Comparison of the N-linked glycosylation of human beta1,3-N-acetylglucosaminyltransferase 2 expressed in insect cells and silkworm larvae.
N-Glycosylation of human beta1,3N-acetylglucosaminyltransferase 2 (beta3GnT2) is essential for its biological function. beta3GnT2 fused to GFP(uv) (GFP(uv)-beta3GnT2) was produced by non-virus expression systems in stably transformed insect cells and silkworm larvae using a recombinant BmNPV bacmid, and purified for analysis of N-glycosylation. The N-glycan structure of beta3GnT2 was identified by glycoamidase A digestion, labeling with 2-aminopyridine (PA), and HPLC mapping. The paucimannosidic N-glycan structure (73.2%) was predominant in stably transformed Trichoplusia ni cells. In contrast, N-glycan with Gal (21.3%) and GlcNAc (16.2%) terminal residues linked to Manalpha(1,3) branch were detected on beta3GnT2 expressed in silkworm larvae. The presence of terminal Gal and bisecting GlcNAc residues such as Galbeta1, 4GlcNAcbeta1, 2Manalpha1,3(GlcNAcbeta1,4)(Manalpha1,6)Manbeta1, 4GlcNAc is not typical structure for lepidopteran insect N-glycosylation. Although allergenic alpha1,3-fucose residues have been found in T. ni cells, only alpha1,6-fucose residues were attached to the beta3GnT2 glycan in silkworm larvae. Therefore, silkworm larvae might be a useful host for producing human glycoproteins.
Dojima
Takashi
T
Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, Ohya Suruga-ku, Shizuoka, Japan.
Nishina
Takuya
T
Kato
Tatsuya
T
Uno
Tsuyoshi
T
Yagi
Hirokazu
H
Kato
Koichi
K
Park
Enoch Y
EY
D000631:Aminopyridines / Q000737:chemistry; D000818:Animals; D012831:Bombyx; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006458:Hemolymph / Q000378:metabolism; D006801:Humans; D007313:Insecta; D037102:Lectins / Q000737:chemistry; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary
D000631:Aminopyridines; D037102:Lectins; D011134:Polysaccharides; D049452:Green Fluorescent Proteins; D017351:N-Acetylglucosaminyltransferases; C428132:B3GNT2 protein, human; C032439:alpha-aminopyridine