Activator-bound C1 is less susceptible to inactivation by C1 inhibition than is fluid-phase C1

Parameters that influence the effective interaction of C1 with the serum regulatory glycoprotein C1 Inhibitor were investigated. C1 that bound to activator particles EAC4 or EA was strikingly less susceptible to inactivation by C1 Inhibitor than was fluid-phase C1. By using the conventional hemolytic assay, the concentrations of C1 Inhibitor required for inhibition of C1 bound to EAC4 were 1000-fold higher than those required for fluid-phase C1. With EA as the activator (and indicator) particle, 17- to 75-fold higher concentrations of C1 Inhibitor were required to inhibit bound vs free C1. These findings suggest that, on binding to these particulate immune complexes, the domain of the C1 molecule capable of interacting with C1 Inhibitor is less available for binding than when C1 is in fluid phase. Alternatively, the conformation of C1 may be altered when bound to EA or EAC4, resulting in a lower association constant of C1 Inhibitor for C1. As assessed by inhibition of classical complement pathway hemolysis, the inhibition of the enzymatic activity of C1 by C1 Inhibitor (both in the fluid phase and particle-bound) was markedly dependent on the concentration of the reactants. Incubation of C1 and C1 Inhibitor at serum concentrations resulted in the inhibition of more than 10 times the amount of C1 hemolytic activity than that which occurred when the same ratio of components was incubated at the more dilute concentrations used in the conventional hemolytic assays. These findings have allowed for the development of a more sensitive and rapid assay for C1 Inhibitor function.     

Activation of C1 by monoclonal antibodies directed against C1q

Eleven monoclonal antibodies directed against the subcomponent C1q of the first component of human complement, C1, were prepared and tested for binding to intact C1q and to the collagenous portion, the C1q stalks. All of the monoclonals bound well to the intact C1q. Eight out of the eleven exhibited strong binding to the collagenous stalks, while three bound very weakly, if at all, to the stalks and, thus, were presumed to bind to the pepsin-sensitive region which includes the C1q heads. For one of the latter monoclonals, this was confirmed by electron microscopy. Five of the monoclonals were purified by C1q affinity chromatography. When tested with C1 reassembled from its subunits, two of these purified monoclonal antibodies markedly enhanced the rate of spontaneous activation.     

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.     

Antibody-independent activation of C1. I. Differences in the mechanism of C1 activation by nonimmune activators and by immune complexes: C1r-independent activation of C1s by cardiolipin vesicles

C1 activation is controlled by the regulatory protein C1-inhibitor (C1-INH). In contrast to immune-complex-induced activation, which is insensitive to C1-INH, antibody-independent activation of C1 is modulated by C1-INH. The mechanisms regulating nonimmune activation were studied with two phospholipids varying in their capacity to activate C1 in the presence of C1-INH: cardiolipin (CL) and phosphatidylglycerol (PG). Whereas C1-INH consistently suppressed activation by PG vesicles, a dose-dependent increase in C1 activation was measured with CL vesicles above 40 mole %. A similar dose-response binding of C1s requiring C1q, but not C1r, was detected only on CL vesicles, but neither on PG vesicles nor on immune complexes. This binding was Ca2+-dependent, suggesting that dimeric C1s is involved and was inhibited by spermine. The C1q-bound C1s was specifically cleaved at 37 degrees C into its active 58 kDa and 28 kDa chains, in the absence of C1r. On the addition of anti-CL antibodies, the C1q-mediated cleavage of C1s by CL vesicles was specifically inhibited. The cleavage of C1r on CL vesicles was also determined. When macromolecular C1 was offered in the presence of C1-INH, C1r cleavage was detected; however, the presence of C1s was a critical factor for C1r activation, because it was required on CL vesicles, but not on immune complexes. These results show that nonimmune activation of C1 presents specific features which distinguish it from immune complex-induced activation. These characteristics varied with the capacity of antibody-independent activators to activate C1 in the presence of C1-INH.     

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 inhibitor cross-linking by tissue transglutaminase

C1 inhibitor, a plasma proteinase inhibitor of the serpin superfamily involved in the regulation of complement classical pathway and intrinsic blood coagulation, has been shown to bind to several components of the extracellular matrix. These reactions may be responsible for C1 inhibitor localization in the perivascular space. In the study reported here, we have examined whether C1 inhibitor could function as a substrate for plasma (factor XIIIa) or tissue transglutaminase. We made the following observations: 1) SDS-polyacrylamide gel electrophoresis and autoradiography showed that C1 inhibitor exposed to tissue transglutaminase (but not to factor XIIIa) incorporated the radioactive amine donor substrate [(3)H]putrescine in a calcium-dependent manner; 2) the maximum stoichiometry for the uptake of [(3)H]putrescine by C1 inhibitor was 1:1; 3) proteolytic cleavage and peptide sequencing of reduced and carboxymethylated [(3)H]putrescine-C1 inhibitor identified Gln(453) (P'9) as the single amine acceptor residue; 4) studies with (125)I-labeled C1 inhibitor showed that tissue transglutaminase was also able to cross-link C1 inhibitor to immobilized fibrin; and 5) C1 inhibitor cross-linked by tissue transglutaminase to immobilized fibrin had inhibitory activity against its target enzymes. Thus, tissue transglutaminase-mediated cross-linking of C1 inhibitor to fibrin or other extracellular matrix components may serve as a mechanism for covalent serpin binding and influence local regulation of the proteolytic pathways inhibited by C1 inhibitor.     

