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PubMed1982

Structural and functional studies in C1q deficiency.

The sera of two brothers were found totally lacking hemolytic C activity. One of them, a 16-yr-old male, presented a severe lupus-like syndrome, whereas the other was apparently healthy. Immunochemical quantitation of C components in both sera showed depressed levels of C1q, whereas the levels of C1r, C1s, and C1 inhibitor were elevated. C4, C3, C5, factor B, and beta 1H levels were in the normal range. Hemolytic C1 activity was totally lacking. C4 titers were elevated (150% of normal). C2 hemolytic activity was about one-third of normal, and the titers of the terminal components C3-C9 were also reduced in the two siblings. Double immunodiffusion against anti-C1q antiserum showed a partial loss of C1q antigenic determinants in the two siblings. Furthermore, the C1q of both siblings was unable to interact with immunoglobulins or to associate with C1r and C1s. Addition of purified human C1q to the sera restored their total C and C1 hemolytic activity. The dose response to the C1q addition was linear, indicating that the functional deficiency was not due to the presence of a serum inhibitor. Although antigenically deficient in comparison with normal C1q, the abnormal C1q appeared to have a larger m.w., as determined by gel chromatography. Investigation of other members of this family suggests a genetically linked disorder, because four out of six siblings had the same dysfunctional C1q in their serum.

Adolescent↗
PubMed1989

Possible mechanisms of degradation of C1q in vivo and in vitro: role of macrophages.

Low levels of C1q may be found in sera of patients with diseases such as systemic lupus erythematosus, serum sickness and bacterial endocarditis. In vitro, C1q can be bound by immune complexes and induce activation of the proenzymes C1r and C1s, resulting in an activated C1 molecule. In the presence of C1-In in the reaction mixture C1r and C1s are dissociated rapidly from the immune complex-bound C1q. This C1q has the possibility to interact with free precursor C1r and C1s to form a new C1 molecule at the surface of the immune complex and induce a new cycle of activation. In vivo, however, such a possibility does not seem to play a major role, because low levels of C1q are found in various diseases which are thought to be mediated by immune complexes. Therefore we studied the clearance rate of C1q in rats in the presence and absence of soluble aggregates of IgG (AIgG). It was found that 125I-C1q is cleared from the circulation of rats with a halflife of 12.4 hrs and increasing concentrations of AIgG enhanced the clearance of 125I-C1q in vivo. Studies in which various organs were analyzed for 125I-C1q indicated that the liver is the main site of clearance and that the Kupffer cells seem to play a major role. These, and other in vitro studies, indicate that in vivo complement activation, may contribute to a higher degree of turnover of C1q in vivo and cause depletion of circulating C1q.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Acquired C1 inhibitor deficiency as a result of an autoantibody to the reactive center region of C1 inhibitor.

An autoantibody that we hypothesize to react with the reactive center of the plasma serine proteinase inhibitor, C1 inhibitor (C1INH), has been found in a patient with acquired C1INH deficiency. The Ab blocks the ability of C1INH to inhibit the hydrolysis of N-carbobenzyloxy-L-lysine thiobenzylester by purified C1s. A cryoprecipitate from the patient's plasma as well as the Ig fraction were able to block C1INH inhibition of C1s. The immunoaffinity purified Ab to C1INH from the patient's plasma Ig fraction neutralizes the inhibitory activity of C1INH in a dose-dependent manner and blocks the ability of normal C1INH to form a complex with C1s. The neutralizing activity of the purified Ab is reversed by a synthetic peptide that corresponds to the amino acid sequence in the P1 to P15 positions of the reactive center of C1INH but not by a 34-amino-acid trypsin peptide or 37-amino-acid elastase peptide derived from the C-terminus of C1INH. Western blot analysis indicated that the Ab is an oligoclonal Ig with kappa light chains.

Adult↗
PubMed1995

Unique C1 inhibitor dysfunction in a kindred without angioedema. II. Identification of an Ala443-->Val substitution and functional analysis of the recombinant mutant protein.

