Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Plant Sci. 2012 Feb;183:14-9. doi: 10.1016/j.plantsci.2011.10.021. Epub 2011 Nov 4.
Dioscorin, the major tuber storage protein of yam, has been shown to possess carbonic anhydrase, trypsin inhibitor, dehydroascorbate reductase, and monodehydroascorbate reductase activities. In the present study, dioscorin from Dioscorea japonica was confirmed as a glycoprotein using the enhanced concanavalin A-peroxidase staining method, and the protein was shown to have both N- and O-glycans. Following the gene cloning, four full-length isoforms of dioscorin were expressed in Escherichia coli and purified by affinity purification and anion-exchange chromatography for structural and biochemical experiments. It was clearly observed that the recombinant dioscorins had carbonic anhydrase, trypsin inhibitor, dehydroascorbate reductase, and monodehydroascorbate reductase activities. However, the dehydroascorbate reductase and monodehydroascorbate reductase activities were markedly decreased in recombinant dioscorins compared with native dioscorin. The decreased activities were closely related to the loss of the glycosylation from the protein.
薯蓣球蛋白是山药的主要块茎贮藏蛋白，已被证明具有碳酸酐酶、胰蛋白酶抑制剂、脱氢抗坏血酸还原酶和单脱氢抗坏血酸还原酶活性。在本研究中，使用增强的伴刀豆球蛋白 A-过氧化物酶染色法证实来自薯蓣的薯蓣球蛋白是一种糖蛋白，并且该蛋白具有 N-和 O-聚糖。在基因克隆之后，四个全长薯蓣球蛋白同工型在大肠杆菌中表达，并通过亲和纯化和阴离子交换层析进行纯化，用于结构和生化实验。清楚地观察到重组薯蓣球蛋白具有碳酸酐酶、胰蛋白酶抑制剂、脱氢抗坏血酸还原酶和单脱氢抗坏血酸还原酶活性。然而，与天然薯蓣球蛋白相比，重组薯蓣球蛋白的脱氢抗坏血酸还原酶和单脱氢抗坏血酸还原酶活性显著降低。活性降低与蛋白质中糖基化的丧失密切相关。
