The effects of N-glycosylation sites and the N-terminal region on the biological function of β1,3-N-acetylglucosaminyltransferase 2 and its secretion

Human β1,3-N-acetylglucosaminyltransferase 2 (β3GnT2) 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 β3GnT2, 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 GFPuv 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 β3GnT 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 (GFPuv-β3GnT2Δ83), 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 β3GnT activity. These results suggest that the N-glycosylation site at Asn219, which is conserved throughout the β1,3-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.