We have determined the cause of an unusual C1 inhibitor abnormality in a large kindred. We previously found that half of serum C1 inhibitor molecules in affected kindred members are normal. The other half complexed with C1s but showed little complex formation with C1r. These molecules also appeared to be relatively resistant to digestion by trypsin. Taken together, the findings suggested that members of this kindred are heterozygous for an unusual C1 inhibitor mutation. Sequencing of genomic DNA from the kindred revealed that thymine has replaced cytosine in the codon for Ala443 (P2 residue) in one C1 inhibitor allele, resulting in substitution with a Val residue. To test the effect of this substitution, a mutant C1 inhibitor containing Ala443-->Val was constructed by site-directed mutagenesis and expressed in COS-1 cells. Both the Ala443-->Val mutant and the wild-type C1 inhibitor complexed completely with C1s, kallikrein, and coagulation Factor XIIa after incubation at 37 degrees C for 60 min. In contrast, the mutant inhibitor failed to complex completely with C1r under the same conditions. Time course analysis showed that the ability of the mutant to complex with C1s is also impaired: although it complexed completely in 60 min, the rate of complex formation during a 0-60-min incubation was decreased compared with wild-type C1 inhibitor. The mutant inhibitor also formed a complex with trypsin, a serine protease that cleaves, and is not inhibited by, wild-type C1 inhibitor. The Ala443-->Val mutation therefore converts C1 inhibitor from a substrate to an inhibitor of trypsin. These studies emphasize the role of the P2 residue in the determination of target protease specificity.

Angioedema↗
PubMed1990

Effects of methylamine and heparin on a rapid chromogenic assay of C1-esterase inhibitor in plasma.

We describe a rapid assay of C1-esterase inhibitor (C1-inh) activity in plasma. After adding purified C1s serine protease (EC 3.4.21.42) in excess to plasma, we determine the residual C1s activity towards a new chromogenic tripeptide, CH3CO-Lys(CbO)-Gly-Arg-pNA. Optimal conditions include the addition of methylamine (final concentration 0.12 mol/L) to reduce the potential inhibitory capacity of alpha 2-macroglobulin towards C1s and the addition of heparin (final concentration 3000 int. units/L) to enhance the reaction of C1s with C1-inh. The correlation with C1-inh antigen concentrations in plasma was excellent. The estimated interassay CV was 4.3%, whereas the intra-assay CV was 2.0% for activity concentrations within the range of normal individuals (means +/- 2 SD: 70-124%), 1.3% at lower concentrations. The method is more convenient, rapid, and precise than previous methods, and C1-inh activity in plasma can be assessed within 30 min. We found that concentrations of C1-inh in plasma were low during open-heart bypass surgery.

Antigens↗
PubMed1981

A C1-inhibitor-complex assay (INCA): a method to detect C1 activation in vitro and in vivo.

A radioimmunoassay (the C1-inhibitor-complex assay, INCA) is described for the detection of complexes that are composed of at least C1s and C1-inhibitor. This INCA is based on demonstrating that C1s and C1-inhibitor (C1-In) are linked: after an incubation with anti-C1s-Sepharose, bound C1sC1-In complexes are detected by 125I-anti-C1-In. C1sC1-In complexes were prepared by the addition of a slight excess of C1s to normal human serum (NHS). As little as 2 ng C1-In bound to C1s was detected. Additional free C1s in serum hardly influenced the detection of C1sC1-In complexes. Complexes presumably composed of C1rC1s(C1-In)2 were generated by the addition of aggregated IgG to NHS. This generation was inhibited by lowering the temperature to 0 degrees C, and by EDTA, and depended on the concentration of aggregated IgG. These complexes had a sedimentation value of approximately 9S. Complexes of C1s and C1-In were also generated in NHS by the addition of DNP-albumin and protein A, but not by zymosan. The INCA was applied to blood samples from normal donors and patients. Sixteen out of 19 samples from patients with acute glomerulonephritis contained increased amounts of C1rC1s(C1-In)2 complexes as compared with the amounts in blood samples from normal donors. The INCA provides a useful tool to assess the activation of C1 in the presence of C1-In, both in vitro and in vivo.

Complement Activation↗
PubMed1983

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.

Animals↗
PubMed1977

Studies of C1 subcomponents in chronic urticaria and angioedema.

C1q, C1r, C1s, C3, C4 and C-1 IA were determined by electroimmunoassay in sera from 150 patients with chronic urticaria or angioedema. Abnormal C1q and C1s levels were found in about 30% of the patients. In seven sera C1r was not measurable due to the appearance of diffuse precipitates. The levels of C3 and/or C4 were decreased in five sera with aberrations of C1 subcomponents in the electroimmunoassay. None of the patients showed reduced C-1 IA levels in the electroimmunoassay. The presence in sera of abnormal C1 subcomponent complexes was studied by crossed immunoelectrophoresis. Sera from 11% of the patients contained C1r-C1s complexes. Increased amounts of alpha2 complexes (C-1r-C-1-S-C-1 IA) were found in 33% of the patients. A major part of the C1q in sera yielding abnormal C1r precipitates had the same electrophoretic mobility as isolated C1q and was not associated with the C1qrs complex. C1 activity in hemolytic tests was low in these sera as well as in sera with decreased C1q levels. In the esterolytic assay for C-1 IA low values were found in 14 patients. Repeated sampling and family studies in appropriate cases gave no evidence for genetically determined deficiencies of C1q, C1r or C-1 IA.

Angioedema↗
PubMed2001

Expression of active human C1 inhibitor serpin domain in Escherichia coli.

Human C1 inhibitor is a highly glycosylated serine protease inhibitor of the serpin family. The protein contains two disulfide bonds. In this study, an N-terminally truncated form of recombinant C1 inhibitor was overexpressed in Escherichia coli strains BL21(DE3) and AD494(DE3), the latter enabling the formation of disulfide bonds within the cytoplasm. With both strains, a major fraction of the recombinant protein produced appeared to be insoluble. However, the soluble fraction of lysates from strain AD494(DE3) inhibited the C1s target protease in functional assays. Recombinant C1 inhibitor produced in this strain also displayed the ability to complex with C1s in vitro. In contrast, lysates from strain BL21(DE3) displayed no C1 inhibitor activity. These data support the notion that glycosylation is not important, whereas disulfide bond formation appears to be essential for the production of an active recombinant C1 inhibitor. Thus, bacterial strains that permit the formation of disulfide bonds may represent a reliable system for the production of recombinant C1 inhibitor. However, a major obstacle to large-scale production will be to produce the protein in a soluble form. Attempts to increase the yield of soluble protein by coexpression of the GroEL/ES chaperonins resulted in an increase in solubility.

Amino Acid Sequence↗
PubMed1973

C2 deficiency. Development of lupus erythematosus.

The study of serum from a patient with C2 deficiency is described. The patient had an episode of pneumococcal meningitis at 5 mo of age with seizures and transient hemiparesis and apparent purpuric skin lesions. He was first admitted to the University of Minnesota Hospitals at 10 yr of age following the discovery of proteinuria accidentally by his mother. Since then he has been admitted repeatedly to this hospital with numerous clinical findings including arthralgia, recurrent abdominal pain, proteinuria, membranous nephropathy, malar butterfly rash, seizures, personality aberrations, and recurrent fever. In June 1971, the patient developed positive DNA and DNP antibodies and positive LE cells. When the C profile was studied before and after recognition of lupus, C1q, C1s, and C4 dropped. C3 levels were elevated as were C5, C6, and C7, C3 proactivator had been reduced in the patient even before he developed lupus. Also because of a traumatic renal biopsy leading to a perirenal hematoma, he required surgery and a blood transfusion. 1 h after blood transfusion, a C2 titer of 23 hemolytic units was detected. Almost immediately levels of C3, C5, C6, and C7 dropped, C8 and C9 remained elevated. The addition of C2 from normal blood permitted dramatic activation of C3. These findings support the view that the rare deficiency in production of C2 predisposes to serious susceptibility to infection, vascular and mesenchymal disease as well as to renal disease and a lupus syndrome.

