CHARACTERIZATION OF GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED NCAM ON MOUSE SKELETAL-MUSCLE CELL-LINE C2C12 - THE STRUCTURE OF THE GPI GLYCAN AND RELEASE DURING MYOGENESIS

The mouse myoblast cell line C2C12 constitutively expressed 160-kDa tr ansmembrane NCAM isoform and 135-kDa GPI-anchored isoform before diffe rentiation, During differentiation into multinucleated myotubes, the c ells newly expressed 150-kDa GPI-anchored isoform and the level of 135 -kDa GPI-anchored isoform increased, Structural analysis of the GPI gl ycan of NCAM, which was purified from C2C12 myotubes after metabolic l abeling with [H-3]inositol, was performed by sequential exoglycosidase digestion and Wistaria floribunda agglutinin-agarose column chromatog raphy, The core GPI glycan structure, Man alpha 1-2Man alpha-Man alpha -GlcNH(2)-myoInositol, was conserved and variations were observed in a dditional mannose and N-acetylgalactosamine residues. Structural analy sis of the GPI glycans of the two GPI-anchored isoforms, GPI-NCAM 135 and GPI-NCAM 150, showed the enhanced attachment of the N-acetylgalact osamine residue to the GPI glycan core of GPI-NCAM 150, These GPI-anch ored NCAM isoforms were released from C2C12 cells during the myoblast differentiation, Release of GPI-anchored NCAMs was observed when C2C12 cells were cultured in a serum-free medium, and inositol but not inos itol phosphate was detected after nitrous acid deamination of the rele ased NCAM. These results suggest that the GPI-anchored NCAM was releas ed from the cell surface by the action of an endogeneous phospholipase D. (C) 1995 Academic Press,Inc.