Differential effect of FBN1 mutations on in vitro proteolysis of recombinant fibrillin-1 fragments

Mutations in the fibrillin-1 gene (FBN1) cause Marfan syndrome (MFS), an au tosomal dominant disorder of connective tissue with highly variable clinica l manifestations. FBN1 contains 47 epidermal growth factor (EGF)-like modul es, 43 of which display a consensus sequence for calcium binding (cbEGF). C alcium binding by cbEGF modules is thought to be essential for the conforma tion and stability of fibrillin-1. Missense mutations in cbEGF modules are the most common mutations found in MFS and generally affect one of the six highly conserved cysteines or residues of the calcium-binding consensus seq uence. We have generated a series of recombinant fibrillin-1 fragments cont aining six cbEGF modules (cbEGF nos. 15-20) with various mutations at diffe rent positions of cbEGF module no. 17, which is known to contain a cryptic cleavage site for trypsin. A mutation affecting a residue of the calcium-bi nding consensus sequence (K1300E) found in a patient with relatively mild c linical manifestations of classic MFS caused a modest increase in susceptib ility to in vitro proteolysis by trypsin, whereas a mutation affecting the sixth cysteine residue of the same cbEGF module (C1320S) reported in a seve rely affected patient caused a dramatic increase in susceptibility to in vi tro proteolysis by trypsin. A mutation at the cryptic cleavage site for try psin abolished sensitivity of wild-type fragments and fragments containing K1300E to trypsin proteolysis. Whereas the relevance of in vitro proteolysi s to the in vivo pathogenesis of MFS remains unclear, our findings demonstr ate that individual mutations in cbEGF modules can affect these modules dif ferentially and may suggest an explanation for some genotype-phenotype rela tionships in MFS.