Spontaneous activation of the first component of human complement (C1) by an intramolecular autocatalytic mechanism

For biochemical characterization, the first component of human complement (C1) was reconstituted from physiologic concentrations of purified C1q, 125I C1r, and 131I C1s. Upon incubation at 37 degrees C, C1 spontaneously activated, as evidenced by the characteristic proteolysis of the C1r and C1s polypeptide chains as detected by SDS-PAGE analysis. This spontaneous C1 activation followed first-order kinetics (t 1/2 = 4 min and k = 0.173 min-1) with an activation energy of 19.1 kcal/mol. Spontaneous C1 activation was unaffected by the general protease inhibitor phenylmethylsulfonylfluoride (PMSF) but reversibly blocked by a known inhibitor of C1 activation, nitrophenylguanidinobenzoate (NPGB). Spontaneous C1 activation was measured at C1 concentrations ranging from 9 to 160 nM (i.e., 0.05 to 1.0 times physiologic concentrations). The data indicate that C1 spontaneously activates by an intramolecular autocatalytic mechanism, for first-order kinetics were observed over the entire concentration range with t 1/2 = 4 min at each concentration. However, the percentage of activable C1 decreased with dilution due to C1 dissociation (i.e., C1qr2s2 leads to C1q + C1r2s2). The observed concentration of C1 that spontaneously activated at each dilution equalled the concentration of C1 present as macromolecular C1. When reconstituted C1 was mixed with normal human serum (NHS) and then incubated at 37 degrees C, spontaneous C1 activation was completely inhibited. Pretreating NHS at 56 degrees C for 30 min destroyed its inhibitory activity. In conclusion, C1 spontaneously autoactivates at 37 degrees C by an intramolecular mechanism. This activation is suppressed in NHS.     

Activation of human C1: analysis with Western blotting reveals slow self-activation

The first component of human complement was separated from C1-INH by sucrose linear gradient ultracentrifugation. Activation of C1 was studied in the absence and presence of immune complexes; activation was monitored by SDS-PAGE and Western blot. When the partially purified native C1 preparation was incubated at 37 degrees C without immune complexes, activated C1s appeared after 30 min in the case of eightfold dilution with respect to the original serum, and after 45 min with 32-fold dilution. Kinetics of appearance of activated C1r was the same as that of activated C1s. From the following results, we concluded that spontaneous activation may be partially due to proteolytic enzymes contaminating the preparation: 1) a nonspecific protease inhibitor, PMSF, completely inhibited spontaneous activation but did not inhibit the activation of C1 by immune complexes; 2) alpha 2-macroglobulin partially inhibited spontaneous activation, and 3) although spontaneous activation in the absence of PMSF was relatively slow, activated C1 accelerated spontaneous activation that was completely blocked by C1-INH. In contrast to spontaneous activation, the partially purified native C1 was rapidly activated by immune complexes: within 5 min almost all C1 was activated by rabbit IgG anti-human IgM-human IgM complexes. These results support conclusions derived from activation studies when using native C1 and hemolytic assays, and do not support those derived from the activation studies with reconstituted C1 and SDS-PAGE analysis. We suggest that the contradictions can be resolved if one assumes that C1 activation can be both an intra- and intermolecular process; which process dominates is determined by the state of C1 and by experimental conditions.     

Kinetics of C1 activation by a monoclonal anti-C1q antibody and its (Fab)2 fragments

Monoclonal antibody 1H11, which binds to the "head" portion of C1q, has been shown to be a strong, stoichiometric activator of C1, the first component of human complement, maximal activation being achieved at a ratio of one antibody-combining site per one C1q head; moreover, this activation occurs even in the presence of C1-inhibitor, as reported previously. In the present paper, the kinetics of activation are shown to be biphasic; that is, a portion of the C1 is activated very rapidly, and the remainder slowly. These two processes can be separated by the order of mixing of preincubated components; thus, only the rapid activation rate is observed if C1q and the monoclonal antibody are preincubated together and are added subsequently to a mixture of C1r2C1S2 and C1-inhibitor. Only the slow activation rate is observed when C1q, C1r2C1S2, and C1-inhibitor are preincubated and are added subsequently to monoclonal antibody 1H11. Similar results are obtained by using either the intact 1H11 antibody or else the (Fab)2 obtained from it by proteolytic digestion and purification. The rapid phase is independent of the concentration of 1H11 over the range employed; the slow phase depends on 1H11 concentration. Plausible activation schemes are presented to explain the two distinct activation rate processes, and kinetic models are developed which provide a reasonable simulation of the experimental data.     

