A truncated form of human alpha 1-acid glycoprotein is useful as a molecular tool for insect glycobiology
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N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (a1AGP) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the a1AGP to be a better model for studying glycosylation. The modified a1AGP (a1AGPΔ) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the a1AGP. Subsequently, we confirmed the detailed profile of N-glycan on the a1AGPΔ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant a1AGPΔ could be usable as a better model glycoprotein of N-glycosylation research in BES.
Abstract
N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (a1AGP) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the a1AGP to be a better model for studying glycosylation. The modified a1AGP (a1AGPΔ) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the a1AGP. Subsequently, we confirmed the detailed profile of N-glycan on the a1AGPΔ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant a1AGPΔ could be usable as a better model glycoprotein of N-glycosylation research in BES.
참고문헌 (23)
- Boscher C, "Glycosylation, galectins and cellular signaling", Curr Opin Cell Biol, 23(4), pp.383~392 (2011)[DOI]
- Cazet A, "Tumour-associated carbohydrate antigens in breast cancer", Breast Cancer Res, 12, pp.204~ (2010)[DOI]
- Ceciliani F, "The acute phase protein alpha1-acid glycoprotein: a model for altered glycosylation during diseases", Curr Protein Pept Sci, 8, pp.91~108 (2007)[DOI]
- Fournier T, "Alpha-1-acid glycoprotein", Biochim Biophys Acta, 1482, pp.157~171 (2000)[DOI]
- Harrison RL, "Protein N-glycosylation in the baculovirus-insect cell expression system and engineering of insect cells to produce "mammalianized" recombinant glycoproteins", Adv Virus Res, 68, pp.159~191 (2006)
- Jarvis DL, "Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production", Virology, 310, pp.1~7 (2003)[DOI]
- Kajiura H, "N-glycosylation and N-glycan moieties of CTB expressed in rice seeds", Mol Biotechnol, 54, pp.784~794 (2013)[DOI]
- Kato T, "Silkworm expression system as a platform technology in life science", Appl Microbiol Biotechnol, 85, pp.459~470 (2010)[DOI]
- Kim YK, "Suppression of b-Nacetylglucosaminidase in N-glycosylation pathway for complex glycoprotein formation in Drosophila S2 cells", Glycobiology, 19, pp.301~308 (2009)
- Lee JM, "Establishment of a Bombyx mori nucleopolyhedrovirus (BmNPV) hyper-sensitive cell line from the silkworm e21 strain", Biotechnol Lett, 34, pp.1773~1779 (2012)[DOI]
- Marchal I, "Glycoproteins from insect cells: sialylated or not?", Biol Chem, 382, pp.151~159 (2001)
- Mon H, "Effective RNA interference in cultured silkworm cells mediated by overexpression of Caenorhabditis elegans SID-1", RNA Biol, 9, pp.40~46 (2012)[DOI]
- Daisuke Morokuma, "Human alpha 1-acid glycoprotein as a model protein for glycoanalysis in baculovirus expression vector system", Journal of Asia-Pacific Entomology, 18(2), pp.303~309 (2015), 한국응용곤충학회
- Oberg F, "Glycosylation increases the thermostability of human aquaporin 10 protein", J Bio Chem, 286(36), pp.31915~31923 (2011)[DOI]
- Ono C, "Construction of the BmNPV T3 bacmid system and its application to the functional analysis of BmNPV he65", J Insect Biotech Sericol, 76, pp.161~167 (2007)
- Pochechueva T, "Tumor-associated glycans and their role in gynecological cancers: accelerating translational research by novel high-throughput approaches", Metabolites, 2(4), pp.913~939 (2012)[DOI]
- Schonfeld DL, "The 1. 8-A crystal structure of <TEX>${\alpha}$<", TEX> 1 -acid glycoprotein (Orosomucoid) solved by UV RIP reveals the broad drug-binding activity of this human plasma lipocalin, 2008, pp.384(2), 393~405
- Soejima Y, "Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system", Cent Euro J Biol, 8(1), pp.1~7 (2013)
- Summers MD, "Milestones leading to the genetic engineering of baculoviruses as expression vector systems and viral pesticides", Adv Virus Res, 68, pp.3~73 (2006)
- Takahashi M, "Core fucose and bisecting GlcNAc, the direct modifiers of the N-glycan core: their functions and target proteins", Carbohydr Res, 344(12), pp.1387~1390 (2009)[DOI]
- Toth AM, "A new insect cell glycoengineering approach provides baculovirus-inducible glycogene expression and increases human-type glycosylation efficiency", J Biotechnol, 182-183, pp.19~29 (2014)[DOI]
- Treuheit MJ, "Analysis of the five glycosylation sites of human alpha 1-acid glycoprotein", Biochemical J, 283, pp.105~112 (1992)[DOI]
- Varki A, "Biological roles of oligosaccharides: all of the theories are correct", Glycobiology, 3(2), pp.97~130 (1993)[DOI]