β,3-Galactosyltransferase β3Gal-T5 Acts on the GlcNAcβ1→3Galβ1→4GlcNAcβ1→R Sugar Chains of Carcinoembryonic Antigen and Other N-Linked Glycoproteins and Is Down-regulated in Colon Adenocarcinomas

Abstract

We attempted to determine whether β1,3-galactosyltransferase β3Gal-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 β3Gal-T cDNAs and studied the relevant glycoconjugates formed. β3Gal-T5 directs synthesis of Lewis type 1 antigens in CHO cells more efficiently than β3Gal-T1, whereas β3Gal-T2, -T3, and -T4 are almost unable to direct synthesis. In the clone expressing Fuc-TIII and β3Gal-T5 (CHO-FT-T5), sialyl-Lewis a synthesis is strongly inhibited by swainsonine but not by benzyl-αGalNAc, and sialyl-Lewis x is absent, although it is detected in the clones expressing Fuc- TIII and β3Gal-T1 (CHO-FT-T1) or Fuc-TIII and β3Gal-T2 (CHO-FT-T2). Endo-β-galactosidase treatment of N-glycans prepared from clone CHO-FT-T5 releases (±NeuAcα2→3)Galβ 1→3[Fucα1→4]GlcNAcβ1→3 Gal but not GlcNAcβ1→3Gal or type 2 chain oligosaccharides, which are found in CHO-FT-T1 cells. This result indicates that β3Gal-T5 expression prevents poly-N-acetyllactosamine and sialyl-Lewis x synthesis on N-glycans. Kinetic studies confirm that β3Gal-T5 prefers acceptors having the GlcNAcβ1→3Gal end, including lactotriosylceramide. Competitive reverse transcriptase mediated-polymerase chain reaction shows that the β3Gal-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 β3Gal-T5 reacts with anti-sialyl-Lewis a and carries type 1 chains on oligosaccharides released by endo-β-galactosidase. We conclude that β3Gal-T5 down-regulation plays a relevant role in determining the cancer-associated glycosylation pattern of N-glycans.