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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

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 (${\alpha}1AGP$) 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.

Keywords

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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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Acknowledgement

Supported by : KAKENHI

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