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.     

Kinetics of interaction of C1 inhibitor with complement C1s

The kinetics of inhibition of the complement serine protease, C1s, by its only known inhibitor, C1 inhibitor, have been measured by a variety of methods. One method continuously monitors the loss of esterolytic activity with a synthetic substrate coupled to a chromogen while another monitors the formation of a stable (covalent) complex by high-pressure size-exclusion chromatography under dissociating conditions. Additional methods employ fluorescence probes to follow the formation of bimolecular complexes but are not expected to distinguish between covalent product and noncovalent (reversible) intermediates. There was good agreement between rate constants obtained by the various methods over a broad range of inhibitor concentrations, suggesting that noncovalent intermediates do not accumulate to a significant extent. The reaction appears to be pure second order with a bimolecular rate constant of 6.0 X 10(4) M-1 s-1 at 30 degrees C, independent of Ca2+, and an activation energy of 11.0 kcal/mol. The rate increases up to 35-fold in the presence of heparin which was shown to bind to all three components (enzyme, inhibitor, and complex) with similar affinity (Kd = 2.0-3.3 microM). The fluorescent probe 1,1'-bis(anilino)-4-,4'-bi(naphthalene)-8,8'-disulfonate [bis(ANS)] bound to the complex with Kd = 0.26 microM under conditions where the individual components had little affinity for the dye, consistent with the generation of one or more hydrophobic binding sites on the protein surface during complex formation.     

Inhibition of bFGF activity by complement C1s: covalent binding of C1s with bFGF

The first complement component C1s formed large aggregates with bFGF when bFGF and C1s were incubated at 37°C overnight. Under non-reducing conditions, a part of the aggregates did not penetrate into 5% polyacrylamide gel in the presence of SDS, and the rest penetrated into 5% gel but not into 12% gel. The aggregates were dissociated into monomers by reducing with 2-mercaptoethanol. Both active and inactive C1s formed aggregates with bFGF. In addition, a portion of bFGF was degraded by active C1s but not by inactive C1s. Aggregates were not formed when 2-mercaptoethanol (2 mM &base;) was added to the incubation mixture. After the incubation with C1s the growth-stimulating activity of bFGF was measured by using human umbilical vein endothelial cells (HUVEC) as indicator cells. The aggregate formation between C1s and bFGF significantly reduced the activity of bFGF. © 1998 John Wiley & Sons, Ltd.     

Selective complement C1s deficiency caused by homozygous four-base deletion in the C1s gene

The complement system plays an important role in defense mechanisms by promoting the adherence of microorganisms to phagocytic cells and lysis of foreign organisms. Deficiencies of the first complement components, C1r/C1s, often cause systemic lupus erythema-tosus-like syndromes and severe pyogenic infections. Up to now no genetic analysis of the C1r/C1s deficiencies has been carried out. In the present work, we report the first genetic analysis of selective C1s deficiency, the patient having a normal amount of C1r. C1s RNA with a normal size was detected in patient’s subcutaneous fibroblasts (YKF) by RNA blot analysis and RT-PCR. The amount of C1s RNA was approximately one-tenth of the RNA from the human chondrosarcoma cell line, HCS2/8. In contrast, the levels of C1r and β-actin RNA of YKF were similar to that of HCS2/8. Sequence analysis of C1s cDNA revealed a deletion at nucleotides 1087–1090 (TTTG), creating a stop codon (TGA) at position 94 downstream of the mutation site. Direct sequencing of the gene between the primers designed on intron 9 and exon 10 indicated the presence of the deletion on exon 10 of the gene. Quantitative Southern blot hybridization suggested the mutation was homozygous. The 4-bp deletion on exon 10 was also found in the patient’s heterozygous mother who had normal hemolytic activity. Received: 6 July 1998 / Accepted: 1 August 1998     

The cleavage of two C1s subunits by a single active C1r reveals substantial flexibility of the C1s-C1r-C1r-C1s tetramer in the C1 complex

The activation of the C1s-C1r-C1r-C1s tetramer in the C1 complex, which involves the cleavage of an Arg-Ile bond in the catalytic domains of the subcomponents, is a two-step process. First, the autolytic activation of C1r takes place, then activated C1r cleaves zymogen C1s. The Arg463Gln mutant of C1r (C1rQI) is stabilized in the zymogen form. This mutant was used to form a C1q-(C1s-C1rQI-C1r-C1s) heteropentamer to study the relative position of the C1r and C1s subunits in the C1 complex. After triggering the C1 by IgG-Sepharose, both C1s subunits are cleaved by the single proteolytically active C1r subunit in the C1s-C1rQI-C1r-C1s tetramer. This finding indicates that the tetramer is flexible enough to adopt different conformations within the C1 complex during the activation process, enabling the single active C1r to cleave both C1s, the neighboring and the sequentially distant one.     

