Davis B G, Lloyd R C, Jones J B
Department of Chemistry, University of Durham, UK.
Bioorg Med Chem. 2000 Jul;8(7):1527-35. doi: 10.1016/s0968-0896(00)00083-3.
Glycoproteins occur naturally as complex mixtures of differently glycosylated forms which are difficult to separate. To explore their individual properties, there is a need for homogeneous sources of carbohydrate-protein conjugates and this has recently prompted us to develop a novel method for the site-selective glycosylation of proteins. The potential of the method was illustrated by site-selective glycosylations of subtilisin Bacillus lentus (SBL) as a model protein. A representative library of mono- and disaccharide MTS reagents were synthesized from their parent carbohydrates and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site. These were the first examples of preparations of homogeneous neoglycoproteins in which both the site of glycosylation and structure of the introduced glycan were predetermined. The scope of this versatile method was expanded further through the combined use of peracetylated MTS reagents and careful pH adjustment to introduce glycans containing different numbers of acetate groups. This method provides a highly controlled and versatile route that is virtually unlimited in the scope of the sites and glycans that may be conjugated, and opens up hitherto inaccessible opportunities for the systematic determination of the properties of glycosylated proteins. This potential has been clearly demonstrated by the determination of detailed glycan structure-hydrolytic activity relationships for SBL. The 48 glycosylated CMMs formed display kcat/KM values that range from 1.1-fold higher than WT to 7-fold lower than WT. The anomeric stereochemistry of the glycans introduced modulates changes in kcat/KM upon acetylation. At positions 62 and 217 acetylation enhances the activity of alpha-glycosylated CMMs but decreases that of beta-glycosylated. This trend is reversed at position 166 where, in contrast, acetylation enhances the kcat/KMs of beta-glycosylated CMMs but decreases those of alpha-glycosylated. Consistent with its surface exposed nature changes at position 156 are more modest, but still allow control of activity, particularly through glycosylation with disaccharide lactose.
糖蛋白天然以不同糖基化形式的复杂混合物存在，难以分离。为了探究它们的个体性质，需要有均一的碳水化合物 - 蛋白质缀合物来源，这促使我们最近开发了一种蛋白质位点选择性糖基化的新方法。该方法的潜力通过将嗜碱芽孢杆菌枯草杆菌蛋白酶（SBL）作为模型蛋白进行位点选择性糖基化得以体现。从其母体碳水化合物合成了代表性的单糖和二糖MTS试剂库，并用于修饰SBL的半胱氨酸突变体，这些突变体位于S2位点的62位、S1位点的156和166位以及S1'位点的217位。这些是制备均一新糖蛋白的首批实例，其中糖基化位点和引入聚糖的结构均预先确定。通过联合使用全乙酰化MTS试剂并仔细调节pH以引入含有不同数量乙酰基的聚糖，进一步扩展了这种通用方法的范围。该方法提供了一条高度可控且通用的途径，在可缀合的位点和聚糖范围方面几乎没有限制，并为系统测定糖基化蛋白质的性质开辟了迄今难以获得的机会。通过测定SBL详细的聚糖结构 - 水解活性关系，已清楚地证明了这种潜力。形成的48种糖基化CMM的kcat/KM值范围从比野生型高1.1倍到比野生型低7倍。引入聚糖的异头立体化学调节乙酰化后kcat/KM的变化。在62和217位，乙酰化增强了α - 糖基化CMM的活性，但降低了β - 糖基化CMM的活性。在166位则相反，乙酰化增强了β - 糖基化CMM的kcat/KM，但降低了α - 糖基化CMM的kcat/KM。与其表面暴露性质一致，156位的变化较为适度，但仍允许对活性进行控制，特别是通过用二糖乳糖进行糖基化。
