Shao Li, Luo Yi, Moloney Daniel J, Haltiwanger Robert
Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, NY 11794-5215, USA.
Glycobiology. 2002 Nov;12(11):763-70. doi: 10.1093/glycob/cwf085.
O-Glucose is an unusual form of posttranslational modification consisting of glucose directly attached to protein through O-linkage. Several serum proteins (factor VII, factor IX, protein Z, and thrombospondin) contain this unique modification on their epidermal growth factor (EGF)-like repeats. Comparison of the glycosylation sites on these proteins revealed a putative consensus sequence for O-glucose modification: C(1)XSXPC(2), where C(1) and C(2) are the first and second conserved cysteines of the EGF repeat. We identify and characterize an enzymatic activity capable of adding glucose to EGF repeats: UDP-glucose: protein O-glucosyltransferase. Using extracts of Chinese hamster ovary cells as the enzyme source, recombinant factor VII EGF repeat as the acceptor, and UDP-[(3)H]glucose as the donor, we show that the activity is linearly dependent on time, enzyme amount, and substrate concentration. As with most glycosyltransferases, metal ions (such as manganese) are required for activity. Analysis demonstrated that the glucose is added in O-linkage to the EGF repeat. Mutation of the serine to alanine in the predicted glycosylation site abrogates glycosylation, as does reduction and alkylation of the EGF repeat, suggesting that the enzyme recognizes not only the consensus sequence but also the 3D structure of the EGF repeat. Detection of O-glucosyltransferase activity in extracts of cell lines from insects to humans and a variety of rat tissues suggests that O-glucose modification is widespread in biology. These studies lay the foundation for future work on the biological role of the O-glucose modification.
O-葡萄糖是一种不寻常的翻译后修饰形式，由葡萄糖通过O-连接直接连接到蛋白质上。几种血清蛋白（因子VII、因子IX、蛋白Z和血小板反应蛋白）在其表皮生长因子（EGF）样重复序列上含有这种独特的修饰。对这些蛋白质上糖基化位点的比较揭示了O-葡萄糖修饰的一个推定共有序列：C(1)XSXPC(2)，其中C(1)和C(2)是EGF重复序列的第一个和第二个保守半胱氨酸。我们鉴定并表征了一种能够将葡萄糖添加到EGF重复序列上的酶活性：UDP-葡萄糖：蛋白质O-葡萄糖基转移酶。以中国仓鼠卵巢细胞提取物作为酶源，重组因子VII EGF重复序列作为受体，UDP-[(3)H]葡萄糖作为供体，我们表明该活性与时间、酶量和底物浓度呈线性相关。与大多数糖基转移酶一样，该活性需要金属离子（如锰）。分析表明，葡萄糖以O-连接的方式添加到EGF重复序列上。预测糖基化位点中的丝氨酸突变为丙氨酸会消除糖基化，EGF重复序列的还原和烷基化也会如此，这表明该酶不仅识别共有序列，还识别EGF重复序列的三维结构。在从昆虫到人类的细胞系提取物以及各种大鼠组织中检测到O-葡萄糖基转移酶活性，表明O-葡萄糖修饰在生物学中广泛存在。这些研究为未来关于O-葡萄糖修饰生物学作用的工作奠定了基础。
