicted from the open reading frame of this cDNA concurs with the amino acid sequence at the amino-terminal end of three proteolytic fragments of purified C9 protein. No long stretches of hydrophobic residues are present, even in the carboxy-terminal half of the molecule which reacts with lipid-soluble photoaffinity probes. Monoclonal antibody epitopes have been mapped by comparing overlapping fragments of C9 molecule to which the antibodies bind on Western blots. Several of these epitopes map to small regions containing other surface features (e.g., proteolytic cleavage sites and N-linked oligosaccharide). The amino-terminal half of C9 is rich in cysteine residues and contains a region with a high level of homology to the LDL receptor cysteine-rich domains. A model for C9 topology based on these findings is proposed.     

Immunoglobulin G antibody bound to the C1q subcomponent of human complement exhibits segmental flexibility

The rotational dynamics of rabbit immunoglobulin G (IgG) anti-dansyl antibodies bound to the C1q subcomponent of human complement were studied by nanosecond fluorescence spectroscopy. Deconvoluted anisotropy decays of IgG-C1q mixtures were fitted to a two-exponential expression and were corrected for the effects of unbound IgG, which was determined with an analytical ultracentrifuge. Compared with the anisotropy parameters for free IgG, the pre-exponential weighting factors and the short correlation time of the C1q-bound antibody were nearly unchanged, and the long correlation time increased by only about 45 nanoseconds. These results, together with rotational diffusion calculations, indicate that the Fab arms of the C1q-bound antibody exhibited considerable flexibility. This finding may have biological relevance because it suggests that C1q can bind to the Fc segments of IgG molecules anchored in an immune complex, even though the angles between the two Fab arms of the different antibodies may vary. The results of this study also support our earlier interpretation that both the short and long correlation times of IgG principally represent flexible motions of the Fab segments.     

Activation of the first component of human complement (C1) by antibody-antigen aggregates.

The activation of subcomponents C1r and C1s in the first component of complement, C1, when bound to antibody-antigen complexes was investigated. Activation was followed both by the splitting of the peptide chains of subcomponents C1r and C1s and by the development of proteolytic activity. For the maximum rate of activation to occur, all components must be present in approximate molar proportions of antibody: C1q:C1r:C1s of 13:1:5:5. For activation of subcomponent C1s, subcomponents C1r or C1r, but not C1r inactivated with iPr2P-F (di-isopropyl phosphorofluorideate), are effective. For activation of subcomponent C1r, subcomponents C1s, C1s or C1s inactivated with iPr2P-F are effective. Subcomponent C1s is activated by C1r, and C1r is activated autocatalytically, probably through the formation of an intermediary C1r. in which the peptide chain is unsplit but a conformational change caused by interaction with the other components has led to the formation of a catalytic site able to split subcomponent C1r to C1r.     

The molecular architecture of human complement component C6

The molecular architecture of human complement component C6 was elucidated at several levels of structural organization. The entire primary structure of C6 was determined by sequencing C6 cDNA that was cloned from a human liver lambda gt11 library. The polypeptide chain of C6 contains 913 amino acids. The protein is homologous with the other terminal components of complement, C7-C9. Specifically, C6 has 29% of its residues identical with C7, and 55 of the 56 cysteines found in C7 match those in C6. The C6 polypeptide chain is cross-linked by 32 disulfide bonds, and most of the cysteines are located in short (34-77 amino acids) discrete segments that exhibit homology with a wide variety of other proteins such as thrombospondin, the low density lipoprotein receptor, epidermal growth factor, and complement factors H and I. C6 is a glycoprotein, and it has two oligosaccharide groups attached to asparagines located near the amino and the carboxyl termini of the molecule. The organization of secondary structural elements in C6 was elucidated using circular dichroism spectroscopy and an empirical method based on sequence analysis. C6 has an estimated 12% alpha-helix, but is comparatively richer in beta-sheet (29%) and beta-turns (21%). Most of the predicted alpha-helical structure resides in a portion of the polypeptide chain that is free of cysteine and which shares homology with C9 and perforin. The tertiary structure of the C6 molecule was visualized by transmission electron microscopy; it has a sickle shape with dimensions of 144 x 66 A. The combined results are discussed and comparisons made with the other late acting components of complement and perforin.     

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.     

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.     

Cloning and sequencing of full-length cDNA encoding the precursor of human complement component C1r.

The sequencing of human liver cDNA clones encoding the entire C1r precursor protein has confirmed the previously determined peptide sequence and has shown that there is a leader peptide which is 17 amino acids long. A residue tentatively identified as beta-hydroxyaspartic acid [Arlaud, Willis & Gagnon (1986) Biochem. J., in the press] located in the C1r A-chain, within an epidermal-growth-factor consensus sequence, was found to be encoded as asparagine. Two sequence elements, tandemly located in the A-chain, are related to a sequence widespread among proteins which interact with C3b or C4b. Structural comparisons between different clones indicate that multiple polyadenylation sites are responsible for the length heterogeneity observed for C1r mRNA from liver and Hep G2 cells.     

Binding of complement component C1q by spectrin

125I-labelled human C1q was found to bind to human spectrin. Scatchard plots for the binding process were non-linear, indicating the possible presence of multiple classes of binding sites for C1q on spectrin. The binding was ionic-strength-dependent; the extent of binding decreased with increasing ionic strength. Chemical modification of arginine and histidine residues on C1q as well as pretreatment of C1q at pH 4.45 or at 56 degrees C reduced its spectrin binding activity. The amount of 125I-labelled C1q bound to immune complexes was reduced by the presence of spectrin. Spectrin was also able to deplete the complement haemolytic activity of human serum in a dose-dependent manner.