A candidate molecule for the matrix assembly receptor to the N-terminal 29-kDa fragment of fibronectin in chick myoblasts.
Myoblast surface proteins with binding activity toward the N-terminal 29-kDa fragment of fibronectin were identified by two different experimental techniques: one involves radioiodination of the cell surface proteins, followed by solubilization with Triton X-100 and affinity purification on a Sepharose column conjugated with the 29-kDa fragment, and the other involves cross-linking of the 29-kDa fragment to the cells metabolically labeled with [35S]methionine, followed by immunoprecipitation with anti-29-kDa IgG. Both approaches revealed that primary cultures of chick myoblasts contain the 66- and 48-kDa proteins that bind to the 29-kDa fragment. These binding proteins were then purified to apparent homogeneity by two successive chromatographies of the solubilized extracts of 12-day-old embryonic muscle on wheat germ agglutinin-agarose and 29-kDa fragment-Sepharose columns. However, the 48-kDa protein was found to be derived from contaminating fibroblasts upon immunoblot analysis of the myogenic cell lines, rat L8E63 and mouse C2A3, and cultured fibroblasts using the antibody raised against the 66-kDa protein. Anti-66-kDa IgG inhibited the binding of the 125I-29-kDa protein to the primary culture of myoblasts in a dose-dependent manner. On the other hand, the same antibody showed little or no effect on the initial binding of 125I-fibronectin to the cell surface, but dramatically inhibited its incorporation into deoxycholate-insoluble matrices. Furthermore, Fab fragments of anti-66-kDa IgG completely blocked the incorporation of fluoresceinated fibronectin into matrices but not its binding to the cell surface. These results suggest that fibronectin matrix assembly is mediated at least in part by the interaction of the 66-kDa protein with the N-terminal type I domain of fibronectin.
Moon
K Y
KY
Department of Molecular Biology, College of Natural Science, Seoul National University, Korea.
Shin
K S
KS
Song
W K
WK
Chung
C H
CH
Ha
D B
DB
Kang
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*
D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D016023:Integrins; D009124:Muscle Proteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface