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

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Abstract

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 complexed with C1s but showed little complex formation with C1r. These molecules also appeared to be relatively resistant to digestion by trypsin. 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 cytosine in the codon for Ala443 (P2 residue) in one C1 inhibitor allele, resulting in substitution with a Val residue. To test the effect of this substitution, a mutant C1 inhibitor containing Ala443-->Val was constructed by site-directed mutagenesis and expressed in COS-1 cells. Both the Ala443-->Val mutant and the wild-type C1 inhibitor complexed completely with C1s, kallikrein, and coagulation Factor XIIa after incubation at 37 degrees C for 60 min. In contrast, the mutant inhibitor failed to complex completely with C1r under the same conditions. Time course analysis showed that the ability of the mutant to complex with C1s is also impaired: although it complexed completely in 60 min, the rate of complex formation during a 0-60-min incubation was decreased compared with wild-type C1 inhibitor. The mutant inhibitor also formed a complex with trypsin, a serine protease that cleaves, and is not inhibited by, wild-type C1 inhibitor. The Ala443-->Val mutation therefore converts C1 inhibitor from a substrate to an inhibitor of trypsin. These studies emphasize the role of the P2 residue in the determination of target protease specificity.

Authors

R Zahedi, J J Bissler, A E Davis 3rd, C Andreadis, J J Wisnieski