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.
Eldering
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E
Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Huijbregts
C C
CC
Lubbers
Y T
YT
Longstaff
C
C
Hack
C E
CE
D000595:Amino Acid Sequence; D000799:Angioedema / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D003172:Complement C1 / Q000378:metabolism; D003174:Complement C1 Inactivator Proteins / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D030342:Genetic Diseases, Inborn / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D003172:Complement C1; D003174:Complement C1 Inactivator Proteins; D011994:Recombinant Proteins