Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin.
Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin.
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细胞外基质成分层粘连蛋白的主要伴刀豆球蛋白 A 反应性寡糖的结构。
DOI：
10.1021/bi00441a034
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Goldstein,IJ
中科院分区：
文献类型：
--
作者：
Knibbs,RN;Perini,F;Goldstein,IJ
修订稿于 1989 年 4 月 19 日收到 摘要：层粘连蛋白是基底膜的一种高分子量 (1000000) 糖蛋白成分，通过凝集素亲和层析，使用 aD-半乳糖基结合凝集素 Griffonia simplicifolia I (GS I)，从 EHS 小鼠肿瘤中分离出与巢蛋白的非共价复合物。在 SDS 存在下，通过 Sephacryl S-1000 从该复合物中除去 Entactin。成分分析表明，亲和纯化的层粘连蛋白含有按重量计25-30％的碳水化合物。甲基化分析表明层粘连蛋白的低聚糖含有双触角链和三触角链、I型血型结构以及3Gal/31、4GlcNAc/31单位的重复序列。通过肼解、再 N-乙酰化和用 NaB3H4 还原，从亲和纯化的层粘连蛋白的天冬酰胺连接的聚糖中衍生出游离寡糖。当在伴刀豆球蛋白 A (Con A)-Sepharose 上通过亲和色谱进行分级时，80% 的寡糖未延迟地通过柱，对应于 20% 寡糖的单峰被吸附，并用 0-30 mM 甲基 aD-吡喃葡萄糖苷的线性梯度特异性洗脱。在 GS I-Sepharose 上对 Con A 反应性寡糖的进一步分级表明，70% 的这些寡糖具有至少一个末端非还原性 αD-吡喃半乳糖基单元。 Con A 反应性寡糖用内切糖苷酶和外切糖苷酶进行连续消化，并通过 Bio-Gel P4 柱上的凝胶过滤色谱法分析反应产物。由此，我们获得了先前未报道的鼠层粘连蛋白上存在的多种结构的证据，包括混合双触角复合物和含有聚（乳糖胺基）重复单元的双触角复合物结构。聚（乳糖胺基）单元出现在双触角链的一个或两个分支上，以及更高支化的血型 I 聚（乳糖胺）结构中。所有唾液酸均以 a2、3 与半乳糖连接的 A-乙酰神经氨酸的形式存在。
Revised Manuscript Received April 19, 1989 abstract: Laminin, a high molecular weight (1000000) glycoprotein component of basement membranes, was isolated from the EHS murine tumor as a noncovalent complex with entactin by lectin affinity chro-matography using the aD-galactosyl binding lectin Griffonia simplicifolia I (GS I). Entactin was removed from this complex by passage over Sephacryl S-1000 in the presence of SDS. Compositional analysis showed that the affinity-purified laminin contained 25-30% carbohydrateby weight. Methylation analysis revealed that theoligosaccharides of laminin contained bi-and triantennary chains, the blood group I structure, and repeating sequences of 3Gal/31, 4GlcNAc/31 units. Free oligosaccharides were derived from the aspara-gine-linked glycans of affinity-purified laminin by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. When fractionated by affinity chromatography on concanavalin A (Con A)-Sepharose, 80% of the oligosaccharides passed through the column unretardedand a single peak corresponding to 20% of the oligosaccharides was adsorbed and specifically eluted with a linear gradient of 0-30 mM methyl aD-glucopyranoside. Further fractionation of the Con A reactive oligosaccharides on GS I-Sepharose demonstrated that 70% of these oligosaccharides possess at least one terminal nonreducing aD-galactopyranosyl unit. TheCon A reactive oligosaccharides were subjected to sequential digestion with endo-and exo-glycosidases, and the reaction products were analyzed by gel filtration chromatography on a column of Bio-Gel P4. We thereby obtained evidence for a variety of structures not previously reported to exist on murine laminin including hybrid biantennary complexand biantennary complex structures containingpoly (lactosaminyl) repeating units. The poly (lactosaminyl) units occur either on one or on both branches of the biantennary chains, as well as in more highly branched blood group I poly (lactosamine) structures. All sialic acid is present as A-acetylneuraminic acid linked a2, 3 to galactose.