Expression cloning of a cDNA encoding UDP-GlcNAc:Gal beta 1-3-GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen.
A cDNA encoding UDP-GlcNAc:Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase (EC 2.4.1.102), which forms critical branches in O-glycans, has been isolated by an expression cloning approach using Chinese hamster ovary (CHO) cells. Increased activity of this enzyme and the concomitant occurrence of the O-glycan core 2 structure [Gal beta 1-3(GlcNAc beta 1-6)GalNAc] has been observed in a variety of biological processes, such as T-cell activation and immunodeficiency due to the Wiskott-Aldrich syndrome and AIDS. Since CHO cells do not express this enzyme, CHO cell lines were established to stably express polyoma large tumor (T) antigen, which enables transient expression cloning. Because the antibody used was found to detect most efficiently the oligosaccharide products attached to leukosialin, the CHO cells were also stably transfected with leukosialin cDNA. By using this particular CHO cell line, a cDNA that encodes a protein determining the formation of the core 2 structure was isolated from an HL-60 cDNA library. The cDNA sequence predicts a protein with type II membrane topology, as has been found for all other mammalian glycosyltransferases cloned to date. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate unequivocally that the cDNA encodes the core 2 beta-1,6-N-acetylglucosaminyltransferase, the enzyme responsible for the formation of Gal beta 1-3(GlcNAc beta 1-6)GalNAc structures. No activity with this enzyme was detected toward the acceptors for other beta 1-6GlcNAc transferases.
Bierhuizen
M F
MF
La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, CA 92037.
Fukuda
M
M
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D051917:Leukosialin; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000378:metabolism*; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000302:isolation & purification; D011120:Polyomavirus / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012795:Sialoglycoproteins / Q000235:genetics*; D013379:Substrate Specificity; D014162:Transfection
D015703:Antigens, CD; D000952:Antigens, Polyomavirus Transforming; D051917:Leukosialin; D009844:Oligosaccharides; D012333:RNA, Messenger; C465014:SPN protein, human; D012795:Sialoglycoproteins; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase