RRC ID 33120
著者 Nomura T, Suganuma M, Higa Y, Kataoka Y, Funaguma S, Okazaki H, Suzuki T, Kobayashi I, Sezutsu H, Fujiyama K.
タイトル Improvement of glycosylation structure by suppression of β-N-acetylglucosaminidases in silkworm.
ジャーナル J Biosci Bioeng
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.
巻・号 119(2)
ページ 131-6
公開日 2015-2-1
DOI 10.1016/j.jbiosc.2014.07.012
PII S1389-1723(14)00288-6
PMID 25193875
MeSH Acetylglucosaminidase / antagonists & inhibitors* Acetylglucosaminidase / isolation & purification Acetylglucosaminidase / metabolism* Animals Animals, Genetically Modified Baculoviridae / genetics Bombyx / enzymology* Bombyx / genetics Bombyx / metabolism Fat Body / metabolism Glycoproteins / biosynthesis* Glycoproteins / chemistry* Glycoproteins / genetics Glycoproteins / metabolism Glycosylation Hemolymph / metabolism Polysaccharides / chemistry* Polysaccharides / metabolism Protein Processing, Post-Translational* Protein Transport RNA Interference Recombinant Proteins / biosynthesis Recombinant Proteins / chemistry Recombinant Proteins / genetics Recombinant Proteins / metabolism
IF 2.366
引用数 11
WOS 分野 FOOD SCIENCE & TECHNOLOGY BIOTECHNOLOGY & APPLIED MICROBIOLOGY
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