Synthesis of O-glycan core 3: characterization of UDP-GlcNAc: GalNAc-R beta 3-N-acetyl-glucosaminyltransferase activity from colonic mucosal tissues and lack of the activity in human cancer cell lines.
UDP-GlcNAc: GalNAc-R beta 3-GlcNAc-transferase (core 3 beta 3-GlcNAc-T, where GlcNAc is N-acetyl-D-glucosamine, GalNAc is N-acetyl-D-galactosamine and T is transferase) is expressed in a tissue-specific fashion and is high in normal colonic tissue, but downregulated in colon cancer. To further study the control of this enzyme, we examined the activity in pig, rat and human colonic tissues, and several human cancer cell lines. The enzyme was difficult to solubilize by detergents and was extremely unstable in the solubilized form. Using synthetic derivatives of the GalNAc-R substrate, we showed that the specificity of the enzyme in normal rat and human colonic mucosa requires all the substituents of the GalNAc-sugar ring of substrates for maximal activity. Core 3 beta 3-GlcNAc-T was significantly influenced by the structure of the aglycon group. None of the inactive substrate derivatives could inhibit the activity. N-Iodoacetamido-galactosamine alpha-benzyl was a weak substrate and significantly inhibited the incorporation of GLcNAc into GalNAc alpha-benzyl by human colonic homogenates. Surprisingly, none of the colonic cancer cell lines or any other cancer and leukaemia cells examined exhibited detectable activity of the enzyme, although a number of other glycosyltransferase activities involved in O-glycan biosynthesis were active. Mixing experiments did not reveal an endogenous inhibitor in HL60 cells or an activator of core 3 beta 3-GlcNAc-T in human colonic mucosa. Thus, the lack of core 3 beta 3-GlcNAc-T in human colonic mucosa. Thus, the lack of core 3 beta 3-GlcNAc-T activity in cancer cell lines may be due to cell transformation or cell culturing.
Vavasseur
F
F
Department of Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada.
Yang
J M
JM
Dole
K
K
Paulsen
H
H
Brockhausen
I
I
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002460:Cell Line; D003106:Colon / Q000201:enzymology; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology*; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*
D017351:N-Acetylglucosaminyltransferases; C046236:acetylgalactosaminyl-O-glycosyl-glycoprotein beta-1,3-N-acetylglucosaminyltransferase