UNIQUE C1 INHIBITOR DYSFUNCTION IN A KINDRED WITHOUT ANGIOEDEMA .2. IDENTIFICATION OF AN ALA(443)-]VAL SUBSTITUTION AND FUNCTIONAL-ANALYSISOF 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 compl exed with C1s but show ed little complex formation with C1r. These mol ecules also appeared to he relatively resistant to digestion by trypsi n. 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 cytosi ne in the codon for Ala(443) (P2 residue) in one C1 inhibitor allele, resulting in substitution with a Val residue. To test the effect of th is substitution, a mutant C1 inhibitor containing Ala(443)-->Val was c onstructed by site-directed mutagenesis and expressed in COS-1 cells. Both the Ala(443)-->Val mutant and the wild-type C1 inhibitor complexe d completely with C1s, kallikrein, and coagulation Factor XIIa after i ncubation at 37 degrees C for 60 min. In contrast, the mutant inhibito r failed to complex completely with C1r under the same conditions. Tim e course analysis showed that the ability of the mutant to complex wit h C1s is also impaired: although it complexed completely in 60 min, th e rate of complex formation during a 0-60-min incubation was decreased compared with mild-type C1 inhibitor. The mutant inhibitor also forme d a complex with trypsin, a serine protease that cleaves, and is not i nhibited by, wild-type C1 inhibitor. The Ala(443)-->Val mutation there fore converts C1 inhibitor from a substrate to an inhibitor of trypsin . These studies emphasize the role of the P2 residue in the determinat ion of target protease specificity.