Initial steps in assembly of microfibrils - Formation of disulfide-cross-linked multimers containing fibrillin-1

Fibrillins are the major constituents of extracellular microfibrils, How fi brillin molecules assemble into microfibrils is not known. Sequential extra ctions and pulse-chase labeling of organ cultures of embryonic chick aortae revealed rapid formation of disulfide-cross-linked aggregates containing f ibrillin-1. These results demonstrated that intermolecular disulfide bond f ormation is an initial step in the assembly process. To identify free cyste ine residues available for intermolecular cross-linking, small recombinant peptides of fibrillin-1 harboring candidate cysteine residues were analyzed . Results revealed that the first four cysteine residues in the unique N te rminus form intramolecular disulfide bonds. One cysteine residue (Cys(204)) in the first hybrid domain of fibrillin-1 was found to occur as a free thi ol and is therefore a good candidate for intermolecular disulfide bonding i n initial steps of the assembly process, Furthermore, evidence indicated th at the comparable cysteine residue in fibrillin-2 (Cys(233)) also occurs as a free thiol. These free cysteine residues in fibrillins are readily avail able for intermolecular disulfide bond formation, as determined by reaction with Ellman's reagent. In addition to these major results, the cleavage si te of the fibrillin-1 signal peptide and the N-terminal sequence of monomer ic authentic fibrillin-1 from conditioned fibroblast medium were determined .