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Fibrillin assembly: dimer formation mediated by amino-terminal sequences

Citation

Ashworth, JL and Kelly, V and Wilson, RR and Shuttleworth, CA and Kielty, CM, Fibrillin assembly: dimer formation mediated by amino-terminal sequences, Journal of Cell Science, 112, (20) pp. 3549-3558. ISSN 0021-9533 (1999) [Refereed Article]

Abstract

We have investigated recombinant fibrillin-1 (profib-1) and fibrillin-2 (glyfib-2) molecules encoding the proline- or glycine-rich regions with flanking domains (exons 9-11), in order to establish whether these sequences might mediate specific molecular recognition events important in fibrillin assembly. Our data demonstrate that both recombinant molecules can form extracellular dimers, but highlight subtle differences in the stability of these dimers. Following expression in COS-1 cells, SDS-PAGE analysis showed that glyfib-2 was present intracellularly as monomers, and extracellularly as monomers and disulphide-bonded dimers. Size fractionation in native non-reducing conditions prior to SDS-PAGE analysis highlighted that glyfib-2 also formed non-covalent associations. In contrast, profib-1 appeared monomeric in cells and medium. Using an in vitro translation system supplemented with semipermeabilised HT1080 cells together with chemical crosslinking, dimers of the fibrillin-1 and fibrillin-2 molecules were detected. Dimerisation was not cell-dependent since molecules translated in the absence of cells dimerised, and was not an intracellular event as judged by proteinase K digestions. A crosslinking and coimmunoprecipitation strategy provided a means of investigating whether molecular chaperones might be involved in preventing dimerisation of translocated molecules. Proteinase K-resistant recombinant molecules 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- and glycine-rich sequences. Differences in solubility and pI were apparent that may contribute to reduced stability of proline-rich region interactions. These studies suggest that extracellular dimer formation mediated by interactions of the proline- and glycine-rich regions may be a crucial early step in the extracellular assembly of fibrillin into microfibrils.

Item Details

Item Type:Refereed Article
Research Division:Biological Sciences
Research Group:Biochemistry and Cell Biology
Research Field:Protein Trafficking
Objective Division:Expanding Knowledge
Objective Group:Expanding Knowledge
Objective Field:Expanding Knowledge in the Medical and Health Sciences
Author:Wilson, RR (Dr Richard Wilson)
ID Code:72437
Year Published:1999
Web of Science® Times Cited:32
Deposited By:Central Science Laboratory
Deposited On:2011-08-26
Last Modified:2011-08-26
Downloads:0

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