Enzymatic Glycoprotein Synthesis: Preparation of Ribonuclease Glycoforms via Enzymatic Glycopeptide Condensation and Glycosylation
Enzymatic Glycoprotein Synthesis: Preparation of Ribonuclease Glycoforms via Enzymatic Glycopeptide Condensation and Glycosylation
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酶促糖蛋白合成：通过酶促糖肽缩合和糖基化制备核糖核酸酶糖型
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发表时间：
1997
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通讯作者：
Chi‐Huey Wong
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作者：
K. Witte;P. Sears;Richard L. Martin;Chi‐Huey Wong
为了研究碳水化合物对糖蛋白结构和功能的影响，必须能够以单一形式合成适当的天然和非天然糖蛋白变体。由于可用的体内技术仅提供不同糖型的异质混合物，因此一直在追求体外酶促方法。使用N-糖蛋白RNase B作为模型系统，除去寡糖，仅留下N-乙酰葡糖胺作为糖基化位点的“标签”。然后使用糖基转移酶来构建独特的碳水化合物部分。为了证明该方法的可行性，酶促合成了一种新的含有支链寡糖、唾液酸化刘易斯X或汞衍生物的核糖核酸酶糖型。此外，单糖基化蛋白质被枯草杆菌蛋白酶BPN '消化成几个较小的片段。通过添加甘油，这些片段被枯草杆菌蛋白酶8397重新连接到全长RNase;这种方法指出了一种新的化学方法。
In order to study the effects carbohydrates have on glycoprotein structure and function, it is imperative to be able to synthesize the appropriate natural and non-natural glycoprotein variants in a single form. Because the available in vivo techniques provide only heterogeneous mixtures of different glycoforms, enzymatic in vitro methodologies have been pursued. Using the N-glycoprotein RNase B as a model system, the oligosaccharide was removed leaving only the N-acetylglucosamine as a “tag” to the site of glycosylation. Glycosyltransferases were then used to build a unique carbohydrate moiety. A new RNase glycoform containing the branched oligosaccharide, sialyl Lewis X or the Hg derivative, was synthesized enzymatically to demonstrate the feasibility of the method. In addition, the monoglycosylated protein was digested into several smaller pieces by subtilisin BPN‘. These fragments were religated by subtilisin 8397 to the full length RNase by addition of glycerol; this method points to a new chemical-en...