Antibodies↗
PubMed1985

Variability in purified dysfunctional C1(-)-inhibitor proteins from patients with hereditary angioneurotic edema. Functional and analytical gel studies.

C1(-)-inhibitor (C1(-)-INH) proteins from normal persons and members of eight different kindred with dysfunctional C1(-)-INH proteins associated with hereditary angioneurotic edema (HANE) were compared with respect to their inhibitory activity against purified preparations of C1s-, plasma kallikrein, activated forms of Hageman factor, and plasmin. Each dysfunctional C1(-)-INH protein showed a unique spectrum of inhibitory activity against these enzymes. Although none of the dysfunctional C1(-)-INH proteins significantly impaired amidolysis by plasmin, all but one inhibited activated Hageman factor. One purified dysfunctional C1(-)-INH (Ta) inhibited purified C1s- to a normal degree. Another C1(-)-INH (Za) had almost seven times as much inhibitory activity as normal C1(-)-INH against activated Hageman factor, but had decreased activity against C1s- and no activity against plasmin. Analyses of mixtures of plasmin and C1(-)-INH proteins in SDS gel electrophoresis revealed variability in the patterns of complex formation and cleavage of dysfunctional proteins after exposure to C1s- and plasmin. Some bound to plasmin and were cleaved, even though none significantly impaired the amidolytic activity of plasmin. Two were cleaved by C1s-, whereas neither normal or other dysfunctional C1(-)-INH were cleaved. Dysfunctional C1(-)-INH proteins from patients with HANE are thus heterogeneous in their inhibitory properties and there must be different structural requirements for the inhibition of the various plasma enzymes that can be regulated by normal C1(-)-INH. The data suggest that in addition to common sites of interactions between these proteases and C1(-)-INH, there are also points of contact that are specific for each protease. Genetic mutations leading to structural changes at some of these sites may have differing effects on the interaction between individual proteases and abnormal C1(-)-INH proteins. These alterations may allow these proteins to serve as probes for structural requirements for inhibitory actions of normal C1(-)-INH.

Angioedema↗
PubMed1997

Probing the structure of C1 with an anti-C1s monoclonal antibody: the possible existence of two forms of C1 in solution.

Anti-human C1s monoclonal antibody H1532, a mouse gamma-1-immunoglobulin elicited by a C1r2C1s2 immunogen, appeared to bind to the beta-domain of C1s by electron microscopy. In agreement with this observation, Western blotting demonstrated good binding to unreduced C1s, but no binding to the alpha or gamma-B domains. When added to solutions of the C1r2C1s2 tetramer, HI532 converted the 8.7 S tetramer into an 18 S complex, which was seen by electron microscopy to be a dimer of parallel C1s x C1r x C1r x C1s molecules cross-linked by two bivalent monoclonal antibodies. If increasing amounts of HI532 were added to C1r2C1s2 followed by addition of equivalent C1q, there was a progressive loss of hemolytic activity, which became zero when two equivalents of antibody HI532 were added. When two equivalents of HI532 were added to serum or C1 reconstituted overnight from purified subcomponents, there was an immediate loss of approximately 50% of the hemolytic activity; thereafter, activity decayed slowly and even after 24 hr, 10-30% of the activity remained. The rapid loss of only 50% of the activity would be readily explained by the existence of two conformations of C1, one of which was rapidly disassembled by antibody, and the other was resistant to disassembly. These two conformations may correspond to two previously proposed structures for the C1 complex.

Animals↗
PubMed1976

In vitro activation of C1s in plasma of patients with hereditary angioneurotic oedema.

