Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae
<p>Abstract</p> <p>Background</p> <p>Sialic acid is a deoxy uronic acid with a skeleton of nine carbons which is mostly found on cell surface in animals. This sialic acid on cell surface performs various biological functions by acting as a receptor for microorganisms, v...
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| Format: | Article |
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BMC
2009-06-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/9/54 |
| _version_ | 1811286272850788352 |
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| author | Ogata Makoto Nakajima Makoto Kato Tatsuya Obara Takakiyo Yagi Hirokazu Kato Koichi Usui Taichi Park Enoch Y |
| author_facet | Ogata Makoto Nakajima Makoto Kato Tatsuya Obara Takakiyo Yagi Hirokazu Kato Koichi Usui Taichi Park Enoch Y |
| author_sort | Ogata Makoto |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Sialic acid is a deoxy uronic acid with a skeleton of nine carbons which is mostly found on cell surface in animals. This sialic acid on cell surface performs various biological functions by acting as a receptor for microorganisms, viruses, toxins, and hormones; by masking receptors; and by regulating the immune system. In order to synthesize an artificial sialoglycoprotein, we developed a large-scale production of rat α2,6-sialyltransferase (ST6Gal1). The ST6Gal1 was expressed in fifth instar silkworm larval hemolymph using recombinant both cysteine protease- and chitinase-deficient <it>Bombyx mori </it>nucleopolyhedrovirus (BmNPV-<it>CP</it><sup>-</sup>-<it>Chi</it><sup>-</sup>) bacmid. The expressed ST6Gal1 was purified, characterized and used for sialylation of asialoglycopolypeptide. We tested the inhibitory effect of the synthesized α2,6-sialoglycopolypeptide on hemagglutination by <it>Sambucus nigra </it>(SNA) lectin.</p> <p>Results</p> <p>FLAG-tagged recombinant ST6Gal1 was expressed efficiently and purified by precipitation with ammonium sulphate followed by affinity chromatography on an anti-FLAG M2 column, generating 2.2 mg purified fusion protein from only 11 silkworm larvae, with a recovery yield of 64%. The purified ST6Gal1 was characterized and its <it>N</it>-glycan patterns were found to be approximately paucimannosidic type by HPLC mapping method. Fluorescently-labelled <it>N</it>-acetyllactosamine (LacNAc) glycoside containing dansyl group was synthesized chemo-enzymatically as high-sensitivity acceptor substrate for ST6Gal1. The acceptor substrate specificity of the enzyme was similar to that of rat liver ST6Gal1. The fluorescent glycoside is useful as a substrate for a highly sensitive picomole assay of ST6Gal1. Asialoglycopolypeptide was regioselectively and quantitatively sialylated by catalytic reaction at the terminal Gal residue to obtain α2,6-sialoglycopolypeptide using ST6Gal1. The α2,6-sialoglycopolypeptide selectively inhibited hemagglutination induced by <it>Sambucus nigra </it>(SNA) lectin, showing about 780-fold higher affinity than the control fetuin. Asialoglycopolypeptide and γ-polyglutamic acid did not affect SNA lectin-mediated hemagglutination.</p> <p>Conclusion</p> <p>The recombinant ST6Gal1 from a silkworm expression system is useful for the sialylation of asialoglycopeptide. The sialylated glycoprotein is a valuable tool for investigating the molecular mechanisms of biological and physiological events, such as cell-cell recognition and viral entry during infection.</p> |
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| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-04-13T02:57:10Z |
| publishDate | 2009-06-01 |
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| series | BMC Biotechnology |
| spelling | doaj.art-d2974a8b34374b93b9c8598c6d9b0b0c2022-12-22T03:05:37ZengBMCBMC Biotechnology1472-67502009-06-01915410.1186/1472-6750-9-54Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvaeOgata MakotoNakajima MakotoKato TatsuyaObara TakakiyoYagi HirokazuKato KoichiUsui TaichiPark Enoch Y<p>Abstract</p> <p>Background</p> <p>Sialic acid is a deoxy uronic acid with a skeleton of nine carbons which is mostly found on cell surface in animals. This sialic acid on cell surface performs various biological functions by acting as a receptor for microorganisms, viruses, toxins, and hormones; by masking receptors; and by regulating the immune system. In order to synthesize an artificial sialoglycoprotein, we developed a large-scale production of rat α2,6-sialyltransferase (ST6Gal1). The ST6Gal1 was expressed in fifth instar silkworm larval hemolymph using recombinant both cysteine protease- and chitinase-deficient <it>Bombyx mori </it>nucleopolyhedrovirus (BmNPV-<it>CP</it><sup>-</sup>-<it>Chi</it><sup>-</sup>) bacmid. The expressed ST6Gal1 was purified, characterized and used for sialylation of asialoglycopolypeptide. We tested the inhibitory effect of the synthesized α2,6-sialoglycopolypeptide on hemagglutination by <it>Sambucus nigra </it>(SNA) lectin.</p> <p>Results</p> <p>FLAG-tagged recombinant ST6Gal1 was expressed efficiently and purified by precipitation with ammonium sulphate followed by affinity chromatography on an anti-FLAG M2 column, generating 2.2 mg purified fusion protein from only 11 silkworm larvae, with a recovery yield of 64%. The purified ST6Gal1 was characterized and its <it>N</it>-glycan patterns were found to be approximately paucimannosidic type by HPLC mapping method. Fluorescently-labelled <it>N</it>-acetyllactosamine (LacNAc) glycoside containing dansyl group was synthesized chemo-enzymatically as high-sensitivity acceptor substrate for ST6Gal1. The acceptor substrate specificity of the enzyme was similar to that of rat liver ST6Gal1. The fluorescent glycoside is useful as a substrate for a highly sensitive picomole assay of ST6Gal1. Asialoglycopolypeptide was regioselectively and quantitatively sialylated by catalytic reaction at the terminal Gal residue to obtain α2,6-sialoglycopolypeptide using ST6Gal1. The α2,6-sialoglycopolypeptide selectively inhibited hemagglutination induced by <it>Sambucus nigra </it>(SNA) lectin, showing about 780-fold higher affinity than the control fetuin. Asialoglycopolypeptide and γ-polyglutamic acid did not affect SNA lectin-mediated hemagglutination.</p> <p>Conclusion</p> <p>The recombinant ST6Gal1 from a silkworm expression system is useful for the sialylation of asialoglycopeptide. The sialylated glycoprotein is a valuable tool for investigating the molecular mechanisms of biological and physiological events, such as cell-cell recognition and viral entry during infection.</p>http://www.biomedcentral.com/1472-6750/9/54 |
| spellingShingle | Ogata Makoto Nakajima Makoto Kato Tatsuya Obara Takakiyo Yagi Hirokazu Kato Koichi Usui Taichi Park Enoch Y Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae BMC Biotechnology |
| title | Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae |
| title_full | Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae |
| title_fullStr | Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae |
| title_full_unstemmed | Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae |
| title_short | Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae |
| title_sort | synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2 6 sialyltransferase expressed in bmnpv bacmid injected silkworm larvae |
| url | http://www.biomedcentral.com/1472-6750/9/54 |
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