Synthesis and processing of glypican during differentiation of skeletal muscle cells.
We identified previously a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan (HSPG) releasable by phosphatidylinositol-specific phospholipase C (PI-PLC) on the surface of differentiated skeletal muscle cells (Campos et al., Eur. J. Biochem. 216, 587-595 (1993)) which is homologous to the HSPG synthesized by fibroblasts and Schwann cells called glypican. In this study we have evaluated the processing, location and amount of this HSPG in skeletal muscle cells during differentiation. Immunoprecipitation of incubation medium obtained from differentiated cells incubated with [35S]sulfate by specific antibodies against glypican isolated from Schwann cells demonstrated that the antisera precipitated an intact HSPG. Immunoblot analysis of the proteins released by PI-PLC after heparitinase treatment revealed the presence of a main band of 64 and a faint band of 62 kDa, whereas the sizes of the core proteins for glypican present in the incubation media were 62 and 59 kDa. Pulse-chase experiments indicated that glypican present in the membrane was spontaneously released into the culture medium with a t1/2 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 differentiation. No changes were detected in the level of the mRNA for glypican. Indirect analysis revealed that in myotubes glypican is present on the cell surface as well as associated with the extracellular matrix (ECM). These results indicate that glypican is present, at least, in two different compartments on the surface of skeletal muscle cells.
Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Santiago/Chile.
E
E
Brandan
D J
DJ
Carey
J
J
Larraín
F
F
Melo
A
A
Campos
D000818:Animals; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000502:physiology*; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis
D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D012333:RNA, Messenger