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Volume 271, Number 52, Issue of December 27, 1996 pp. 33284-33292
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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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

(Received for publication, July 29, 1996, and in revised form, September 27, 1996)

From the Departments of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706

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 ~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 ~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.

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