Lectin-resistant Myoblasts

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Degree Name

Doctor of Philosophy


Several lectin-resistant mutants were isolated from a permanent rat skeletal myoblast cell line with the objectives of determining the role of cell surface carbohydrate in myoblast differentiation and of determining the mode of action of several lectins and how resistance to them is achieved. Mutants were isolated in single- and multi-step selections and were found to fall into four phenotypic groups named after the lectin used in their selection: concanavalin A-resistant (ConA('R)), phytohemagglutinin-resistant (PHA('R)), wheat germ agglutinin-resistant, type I (WGA('RI)), and wheat germ agglutinin-resistant, type II (WGA('RII)).;The WGA('RII) mutants were about eight-fold more resistant to WGA than the wild type (WT). They bound at least as much WGA as the WT at low temperature but internalized less WGA at 37(DEGREES)C than the WT. Metabolic and lactoperoxidase labelling revealed no changes upon polyacrylamide gel electrophoresis. Complex glycopeptides isolated from the cell surface were of masses suggestive of triantennary and higher order structures. They were similar to those of the WT except for an acquired resistance to sialidase digestion unless first treated with base.;The WGA('RI) mutants bound less WGA and internalized less WGA than the WT. Polyacrylamide gel electrophoresis revealed a shift to a lower molecular mass of at least one band. The complex glycopeptides had lost their terminal sialic acid and galactose residues. Representative clones of WGA('RI) alone among the LEC('R) mutants isolated differentiated into myotubes.;WGA('RI), WGA('RII) and WT had the same high mannose oligosaccharides: Man(,8)GlcNAc(,2), Man(,7)GlcNAc(,2) and Man(,6)GlcNAc(,2). The WGA('R) mutants showed no decreases in the following: UDP-hexose, UDP-N-acetylhexosamine and CMP-sialic acid pools, uptake of UDP-Gal into the Golgi apparatus, synthesis of glycosaminoglycan, UDP-galactose: N-acetylglucosamine galactosyltransferase activity and UDP-N-acetylglucosamine: ribonuclease B N-acetyl-acetylglucosaminyltransferase activity or increases in (beta)-galactosidase activity.;The ability of tunicamycin to inhibit myotube formation and the ability of N-acetylglucosamine to reverse this inhibition was also described.;The results indicated that cell surface carbohydrate (i.e. lectin receptors) have only an indirect and dispensable role in myotube formation.

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