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Glycobiology Advance Access published online on December 17, 2002
Glycobiology, doi:10.1093/glycob/cwg021
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Submitted on August 7, 2002
Revised on October 7, 2002
Accepted on October 7, 2002

© 2002 Oxford University Press

ORIGINAL ARTICLES

Plant cultured cells expressing human {beta}1,4-galactosyltransferase secrete glycoproteins with galactose-extended N-linked glycans

Ryo Misaki 1, Yoshinobu Kimura 2, Nirianne Q. Palacpac 1, Shohei Yoshida 1, Kazuhito Fujiyama 1*, Tatsuji Seki 1

1 The International Center for Biotechnology, Osaka University, Yamada-oka 2-1, Suita-shi, Osaka 565-0871, Japan
2 Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushima-naka 1-1-1, Okayama 700-8530, Japan

* To whom correspondence should be addressed. E-mail: fujiyama{at}icb.osaka-u.ac.jp.

Abstract

Previously, we generated transgenic tobacco BY2 suspension-cultured cells (GT6 cells) that produced human {beta}1,4-galactosyltransferase. In this study, we analyzed the N-glycan structures of glycoproteins secreted from GT6 cells to the spent medium. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by the combined use of the two-dimensional PA sugar chain mapping, MS/MS analysis, and exoglycosidase digestion. The distribution of proposed N-glycan structures of GT6 secreted glycoproteins (GalGNM5 (26.8%), GalGNM4 (18.4%), GalGNM3 (19.6%) and GalGNM3X (35.2%)) is different from that found in intracellular glycoproteins (M7A (9.3%), M7B (15.9%), M6B (19.5%), M5 (1.4%), M3X (6.6%), GalGNM5 (35.5%) and GalGNM3 (11.8%)) (Palacpac et al., 1999a). In vitro, sialic acid was transferred to sugar chains of extracellular glycoproteins from the GT6 spent medium. The results suggested that sugar chains of extracellular glycoproteins from the GT6 spent medium are candidates for substrates of sialic acid transfer.


Key words: N-glycan, plant suspension-cultured cell, extracellular, glycan synthetic pathway
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