Mukasa R, Umeda M, Endo T, Kobata A, Inoue K
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Arch Biochem Biophys. 1995 Apr 1;318(1):182-90. doi: 10.1006/abbi.1995.1219.
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.
小鼠成肌细胞系C2C12在分化前组成性表达160 kDa跨膜神经细胞黏附分子（NCAM）异构体和135 kDa糖基磷脂酰肌醇（GPI）锚定异构体。在分化为多核肌管的过程中，细胞新表达了150 kDa GPI锚定异构体，且135 kDa GPI锚定异构体的水平增加。在用[3H]肌醇进行代谢标记后，从C2C12肌管中纯化得到NCAM的GPI聚糖，并通过外切糖苷酶顺序消化和紫藤凝集素-琼脂糖柱色谱法进行结构分析。核心GPI聚糖结构，即Manα1-2Manα-Manα-GlcNH2-肌醇，得以保留，且在额外的甘露糖和N-乙酰半乳糖胺残基上观察到了变异。对两种GPI锚定异构体GPI-NCAM 135和GPI-NCAM 150的GPI聚糖进行结构分析表明，N-乙酰半乳糖胺残基与GPI-NCAM 150的GPI聚糖核心的连接增强。这些GPI锚定的NCAM异构体在成肌细胞分化过程中从C2C12细胞中释放出来。当C2C12细胞在无血清培养基中培养时，观察到GPI锚定的NCAMs释放，并且在对释放的NCAM进行亚硝酸脱氨后检测到肌醇而非肌醇磷酸。这些结果表明，GPI锚定的NCAM是通过内源性磷脂酶D的作用从细胞表面释放的。
