SYNTHESIS AND PROCESSING OF GLYPICAN DURING DIFFERENTIATION OF SKELETAL-MUSCLE CELLS

We identified previously a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan can (HSPG) releasable by phosphatidylino sitol-specific phospholipase C (PI-PLC) on the surface of differentiat ed skeletal muscle cells (Campos et al., Eur. J. Biochem. 216, 587-595 (1993)) which is homologous to the HSPG synthesized by fibroblasts an d Schwann cells called glypican. In this study we have evaluated the p rocessing, location and amount of this HSPG in skeletal muscle cells d uring differentiation. Immunoprecipitation of incubation medium obtain ed from differentiated cells incubated with [S-35]sulfate by specific antibodies against glypican isolated from Schwann cells demonstrated t hat the antisera precipitated an intact HSPG. Immunoblot analysis of t he proteins released by PI-PLC after hoparitinase treatment revealed t he presence of a main band of 64 and a faint band of 62 kDa, whereas t he sizes of the core proteins for glypican present in the incubation m edia were 62 and 59 kDa. Pulse-chase experiments indicated that glypic an present in the membrane was spontaneously released into the culture medium with a t(1/2) of 12 h. The level of expression of glypican was analyzed during in vitro differentiation. The specific amount of the PI-PLC releasable HSPG increased about fourfold during cell differenti ation, No changes were detected in the level of the mRNA for glypican. Indirect analysis revealed that in myotubes glypican is present on th e cell surface as well as associated with the extracellular matrix (EC M). These results indicate that glypican is present, at least, in two different compartments on the surface of skeletal muscle cells.