Plant cultured cells expressing human beta1,4-galactosyltransferase secrete glycoproteins with galactose-extended N-linked glycans.
Previously, we generated transgenic tobacco BY2 suspension-cultured cells (GT6 cells) that produced human beta1,4-galactosyltransferase. In this study, we analyze 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 (PA). The pyridylaminated glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by the combined use of 2D 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%]). In vitro, sialic acid was transferred to sugar chains of extracellular glycoproteins from the GT6 spent medium. The results suggest that sugar chains of extracellular glycoproteins from the GT6 spent medium are candidates for substrates of sialic acid transfer.
Misaki
Ryo
R
International Center for Biotechnology, Osaka University, Yamada-oka 2-1, Suita-shi, Osaka 565-0871, Japan.
Kimura
Yoshinobu
Y
Palacpac
Nirianne Q
NQ
Yoshida
Shohei
S
Fujiyama
Kazuhito
K
Seki
Tatsuji
T
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned / Q000737:chemistry; D005690:Galactose / Q000032:analysis*; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014026:Nicotiana / Q000166:cytology / Q000235:genetics*
D017077:Culture Media, Conditioned; D006023:Glycoproteins; D011994:Recombinant Proteins; D005700:Galactosyltransferases; C119701:beta-1,4-galactosyltransferase I; D019158:N-Acetylneuraminic Acid; D005690:Galactose