title: The effects of N-glycosylation sites and the N-terminal region on the biological function of beta1,3-N-acetylglucosaminyltransferase 2 and its secretion.
abstract: Human beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is thought to be an enzyme that extends the polylactosamine acceptor chains, but its function and structure analysis are unknown. To obtain insight into the structure of beta3GnT2, the effects of N-glycosylation on its biological function were evaluated using the addition of inhibitors, site-directed mutagenesis of potential N-glycosylation sites, and deletion of its N-terminal region using a fusion protein with GFP(uv) in a baculovirus expression system. Four of five potential N-glycosylation sites were found to be occupied, and their biological function and secretion were inhibited with the treatment of N-glycosylation inhibitor, tunicamycin. The N-glycosylation at Asn219 was necessary for the beta3GnT activity; moreover, N-glycosylation at Asn127 and Asn219 was critical for efficient protein secretion. When Ser221 was replaced with Thr, fusion protein was expressed as a single band, indicating that the double band of the expressed fusion protein was due to the heterogeneity of the glycosylation at Asn219. The truncated protein consisting of amino acids 82-397 (GFP(uv)-beta3GnT2Delta83), which lacked both one N-glycosylation site at Asn79 and the stem region of glycosyltransferase, was expressed as only a small form and showed no beta3GnT activity. These results suggest that the N-glycosylation site at Asn219, which is conserved throughout the beta1,3-glycosyltransferase family, is indispensable not only with regard to its biological function, but also to its secretion. The N-terminal region, which belongs to a stem region of glycosyltransferase, might also be important to the active protein structure.
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Kato
authors.forename: Mami
authors.initials: M
authors.lastname: Suzuki
authors.forename: Takeomi
authors.initials: T
authors.lastname: Murata
authors.forename: Enoch Y
authors.initials: EY
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D006031:Glycosylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D017351:N-Acetylglucosaminyltransferases; C428132:B3GNT2 protein, human