beta 1,3-Galactosyltransferase beta 3Gal-T5 acts on the GlcNAcbeta 1-->3Galbeta 1-->4GlcNAcbeta 1-->R sugar chains of carcinoembryonic antigen and other N-linked glycoproteins and is down-regulated in colon adenocarcinomas.
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.
Salvini
R
R
Department of Biochemistry, University of Pavia, via Taramelli 3B, 27100 Pavia, Italy.
Bardoni
A
A
Valli
M
M
Trinchera
M
M
D000230:Adenocarcinoma / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000368:Aged; D000818:Animals; D016466:CHO Cells; D002272:Carcinoembryonic Antigen / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D015536:Down-Regulation; D005260:Female; D005647:Fucosyltransferases / Q000096:biosynthesis; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D005753:Gastric Mucosa / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007983:Lewis Blood Group Antigens / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000080506:Sialyl Lewis X Antigen; D014162:Transfection
D002272:Carcinoembryonic Antigen; D006023:Glycoproteins; D007983:Lewis Blood Group Antigens; D009844:Oligosaccharides; D011134:Polysaccharides; D000080506:Sialyl Lewis X Antigen; D005647:Fucosyltransferases; D005700:Galactosyltransferases; C120745:UDP-galactose N-acetylglucosaminyl-1-3-N-acetylgalactosamine beta-1,3-galactosyltransferase; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase