Sialomucins are the dominant components of the cell surfaces of some carcinoma ascites cells and have been postulated to inhibit recognition of tumours by the immune system. The sialomucin ASGP-1 (ascites sialoglycoprotein-1) of the 13762 rat mammary adenocarcinoma is associated with the cell surface as a complex with a concanavalin-A-binding glycoprotein called ASGP-2. This sialomucin complex has been purified from ascites cell microvilli by extraction with Triton X-100 and CsCl density-gradient centrifugation. ASGP-1 (which has been purified previously) and ASGP-2 were dissociated in 6 M-guanidine hydrochloride and separated by gel filtration. The molecular mass of the undenatured detergent complex of ASGP-2, estimated by gel filtration and velocity sedimentation in Triton X-100, was 148 kDa. Since the apparent molecular mass by SDS/polyacrylamide-gel electrophoresis was about 120 kDa, ASGP-2 must be a monomer as extracted from the membrane. Studies of its chemical composition indicate that it contains about 45% carbohydrate by weight, including both mannose and galactosamine. Alkaline borohydride treatment of ASGP-2 converted approx. half of the N-acetylgalactosamine to N-acetylgalactosaminitol, demonstrating the presence of O-linked oligosaccharides. Analyses of mannose-labelled Pronase glycopeptides from ASGP-2 by lectin-affinity chromatography on concanavalin A and leucocyte-agglutinating phytohaemagglutinin suggested that 40% of the label was present in high-mannose/hybrid oligosaccharides, 20% in triantennary oligosaccharides substituted on the C-2 and C-4 mannose positions and 40% in tri- or tetra-antennary oligosaccharides substituted on C-2 and C-6. The presence of polylactosamine sequences on these oligosaccharides was suggested by lectin blots and by precipitation from detergent extracts with tomato lectin. From chemical analyses and lectin-affinity studies, we estimate that ASGP-2 contains four high-mannose and 13 complex N-glycosylated oligosaccharides, plus small amounts of polylactosamine and O-linked oligosaccharides. The presence of four different classes of oligosaccharides on this glycoprotein suggests that it will be an interesting model system for biosynthetic comparisons of the different glycosylation pathways.

译文

唾液球蛋白是某些癌腹水细胞细胞表面的主要成分,并被认为可以抑制免疫系统对肿瘤的识别。13762只大鼠乳腺腺癌的唾液酸核素ASGP-1 (腹水sialoglycoprotein-1) 与细胞表面相关,与称为ASGP-2的伴刀豆球蛋白a结合糖蛋白的复合物。通过用Triton X-100和CsCl密度梯度离心提取,从腹水细胞微绒毛中纯化了这种唾液酸苷复合物。将ASGP-1 (先前已纯化) 和ASGP-2在6 m-胍盐酸盐中解离并通过凝胶过滤分离。通过凝胶过滤和Triton X-100中的速度沉降估算,ASGP-2的未变性洗涤剂复合物的分子量为148 kDa。由于通过SDS/聚丙烯酰胺-凝胶电泳的表观分子量为约120 kDa,ASGP-2必须是从膜中提取的单体。对其化学成分的研究表明,它含有约45% 重量的碳水化合物,包括甘露糖和半乳糖胺。碱性硼氢化物处理ASGP-2转化约。N-乙酰半乳糖胺至N-乙酰半乳糖胺的一半,证明存在O-连接的寡糖。通过刀豆球蛋白A和白细胞凝集植物血凝素上的凝集素亲和色谱分析来自ASGP-2的甘露糖标记的链霉蛋白酶糖肽,表明该标记的40% 存在于高甘露糖/杂交寡糖中,在C-2和C-4甘露糖位置上取代的三天线寡糖中的20% 和在C-2和C-6上取代的三或四天线寡糖中的40%。通过凝集素印迹和番茄凝集素从洗涤剂提取物中沉淀出来,表明这些寡糖上存在聚乳糖胺序列。根据化学分析和凝集素亲和力研究,我们估计ASGP-2含有四种高甘露糖和13种复杂的N-糖基化寡糖,以及少量的聚乳糖胺和O-连接寡糖。该糖蛋白上存在四种不同类别的寡糖,这表明它将是用于不同糖基化途径的生物合成比较的有趣模型系统。

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