Control of C1 activation by nascent C3b and C4b: a mechanism of feedback inhibition

We have demonstrated that immune complexes turn over C1, i.e., limiting quantities of immune complexes activate an excess of C1. This was readily apparent in a system of purified C1 and C1-inhibitor (C1-In) but not in normal human serum (NHS). The following results indicate that C3 and C4 are the serum factors responsible for the inhibition of C1 turnover by immune complexes. 1) In a purified protein system composed of C1 and C1-In at pH 7.5, ionic strength 0.14 M, doses of immune complexes that activated all the C1 in 60 min at 37 degrees C yielded no detectable C1 activation when C2, C3, and C4 were also present. All proteins were at their physiologic concentrations. Activation was quantified by SDS-PAGE analysis and hemolytic titration 2) In order to inactivate C3 and C4, NHS was treated with 50 mM methylamine (MeAm) for 15 min at 37 degrees C, after which the MeAm was removed by dialysis. The activities of C1, C2, and C1-In were unaffected by this treatment. Doses of immune complexes that consumed no C1 in NHS, consumed all the C1 in MeAm-treated NHS (MeAm-NHS). 3) Reconstitution of MeAm-NHS with physiologic concentrations of C3 and C4 rendered the serum again resistant to excessive C1 consumption by immune complexes. Immune complexes used in these studies included EA-IgG, EA-IgM, tetanus-human anti-tetanus, and aggregated human IgG. There appeared to be specificity to the inhibition reaction since C4 by itself could inhibit C1 consumption by EA-IgM, whereas the presence of C3 was also required to control EA-IgG. Finally, N-acetyl-L-tyrosine was added to NHS at a final concentration of 30 mM. This nucleophile did not interact with native C3 or C4, nor did it directly activate C1. However, upon the addition of low doses of immune complexes, acetyl tyrosine did yield uncontrolled C1 activation, presumably by binding nascent C3b and C4b and thereby blocking their attachment to the immune complexes. We conclude that in NHS there is a mechanism of feedback inhibition by which nascent C3b and C4b inhibit C1 turnover by immune complexes. This mechanism of control might be physiologically important in that it prevents excessive complement activation by low concentrations of immune complexes.     

Purification of a proteinase inhibitor from bovine serum with C1-inhibitor activity

This report describes the purification of a novel proteinase inhibitor from bovine serum. This protein was purified to apparent homogeneity employing affinity binding to sulfated dextran and precipitation by ammonium sulfate, followed by sequential chromatography on DEAE-cellulose, heparin-Sepharose and Sephacryl S-200. Quantitative enzyme-linked immunosorbent assays revealed that the concentration of this inhibitor is approximately 3 microM in bovine serum. The inhibitor is a single polypeptide chain with an estimated Mr of 83,000 as determined by SDS-polyacrylamide gel electrophoresis. An aspartic acid was found at the amino terminus of the protein; N-terminal amino acid sequence data indicated that there was no significant homology with other reported amino acid sequences. This bovine inhibitor covalently complexed the human proteinases C1-r, C1-s, factor XIIa and plasma kallikrein, which are also complexed and inactivated by human C1-inhibitor. In addition, the bovine inhibitor complexed and inactivated bovine chymotrypsin, a feature which functionally distinguishes it from human C1-inhibitor. Although the bovine inhibitor appears functionally very similar to C1-inhibitor, we found no evidence for structural homology with the human counterpart.     

The dissociation properties of native C1

The first component of human complement (C1) readily dissociates under physiologic conditions into two subunits - C1q and C1r₂C1s₂. The equilibrium constant for this reaction has been determined for native C1 in fresh normal human serum by hemolytic titration. Standard technology was modified to simulate physiologic conditions. Furthermore, assays were carried out at numerous poncentrations of sensitized erythrocytes, thereby allowing the calculation of the percent of associated C1 at different total C1 concentration. Increased C1 dissociation was observed with dilution. From these data, an association constant of 4.5 × 10⁸ M^?1 was calculated for native C1. Thus in normal human serum approximately ten percent of the C1 is present as free C1q and C1r₂C1s₂.     

Characterization of carbohydrate chains of C1-inhibitor and of desialylated C1-inhibitor.

