Miyazaki Takatsugu, Kato Tatsuya, Park Enoch Y
Laboratory of Biotechnology, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan; Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
Laboratory of Biotechnology, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan; Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
J Biosci Bioeng. 2018 Jul;126(1):15-22. doi: 10.1016/j.jbiosc.2018.01.011. Epub 2018 Mar 1.
β-1,2-N-Acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi-localized type II transmembrane enzyme that catalyzes the transfer of N-acetylglucosamine to the 6-arm of the trimanosyl core of N-glycans, an essential step in the conversion of oligomannose-type to complex-type N-glycans. Despite its physiological importance, there have been only a few reports on the heterologous expression and structure-function relationship of this enzyme. Here, we constructed a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system and expressed human GnTII (hGnTII) lacking the N-terminal cytosolic tail and transmembrane region. The recombinant hGnTII was purified from silkworm larval hemolymph in two steps by using tandem affinity purification tags, with a yield of approximately 120 μg from 10 mL hemolymph, and exhibited glycosyltransferase activity and strict substrate specificity. The enzyme was found to be N-glycosylated by the enzymatic cleavage of glycans, while hGnTII expressed in insect cells had not been reported to be glycosylated. Although insects typically produce pauci-mannosidic-type glycans, the structure of N-glycans in the recombinant hGnTII was suggested to be of the complex type, and the removal of the glycans did not affect the enzymatic activity.
β-1,2-N-乙酰葡糖胺基转移酶II（GnTII，EC 2.4.1.143）是一种定位于高尔基体的II型跨膜酶，它催化N-乙酰葡糖胺转移至N-聚糖三甘露糖核心的6臂上，这是寡甘露糖型N-聚糖转变为复合型N-聚糖的关键步骤。尽管其具有重要的生理学意义，但关于该酶的异源表达及结构-功能关系的报道却很少。在此，我们构建了基于家蚕的家蚕核型多角体病毒杆粒表达系统，并表达了缺失N端胞质尾和跨膜区的人GnTII（hGnTII）。通过使用串联亲和纯化标签，从家蚕幼虫血淋巴中分两步纯化重组hGnTII，从10 mL血淋巴中获得的产量约为120 μg，并且该重组酶表现出糖基转移酶活性和严格的底物特异性。通过聚糖的酶切发现该酶被N-糖基化，而此前尚未有报道称在昆虫细胞中表达的hGnTII会发生糖基化。尽管昆虫通常产生寡甘露糖型聚糖，但重组hGnTII中N-聚糖的结构被认为是复合型的，并且聚糖的去除并不影响酶活性。
