Enhanced laminin binding by α-dystroglycan after enzymatic deglycosylation
Enhanced laminin binding by α-dystroglycan after enzymatic deglycosylation
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DOI：
10.1042/bj20050375
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发表时间：
2005-08-15
影响因子：
4.1
通讯作者：
Ervasti, JM
中科院分区：
文献类型：
--
作者：
Combs, AC;Ervasti, JM
碳水化合物的修饰显然对α-营养不良多糖的功能很重要，但其组成和结构仍然知之甚少。在本研究中，我们描述了旨在鉴定对其与层粘连蛋白-1和碳水化合物依赖的单抗(单抗)IIH6和VIA4(T)结合至关重要的α-营养不良寡糖的实验。我们用一系列连接特异的内切和外切糖苷酶来消化高度纯化的骨骼肌α-肌营养不良聚糖，通过凝集素与特定糖表位的反应性的丧失/获得来验证它们对α-肌营养不良聚糖的作用。值得注意的是，节杆菌唾液酸酶、β(1-4)半乳糖苷酶和β-N-乙酰氨基葡萄糖苷酶的组合消化大大降解了高纯度α-肌营养不良聚糖上的SiaAα2-3Galβ1-4GlcNAcβ1-2man多糖，但仍显示出增强的IIH6、VIA4(1)和层粘连蛋白-1结合活性。另外的结果表明，α-营养不良聚糖可能被唾液酸以外的其他阴离子糖修饰，这表明罕见的α-连接的GlcNAc部分可能阻止其与现有酶的完全脱糖作用。
Carbohydrate modifications are clearly important to the function of a-dystroglycan but their composition and structure remain poorly understood. In the present study, we describe experiments aimed at identifying the alpha-dystroglycan oligosaccharides important for its binding to laminin-1 and carbohydrate-dependent mAbs (monoclonal antibodies) IIH6 and VIA4(t). We digested highly purified skeletal muscle alpha-dystroglycan with an array of linkage-specific endo- and exoglycosidases, which were verified for action on alpha-dystroglycan by loss/gain of reactivity for lectins with defined glyco-epitopes. Notably, digestion with a combination of Arthrobacter ureafaciens sialidase, beta(1-4)galactosidase and beta-N-acetylglucosaminidase substantially degraded SiaA alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man glycans on highly purified alpha-dystroglycan that nonetheless exhibited enhanced IIH6, VIA4(1) and laminin-1 binding activity. Additional results indicate that alpha-dystroglycan is probably modified with other anionic sugars besides sialic acid and suggest that rare alpha-linked GlcNAc moieties may block its complete deglycosylation with currently available enzymes.