Chemo-enzymatic synthesis of eel calcitonin glycosylated at two sites with the same and different carbohydrate structures
Chemo-enzymatic synthesis of eel calcitonin glycosylated at two sites with the same and different carbohydrate structures
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DOI：
10.1016/j.carres.2005.11.015
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发表时间：
2006-02-06
影响因子：
3.1
通讯作者：
Yamamoto, K
中科院分区：
文献类型：
--
作者：
Haneda, K;Takeuchi, M;Yamamoto, K
天然存在的糖肽和糖蛋白通常含有一个以上的糖基化位点，并且所连接的碳水化合物的结构通常因位点而异。因此，需要用于制备在多个位点糖基化的肽和蛋白质（可能具有不同的碳水化合物结构）的合成方法。在这里，我们报告了一种化学酶的方法来实现这一点。通过用冬季毛霉内切-β-N-乙酰氨基葡萄糖苷酶处理，将复合型寡糖引入到在不同位点含有两个N-乙酰-D-氨基葡萄糖（GlcNAc）残基的降钙素衍生物中。使用这种酶促转糖基化反应，产生三种糖肽，一种降钙素衍生物在两个位点具有相同的复合型碳水化合物，两种降钙素衍生物各具有一种复合型碳水化合物和一种GlcNAc。从具有一种复合型碳水化合物和一种GlcNAc的衍生物开始，使用来自原激素节杆菌的另一种内切-β-N-乙酰氨基葡糖苷酶将高甘露糖型寡糖成功地转移到剩余的GlcNAc。因此，我们能够获得在单个分子中不仅含有两种复合型碳水化合物，而且含有复合型和高甘露糖型寡糖的糖肽。利用所得到的糖基化降钙素衍生物，研究了二-N-糖基化对降钙素结构和活性的影响。从单-N-糖基化降钙素衍生物的结果可以预测该效应。(c)2005爱思唯尔有限公司保留所有权利。
Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-D-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-beta-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives. (c) 2005 Elsevier Ltd. All rights reserved.