Differentiation defective mutants of skeletal myoblasts altered in a gelatin-binding glycoprotein.
We have previously described a myoblast cell surface glycoprotein of the molecular mass 46,000 (gp46), which is associated with myoblast differentiation. In this report we demonstrate that gp46 binds specifically to gelatin-Sepharose and in this respect is similar to a glycoprotein of the molecular mass 47,000, which has earlier been described as a cell surface localized protein in mouse parietal endoderm cells and in chick embryo fibroblasts. To ascertain the relationship of gp46 to myoblast differentiation, wild-type L6 myoblasts, as well as two concanavalin A (ConA) resistant, differentiation-negative, myoblast mutants (D-1 and C-8), were examined for gp46 expression. In the mutant designated D-1, which has a defect in dolichol mannosyl transferase, both mannose incorporation into gp46 and ConA binding to gp46 was reduced compared with L6, without markedly affecting the gelatin adhesion properties of gp46. Western blotting with a monoclonal antibody against gp46 was used to show that the expression of gp46 was normal in D-1 but was reduced in mutant C-8 compared with L6. Reduction occurred both in the plasma membrane and endoplasmic reticulum fractions of C-8 compared with wild-type L6. In L6 myoblasts, the expression of gp46 remained constant during myoblast replication and fusion but decreased markedly postfusion. In the nonfusing myoblast mutants D-1 and C-8 and in wild-type L6 cells that were prevented from fusing by treatment with 5-bromo-2'-deoxyuridine, the expression of gp46 remained invariant. We suggest that collagen interactions, mediated by gp46, are important for normal rat skeletal muscle differentiation.
Department of Biochemistry, Queen's University, Kingston, Ont., Canada.
G A
GA
Cates
D
D
Nandan
A M
AM
Brickenden
B D
BD
Sanwal
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002448:Cell Adhesion; D002454:Cell Differentiation; D002460:Cell Line; D003208:Concanavalin A / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism*; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D009132:Muscles / Q000166:cytology* / Q000378:metabolism; D009154:Mutation; D010449:Peptide Mapping; D011485:Protein Binding; D051381:Rats
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D003208:Concanavalin A; D005780:Gelatin