The serum of patients with hereditary angioneurotic oedema contains small amounts of inhibitor of Cl-esterase (Cl-s) which is usually present in appreciable amounts in the serum of healthy individuals. In the citrated plasma of these patients in remission Cl-s activity was not detectable, but prolonged incubation with various alcohols, detergents, acetone, phenols and metal chelating agents generated the enzyme. Normal plasma did not respond to these reagents. The generation of Cl-s by alcohol and EDTA was inhibited by addition of natural inhibitor of Cl-s but not by soyabean trypsin inhibitor, trasylol and hexadimethrine bromide. Hexadimethrine bromide did not block the generation when experiments were carried out in silicon coated glassware. Incubation with kaolin and kallikrein generated Cl-s in remission plasma. Density gradient centrifugation studies showed that alcohol caused at least partial dissociation of Cl in remission plasma but not in normal plasma. This effect was similar to that of EDTA described in earlier reports. The possible explanations for the findings are discussed.

Alcohols↗
PubMed1994

C1 inhibitor and diagnosis of hereditary angioedema in newborns.

Symptoms of hereditary angioedema may present during the child's first years. Attacks may be a particular threat to the narrower airway of the child. An early diagnosis is most valuable because effective C1 inhibitor (C1 INH) concentrate is available. We present a reference area for the antigenic and functional determination of C1 INH by using uncontaminated umbilical cord blood from 80 normal newborns collected by puncturing vessels in the newly delivered placenta. We examined two full-term babies (1 and 2) from mothers with hereditary angioedema type I the same way. The concentration of C1 INH antigen was determined by radial immunodiffusion. The C1 INH functional assay was based on the addition of a known quantity of C1s, which enzymatically splits a chromogenic substrate. The test was performed in the presence of methylamine and heparin in a kinetic microtiter plate assay. Citrated plasma was used in both assays. The data obtained in the 80 cord blood samples (2.5-97.5 percentile) were 0.11-0.22 g/L for C1 INH antigen (adults, 0.15-0.33 g/L) and 47.2-85.9% for C1 INH function (percentage of adults). In cord blood, baby 1 had an antigenic value of 0.12 g/L (7.5 percentile) and C1 INH function of 61.8% (42 percentile). The corresponding values for baby 2 in cord blood were less than 0.05 g/L (0.106 g/L < 2.5 percentile) and 34.3% (12.9% < 2.5 percentile). Baby 2 had markedly lower C4 values yet much higher C4 activation products than baby 1. At 4 mo, baby 1 had an antigenic C1 INH value of 0.24 g/L.(ABSTRACT TRUNCATED AT 250 WORDS)

Angioedema↗
PubMed1993

C1 inhibitor functional deficiency in systemic lupus erythematosus (SLE).

C1 inhibitor (C1-inh) was assayed in eight SLE patients presenting with consistently low levels of intact C4. C1-inh antigenic levels were normal in all patients; however, the function of the C1-inh tested against C1s and C1r was variable and outside the normal functional range in seven of the eight patients. The molecular weight of patients' C1-inh protein was 105 kD, corresponding to the size of the intact molecule. The C1-inh gene was analysed in all patients. Restriction fragments generated with TaqI, PstI and HgiAI gave no indication of a major C1-inh gene rearrangement. Direct genomic sequencing of exon VIII revealed three polymorphic point mutations, but there were no changes from the normal gene in or around the reactive-centre residue of C1-inh. Furthermore, we found no evidence for a C1-inh autoantibody in patients which could affect normal C1-inh function in vitro. These results indicate that the etiology of C1-inh dysfunction in SLE is heterogeneous and distinct from that reported in either hereditary or acquired angioedema.

Base Sequence↗
PubMed1992

Characterization of recombinant C1 inhibitor P1 variants.