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.     

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.     

Calcium-linked self-association of human complement C1s.

The weight-average molecular weight of C1s, an activated serine protease subcomponent of human complement C1, has been measured by means of sedimentation equilibrium over a wide range of both protein and calcium ion concentrations. The combined data may be accounted for quantitatively by a simple model for Ca(2+)-dependent self-association of C1s to a dimer. According to this model, the monomer contains a single Ca2+ binding site with K approximately equal to 3 x 10(5) M-1, and the dimer contains three independent Ca binding sites, two having a Ca2+ affinity lower than that of the monomer (K approximately equal to 3 x 10(4) M-1). The third binding site in the dimer, which presumably lies at the interface between the two amino-terminal alpha domains, has a higher Ca2+ affinity (K approximately equal to 1 x 10(8) M-1) and provides the driving force for C1s dimerization in the presence of calcium.     

Biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component C1s

Complement activation has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury, acute respiratory distress syndrome, and acute transplant rejection. Even though the complement cascade provides several protein targets for potential therapeutic intervention only two complement inhibitors have been approved so far for clinical use including anti-C5 antibodies for the treatment of paroxysmal nocturnal hemoglobinuria and purified C1-esterase inhibitor replacement therapy for the control of hereditary angioedema flares. In the present study, optimization of potency and physicochemical properties of a series of thiophene amidine-based C1s inhibitors with potential utility as intravenous agents for the inhibition of the classical pathway of complement is described.     

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.     

C1 dissociation. Spontaneous generation in human serum of a trimer complex containing C1 inactivator, activated C1r, and zymogen C1s

Activation of the C1 complex in the presence of C1 inactivator (C1 IA) is known to result in the formation of tetramer C1 IA-C1r-C1s-C1 IA complexes that are dissociated from C1q. Both C1r and C1s of the tetramers are present in their activated forms. The present investigation concerned the generation of trimer complexes containing C1 IA, activated C1r, and zymogen C1s (C1 IA-C1r-C1s). C1 IA-C1r-C1s were released from C1q and were formed in high concentration during prolonged incubation (1 to 3 days) of normal serum at 37 degrees C without addition of activators. By contrast, dissociation of C1 with formation of C1 IA-C1r-C1s-C1 IA was complete within 30 min at 37 degrees C, when the serum was treated with heat-aggregated IgG (1 g/liter). On size exclusion chromatography (TSK-4000), C1 IA-C1r-C1s and C1 IA-C1r-C1s-C1 IA emerged with apparent m.w. of 320,000 and 460,000, respectively. The composition of the complexes was examined by absorption of serum with F(ab')2 anti-C1s- or anti-C1r-coated Sepharose beads. Eluates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with immunoblotting. Under nonreducing conditions, heat-aggregated IgG-treated serum showed high concentrations of C1 IA-C1r (m.w. 202,000) and C1 IA-C1s (m.w. 194,000), while serum incubated at 37 degrees C without activators showed high concentrations of C1 IA-C1r but no C1 IA-C1s. Under reducing conditions, heat-aggregated IgG-treated serum showed m.w. 120,000 and 110,000 complexes of C1 IA and the C1r and C1s light chains, respectively. Uncleaved C1s and the m.w. 120,000 complex was found in serum that was incubated at 37 degrees C without activators. Consistent with results obtained by size exclusion chromatography, analysis by crossed immunoelectrophoresis and by electroimmunoassay showed that C1s could be released from C1 IA-C1r-C1s in the presence of EDTA.     

Identification of the C1q-binding Sites of Human C1r and C1s: A REFINED THREE-DIMENSIONAL MODEL OF THE C1 COMPLEX OF COMPLEMENT*