Carbohydrate chains of C1-inhibitor were identified with a binding assay using different lectins. Lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA) that are specific for sialic acids bound to C1-inhibitor. Lectin from Datura stramonium (DSA) reacted also with the inhibitor indicating complex and hybrid sugar structures. C1-inhibitor was enzymatically desialylated and reexamined for lectin binding. SNA and MAA did not react anymore, but in addition to DSA, peanut agglutinin, which can bind to carbohydrate chains after sialic acids are removed, bound to desialylated C1-inhibitor. C1-inhibitor contains about 30 sialic acid residues per molecule. SDS-polyacrylamide gel electrophoresis showed that desialylated C1-inhibitor had a faster mobility than native C1-inhibitor. The N-terminal sequence of desialylated C1-inhibitor was the same as of native C1-inhibitor and no change in the inhibition of human plasma kallikrein was observed.     

Regulation of the mannan-binding lectin pathway of complement on Neisseria gonorrhoeae by C1-inhibitor and alpha 2-macroglobulin

We examined complement activation by Neisseria gonorrhoeae via the mannan-binding lectin (MBL) pathway in normal human serum. Maximal binding of MBL complexed with MBL-associated serine proteases (MASPs) to N. gonorrhoeae was achieved at a concentration of 0.3 microg/ml. Preopsonization with MBL-MASP at concentrations as low as 0.03 microg/ml resulted in approximately 60% killing of otherwise fully serum-resistant gonococci. However, MBL-depleted serum (MBLdS) reconstituted with MBL-MASP before incubation with organisms (postopsonization) failed to kill at a 100-fold higher concentration. Preopsonized organisms showed a 1.5-fold increase in C4, a 2.5-fold increase in C3b, and an approximately 25-fold increase in factor Bb binding; enhanced C3b and factor Bb binding was classical pathway dependent. Preopsonization of bacteria with a mixture of pure C1-inhibitor and/or alpha(2)-macroglobulin added together with MBL-MASP, all at physiologic concentrations before adding MBLdS, totally reversed killing in 10% reconstituted serum. Reconstitution of MBLdS with supraphysiologic (24 microg/ml) concentrations of MBL-MASP partially overcame the effects of inhibitors (57% killing in 10% reconstituted serum). We also examined the effect of sialylation of gonococcal lipooligosaccharide (LOS) on MBL function. Partial sialylation of LOS did not decrease MBL or C4 binding but did decrease C3b binding by 50% and resulted in 80% survival in 10% serum (lacking bacteria-specific Abs) even when sialylated organisms were preopsonized with MBL. Full sialylation of LOS abolished MBL, C4, and C3b binding, resulting in 100% survival. Our studies indicate that MBL does not participate in complement activation on N. gonorrhoeae in the presence of "complete" serum that contains C1-inhibitor and alpha(2)-macroglobulin.     

Activation of human C1r: Western blot analysis reveals slow and dose-dependent activation

Spontaneous activation of C1r in the presence of EDTA was examined by a Western blot. Partially purified native C1r was prepared by ultracentrifugation of fresh serum in 10 to 30% sucrose gradient; final concentration of C1r was one-sixth of the original serum. C1(-)-INH was not detectable by a single radial immunodiffusion (less than 0.5% of serum). The results demonstrated that 1) the rate of spontaneous activation of C1r was slow (less than 10% in 30 min); 2) it was concentration-dependent; 3) it was enhanced by activated C1r; and 4) it was almost completely suppressed by serine protease inhibitors up to 1 h. These results were inconsistent with an intramolecular autoactivation model of C1r in the fluid phase and suggested intermolecular activation by contaminating protease or activated C1r.     

C1-inhibitor gene nucleotide insertion causes type II hereditary angio-oedema

The polymerase chain reaction and nucleotide sequence analysis have been used to characterise a three nucleotide insertion in the eighth exon of one allele of the C1-inhibitor gene between nucleotides 16749 and 16750 in a kindred with type II hereditary angio-oedema (HAE). The effect of the resulting C1-inhibitor amino acid sequence alteration is discussed. This represents the first report of a nucleotide insertion in the C1-inhibitor gene causing type II HAE.     

Time dependence of activation of muscle AMP-aminohydrolase by substrate and potassium ion

The kinetics of AMP-aminohydrolase, which under steady state conditions shows a typical sigmoid dependence of initial velocities versus substrate concentration, have been examined by rapid mixing methods. Using this technique it was observed that when substrate or substrate plus activator (K(+)) were mixed with enzyme, the rate of appearance of product markedly increased during the first few tenths of a second. The time course of this change in rate was taken to reflect the progress of activation by substrate or by K(+). On the other hand, addition of activator to enzyme prior to mixing with substrate gave process curves for the formation of product consistent with normal Michaelis-Menten behaviour.Under the conditions where the reaction was examined, the enzyme at time zero had less than 10% of the activity of the fully active enzyme. The time course for activation with K(+) followed a first order process with a rate constant of 10.6 sec(-1) at 20 degrees C. A simple mechanism consistent with the data and capable of explaining the sigmoid dependence of initial velocities versus substrate concentrations observed in steady state kinetics was proposed.     