Twelve human C1 inhibitor P1 variants were constructed by site-directed mutagenesis of the codon for arginine 444 and were expressed in COS-1 cells to analyze the functional properties. The ability to bind to target proteases, as well as potential substrate-like behavior, was investigated with radioimmunoassays. The P1-Lys variant retained binding capacity toward C1s, plasmin, and kallikrein. In addition, complex formation with C1s was detected for P1-Asn and P1-His. All other P1 substitutions resulted in C1 inhibitor variants that neither complexed with nor were inactivated by C1s, kallikrein, beta-factor XIIa, or plasmin. Electrophoretic studies confirmed that P1-Lys and P1-His can form sodium dodecyl sulfate-resistant complexes with C1s. In contrast, the C1s-P1-Asn complex dissociated upon addition of sodium dodecyl sulfate. Kinetic experiments by the method of progress curves generated association rate constants (kon) with C1s of 4.2 x 10(4) M-1 s-1 for recombinant wild-type C1 inhibitor and 1.7 x 10(4) M-1 s-1 for P1-Lys. For P1-Asn and P1-His, kon was decreased approximately 100-fold. The results from inhibition experiments were compatible with a model of reversible inhibition, although the observed dissociation rate for wild-type C1 inhibitor is too low (1-2 x 10(-6) s-1) to be physiologically relevant. The overall inhibition constant (Ki) was estimated to be 0.03 nM. With P1-Asn, reversible inhibition could be demonstrated directly upon dilution of preformed complexes; the observed dissociation rate constant was 3.2 x 10(-4) s-1; and Ki increased to approximately 380 nM. These findings are discussed in relation to inhibitor specificity and inhibition mechanism.

Amino Acid Sequence↗
PubMed1975

Studies on human plasma C1 inactivator-enzyme interactions. II. Structural features of an abnormal C1 inactivator from a kindred with hereditary angioneurotic edema.

The function and several of the structural features of the C1 inactivator protein isolated from the plasma of a mother and daughter with the variant form of hereditary angioneurotic edema have been examined. These abnormal inhibitors shared immunologic identity with the normal C1 inactivator protein; however, they were inactive in inhibiting the functional activity of C1s. Analysis of the abnormal inhibitors by sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis suggested that each consisted of a single polypeptide chain, the mobility of which was slower than that of the normal C1 inactivator. The apparent molecular weight of the patients' inhibitors was 109,000 daltons as contrasted to 105,000 daltons, that of the normal C1 inactivator. The abnormal inhibitors failed to form a complex with C1s or plasmin as analyzed by SDS-acrylamide gels. The large proteolytic derivatives resulting from the plasmin- and trypsin-induced degradation of the abnormal inhibitors were approximately 3,000 daltons heavier than the corresponding products derived from normal C1 inactivator. Thus, the structural abnormality identified appeared to be a property of the core molecule. Treatment of the inhibitors with neuraminidase failed to demonstrate a difference between the normal and patient-derived C1 inactivator molecule. Neither were major differences found between the amino acid composition of the defective and normal inhibitors; however, the acidic amino acids tended to be higher in the patients' inhibitors, and the phenylalanine content lower. Thus, these studies have identified both structural and functional abnormalities in the C1 inactivator protein isolated from two related patients with hereditary angioneurotic edema. Examination of the interaction between endopeptidases and the inhibitors has further delineated the abnormal structural features.

Amino Acids↗
PubMed1981

The role of sialic acid in the functional activity and the hepatic clearance of C1-INH.

The immunochemical properties, catabolic clearance, and organ distribution of highly purified radiolabeled human C1-INH were studied after treatment with glycosidases. The antigenicity and C1s inhibitory activity of C1-INH were unimpaired after removal of sialic acid residues (60%) or sialic acid and galactose residues (60 and 20%, respectively) with immobilized neuraminidase and beta-galactosidase. Treatment of C1-INH with neuraminidase was associated with increased cathodal migration of the glycoprotein. In vivo studies in the rabbit, however, showed that removal of sialic acid residues resulted in rapid blood clearance of C1-INH and enhanced localization of the asialo C1-INH in the liver. Subsequent removal of penultimate galactose residues returned the survival time in the circulation and organ distribution to near normal. Additionally, simultaneous infusion of asialo-C1-INH with asialo alpha 1-acid glycoprotein, a protein known to bind to hepatic asialoglycoprotein receptors, dramatically extended the intravascular survival time of asialo C1-INH. In agreement with the observations on other plasma glycoproteins, these results indicate that desialylation of C1-INH results in rapid clearance by hepatic asialoglycoprotein receptors.

Animals↗