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 transmembrane NCAM isoform and 135-kDa GPI-anchored isoform before differentiation. During differentiation into multinucleated myotubes, the cells newly expressed 150-kDa GPI-anchored isoform and the level of 135-kDa GPI-anchored isoform increased. Structural analysis of the GPI glycan of NCAM, which was purified from C2C12 myotubes after metabolic labeling with [3H]inositol, was performed by sequential exoglycosidase digestion and Wistaria floribunda agglutinin-agarose column chromatography. The core GPI glycan structure, Man alpha 1-2Man alpha-Man alpha-GlcNH2-myoInositol, was conserved and variations were observed in additional mannose and N-acetylgalactosamine residues. Structural analysis of the GPI glycans of the two GPI-anchored isoforms, GPI-NCAM 135 and GPI-NCAM 150, showed the enhanced attachment of the N-acetylgalactosamine residue to the GPI glycan core of GPI-NCAM 150. These GPI-anchored 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 inositol phosphate was detected after nitrous acid deamination of the released NCAM. These results suggest that the GPI-anchored NCAM was released from the cell surface by the action of an endogeneous phospholipase D.
Mukasa
R
R
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Umeda
M
M
Endo
T
T
Kobata
A
A
Inoue
K
K
D000818:Animals; D002240:Carbohydrate Sequence; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D003470:Culture Media; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D007294:Inositol / Q000032:analysis; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism
D015816:Cell Adhesion Molecules, Neuronal; D003470:Culture Media; D017261:Glycosylphosphatidylinositols; D011134:Polysaccharides; D007294:Inositol