β1,3-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 ␤1,3-galactosyltransferase ␤3Gal-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 ␤3Gal-T cDNAs and studied the relevant glycoconjugates formed. ␤3Gal-T5 directs synthesis of Lewis type 1 antigens in CHO cells more efficiently than ␤3Gal-T1, whereas ␤3Gal-T2, -T3, and -T4 are almost unable to direct synthesis. In the clone expressing Fuc-TIII and ␤3Gal-T5 (CHO-FT-T5), sialyl-Lewis a synthesis is strongly inhibited by swainsonine but not by benzyl-␣-GalNAc, and sialyl-Lewis x is absent, although it is detected in the clones expressing Fuc-TIII and ␤3Gal-T1 (CHO-FT-T1) or Fuc-TIII and ␤3Gal-T2 (CHO-FT-T2). Endo-␤-galactosidase treatment of Nglycans prepared from clone CHO-FT-T5 releases (؎NeuAc␣233)Gal␤133[Fuc␣134]GlcNAc␤133Gal but not GlcNAc␤133Gal or type 2 chain oligosaccharides, which are found in CHO-FT-T1 cells. This result indicates that ␤3Gal-T5 expression prevents poly-Nacetyllactosamine and sialyl-Lewis x synthesis on Nglycans. Kinetic studies confirm that ␤3Gal-T5 prefers acceptors having the GlcNAc␤133Gal end, including lactotriosylceramide. Competitive reverse transcriptase mediated-polymerase chain reaction shows that the ␤3Gal-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 ␤3Gal-T5 reacts with anti-sialyl-Lewis a and carries type 1 chains on oligosaccharides released by endo-␤-galactosidase. We conclude that ␤3Gal-T5 down-regulation plays a relevant role in determining the cancer-associated glycosylation pattern of N-glycans.Type 1 chain oligosaccharides found in N-and O-glycans, as well as in glycolipids, contain the distinctive Gal␤133GlcNAc disaccharide as their core structure. It is synthesized by ␤1,3-galactosyltransferases (

Comparative Analysis of Retroviral and Native Promoters Driving Expression of β1,3-Galactosyltransferase β3Gal-T5 in Human and Mouse Tissues

“…Human bile duct carcinoma cells HuCC-T1 (Japanese Collection of Research Bioresources 0425) were cultured in RPMI 1640 medium containing the same supplements as above. Human colon adenocarcinoma cells Caco-2 and SW-1116 and human gastric carcinoma cells MKN-45 were cultured as previously described (3,16). Human ileum and colon samples were collected at surgery and kindly donated by Dr. Filippo Mare (Hospital di Circolo, Varese, Italy).…”

“…Mouse total RNA was purchased from Clontech. Total and poly(A) ϩ RNAs were prepared from human tissues or cell lines as previously reported (3,16).…”

“…␤3Gal-T5 is highly expressed in human colon mucosa, down-regulated in colon cancers (3), and large amounts of its reaction products are found in the bile and small intestine (4,5). In some colon cancer cells, the first exon (exon 1) in the 5Ј-UTR of the ␤3Gal-T5 transcript is under the control of a promoter located ϳ150 bp upstream and regulated primarily by the transcription factors HNF-1 and Cdx (6).…”

“…␤1,3-Galactosyltransferase 5 (␤3Gal-T5) 3 is a late-acting glycosyltransferase responsible for the synthesis of type 1 chain carbohydrates, including Lewis antigens, in human epithelial cells (1,2). ␤3Gal-T5 is highly expressed in human colon mucosa, down-regulated in colon cancers (3), and large amounts of its reaction products are found in the bile and small intestine (4,5).…”

“…Section 1734 solely to indicate this fact. 3 The abbreviations used are: ␤3Gal-T5, ␤1,3-galactosyltransferase 5; CBF, CCAAT-binding factor; HNF-1, hepatocyte nuclear factor 1; LTR, long terminal repeat; RT, reverse transcription; RACE, rapid amplification of cDNA ends; EMSA, electrophoretic mobility shift assay. …”