Complement-subcomponent-C1-inhibitor synthesis by human monocytes.

By using a radioimmunoassay, C1-inhibitor was found to accumulate in the supernatants of human monocyte cultures. The production of this protein was inhibited reversibly by cycloheximide. When C1-inhibitor synthesis was compared with C2 synthesis, it was found that C1-inhibitor synthesis continued, whereas synthesis of C2 appeared to cease after about 7 days in culture. Immunoprecipitation of supernatants of monocyte cultures that had been pulsed with [35S]methionine showed a specific band with an Mr of 105 000. Immunoprecipitates of the lysates revealed a band of Mr 83 000; this was thought to represent a partially or non-glycosylated precursor of C1-inhibitor. C1-inhibitor produced by the monocytes was shown, by using a haemolytic assay, to be functionally active. However, the functional activity of C1-inhibitor was reduced by only 44% in the presence of cycloheximide, whereas the concentration of this protein in cycloheximide-treated culture supernatants fell by more than 93%. This finding suggests that monocytes secrete a second molecule, which inhibits C1 activity but is distinct from classical C1-inhibitor.     

Recombinant human complement subcomponent C1s lacking beta-hydroxyasparagine, sialic acid, and one of its two carbohydrate chains still reassembles with C1q and C1r to form a functional C1 complex.

In contrast to the human serum protein which is approximately one-half erythro-beta-hydroxyasparagine at asparagine 134 [Theilens et al. (1990) Biochemistry 29, 3570-3578], recombinant C1s expressed by insect cells after infection with recombinant baculovirus entirely lacks posttranslational modification at asparagine 134. It is also incompletely glycosylated, lacking, at least, sialic acid. Site-directed mutagenesis of one of the two sites of carbohydrate attachment (Asn 159 to Gln 159) yields a faster migrating recombinant C1s still abundantly secreted. Furthermore, the mutated protein displays good hemolytic activity when reassembled with C1q and either human serum or recombinant C1r, demonstrating that these posttranslational modifications are not critical for any of the multiple interactions between C1s and C1q, C1r, C2, and C4 required for reassembly of the C1 complex, activation, and initiation of the classical complement pathway. The 4.0S recombinant C1s dimerizes to yield 5.6S C1s2 in the presence of Ca2+ and forms the 9.1S C1s-C1r-C1r-C1s tetramer upon the addition of human serum C1r and the 15.6S C1 complex upon the addition of C1q to the tetramer. The recombinant C1s and human serum C1s have identical N-terminal amino acid sequences, indicating proper recognition by the insect signal peptidase. The recombinant C1s is secreted and isolated as the unactivated zymogen, and it may be activated by human serum C1r which cleaves at Arg422-Ile423 to yield the characteristic heavy and light chains. A very tight complex is formed between C1-inhibitor and the light chain of recombinant C1s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence and acute phase regulation of rat alpha 1-inhibitor III messenger RNA

cDNA clones coding for the plasma proteinase inhibitor alpha 1-inhibitor III were isolated from an acute phase rat liver library. The isolates could be divided into four groups with characteristic BamHI restriction fragment patterns. The identity of the prototype clone pRLA1I3/2J was established by comparison with the published amino acid sequence of the purified protein. It codes for a 1477-amino acid precursor polypeptide with a 24-residue signal peptide. The mature protein shares 58% overall sequence identity with rat alpha 2-macroglobulin and contains a typical internal thiolester sequence. Twenty-two of its twenty-three cysteinyl residues are conserved with alpha 2-macroglobulin implying similar tertiary structure. However, the prototype alpha 1-inhibitor III sequence differed significantly from the rat and human alpha 2-macroglobulin sequences in its bait region suggesting alpha 1-inhibitor III possesses proteinase inhibitory specificities different from those of alpha 2-macroglobulin. The variant alpha 1-inhibitor III clone pRLA1I3/2J from a second cDNA group also differed from the prototype in the bait region coding sequence, although both specify similar signal peptides and NH2 termini. The observation of variant cDNA classes suggests that acute phase rat livers produce a heterogeneous mixture of alpha 1-inhibitor III mRNA molecules. Evidence was obtained for the presence of at least four different alpha 1-inhibitor III-related genes in the rat genome. During the first 24 h of an acute phase response the abundance of hepatic alpha 1-inhibitor III mRNA was decreased 3-4-fold. This decrease was of the same order of magnitude as the reported reduction of the corresponding plasma protein concentration, suggesting that in the early phase of the acute inflammatory response the plasma concentration of this protein is mainly controlled through the abundance of its hepatic mRNA.     

