Bierhuizen M F, Fukuda M
La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9326-330. doi: 10.1073/pnas.89.19.9326.
A cDNA encoding UDP-GlcNAc:Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase (EC 2.4.1.102), which forms critical branches in O-glycans, has been isolated by an expression cloning approach using Chinese hamster ovary (CHO) cells. Increased activity of this enzyme and the concomitant occurrence of the O-glycan core 2 structure [Gal beta 1-3(GlcNAc beta 1-6)GalNAc] has been observed in a variety of biological processes, such as T-cell activation and immunodeficiency due to the Wiskott-Aldrich syndrome and AIDS. Since CHO cells do not express this enzyme, CHO cell lines were established to stably express polyoma large tumor (T) antigen, which enables transient expression cloning. Because the antibody used was found to detect most efficiently the oligosaccharide products attached to leukosialin, the CHO cells were also stably transfected with leukosialin cDNA. By using this particular CHO cell line, a cDNA that encodes a protein determining the formation of the core 2 structure was isolated from an HL-60 cDNA library. The cDNA sequence predicts a protein with type II membrane topology, as has been found for all other mammalian glycosyltransferases cloned to date. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate unequivocally that the cDNA encodes the core 2 beta-1,6-N-acetylglucosaminyltransferase, the enzyme responsible for the formation of Gal beta 1-3(GlcNAc beta 1-6)GalNAc structures. No activity with this enzyme was detected toward the acceptors for other beta 1-6GlcNAc transferases.
编码UDP - N - 乙酰葡糖胺：β1,3 - 半乳糖基 - N - 乙酰半乳糖胺 - R（从GlcNAc到GalNAc）β1,6 - N - 乙酰葡糖胺转移酶（EC 2.4.1.102）的cDNA已通过使用中国仓鼠卵巢（CHO）细胞的表达克隆方法分离出来，该酶在O - 聚糖中形成关键分支。在多种生物学过程中，如T细胞活化以及因威斯科特 - 奥尔德里奇综合征和艾滋病导致的免疫缺陷，都观察到了这种酶活性的增加以及O - 聚糖核心2结构[β1,3 - 半乳糖（β1,6 - N - 乙酰葡糖胺） - N - 乙酰半乳糖胺]的同时出现。由于CHO细胞不表达这种酶，因此建立了稳定表达多瘤大肿瘤（T）抗原的CHO细胞系，这使得能够进行瞬时表达克隆。因为发现所使用的抗体能最有效地检测附着在白细胞唾液酸蛋白上的寡糖产物，所以CHO细胞也被稳定转染了白细胞唾液酸蛋白cDNA。通过使用这种特定的CHO细胞系，从HL - 60 cDNA文库中分离出了一个编码决定核心2结构形成的蛋白质的cDNA。该cDNA序列预测的蛋白质具有II型膜拓扑结构，这与迄今为止克隆的所有其他哺乳动物糖基转移酶的情况相同。将推测的催化结构域作为与蛋白A的IgG结合结构域的融合蛋白进行表达，使我们能够明确证明该cDNA编码核心2 β1,6 - N - 乙酰葡糖胺转移酶，即负责形成β1,3 - 半乳糖（β1,6 - N - 乙酰葡糖胺） - N - 乙酰半乳糖胺结构的酶。未检测到该酶对其他β1,6 - N - 乙酰葡糖胺转移酶的受体有活性。
