A truncated form of human alpha 1-acid glycoprotein is useful as a molecular tool for insect glycobiology

  • Morokuma, Daisuke (Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences) ;
  • Hino, Masato (Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences) ;
  • Tsuchioka, Miho (Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences) ;
  • Masuda, Akitsu (Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences) ;
  • Mon, Hiroaki (Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences) ;
  • Fujiyama, Kazuhito (The International Center for Biotechnology, Osaka University) ;
  • Kajiura, Hiroyuki (Department of Biotechnology, College of Life Sciences, Ritsumeikan University) ;
  • Kusakabe, Takahiro (Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences) ;
  • Lee, Jae Man (Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences)
  • Received : 2017.09.04
  • Accepted : 2018.03.01
  • Published : 2018.03.31


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 (${\alpha}1AGP$) 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 ${\alpha}1AGP$ to be a better model for studying glycosylation. The modified ${\alpha}1AGP$ (${\alpha}1AGP{\Delta}$) 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 ${\alpha}1AGP$. Subsequently, we confirmed the detailed profile of N-glycan on the ${\alpha}1AGP{\Delta}$ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant ${\alpha}1AGP{\Delta}$ could be usable as a better model glycoprotein of N-glycosylation research in BES.


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Fig. 1. (A) Schematic representation of the α1AGP? in this study.The 90 amino acid sequences were deleted from C-terminus ofhuman α1AGP. Artificial N-glycosylation sites (2N) were addedN-terminal of 93 amino acid sequences of deleted α1AGP. (B) 151Amino acid sequences of the recombinant α1AGP?. 30K signalpeptides and 2N sites were added in N-terminus, and Histidine-tag (His x 8) and TEV protease cleavage site in C-terminus. Theasparagine residues (N) to which N-glycan may be added indicatedby bold letters. The amino acid numbers of asparagine as N-glycansites is shown at the bottom.

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Fig. 2. Expression of the recombinant α1AGP? in cultured silkwormcells. Time courses of the expression of the α1AGP protein inBme21 cells, BmN4 cells and BmN4 SID-1 cells (A). The cells andculture medium were collected at 2, 3, 4 days post-infection (DPI).The recombinant α1AGP? was detected by Western blotting usingHis-Probe.

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Fig. 3. (A) Purifcation of human α1AGP from larval haemolymph.The Histidine tagged α1AGP? protein was purifed through nickelaffinity chromatography as described in Materials and methods.Each fraction was resolved on 15% SDS-PAGE and visualized byCoomassie Brilliant Blue (CBB) R-250. IP: input; FT: fow-throughfraction; W: wash fraction; Lane No.1~3: eluent fraction (100mMimidazole); Lane No.4~9: eluent fraction (500mM imidazole). (B)Characterization of N-glycan structures of the α1AGP secreted insilkworm larval haemolymph. The purified recombinant α1AGPor α1AGP? form silkworm larvae as indicated in Materials andmethods were incubated with (+) or without (?) PNGaseF for 1 hat 37 °C. After reaction, each mixture was resolved on 15% SDS-PAGE and visualized by CBB R-250, His-Probe, or Concanavalin A(ConA).

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Fig. 4. (A) The purifed α1AGP? was separated into multiple bandsin 15% SDS-PAGE and visualized by CBB R-250. The multiplebands were assigned Band 1 ~ Band 8 from the top. Band 1 is thesmear portion at the top of Band 2. (B) The degree of glycosylationof each band 1~ 8 analyzed by LC/MS. At the schematic diagram ofN-glycan, the open square, open circle and flled triangle representGlcNAc, mannose and fucose, respectively. The frequency ofaddition of N-glycans at each site is indicated by shading of thediagrams. Approximate number of sugar chains attached to eachband is shown on the right.

Table 1. The ratio of N-glycan structure for each sugar chain binding site.

