title: Cross-linking of the NH2-terminal region of fibronectin to molecules of large apparent molecular mass. Characterization of fibronectin assembly sites induced by the treatment of fibroblasts with lysophosphatidic acid.
abstract: Cell surface molecules on adherent cells that bind 125I-labeled fibronectin or its 70-kDa N-terminal fragment were identified by cross-linking with factor XIIIa and by photoaffinity labeling. Such cross-linking caused the 70-kDa fragment to become associated irreversibly to cell layers and was greater in cells treated with lysophosphatidic acid, an enhancer of fibronectin assembly and strong modulator of cell shape. Cross-linking of the 70-kDa fragment with factor XIIIa was to molecules that migrated in discontinuous sodium dodecyl sulfate-polyacrylamide gels at the top of the 3.3% stacking gel and near the top of the separating gel. Estimated sizes of these large apparent molecular mass molecules (LAMMs) were >>3 MDa and approximately 3 MDa. The label in 70-kDa fragment conjugated with 125I-sulfosuccinimidyl 2-(p-azidosalicylamido)-1, 3'-dithiopropionate was associated with >>3-MDa LAMMs without reduction and with approximately 3-MDa LAMMs after reduction and transfer of the cleavable label. The LAMMs were expressed on monolayer cells shortly after adherence, required both 1% Triton X-100 and 2 M urea for efficient extraction, and were susceptible to digestion with trypsin but not to cathepsin D digestion. Complexes of 125I-70-kDa fragment and LAMMs were also susceptible to limited acid digestion and Glu-C protease digestion but were not cleaved by chondroitin lyase or heparitinase. Neither the uncleaved complexes nor the cleavage products were immunoprecipitated with anti-fibronectin antibodies directed toward epitopes outside the 70-kDa region. Thus, cell surface molecules that are either very large or not dissociated in sodium dodecyl sulfate comprise the labile matrix assembly sites for fibronectin.
authors.affiliation: Departments of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Zhang
authors.forename: D F
authors.initials: DF
authors.lastname: Mosher
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism; D001386:Azides / Q000494:pharmacology; D002103:Cadaverine / Q000031:analogs & derivatives / Q000378:metabolism; D002808:Chondroitin Lyases / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D005561:Formates / Q000378:metabolism; D006801:Humans; D008246:Lysophospholipids / Q000378:metabolism*; D008970:Molecular Weight; D012516:Osteosarcoma / Q000378:metabolism; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism; D013388:Succinimides / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D001386:Azides; D003432:Cross-Linking Reagents; D005353:Fibronectins; D005456:Fluorescent Dyes; D005561:Formates; D008246:Lysophospholipids; D013388:Succinimides; C030544:formic acid; C063086:sulfosuccinimidyl 3-((2-(4-azidosalicylamido)ethyl)dithio)propionate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D002808:Chondroitin Lyases; C008542:monodansylcadaverine; D002103:Cadaverine