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PubMed2000

Mouse complement components C4 and Slp act synergistically in a homologous hemolytic C4 assay.

Goals of the present study were to compare the hemolytic activities of mouse C4 and Slp in a homologous system and to study a possible interaction between these proteins during complement activation. As reagents for mouse C4 and Slp, we used serum of C4(- / -) knockout C57BL / 6 (C4(-) / Slp(-)) mice and sensitized rabbit erythrocytes as target cells. Sera to be tested contained none, either of the two or both proteins. We found that C4(-) / Slp(+) serum has some hemolytic C4 activity, but less than C4(+) / Slp(-) serum. Comparing C4 activities of C4(+) / Slp(-) and C4(+) / Slp(+) sera, we found a threefold enhanced activity in double-positive serum. Hemolytic C4 levels of mixtures of solely C4- and Slp-sufficient sera did not overlap with expected C4 levels, but rather these sera showed synergy. This explains the enhanced activity of double-positive serum. Similar results were observed for total complement activation. In conclusion, Slp has measurable, but poor C4 activity as compared with mouse C4. Using our homologous system, we showed that the enhanced classical pathway activity of double-positive sera is most probably based on synergy between C4 and Slp. Our results answer an old question as to why C4(+) / Slp(+) mice have higher complement levels than C4(+) / Slp(-) mice.

Animals↗
PubMed1996

The reaction mechanism of the internal thioester in the human complement component C4.

A key step in the elimination of pathogens from the body is the covalent binding of complement proteins C3 and C4 to their surfaces. Proteolytic activation of these proteins results in a conformational change, and an internal thioester is exposed which reacts with amino or hydroxyl groups on the target surface to form amide or ester bonds, or is hydrolysed. We report here that the binding of the human C4A isotype involves a direct reaction between amino-nucleophiles and the thioester. A two-step mechanism is used by the C4B isotype. The histidine at position 1,106(aspartic acid in C4A) first attacks the thioester to form an acyl-imidazole intermediate. The released thiol then acts as a base to catalyse the transfer of the acyl group to amino- and hydroxyl-nucleophiles, including water.

Amino Acid Sequence↗
PubMed1981

Intramolecular general acid catalysis in the binding reactions of alpha 2-macroglobulin and complement components C3 and C4.

The complement system proteins C3 and C4 and the plasma protease inhibitor alpha 2-macroglobulin, when activated by limited proteolysis, can bind covalently to other macromolecules. The three proteins also exhibit an unusual internal peptide-bond cleavage reaction when denatured. The covalent binding reaction is likely to occur by a transacylation mechanism involving an internal thiolester in the three proteins. However, the activated species of these proteins are much more reactive than simple thiolesters. Studies of molecular models of the thiolester region in C3 show that an intramolecular acid catalysis mechanism can both account for the exceptional reactivity of the activated form of these proteins and provide an explanation for the denaturation-induced peptide bond cleavage.

Amino Acid Sequence↗
PubMed1993

The interaction of soluble human complement receptor type 1 (sCR1, BRL55730) with human complement component C4.

Human CR1 is a membrane-bound protein which plays an important role in the control of the human complement system. In addition to its involvement in the processing and clearance of immune complexes with C3b or C4b on their surface, CR1 acts as a cofactor for the proteolysis of C3b and C4b by Factor I. sCR1 is a recombinant, soluble form of CR1 which retains the cofactor activities of CR1, and is of potential therapeutic value for the suppression of complement-mediated tissue damage in vivo. An assay has been established using microtitre plates to explore the binding of sCR1 to the two isotypes of C4, C4A and C4B, and to C4 fragments. Specific binding of 125I-sCR1 to C4b and ammonia-treated C4 has been demonstrated. The binding of 125I-sCR1 to ammonia-treated C4 is dependent on pH and ionic strength, decreasing with an increase in pH and with an increase in ionic strength. At physiological ionic strength, up to twice as much 125I-sCR1 bound to ammonia-treated C4A as bound to ammonia-treated C4B. This preference of sCR1 for binding to the C4A isotype has implications for the clinical association of immune complex disease with C4A null alleles.

Ammonia↗
PubMed1993

Covalent binding properties of the human complement protein C4 and hydrolysis rate of the internal thioester upon activation.

