Functional model of subcomponent C1 of human complement

The domain organization of the zymogen subunits of the first component of human complement C1s, C1r2 and the complex C1s-C1r2-C1s was studied by electron microscopy. In the absence of Ca2+, monomeric C1s 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 CIs. The subunit C1r is a stable dimer in the presence or absence of Ca2+. This dimer C1r2 was visualized as composed of two dumb-bells of dimensions similar to those observed for C1s. 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 C1rII2, in which part of the A-chain is removed, the inner globular domains were assigned to the catalytic B-chains. This characteristic structure of C1r2 is readily recognized in the central portion of the thread-like 54 nm long C1s-C1r2-C1s complex formed in the presence of Ca2+. By affinity-labeling of C1s with biotin and visualization of avidin-ferritin conjugates in the reconstituted complex, it was demonstrated that C1s forms the outer portion of the complex. A detailed model of C1s-C1r2-C1s is proposed, according to which two C1s monomers bind to the outer globes of C1r2 by contacts between their heavy chains and those of C1r. According to this model the catalytic domains of C1r are located in the center and those of C1s at the very tips of the C1s-C1r2-C1s complex. On the basis of the structure of C1s-C1r2-C1s, 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.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

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.     

Human complement subcomponent C2: purification and proteolytic cleavage in fluid phase by C1s, C1r2-C1s2 and C1

Photosynthetic membranes contain considerable regions of high surface curvature, notably at their margins, where the average radius of curvature is about 10 nm. The proportion of total membrane lipid in the outer and inner thylakoid margin monolayers is estimated at 21% and 13%, respectively. The major thylakoid lipid, monogalactosyldiacylglycerol, is roughly cone-shaped and will not form complete lamellar bilayer phases, even in combination with other thylakoid lipids. It is proposed that this galactolipid plays a role in: (a) stabilising regions of concave curvature in thylakoids; and (b) packaging hydrophobic proteins in planar bilayer regions by means of inverted micelles. This model predicts substantial asymmetries in the distribution of lipids both across and along the thylakoid bilayer plane.     

Hydroxylysine-linked glycosides of human complement subcomponent C1q and various collagens.

1. Human C1q, a subcomponent of the first component of complement, contains 67 disaccharides (glucosylgalactose) and 2.4 monosaccharides (galactose) linked to hydroxylysine in one molecule. It was found that 82.6% of the hydroxylsine residues were glycosylated. The suggestion of the possible existence of glucosylgalactosylhydroxylysine reported previously [Yonemasu, Stroud, Niedermeir & Butler (1971) Biochem. Biophys. Res. Commun. 43, 1388--1394] was confirmed. 2. The hydroxylysine-glycosides are not detected in the C-terminal, non-collagen-like, globular regions, but only in the collagen-like regions in the subcomponent C1q molecule. 3. Alpha 1(I) and alpha 2 in pig skin, alpha 1(II) in bovine cartilage and alpha 1(III) in bovine skin collagens contain 2.0, 2.2, 13.2 and 2.0 residues of hydroxylysine-glycosides per molecule, respectively. The percentage of hydroxylysine residues glycosylated in each of these chains is relatively low (on average 38%). 4. Neither the high percentage of hydroxylysine residues glycosylated nor the high values for the ratios of disaccharides to monosaccharides in the subcomponent C1q resembles that in alpha 1(I), alpha 2, alpha 1(II) and alpha 1(III). 5. Similarities between the extent of glycosylation of hydroxylysine residues in collagen-like regions in the subcomponent C1q molecule and that of the collagenous constituents of human glomerular basement membranes, aortic intima, skin A- and B-chains and of bovine anterior lens capsule are discussed.     

Calcium-sensitive thermal transitions and domain structure of human complement subcomponent C1r

Fluorescent probes and other methods have been used to investigate the thermal stability of activated C1r and functionally intact fragments isolated from tryptic digests of the protein. This enzyme exhibits two irreversible transitions that differ with respect to their sensitivity to metal ions. The high-temperature transition occurs with a midpoint near 53 degrees C in 0.02 M tris(hydroxymethyl)aminomethane buffer and 0.15 M NaCl, pH 7.4. It is relatively insensitive to Ca2+ and ionic strength and is accompanied by a loss of catalytic activity. The low-temperature transition is most easily observed in the presence of ethylenediaminetetraacetic acid and is completely abolished by 100 microM Ca2+. Its midpoint varies between 26 degrees C at low ionic strength and 40 degrees C in the presence of 0.5 M NaCl. The low-temperature transition results in extensive polymerization of the protein without loss of the esterolytic activity or the ability to react with C1 inhibitor; however, the ability to reconstitute hemolytically active C1 or even bind to C1s in the presence of Ca2+ is destroyed. A highly purified N-terminal fragment generated by tryptic digestion of C1r in the presence of Ca2+ retained its ability to interact with C1s, disrupting the formation of C1s dimers in the presence of Ca2+. In the absence of Ca2+, this fragment displays only a low-temperature transition that is very similar to the one observed with the whole protein and that destroys its ability to bind to C1s. Addition of Ca2+ stabilizes this fragment, shifting the midpoint of its melting transition upward by more than 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete amino acid sequence of the catalytic chain of human complement subcomponent C1-r

