Isolation and partial characterization of ascites sialoglycoprotein-2 of the cell surface sialomucin complex of 13762 rat mammary adenocarcinoma cells.
Isolation and partial characterization of ascites sialoglycoprotein-2 of the cell surface sialomucin complex of 13762 rat mammary adenocarcinoma cells.
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13762 只大鼠乳腺癌细胞表面唾液酸粘蛋白复合物腹水唾液酸糖蛋白 2 的分离和部分表征。
DOI：
10.1042/bj2650121
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Carraway,KL
中科院分区：
文献类型：
--
作者：
Hull,SR;Sheng,Z;Vanderpuye,O;David,C;Carraway,KL
唾液酸粘蛋白是一些癌腹水细胞的细胞表面的主要成分，并且被假定抑制免疫系统对肿瘤的识别。13762大鼠乳腺癌的唾液粘蛋白ASGP-1（腹水唾液糖蛋白-1）与细胞表面结合，作为与伴刀豆球蛋白A结合糖蛋白ASGP-2的复合物。该唾液粘蛋白复合物已通过Triton X-100和CsCl密度梯度离心的提取从腹水细胞微绒毛中纯化。ASGP-1（已纯化）和ASGP-2在6 M盐酸胍中解离，并通过凝胶过滤分离。通过凝胶过滤和Triton X-100中的速度沉降估计，ASGP-2的未变性洗涤剂复合物的分子量为148 kDa。由于SDS/聚丙烯酰胺凝胶电泳的表观分子量约为120 kDa，ASGP-2必须是从膜中提取的单体。其化学成分的研究表明，它含有约45%的碳水化合物重量，包括甘露糖和半乳糖胺。ASGP-2的碱性硼氢化物处理转化了约10%。N-乙酰半乳糖胺的一半转化为N-乙酰半乳糖胺醇，证明存在O-连接的寡糖。甘露糖标记的链霉蛋白酶糖肽从ASGP-2的凝集素亲和层析伴刀豆球蛋白A和白细胞凝集的植物血凝素的分析表明，40%的标签是存在于高甘露糖/杂合寡糖，20%的三触角寡糖取代的C-2和C-4甘露糖的位置和40%的三或四触角寡糖取代的C-2和C-6。通过凝集素印迹和用番茄凝集素从洗涤剂提取物中沉淀，表明这些寡糖上存在聚乳糖胺序列。从化学分析和凝集素亲和性研究，我们估计ASGP-2含有4个高甘露糖和13个复杂的N-糖基化低聚糖，加上少量的聚乳糖胺和O-连接的低聚糖。该糖蛋白上存在四种不同类型的寡糖，这表明它将是一个有趣的模型系统，用于不同糖基化途径的生物合成比较。
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.