Fibrillin assembly: dimer formation mediated by amino-terminal sequences

We have investigated recombinant fibrillin-1 (profib-1) and fibrillin-2 (gl yfib-2) molecules encoding the proline- or glycine-rich regions with flanki ng domains (exons 9-11), in order to establish whether these sequences migh t mediate specific molecular recognition events important in fibrillin asse mbly Our data demonstrate that both recombinant molecules can form extracel lular dimers, but highlight subtle differences in the stability of these di mers, Following expression in COS-1 cells, SDS-PAGE analysis showed that gl yfib-2 was present intracellularly as monomers, and extracellularly as mono mers and disulphide-bonded dimers, Size fractionation in native non-reducin g conditions prior to SDS-PAGE analysis highlighted that glyfib-2 also form ed non-covalent associations. In contrast, profib-1 appeared monomeric in c ells and medium. Using an in vitro translation system supplemented with sem ipermeabilised HT1080 cells together with chemical crosslinking, dimers of the fibrillin-1 and fibrillin-2 molecules were detected. Dimerisation was n ot cell-dependent since molecules translated in the absence of cells dimeri sed, and was not an intracellular event as judged by proteinase K digestion s. A crosslinking and coimmunoprecipitation strategy provided a means of in vestigating whether molecular chaperones might be involved in preventing di merisation of translocated molecules, Proteinase K-resistant recombinant mo lecules associated rapidly with BiP, and thereafter with protein disulphide isomerase and calreticulin, Differences between the two fibrillin isoforms in ability to form stable dimers prompted investigation of the proline- an d glycine-rich sequences. Differences in solubility and pI were apparent th at may contribute to reduced stability of proline-rich region interactions. These studies suggest that extracellular dimer formation mediated by inter actions of the proline- and glycine-rich regions may be a crucial early ste p in the extracellular assembly of fibrillin into microfibrils.