Murine complement component C4 and sex-limited protein: identification of amino acid residues essential for C4 function.
Murine sex-limited protein (Slp) is an isotype of murine complement component C4 that shares 95% sequence identity with C4 as well as the intramolecular thioester necessary for C4 function but has no complement activity. Slp is nonfunctional at least in part because it is not cleaved by the activated form of complement protease C1s (C1s), which proteolytically activates C4 in the classical complement pathway. Slp is also distinct from C4 in that its expression in some mouse strains is under testosterone control. In the present studies, we used site-directed mutagenesis of C4 and expression of the mutant proteins in cultured cells to identify the amino acid substitutions in Slp that are responsible for resistance to C1s cleavage. We focused on sequence changes immediately downstream of the cleavage site in C4 because the arginine at that site is conserved in Slp, but the downstream sequences diverge substantially, with six differences in the first 7 residues followed by a 3-residue deletion in Slp. We found that a C4 mutant carrying only the 3-residue deletion is not cleaved by C1s and has essentially no hemolytic activity, whereas a mutant carrying only the six replacement changes is cleaved by C1s and has normal hemolytic activity. Both mutants have intact thioesters. A third mutant in which two acidic residues in the segment deleted in Slp were replaced by aliphatic residues is also cleaved by C1s, has an intact thioester group, and has normal hemolytic activity. These results indicate that the downstream mutations are responsible for the resistance of Slp to C1s cleavage and suggest that the length rather than the specific sequence of this segment is critical in determining susceptibility to the protease.
Ogata
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RT
Medical Biology Institute, La Jolla, CA 92037.
Cooper
N R
NR
Bradt
B M
BM
Mathias
P
P
Picchi
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D003181:Complement C4 / Q000235:genetics / Q000502:physiology*; D004247:DNA / Q000235:genetics; D006461:Hemolysis; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid
D001798:Blood Proteins; C018386:C4a protein, mouse; D003181:Complement C4; D015345:Oligonucleotide Probes; D004247:DNA