Differential Vicia villosa agglutinin reactivity identifies three distinct dystroglycan complexes in skeletal muscle.

We present evidence for the expression of three alpha-dystroglycan glycoforms in skeletal muscle cells, including two minor glycoforms marked by either patent or latent reactivity with the N-acetylgalactosamine-specific lectin Vicia villosa agglutinin. Both minor glycoforms co-isolated with beta-dystroglycan, but not with other dystrophin/utrophin-glycoprotein complex components, suggesting that they may perform distinct or modified cellular functions. We also confirmed that both patent and latent V. villosa agglutinin-reactive alpha-dystroglycan glycoforms are expressed in C2C12 myotubes. However, we found that the combined effect of saturating concentrations of V. villosa agglutinin and laminin-1 were strictly additive with respect to acetylcholine receptor cluster formation in C2C12 myotubes, which suggests that laminin-1 and V. villosa agglutinin do not compete for the same binding site on the cell surface. Finally, although beta-N-acetylhexosaminidase digestion dramatically inhibited agrin-, V. villosa agglutinin-, and laminin-1-induced acetylcholine receptor clustering in C2C12 myotubes, treatment with this enzyme had no effect on the amount of alpha-dystroglycan that was bound to V. villosa agglutinin-agarose. We conclude that alpha-dystroglycan is not the V. villosa agglutinin receptor implicated in acetylcholine receptor cluster formation. However, our data provide new support for the hypothesis that different glycoforms of alpha-dystroglycan may perform distinct functions even within the same cell.

Differential heparin sensitivity of alpha-dystroglycan binding to laminins expressed in normal and dy/dy mouse skeletal muscle.

The alpha-dystroglycan binding properties of laminins extracted from fully differentiated skeletal muscle were characterized. We observed that the laminins expressed predominantly in normal adult rat or mouse skeletal muscle bound alpha-dystroglycan in a Ca2+-dependent, ionic strength-sensitive, but heparin-insensitive manner as we had observed previously with purified placental merosin (Pall, E. A., Bolton, K. M., and Ervasti, J. M. 1996 J. Biol. Chem. 271, 3817-3821). Rat skeletal muscle laminins partially purified by heparin-agarose affinity chromatography also bound alpha-dystroglycan without sensitivity to heparin. We also confirm previous studies of dystrophic dy/dy mouse skeletal muscle showing that the alpha2 chain of merosin is reduced markedly and that the laminin alpha1 chain is not up-regulated detectably. However, we further observed a quantitative decrease in the expression of laminin beta/gamma chain immunoreactivity in alpha2 chain-deficient dy/dy skeletal muscle and reduced alpha-dystroglycan binding activity in laminin extracts from dy/dy muscle. Most interestingly, the alpha-dystroglycan binding activity of residual laminins expressed in merosin-deficient dy/dy skeletal muscle was inhibited dramatically (69 +/- 19%) by heparin. These results identify a potentially important biochemical difference between the laminins expressed in normal and dy/dy skeletal muscle which may provide a molecular basis for the inability of other laminin variants to compensate fully for the deficiency of merosin in some forms of muscular dystrophy.