A single base deletion from the C1-inhibitor gene causes type I hereditary angio-oedema.

RFLP analysis, the polymerase chain reaction and nucleotide sequencing have been used to characterise a C1-inhibitor gene mutation responsible for type I hereditary angio-oedema (HAE). A single base deletion (C-16698) from the eighth exon of the C1-inhibitor gene alters the reading frame of the exon and generates a premature translation termination codon. This represents the first report of this form of C1-inhibitor gene mutation in type I HAE.     

The native metastable fold of C1-inhibitor is stabilized by disulfide bonds

C1-inhibitor is a member of the serpin family of proteinase inhibitors and is an important inhibitor of complement and contact system proteinases. The native protein has the characteristic serpin feature of being in a kinetically trapped metastable state rather than in the most stable state it could adopt. A consequence of this is that it readily forms loop-sheet dimers and polymers, by a mechanism believed to be the same as observed with other serpins. An unusual feature of C1-inhibitor is that it has a unique amino-terminal domain, of unknown function, held to the serpin domain by two disulfide bonds not found in other serpins. We report here that reduction of these bonds by DTT, causes a conformational change such that the reactive center loop inserts into beta-sheet A. This form of C1-inhibitor is less stable to heat and urea than the native protein, and is more susceptible to extensive degradation by trypsin. These data show that the disulfide bonds in C1-inhibitor are required for the protein to be stabilized in the metastable state with the reactive center loop expelled from beta-sheet A.     

Increased plasma concentrations of C9, C1-inhibitor and alpha 1-protease inhibitor associated with cigarette smoking

The association between various parameters of acute and chronic smoking status and plasma levels of three proteins, C9, C1-inhibitor (C1-INH) and alpha 1-protease inhibitor (alpha 1-PI) were determined for 49 male cigarette smokers and 49 age-matched nonsmokers (mean age = 32.2 years). The mean number of cigarettes smoked was 28.7 per day while the cumulative consumption was only 18.1 pack-years. Plasma levels of all three proteins were significantly higher in the smokers than nonsmokers. Plasma C9 and alpha 1-PI concentrations correlated with cumulative cigarette consumption and plasma nicotine concentrations. While C1-INH concentration did not correlate with either cumulative cigarette consumption or plasma nicotine concentration, it correlated significantly with serum thiocyanate concentration. No consistent correlation was found between plasma concentration of these proteins and parameters of pulmonary function.     

Biosynthesis,intracellular targeting,and degradation of the EAAC1 glutamate/aspartate transporter in C6 glioma cells

Rat C6 glioma cells were used as a model system to study the biosynthesis, intracellular targeting, and degradation of the EAAC1 transporter, a sodium-dependent glutamate/aspartate transport protein that encodes System X(-)A,G activity. At steady state, nearly 70% of the EAAC1 transporter was located at the cell surface. The newly synthesized EAAC1 protein was co-translationally N-glycosylated with high mannose oligosaccharide chains that were processed into complex-type sugar chains as the protein matured. The final maturation steps for EAAC1 protein coincided with its plasma membrane arrival, which was first detected at about 45 min after the initial synthesis. The newly synthesized EAAC1 protein was protected from degradation during the maturation and targeting process, as well as during the first 5 h after plasma membrane arrival. After this initial lag period, both the newly synthesized transporter and the total cellular EAAC1 pool were degraded by first order kinetics with a half-life of 6 h. These results represent the first analysis of the synthesis and degradation of the EAAC1 amino acid transporter.     

Inactivation of human plasma C1-inhibitor by human PMN leucocyte matrix metalloproteinases

Highly purified human polymorphonuclear (PMN) leucocyte matrix metalloproteinases, collagenase and gelatinase, cleaved human plasma C1-inhibitor at the carboxyl site of Ala⁴³⁹ (P₆). This led to a concomitant loss of C1-inhibitor activity. An additional cleavage site at the carboxyl site of Ser⁴⁴¹ (P₄), was observed during PMN leucocyte gelatinase-induced inactivation, and a minor fragment of the plasma C1-inhibitor was generated.