Tsuji T, Osawa T
J Biochem. 1986 Nov;100(5):1387-98. doi: 10.1093/oxfordjournals.jbchem.a121845.
Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.
人血小板膜糖蛋白IIb（GPIIb）和IIIa（GPIIIa）被认为是纤维蛋白原受体的亚基，通过在伴刀豆球蛋白A（Con A）-琼脂糖柱上进行亲和层析，随后进行制备性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，从经Triton X-100增溶的血小板膜中纯化得到。对纯化的糖蛋白进行成分分析表明，GPIIb和GPIIIa分别含有15%和18%（重量）的碳水化合物，其由半乳糖、甘露糖、葡糖胺、岩藻糖和唾液酸组成。这表明这些糖蛋白含有N-连接的碳水化合物链。通过肼解从每种糖蛋白中释放出碳水化合物链，然后在Mono Q柱上通过离子交换层析进行分离。从每种糖蛋白中获得了单唾液酸化、双唾液酸化和三唾液酸化以及中性寡糖组分。通过成分和甲基化分析以及外切糖苷酶消化对这些寡糖的结构进行了研究，并且还检测了它们与固定化凝集素的反应性。中性寡糖占从GPIIb释放的总寡糖的约14%，占从GPIIIa释放的总寡糖的约52%，发现它们属于高甘露糖型，因为它们含有5或6个甘露糖残基。另一方面，发现酸性寡糖的主要部分由典型的双天线和三天线复合型糖链组成，还检测到少量的四天线复合型糖链，以及在外围部分的N-乙酰葡糖胺残基处带有岩藻糖基残基或在核心部分的β-甘露糖基残基处带有平分型N-乙酰葡糖胺的复合型糖链。总之，我们发现GPIIb主要含有复合型糖链，而高甘露糖型糖链是GPIIIa中的主要碳水化合物单元，并且检测到的GPIIb和GPIIIa碳水化合物部分的差异是定量的而非定性的。
