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

Abstract

We have determined the cause of an unusual Cl inhibitor abnormality in a large kindred. We previously found that half of serum Cl inhibitor molecules in affected kindred members are normal. The other half complexed with Cls but showed little complex formation with Clr. These mole- cules also appeared to be relatively resistant to digestion by trypsin. Taken together, the findings suggested that mem- bers of this kindred are heterozygous for an unusual Cl inhibitor mutation. Sequencing of genomic DNA from the kindred revealed that thymine has replaced cytosine in the codon for Ala443 (P2 residue) in one Cl inhibitor allele, resulting in substitution with a Val residue. To test the effect of this substitution, a mutant Cl inhibitor containing Ala"3--Val was constructed by site-directed mutagenesis and expressed in COS-1 cells. Both the Ala'3--Val mutant and the wild-type Cl inhibitor complexed completely with Cls, kallikrein, and coagulation Factor XIIa after incuba- tion at 37C for 60 min. In contrast, the mutant inhibitor failed to complex completely with Clr under the same condi- tions. Time course analysis showed that the ability of the mutant to complex with Cls is also impaired: although it complexed completely in 60 min, the rate of complex forma- tion during a 0-60-min incubation was decreased compared with wild-type Cl inhibitor. The mutant inhibitor also formed a complex with trypsin, a serine protease that cleaves, and is not inhibited by, wild-type Cl inhibitor. The Ala"3-Val mutation therefore converts Cl inhibitor from a substrate to an inhibitor of trypsin. These studies empha- size the role of the P2 residue in the determination of target protease specificity. (