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Table 1. Continued

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Table 1. Continued

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Table 1. Continued

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Supported by : KAKENHI


  1. Boscher C, Dennis JW, Nabi, IR (2011) Glycosylation, galectins and cellular signaling. Curr Opin Cell Biol 23(4), 383-92.
  2. Cazet A, Julien S, Bobowski M, Burchell J, Delannoy P (2010) Tumour-associated carbohydrate antigens in breast cancer. Breast Cancer Res 12, 204.
  3. Ceciliani F, Pocacqua V (2007) The acute phase protein alpha1-acid glycoprotein: a model for altered glycosylation during diseases. Curr Protein Pept Sci 8, 91-108.
  4. Fournier T, Medjoubi-NN, Porquet D (2000) Alpha-1-acid glycoprotein. Biochim Biophys Acta 1482, 157-71.
  5. Harrison RL, Jarvis DL (2006) Protein N-glycosylation in the baculovirus-insect cell expression system and engineering of insect cells to produce "mammalianized" recombinant glycoproteins. Adv Virus Res 68, 159-191.
  6. Jarvis DL (2003) Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production. Virology 310, 1-7.
  7. Kajiura H, Wasai M, Kasahara S, Takaiwa F, Fujiyama K (2013) N-glycosylation and N-glycan moieties of CTB expressed in rice seeds. Mol Biotechnol 54, 784-794.
  8. Kato T, Kajikawa M, Maenaka K, Park EY (2010) Silkworm expression system as a platform technology in life science. Appl Microbiol Biotechnol 85, 459-470.
  9. Kim YK, Kim KR, Kang DG, Jang SY, Kim YH, Cha HJ (2009) Suppression of b-Nacetylglucosaminidase in N-glycosylation pathway for complex glycoprotein formation in Drosophila S2 cells. Glycobiology 19, 301-308.
  10. Lee JM, Kawakami N, Mon H, Mitsunobu H, Iiyama K, Ninaki S, et al. (2012) Establishment of a Bombyx mori nucleopolyhedrovirus (BmNPV) hyper-sensitive cell line from the silkworm e21 strain. Biotechnol Lett 34, 1773-1779.
  11. Marchal I, Jarvis DL, Cacan R, Verbert A (2001) Glycoproteins from insect cells: sialylated or not? Biol Chem 382, 151-159.
  12. Mon H, Kobayashi I, Ohkubo S, Tomita S, Lee JM, Sezutsu H, et al. (2012) Effective RNA interference in cultured silkworm cells mediated by overexpression of Caenorhabditis elegans SID-1. RNA Biol 9, 40-46.
  13. Morokuma D, Xu J, Mon H, Hirata K, Hino M, Kuboe S, et al. (2015) Human alpha 1-acid glycoprotein as a model protein for glycoanalysis in baculovirus expression vector system. J Asia Pac Entomol 18(2), 303-309.
  14. Oberg F, Sjohamn J, Fischer G, Moberg A, Pedersen A, Neutze R, et al. (2011) Glycosylation increases the thermostability of human aquaporin 10 protein. J Bio Chem 286(36), 31915-23.
  15. Ono C, Nakatsukasa T, Nishijima Y, Asano S, Sahara K, Bando H (2007) Construction of the BmNPV T3 bacmid system and its application to the functional analysis of BmNPV he65. J Insect Biotech Sericol 76, 161-167.
  16. Pochechueva T, Jacob F, Fedier A, Heinzelmann-schwarz V (2012) Tumor-associated glycans and their role in gynecological cancers: accelerating translational research by novel high-throughput approaches. Metabolites 2(4), 913-39.
  17. Schonfeld DL, Ravelli RB, Mueller U, Skerra A (2008) The 1. 8-A crystal structure of ${\alpha}$ 1 -acid glycoprotein (Orosomucoid) solved by UV RIP reveals the broad drug-binding activity of this human plasma lipocalin. J Mol Biol 384(2), 393-405.
  18. Soejima Y, Lee JM, Nagata Y, Mon H, Iiyama K, Kitano H, et al. (2013) Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system. Cent Euro J Biol 8(1), 1-7.
  19. Summers MD (2006) Milestones leading to the genetic engineering of baculoviruses as expression vector systems and viral pesticides. Adv Virus Res 68, 3-73.
  20. Takahashi M, Kuroki Y, Ohtsubo K, Taniguchi N (2009) Core fucose and bisecting GlcNAc, the direct modifiers of the N-glycan core: their functions and target proteins. Carbohydr Res 344(12), 1387-1390.
  21. Toth AM, Kuo CW, Khoo KH, Jarvis DL (2014) A new insect cell glycoengineering approach provides baculovirus-inducible glycogene expression and increases human-type glycosylation efficiency. J Biotechnol 182-183, 19-29.
  22. Treuheit MJ, Costello CE, Halsall HB (1992) Analysis of the five glycosylation sites of human alpha 1-acid glycoprotein. Biochemical J 283 (Pt 1), 105-112.
  23. Varki A (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3(2), 97-130.