The C1 complex of complement is assembled from a recognition protein C1q and C1s-C1r-C1r-C1s, a Ca²⁺-dependent tetramer of two modular proteases C1r and C1s. Resolution of the x-ray structure of the N-terminal CUB₁-epidermal growth factor (EGF) C1s segment has led to a model of the C1q/C1s-C1r-C1r-C1s interaction where the C1q collagen stem binds at the C1r/C1s interface through ionic bonds involving acidic residues contributed by the C1r EGF module (Gregory, L. A., Thielens, N. M., Arlaud, G. J., Fontecilla-Camps, J. C., and Gaboriaud, C. (2003) J. Biol. Chem. 278, 32157–32164). To identify the C1q-binding sites of C1s-C1r-C1r-C1s, a series of C1r and C1s mutants was expressed, and the C1q binding ability of the resulting tetramer variants was assessed by surface plasmon resonance. Mutations targeting the Glu¹³⁷-Glu-Asp¹³⁹ stretch in the C1r EGF module had no effect on C1 assembly, ruling out our previous interaction model. Additional mutations targeting residues expected to participate in the Ca²⁺-binding sites of the C1r and C1s CUB modules provided evidence for high affinity C1q-binding sites contributed by the C1r CUB₁ and CUB₂ modules and lower affinity sites contributed by C1s CUB₁. All of the sites implicate acidic residues also contributing Ca²⁺ ligands. C1s-C1r-C1r-C1s thus contributes six C1q-binding sites, one per C1q stem. Based on the location of these sites and available structural information, we propose a refined model of C1 assembly where the CUB₁-EGF-CUB₂ interaction domains of C1r and C1s are entirely clustered inside C1q and interact through six binding sites with reactive lysines of the C1q stems. This mechanism is similar to that demonstrated for mannan-binding lectin (MBL)-MBL-associated serine protease and ficolin-MBL-associated serine protease complexes.The classical pathway of complement, a major component of innate immune defense against pathogens and altered self, is triggered by C1, a 790-kDa Ca²⁺-dependent complex assembled from a recognition protein C1q and C1s-C1r-C1r-C1s, a tetramer of two modular proteases, C1r and C1s, that respectively mediate activation and proteolytic activity of the complex (1–3). C1q has the overall shape of a bunch of tulips and comprises six heterotrimeric collagen-like triple helices that assemble through their N-terminal moieties to form a “stalk” and then diverge to form individual “stems,” each prolonged by a C-terminal globular recognition domain (4). C1r and C1s are homologous modular proteases each comprising, starting from the N-terminal end, a C1r/C1s, sea urchin EGF² (uEGF), bone morphogenetic protein (CUB) module (5), an EGF-like module (6), a second CUB module, two complement control protein modules (7), and a serine protease domain. This modular structure is shared by the mannan-binding lectin-associated serine proteases (MASPs), a group of enzymes that associate with mannan-binding lectin (MBL) and the ficolins and thereby trigger activation of the lectin pathway of complement (8).Assembly of the C1s-C1r-C1r-C1s tetramer involves Ca²⁺-dependent heterodimeric C1r-C1s interactions between the CUB₁-EGF segments of each protease (9–12). Similarly, MASP-1, MASP-2, MASP-3, and mannan-binding lectin-associated protein 19 (MAp19), an alternative splicing product of the MASP-2 gene comprising the N-terminal CUB₁-EGF segment of MASP-2, all associate as homodimers through their N-terminal CUB₁-EGF moieties (13–15). The structures of human C1s CUB₁-EGF, human MAp19, human MASP-1/3 CUB₁-EGF-CUB₂, and rat MASP-2 CUB₁-EGF-CUB₂ have been solved by x-ray crystallography (16–19), revealing that these domains all associate as head-to-tail homodimers through a highly conserved interface involving interactions between the CUB₁ module of one monomer and the EGF module of its counterpart. In addition, all CUB modules contained in these structures were found to contain a hitherto unrecognized Ca²⁺-binding site involving three conserved acidic residues (Glu⁴⁵, Asp⁵³, and Asp⁹⁸ in C1s), defining a novel CUB module subset diverging from the type originally described in the spermadhesins (20).Mutagenesis studies have recently established that assembly of the MBL- and ficolin-MASP complexes involves a major electrostatic interaction between two acidic Ca²⁺ ligands from the MASP CUB modules and a conserved lysine located in the collagen fibers of MBL and ficolins (16, 18, 21, 22). In the case of C1, a hypothetical model of the C1q/C1r/C1s interface, involving interaction between acidic residues mainly contributed by the C1r EGF module and unmodified lysine residues also located in the collagen-like stems of C1q, was derived from the x-ray structure of the C1s CUB₁-EGF interaction domain (16, 23). The aim of this work was to use site-directed mutagenesis to delineate the sites of C1r and C1s involved in the interaction between C1s-C1r-C1r-C1s and C1q. Our data rule out our previous interaction model and provide evidence that C1 assembly involves the same basic Ca²⁺-dependent mechanism as demonstrated in the case of MBL-MASP and ficolin-MASP complexes.     

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.