Salvini R, Bardoni A, Valli M, Trinchera M
Department of Biochemistry, University of Pavia, via Taramelli 3B, 27100 Pavia, Italy.
J Biol Chem. 2001 Feb 2;276(5):3564-73. doi: 10.1074/jbc.M006662200. Epub 2000 Oct 31.
We attempted to determine whether beta1,3-galactosyltransferase beta3Gal-T5 is involved in the biosynthesis of a specific subset of type 1 chain carbohydrates and expressed in a cancer-associated manner. We transfected Chinese hamster ovary (CHO) cells expressing Fuc-TIII with beta3Gal-T cDNAs and studied the relevant glycoconjugates formed. beta3Gal-T5 directs synthesis of Lewis type 1 antigens in CHO cells more efficiently than beta3Gal-T1, whereas beta3Gal-T2, -T3, and -T4 are almost unable to direct synthesis. In the clone expressing Fuc-TIII and beta3Gal-T5 (CHO-FT-T5), sialyl-Lewis a synthesis is strongly inhibited by swainsonine but not by benzyl-alpha-GalNAc, and sialyl-Lewis x is absent, although it is detected in the clones expressing Fuc-TIII and beta3Gal-T1 (CHO-FT-T1) or Fuc-TIII and beta3Gal-T2 (CHO-FT-T2). Endo-beta-galactosidase treatment of N- glycans prepared from clone CHO-FT-T5 releases (+/-NeuAcalpha2-->3)Galbeta1-->3[Fucalpha1-->4]GlcNAcbeta1-->3Gal but not GlcNAcbeta1-->3Gal or type 2 chain oligosaccharides, which are found in CHO-FT-T1 cells. This result indicates that beta3Gal-T5 expression prevents poly-N-acetyllactosamine and sialyl-Lewis x synthesis on N-glycans. Kinetic studies confirm that beta3Gal-T5 prefers acceptors having the GlcNAcbeta1-->3Gal end, including lactotriosylceramide. Competitive reverse transcriptase mediated-polymerase chain reaction shows that the beta3Gal-T5 transcript is expressed in normal colon mucosa but not or poorly in adenocarcinomas. Moreover, recombinant carcinoembryonic antigen purified from a CHO clone expressing Fuc-TIII and beta3Gal-T5 reacts with anti-sialyl-Lewis a and carries type 1 chains on oligosaccharides released by endo-beta-galactosidase. We conclude that beta3Gal-T5 down-regulation plays a relevant role in determining the cancer-associated glycosylation pattern of N-glycans.
我们试图确定β1,3-半乳糖基转移酶β3Gal-T5是否参与1型链碳水化合物特定亚群的生物合成，并以与癌症相关的方式表达。我们用β3Gal-T cDNA转染表达Fuc-TIII的中国仓鼠卵巢（CHO）细胞，并研究形成的相关糖缀合物。β3Gal-T5在CHO细胞中指导Lewis 1型抗原的合成比β3Gal-T1更有效，而β3Gal-T2、-T3和-T4几乎无法指导合成。在表达Fuc-TIII和β3Gal-T5的克隆（CHO-FT-T5）中，唾液酸化Lewis a的合成受到swainsonine的强烈抑制，但不受苄基-α-GalNAc的抑制，且未检测到唾液酸化Lewis x，尽管在表达Fuc-TIII和β3Gal-T1（CHO-FT-T1）或Fuc-TIII和β3Gal-T2（CHO-FT-T2）的克隆中可检测到。用内切β-半乳糖苷酶处理从克隆CHO-FT-T5制备的N-聚糖，可释放出（±NeuAcalpha2→3）Galbeta1→3[Fucalpha1→4]GlcNAcbeta1→3Gal，但不能释放GlcNAcbeta1→3Gal或2型链寡糖，后者存在于CHO-FT-T1细胞中。这一结果表明，β3Gal-T5的表达可阻止N-聚糖上多聚N-乙酰乳糖胺和唾液酸化Lewis x的合成。动力学研究证实，β3Gal-T5更喜欢具有GlcNAcbeta1→3Gal末端的受体，包括乳糖三糖神经酰胺。竞争性逆转录酶介导的聚合酶链反应表明β3Gal-T5转录本在正常结肠黏膜中表达，但在腺癌中不表达或表达较弱。此外，从表达Fuc-TIII和β3Gal-T5的CHO克隆中纯化的重组癌胚抗原与抗唾液酸化Lewis a反应，并在经内切β-半乳糖苷酶释放的寡糖上带有1型链。我们得出结论，β3Gal-T5的下调在决定N-聚糖的癌症相关糖基化模式中起相关作用。