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.     

Phosphorylation of laminin-1 by protein kinase C

Several extracellular proteins have been reported to be phosphorylated. Previous studies of our laboratory indicated that laminin-1 can be phosphorylated by protein kinase A (PKA). Moreover, it has been reported that protein kinase C (PKC), although known to be intracellular, can phosphorylate extracellular proteins in the case of cellular damage and/or platelet activation. In the present study we examined the possibility of laminin-1 serving as a substrate of PKC. Amino acid analysis revealed that laminin-1 is phosphorylated by this enzyme on serine residues. Self assembly, heparin binding, and cell attachment on the phosphorylated molecule were then studied. Phosphorylated laminin-1 showed an increased and more rapid self assembly than the non-phosphorylated molecule. Heparin binding and cell attachment experiments indicated enhanced heparin and cell binding capacity of the phosphorylated molecule in comparison to the non- phosphorylated control. These results indicate that laminin-1 can be phosphorylated by protein kinase C. Furthermore, phosphorylation by protein kinase C seems to alter several properties of the molecule, though, the in vivo significance of this phenomenon remains to be studied.     

Antibody-independent C1 activation by E. coli

Antibody-independent interactions of C1 with several E. coli strains were examined. Purified C1 was directly activated by the semi-rough mutant E. coli J-5, its parental wild-type strain, E. coli 0111:B4, and two clinical isolates, E. coli (P) and E. coli (A), in the absence of C1 inhibitor. E. coli J-5 activated C1 about 10-fold more rapidly and bound approximately threefold more C1 than the other strains. E. coli J-5, but not the other strains, also bound C1s2, provided that the subcomponent was offered to the bacteria in the presence of C1q and calcium; such binding was thus independent of the presence or absence of C1r2. After C1 activation in the absence of C1 inhibitor, activated C1s spontaneously dissociated from E. coli 0111:B4, (P), and (A), but remained associated with E. coli J-5. The regulatory protein C1 inhibitor prevented C1 activation by the weaker activators, E. coli strains 0111:B4, (P), and (A), but had no effect on C1 activation by E. coli J-5. Although C1 inhibitor thus failed to modulate C1 activation by E. coli J-5, it did block the enzymatic activity of activated C1 bound to this strain. Analyses of the molecular processes involved revealed differences with other systems. In the presence of C1 inhibitor, the C1s subunit of C1 activated by E. coli J-5 underwent further cleavage with the release into the supernatant of C1s fragments and complexes of C1 inhibitor with light chain fragments. Such fragments were not disulfide-linked to the remainder of the C1s molecule. The bulk of the heavy chain remained adherent to the surface of E. coli J-5. This finding documents the presence of a binding site for activated C1s on the surface of E. coli J-5 and localizes this site to the heavy chain. These studies thus indicate that several E. coli strains are direct C1 activators. Furthermore, E. coli J-5 provides another example of a direct C1 activator having binding sites not only for C1q but also for dimeric C1s. The studies also show that there are multiple properties of particles which determine the ability to activate C1, the rate of activation, the possibility of regulation of the activation process by C1 inhibitor, and the fate of activated C1.     

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.     

C1q (c1) receptor on human platelets: inhibition of collagen-induced platelet aggregation by C1q (C1) molecules.

Hemolytically active human C1q incubated with EA before the addition of complement inhibited the immune hemolysis. On the contrary, heat-inactivated preparation (30 min 56 degrees C) was ineffective. Preincubation of EA with bovine collagen also resulted in a decreased hemolysis. When aggregation was measured by a turbidimetric method in citrated human platelet-rich plasma, it was found that hemolytically active human C1q (C1) alone does not induce platelet aggregation. However, in its presence the platelets failed to aggregate or exhibited a significantly reduced aggregation response to bovine collagen. The inhibition by C1q depended on the preincubation time with platelets. Heat treatment (30 min 56 degrees C) destroyed the inhibitory action of C1q (C1). The effect of C1q proved to be highly specific because different C1q preparations at their inhibitory doses in collagen-induced platelet aggregation did not influence the response to other aggregating agents (bovine thrombin, ADP, horse anti-human thymocyte globulin, goat anti-baboon platelet antiserum). The results prove that collagen and C1q are capable of binding to the same site(s); namely, to those of EA and human platelets; furthermore, they suggest the presence of a receptor for C1q (C1) on human platelets.     

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     

Characterization of C1q, C1s and C-1 Inh synthesized by stimulated human monocytes in vitro

C1q, C1s and C1 Inh synthesized and secreted by human monocytes were characterized by SDS-PAGE. C1q is formed of three chains A (Mr approximately 35 000), B (Mr approximately 33 000) and C (Mr approximately 25 000) which are associated in two subunits A-B and C-C. It appears identical to C1q purified from plasma. C1s is secreted as a non-activated, monocatenar protein of Mr approximately 87 000 identical to proenzymic C1s from plasma. Secreted C1 Inh (Mr approximately 100 000) has a slightly higher Mr than purified plasmatic C1 Inh. Monensin treatment of the cells favours the intracytoplasmic accumulation of products at various glycosylation stages.