Edge A S, Kahn C R, Spiro R G
Department of Medicine, Harvard Medical School, Boston, Massachusetts.
Endocrinology. 1990 Oct;127(4):1887-95. doi: 10.1210/endo-127-4-1887.
The insulin receptor was immunoprecipitated from cultured human lymphocytes (IM-9) and rat hepatocytes (Fao) after biosynthetic labeling with [3H]glucosamine or [3H]mannose, and the nature of the carbohydrate units was investigated. Digestion of the receptor from IM-9 lymphocytes with E. freundii endo-beta-galactosidase increased the migration of the insulin receptor alpha- and beta-subunits on sodium dodecyl sulfate-polyacrylamide gels and sharpened the electrophoretic bands; the alpha-subunit was converted from an apparent mol wt (Mr) of 123,000 to a Mr of 118,000, and the beta-subunit from a Mr of 92,000 to 89,000. The susceptibility of the insulin receptor to this enzyme indicates that its carbohydrate units contain poly-N-acetyllactosamine sequences. Affinity chromatography of receptor glycopeptides on Concanavalin-A-Sepharose revealed that the poly-N-acetyllactosamine units were attached to multiantennary glycopeptides that accounted for over 75% of the [3H]glucosamine incorporated into the IM-9 lymphocyte insulin receptor; the remaining radioactivity was present in polymannose units (primarily Man8GlcNAc2) and biantennary complex saccharides. Several differences in the carbohydrate chains of the insulin receptor from the Fao and IM-9 cells indicated that glycosylation was cell specific despite the occurrence of poly-N-acetyllactosamine chains in both cell types. The IM-9 lymphocyte receptor glycopeptides were larger (Mr, 3,200-9,500) and more susceptible to endo-beta-galactosidase than those from the Fao receptor (Mr, 3,000-5,000). Moreover, the released saccharides from the Fao receptor were found by exoglycosidase digestions and chromatographic comparison to standards to contain terminal sialic acid in both alpha 2----3 and alpha 2----6 linkage to galactose, whereas the IM-9 carbohydrate units contained only alpha 2----3-linked sialic acid.
在用[³H]葡糖胺或[³H]甘露糖进行生物合成标记后，从培养的人淋巴细胞（IM-9）和大鼠肝细胞（Fao）中免疫沉淀胰岛素受体，并研究碳水化合物单元的性质。用弗氏埃希菌内切β-半乳糖苷酶消化IM-9淋巴细胞中的受体，可增加胰岛素受体α亚基和β亚基在十二烷基硫酸钠-聚丙烯酰胺凝胶上的迁移率，并使电泳条带变锐；α亚基的表观分子量（Mr）从123,000转变为118,000，β亚基的Mr从92,000转变为89,000。胰岛素受体对该酶的敏感性表明其碳水化合物单元含有多聚N-乙酰乳糖胺序列。在伴刀豆球蛋白A-琼脂糖上对受体糖肽进行亲和层析显示，多聚N-乙酰乳糖胺单元连接到多天线糖肽上，这些多天线糖肽占掺入IM-9淋巴细胞胰岛素受体中的[³H]葡糖胺的75%以上；其余放射性存在于多聚甘露糖单元（主要是Man8GlcNAc2）和双天线复合糖中。Fao细胞和IM-9细胞胰岛素受体碳水化合物链的几个差异表明，尽管两种细胞类型中都存在多聚N-乙酰乳糖胺链，但糖基化具有细胞特异性。IM-9淋巴细胞受体糖肽比Fao受体的糖肽更大（Mr为3,200 - 9,500），且对内切β-半乳糖苷酶更敏感（Mr为3,000 - 5,000）。此外，通过外切糖苷酶消化和与标准品的色谱比较发现，Fao受体释放的糖类在与半乳糖的α2→3和α2→6连接中均含有末端唾液酸，而IM-9碳水化合物单元仅含有α2→3连接的唾液酸。
