Prieto P A, Larsen R D, Cho M, Rivera H N, Shilatifard A, Lowe J B, Cummings R D, Smith D F
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
J Biol Chem. 1997 Jan 24;272(4):2089-97. doi: 10.1074/jbc.272.4.2089.
The human H(O) blood group is specified by the structure Fucalpha1-2Galbeta1-R, but the factors regulating expression of this determinant on cell surface glycoconjugates are not well understood. To learn more about the regulation of H blood group expression, cDNA encoding the human H-type GDPFuc:beta-D-galactoside alpha1, 2-fucosyltransferase (alpha1,2FT) was stably transfected into Chinese hamster ovary (CHO) cells. The new cell line, designated CHO(alpha1,2)FT, expressed surface neoglycans containing the H antigen. The structures of the fucosylated neoglycans in CHO(alpha1, 2)FT cells and the distribution of these glycans on glycoproteins were characterized. Seventeen percent of the [3H]Gal-labeled glycopeptides from CHO(alpha1,2)FT cells bound to the immobilized H blood group-specific lectin Ulex europaeus agglutinin-I (UEA-I), whereas none from parental CHO cells bound to the lectin. The glycopeptides from CHO(alpha1,2)FT cells binding to UEA-I contained polylactosamine [3Galbeta1-4GlcNAcbeta1-]n with the terminal sequence Fucalpha1-2Galbeta1- 4GlcNAc-R. Fucosylation of the polylactosamine sequences on complex-type N-glycans in CHO(alpha1, 2)FT cells caused a decrease in both sialylation and length of polylactosamine. Unexpectedly, only small amounts of terminal fucosylation was found in diantennary complex-type N-glycans. The O-glycans and glycolipids were not fucosylated by the H-type alpha1, 2FT. Two major high molecular weight glycoproteins, one of which was shown to be the lysosome-associated membrane glycoprotein LAMP-1, preferentially contained the H-type structure and were bound by immobilized UEA-I. These results demonstrate that in CHO cells the expressed H-type alpha1,2FT does not indiscriminately fucosylate terminal galactosyl residues in complex-type N-glycans, but it favors glycans containing polylactosamine and dramatically alters their length and sialylation.
人类H(O)血型由结构Fucα1-2Galβ1-R决定，但调节这种决定簇在细胞表面糖缀合物上表达的因素尚不清楚。为了更多地了解H血型表达的调控，编码人类H型GDPFuc:β-D-半乳糖α1,2-岩藻糖基转移酶（α1,2FT）的cDNA被稳定转染到中国仓鼠卵巢（CHO）细胞中。新的细胞系，命名为CHO(α1,2)FT，表达含有H抗原的表面新糖链。对CHO(α1,2)FT细胞中岩藻糖基化新糖链的结构及其在糖蛋白上的分布进行了表征。来自CHO(α1,2)FT细胞的17%的[3H]Gal标记糖肽与固定化的H血型特异性凝集素欧洲荆豆凝集素-I（UEA-I）结合，而亲本CHO细胞的糖肽均不与该凝集素结合。来自CHO(α1,2)FT细胞且与UEA-I结合的糖肽含有多乳糖胺[3Galβ1-4GlcNAcβ1-]n，其末端序列为Fucα1-2Galβ1-4GlcNAc-R。CHO(α1,2)FT细胞中复合型N-聚糖上多乳糖胺序列的岩藻糖基化导致唾液酸化和多乳糖胺长度均减少。出乎意料的是，在二天线复合型N-聚糖中仅发现少量的末端岩藻糖基化。O-聚糖和糖脂未被H型α1,2FT岩藻糖基化。两种主要的高分子量糖蛋白，其中一种被证明是溶酶体相关膜糖蛋白LAMP-1，优先含有H型结构并被固定化的UEA-I结合。这些结果表明，在CHO细胞中，表达的H型α1,2FT不会无差别地将复合型N-聚糖中的末端半乳糖基残基岩藻糖基化，而是倾向于含有多乳糖胺的聚糖，并显著改变其长度和唾液酸化程度。
