Research article

Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae

Makoto Ogata¹ , Makoto Nakajima¹ , Tatsuya Kato¹ , Takakiyo Obara¹ , Hirokazu Yagi² , Koichi Kato^2,3,4,5 , Taichi Usui^1,6 and Enoch Y Park^1,6

¹Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8529, Japan

²Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan

³Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of National Science, 5-1 Higashiyama Myodaiji, Okazaki 444-8787, Japan

⁴GLYENCE Co., Ltd., 2-22-8 Chikusa, Chikusa-ku, Nagoya 464-0858, Japan

⁵The Glycoscience Institute, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan

⁶Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8529, Japan

author email corresponding author email

BMC Biotechnology 2009, 9:54doi:10.1186/1472-6750-9-54

Published:	5 June 2009

Abstract

Background

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 Bombyx mori nucleopolyhedrovirus (BmNPV-CP^--Chi^-) 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 Sambucus nigra (SNA) lectin.

Results

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 N-glycan patterns were found to be approximately paucimannosidic type by HPLC mapping method. Fluorescently-labelled N-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 Sambucus nigra (SNA) lectin, showing about 780-fold higher affinity than the control fetuin. Asialoglycopolypeptide and γ-polyglutamic acid did not affect SNA lectin-mediated hemagglutination.

Conclusion

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.