Characterisation of fibrillin-1 cDNA clones in a human fibroblast cell line that assembles microfibrils.

Fibrillin-1 is a large extracellular glycoprotein which is a major structural component of 10-12 nm microfibrils. Defects in human fibrillin-1 give rise to the autosomal dominant connective tissue disease the Marfan syndrome and related disorders. Previous studies examining the biosynthesis and secretion of recombinant fibrillin-1 fragments have been performed in cell lines which do not assemble fibrillin into extracellular 10-12 nm microfibrils. Conflicting data have been obtained regarding N-terminal processing. In this study we have characterised a human fibroblast cell line MSU-1.1 which shows a similar endogenous fibrillin-1 pulse chase profile to primary human dermal fibroblasts and produces microfibrils. Expression of a approximately 50 kDa N-terminal recombinant peptide in MSU-1.1 resulted in efficient secretion of this peptide into conditioned media, N-terminal sequence analysis of the purified peptide identified 2 protease cleavage sites and a presumed signal peptidase site. Together these data identify the natural leader sequence of fibrillin-1 and the presence of two processing sites in the N-terminus of fibrillin-1. The identification of an N-terminal processing site in recombinant fibrillin-1 similar to that obtained in a previous study which used an HT1080 fibrosarcoma host cell line excludes defective N-terminal processing as the cause of the assembly defect in this cell line. A full length normal and mutant fibrillin cDNA (approximately 8.6 kb) was constructed and stable integration of each into MSU1.1 led to RNA transcription at approximately 5% of endogenous levels. This is the first report of transcription from the full length fibrillin-1 cDNA. The low levels of transcription achieved, suggest that additional upstream and downstream DNA sequence elements will be required for high levels of full length fibrillin-1 cDNA expression.