title: Improvement of glycosylation structure by suppression of β-N-acetylglucosaminidases in silkworm.
abstract: The baculovirus-silkworm recombinant protein expression system is an excellent method for achieving high-level expression and post-translational modifications, especially glycosylation. However, the presence of paucimannosidic-type N-glycan in glycoproteins restricts their clinical use. Paucimannosidic-type N-glycan is produced by insect-specific membrane-binding-type β-N-acetylglucosaminidase (GlcNAcase). In the silkworm, BmGlcNAcase1, BmGlcNAcase2, and BmFDL are membrane-binding-type GlcNAcases. We investigated the localization of these GlcNAcases and found that BmFDL and BmGlcNAcase2 were mainly located in the fat body and hemolymph, respectively. The fat body is the main tissue of recombinant protein expression by baculovirus, and many glycoproteins are secreted into the hemolymph. These results suggest that inhibition of BmFDL and BmGlcNAcase2 could increase GlcNAc-type N-glycan levels. We therefore injected a GlcNAcase inhibitor into silkworms to investigate changes in the N-glycan structure of the glycoprotein expressed by baculovirus; modest levels of GlcNAc-type N-glycan were observed (0.8% of total N-glycan). Next, we generated a transgenic silkworm in which RNA interference (RNAi) reduced the BmFDL transcript level and enzyme activity to 25% and 50%, respectively, of that of the control silkworm. The proportion of GlcNAc-type N-glycan increased to 4.3% in the RNAi-transgenic silkworm. We conclude that the structure of N-glycan can be changed by inhibiting the GlcNAcases in silkworm.
authors.affiliation: Protein Development Center, Sysmex Corporation, 1548 Simo-okudomi, Sayama, Saitama 350-1332, Japan; The International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-8071, Japan. Electronic address: Nomura.Tsuyoshi@sysmex.co.jp.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Nomura
authors.affiliation: Protein Development Center, Sysmex Corporation, 1548 Simo-okudomi, Sayama, Saitama 350-1332, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Suganuma
authors.affiliation: Protein Development Center, Sysmex Corporation, 1548 Simo-okudomi, Sayama, Saitama 350-1332, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Higa
authors.affiliation: Protein Development Center, Sysmex Corporation, 1548 Simo-okudomi, Sayama, Saitama 350-1332, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Kataoka
authors.affiliation: Protein Development Center, Sysmex Corporation, 1548 Simo-okudomi, Sayama, Saitama 350-1332, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Funaguma
authors.affiliation: Protein Development Center, Sysmex Corporation, 1548 Simo-okudomi, Sayama, Saitama 350-1332, Japan.
authors.forename: Hironobu
authors.initials: H
authors.lastname: Okazaki
authors.affiliation: Protein Development Center, Sysmex Corporation, 1548 Simo-okudomi, Sayama, Saitama 350-1332, Japan.
authors.forename: Takeo
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Transgenic Silkworm Research Unit, Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Kobayashi
authors.affiliation: Transgenic Silkworm Research Unit, Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Sezutsu
authors.affiliation: The International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-8071, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Fujiyama
mesh_terms: D000118:Acetylglucosaminidase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D030801:Animals, Genetically Modified; D016367:Baculoviridae / Q000235:genetics; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005216:Fat Body / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006458:Hemolymph / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D034622:RNA Interference; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D000118:Acetylglucosaminidase
keywords: Baculovirus; Fused lobes; N-Glycan processing; RNA interference; Transgenic silkworm; β-N-Acetylglucosaminidase