Substrate specificity and distribution of UDP-GalNAc:sialylparagloboside N-acetylgalactosaminyltransferase in the human stomach.
The detailed substrate specificity of the UDP-GalNAc:sialylparagloboside N-acetylgalactosaminyltransferase to form the Sd(a+) blood group active carbohydrate determinant GalNAc beta 1-4(NeuAc alpha 2-3)Gal was studied using a membrane fraction prepared from human gastric fundic mucosa. Various sialosylated oligosaccharides and gangliosides were examined as acceptor substrates. Oligosaccharide substrates were fluorescence-labelled with 2-aminopyridine, and the transferase activity was quantified by h.p.l.c. using a reversed-phase column. The structures of the products were determined by glycosidase degradation and proton n.m.r. 3'-Sialyl-lactose (II3NeuAcLac), 3'-sialyl-lactotetraose (IV3NeuAcLc4), and 3'-sialyl-lactoneotetraose (IV3NeuAcnLc4) were good substrates for the beta 1-4GalNAc transferase in gastric fundic mucosa, but 6'-sialyl-lactoneotetraose (IV6NeuAcnLc4) or 6'-sialyl-lactose (II6NeuAcLac) were not. Gangliosides with a terminal NeuAc alpha 2-3Gal residue such as GM3, sialylparagloboside, GM1b and GD1a were also studied. The activity of beta 1-4GalNAc transfer to sialylparagloboside was much higher than that to GM2, GM1b or GD1a in spite of them having the same terminal residue. Measurement of the activity of the beta 1-4GalNAc transferase in biopsy specimens demonstrated that the activity was localized in gastric fundic mucosa and was absent in pyloric mucosa, intestinal metaplasia and gastric cancer tissue. Thus the beta 1-4GalNAc transferase present specifically in fundic mucosa required a NeuAc alpha 2-3Gal residue connected to either type-1-chain or type-2-chain oligosaccharides. In glycolipids, the acceptor specificity was restricted to NeuAc alpha 2-3Gal beta 1-4GlcNAc because the NeuAc alpha 2-3Gal beta 1-3GalNAc structure in ganglio-series glycolipids was not a good acceptor substrate.
Dohi
T
T
Division of Clinical Biochemistry, National Medical Center, Tokyo, Japan.
Nishikawa
A
A
Ishizuka
I
I
Totani
M
M
Yamaguchi
K
K
Nakagawa
K
K
Saitoh
O
O
Ohshiba
S
S
Oshima
M
M
D000631:Aminopyridines; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D005732:Gangliosides / Q000378:metabolism; D005748:Gastric Fundus / Q000201:enzymology; D005753:Gastric Mucosa / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000032:analysis* / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D014018:Tissue Distribution
D000631:Aminopyridines; D005456:Fluorescent Dyes; D005732:Gangliosides; D009844:Oligosaccharides; C025447:sialogangliosides; C016735:sialooligosaccharides; D017350:N-Acetylgalactosaminyltransferases; C078172:sialylparagloboside N-acetylgalactosaminyltransferase; D006026:Glycoside Hydrolases; C032439:alpha-aminopyridine