The amino acid sequence of human C1-r b chain hs been determined, from sequence analysis performed on fragments obtained by CNBr cleavage, dilute acid hydrolysis, tryptic cleavage of the succinylated protein, and subcleavages by staphylococcal protease. The polypeptide chain contains 242 amino acids (Mr 27 096), and the sequence shows strong homology with other mammalian serine proteases. The histidine, aspartic acid, and serine residues of the active site (His-57, Asp-102, and Ser-195 in bovine chymotrypsinogen) are located at positions 39, 94, and 191, respectively. The chain which lacks the "histidine-loop" disulfide bridge, contains five half-cystine residues, of which four (positions 157-176 and 187-217) are homologous to residues involved in disulfide bonds generally conserved in serine proteases, whereas the half-cystine residue at position 114 is likely to be involved in the single disulfide bridge connecting the catalytic b chain to the n-terminal a chain. Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 51 and 118.     

The enzymatic nature of human c1r: a subcomponent of the first component of complement.

The esterase activity of the C1r subcomponent of the first component of complement has been investigated. C1r was found to hydrolyze two amino acid methyl esters; N-acetyl-L-arginine methyl ester and N-acetyl-glycyl-L-lysine methyl ester, and two amino acid p-nitrophenyl esters, N-carbobenzyloxy-L-tyrosine-p-nitrophenyl ester and N alpha-carbobenzyloxy-L-lysine-p-nitrophenyl ester. A detailed kinetic analysis of the hydrolysis of N-Z-L-Tyr-ONp by C1r revealed that the enzymatic activity per microgram of protein decreased as the C1r concentration was increased. The loss of activity suggested that above 0.5 micron C1r was undergoing aggregation with a loss of active sites. Similarly, when C1r was titrated with the active site titrant p-nitrophenyl-P'-guanidinobenzoate the number of titratable sites per milligram of protein decreased with increasing protein concentration. The hydrolysis of N-Z-L-Tyr-ONp by C1r was inhibited by several synthetic inhibitors including phenylmethanesulfonylfluoride, p-amidinophenylmethanesulfonylfluoride, diisopropylfluorophosphate, and p-tosyl-L-lysine-chloromethyl ketone. However, the peptide esterase inhibitors Trasylol, hirudin, leupeptin, and C1 esterase inhibitor had no effect on the esterase activity of C1r.     

The role of C1 esterase inhibitor in the activation of C1r, a subcomponent of the first component of complement from human plasma.

Clr was isolated from human serum by DEAE-cellulose column chromatography in the presence of EDTA. The isolated Clr did not hydrolyze N(alpha)-acetyl-L-arginine methyl ester, unless activated by brief treatment with trypsin [EC 3.4.21.4]. On thecolumn, the C1 esterase inhibitor activity was found to coincide with Clr but not C1s (another subcomponent of the first component) C1r was isolated from the euglobulin fraction of human serum by DEAE-cellulose column chromatograph. On Sephadex G-200 column chromatography, Clr was eluted in the void volume, whereas Clr was eluted in a position corresponding to a molecular weight of 140,000-160,000. The results indicated that, on activation, Clr was converted to an enzyme of lower molecular weight...     

The purification and characterization of subcomponent C1s of the first component of bovine complement.

Bovine C1s, a subcomponent of the first component of complement, was purified in good yield by a combination of euglobulin precipitation and ion-exchange and molecular-sieve chromatography. Approx. 10 mg can be obtained from 3 litres of serum, representing a yield of 11%. The C1s is obtained in zymogen form, with a mol.wt. of 85000-88000, determined by gel filtration and SDS/polyacrylamide-gel electrophoresis. It is haemolytically active when tested with human C1q and C1r. Activation can be achieved by incubation with human C1r, resulting in cleavage of the C1s chain into two chains of 65000 and 27000 mol.wt. and the generation of an isoleucine N-terminal residue on the smaller chain. Active C1s binds an equimolar amount of di-isopropyl phosphorfluoridate to the smaller chain, which is the C-terminal part in the zymogen. The chains can be separated by ion-exchange in 8 M-urea. All of these characteristics show that bovine C1s is very similar to its human counterpart.     

Proteolytic cleavage of an activated subcomponent of the first component of rabbit complement, C1s

When rabbit C1 purified by affinity chromatography on IgG-Sepharose 6B was chromatographed on DEAE-cellulose in the presence of ethylenediaminetetraacetate, C1s was isolated as two forms, C1s(I) and C1s(II), having different molecular weights. On the other hand, incubation of the C1 with soybean trypsin inhibitor before the chromatography resulted in the isolation of C1s(I) alone, indicating that, during the purification, C1s(II) was derived from C1s(I) by proteolytic cleavage of C1s(I) by a contaminating protease, probably plasmin [EC 3.4.21.7]. In fact, C1s(I) was completely converted to C1s(II) or a C1s(II)-like fragment by highly purified plasmin. Analysis of the polypeptide chain structures revealed that C1s(I), which consisted of H and L chains with molecular weights of 70,000 and 36,000, respectively, was converted to C1s(II) by cleavage of the H chain, since C1s(II) consisted of two chains each with a molecular weight of 37,000. This conversion proceeded without any alteration in C1 esterase activity, but was accompanied by loss of the ability to form C1r-C1s complex.     

Assembly and enzymatic properties of the catalytic domain of human complement protease C1r.

The catalytic properties of C1r, the protease that mediates activation of the C1 complex of complement, are mediated by its C-terminal region, comprising two complement control protein (CCP) modules followed by a serine protease (SP) domain. Baculovirus-mediated expression was used to produce fragments containing the SP domain and either 2 CCP modules (CCP1/2-SP) or only the second CCP module (CCP2-SP). In each case, the wild-type species and two mutants stabilized in the proenzyme form by mutations at the cleavage site (R446Q) or at the active site serine residue (S637A), were produced. Both wild-type fragments were recovered as two-chain, activated proteases, whereas all mutants retained a single-chain, proenzyme structure, providing the first experimental evidence that C1r activation is an autolytic process. As shown by sedimentation velocity analysis, all CCP1/2-SP fragments were dimers (5.5-5.6 S), and all CCP2-SP fragments were monomers (3.2-3.4 S). Thus, CCP1 is essential to the assembly of the dimer, but formation of a stable dimer is not a prerequisite for self-activation. Activation of the R446Q mutants could be achieved by extrinsic cleavage by thermolysin, which cleaved the CCP2-SP species more efficiently than the CCP1/2-SP species and yielded enzymes with C1s-cleaving activities similar to their active wild-type counterparts. C1r and its activated fragments all cleaved C1s, with relative efficiencies in the order C1r < CCP1/2-SP < CCP2-SP, indicating that CCP1 is not involved in C1s recognition.     

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.     

Chemical and hydrodynamic characterization of the human leucocyte receptor for complement subcomponent C1q.

A procedure for preparation of the receptor for complement subcomponent Clq from human tonsil lymphocytes and the monocytic cell line U937 was developed. The procedure is suitable for isolation of several hundred micrograms of the receptor, Clq-R, and has yielded sufficient material for chemical and hydrodynamic characterization. Clq-R from tonsil lymphocytes behaves identically with that from U937 cells. Clq-R has a monomer Mr of 56,000, and is an acidic glycoprotein containing about 17% carbohydrate. The polypeptide chain length is estimated to be 416-448 amino acid residues, with two or three sites for N-linked glycosylation. Detergent-solubilized Clq-R exists as an elongated dimer (f/fo = 1.8), and does not bind a significant weight of detergent. The radioiodinated isolated receptor binds specifically and saturably to solid-phase Clq, but not to collagen, IgG, bovine serum albumin or complement component C3.     

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.     

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.     

Interaction of human plasma kallikrein and its light chain with C1 inhibitor

The light chain of human plasma kallikrein contains the enzymatic active site. The inactivation of kallikrein and of its isolated light chain by C1 inhibitor was investigated to assess the functional contributions of the heavy-chain region of kallikrein and of high molecular weight kininogen to this reaction. The second-order rate constants for the inactivation of kallikrein or its light chain were respectively 2.7 X 10(6) and 4.0 X 10(6) M -1 min -1. High molecular weight kininogen did not influence the rate of kallikrein inactivation. The nature of the complexes formed between kallikrein or its light chain and C1 inhibitor was studied by using sodium dodecyl sulfate (SDS) gradient polyacrylamide slab gel electrophoresis. Kallikrein as well as its light chain combined with C1 inhibitor to form stable stoichiometric complexes that were not dissociated by SDS and that exhibited apparent molecular weights (Mr's) of 185 000 and 135 000, respectively, on nonreduced SDS gels. Reduction of the kallikrein-C1 inhibitor complex gave a band at Mr 135 000 that comigrated with the complex seen for the light chain-C1 inhibitor complex. During the inactivation of both kallikrein and its light chain, a Mr 94 000 fragment of C1 inhibitor was formed which was unable to inactivate or bind kallikrein or its light chain. Kallikrein inactivated by diisopropyl phosphofluoridate did not form SDS-stable complexes with C1 inhibitor. These results demonstrate that the functional binding site for C1 inhibitor is localized in the light chain of kallikrein.(ABSTRACT TRUNCATED AT 250 WORDS)