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 structur al component of 10-12 nm microfibrils. Defects in human fibrillin-1 give ri se to the autosomal dominant connective tissue disease the Marfan syndrome and related disorders. Previous studies examining the biosynthesis and secr etion of recombinant fibrillin-1 fragments have been performed in cell line s which do not assemble fibrillin into extracellular 10-12 am microfibrils. Conflicting data have been obtained regarding N-terminal processing. In th is study we have characterised a human fibroblast cell line MSU-1.1 which s hows a similar endogenous fibrillin-1 pulse-chase profile to primary human dermal fibroblasts and produces microfibrils. Expression of a similar to 50 kDa N-terminal recombinant peptide in MSU-1.1 resulted in efficient secret ion of this peptide into conditioned media. N-terminal sequence analysis of the purified peptide identified 2 protease cleavage sites and a presumed s ignal peptidase site. Together these data identify the natural leader seque nce of fibrillin-1 and the presence of two processing sites in the N-termin us of fibrillin-1. The identification of an N-terminal processing site in r ecombinant fibrillin-1 similar to that obtained in a previous study which u sed an HT1080 fibrosarcoma host cell line excludes. defective N-terminal pr ocessing as the cause of the assembly defect in this cell line. A full length normal and mutant fibrillin cDNA (similar to 8.6 kb) was cons tructed and stable integration of each into MSU1.1 led to RNA transcription at similar to 5% of endogenous levels. This is the first report of transcr iption from the full length fibrillin-1 cDNA. The low levels of transcripti on achieved suggest that additional upstream and downstream DNA sequence el ements will be required for high levels of full length fibrillin-1 cDNA exp ression. (C) 2000 Elsevier Science Ltd. All rights reserved.