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

被引:43
作者
Zhang, QH
Mosher, DF
机构
[1] UNIV WISCONSIN,DEPT MED,MADISON,WI 53706
[2] UNIV WISCONSIN,DEPT BIOMOL CHEM,MADISON,WI 53706
关键词
D O I
10.1074/jbc.271.52.33284
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Cell surface molecules on adherent cells that bind I-125-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 similar to 3 MDa. The label in 70-kDa fragment conjugated with I-125-sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3'-dithiopropionate was associated with >>3-MDa LAMMs without reduction and with similar to 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 I-125-70-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.
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收藏
页码:33284 / 33292
页数:9
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