Effect of mutations within the coding region of C1-INH gene on protein function in families with HAE

C1 esterase inhibitor (C1-INH) is a serine protease inhibitor (serpin). Serpins are a superfamily of proteins that fold into a conserved structure and utilize a unique suicide substrate-like inhibitory mechanism. The native structure has a metastable conformation that is required for serpin inhibitory activity. When the target protease recognises and cleaves the scissile bond in the reactive site loop (RSL), this flexible loop, initially exposed to the solvent, starts to insert into -sheet A and bring the covalently bound protease with it. Upon complete loop insertion the proteinase is translocated by over 70 Å, and its active site distorted. The energy needed to complete the inhibitory process comes from the higher stability of the locked-in conformation compared with the native-like conformation. The picture of C1-INH looks like that of other serpins. However, C1-INH tertiary structure is not experimentally known and systematic functional and structural studies on this inhibitor are not abundant. We are now screening for mutations in C1-INH gene a case list of 471 HAE patients belonging to 179 independent families. At present we have identified 132 different mutations in C1-INH gene. In order to gain insights onto the structure-function relationship of C1-INH, we selected those mutations affecting the mechanism of loop insertion (delThr280, Tyr308Asn, Arg378Cys, Leu427Pro/Arg) to be expressed in Pichia pastoris and functionally characterized. The requirement for a metastable conformation in the native/active state renders serpins extremely sensitive to point mutations resulting in conformational instability and polymerization. The deleterious effects of serpins polymerization are well known (Alzheimer, Parkinson diseases, etc), while effects on minor conformational changes are just now revealing to act as signals for a range of physiological responses. The structural and functional properties of these targeted mutants could contribute to elucidate the biological activities of serpins in general, providing useful insights into the pathogenesis of conformational diseases and could also identify genotype-phenotype correlates in HAE to be exploited for new therapeutic strategies.