The complement proteins C3 and C4 have an internal thioester. Upon activation on the surface of a target cell, the thioester becomes exposed and reactive to surface-bound amino and hydroxyl groups, thus allowing covalent deposition of C3 and C4 on these targets. The two human C4 isotypes, C4A and C4B, which differ by only four amino acids, have different binding specificities. C4A binds more efficiently than C4B to amino groups, and C4B is more effective than C4A in binding to hydroxyl groups. By site-directed mutagenesis, the four residues in a cDNA clone of C4B were modified. The variants were expressed and their binding properties studied. Variants with a histidine residue at position 1106 showed C4B-like binding properties, and those with aspartic acid, alanine, or asparagine at the same position were C4A-like. These results suggest that the histidine is important in catalyzing the reaction of the thioester with water and other hydroxyl group-containing compounds. When substituted with other amino acids, this reaction is not catalyzed and the thioester becomes apparently more reactive with amino groups. This interpretation also predicts that the stability of the thioester in C4A and C4B, upon activation, will be different. We measured the time course of activation and binding of glycine to C4A and C4B. The lag in the binding curve behind the activation curve for C4A is significantly greater than that for C4B. The hydrolysis rates (k0) of the thioester in the activated proteins were estimated to be 0.068 s-1 (t1/2 of 10.3 s) for C4A and 1.08 s-1 (t1/2 of 0.64 s) for C4B. These results indicate that the difference in hydrolysis rate of the thioester accounts, at least in part, for the difference in the binding properties of C4A and C4B.

Amino Acid Sequence↗
PubMed1995

The effect of residue 1106 on the thioester-mediated covalent binding reaction of human complement protein C4 and the monomeric rat alpha-macroglobulin alpha 1 I3.

The histidine at position 1106 of the C4B isotype of human complement is involved in catalyzing the covalent binding of the thioester to glycerol and water. By replacing the histidine with other residues, it was found that tyrosine is also capable of mediating the reaction. We propose that they act as nucleophiles by first attacking the thioester, upon activation, to form acyl intermediates, which subsequently react with the hydroxyl groups of glycerol or water. The monomeric alpha-macroglobulin, alpha 1I3 of the rat, was also studied. Unlike alpha 2-macroglobulin, which is a tetramer, alpha 1I3 has binding properties similar to those of C4A.

Amino Acid Sequence↗
PubMed1992

The coding sequence of the hemolytically inactive C4A6 allotype of human complement component C4 reveals that a single arginine to tryptophan substitution at beta-chain residue 458 is the likely cause of the defect.

The C4A6 allotype of the human complement component C4 is known to be defective in C5 binding within the C5 convertase. To characterize the position and nature of the molecular defect in the C4A6 allotype we have isolated the C4A6 gene from a cosmid genomic DNA library. Direct sequencing of a 4.4-kb region of the gene covering exons 17 to 31 and encoding the C4d fragment and most of the rest of the alpha chain of C4 revealed that the C4A6 allele encodes the A isotypic residues Pro Cys-Leu Asp at positions 1101, 1102, 1105, and 1106 and the same residues as the C4A3 alpha gene at the polymorphic positions 1054 (Asp), 1157 (Asn), 1182 (Thr), 1188 (Val), 1191 (Leu) and 1267 (Ala). In addition the C4A6 allele was shown to encode a Pro at the previously characterized polymorphic position 707 in the C4a peptide where the C4A3 alpha allele encodes a Leu. The remaining 26 exons of the C4A6 gene were analyzed by detecting nucleotide mismatches in C4A6/C4A3 and C4A6/C4B1 DNA heteroduplexes using the chemical cleavage of mismatch technique. The regions around detected mismatches were sequenced. In total seven nucleotide differences were defined on comparison of the C4A6 and other C4 sequences, of which three were present in exons. Two of these resulted in amino acid changes. One of the amino acid differences is a known polymorphism in C4, a Tyr/Ser substitution at position 328 in the beta-chain. The second amino acid difference caused by a C to T transition in the first base of the codon for amino acid residue 458 was the only one shown to be specific to the C4A6 allotype. The C4A6 allotype contains a Trp residue at this position in the beta-chain instead of the Arg residue found in all other C4A and C4B allotypes so far characterized. We propose that this Arg to Trp substitution at beta-chain residue 458 is responsible for the inability of C4A6 to bind C5 in the C5 convertase.

Alleles↗