Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat alpha2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae.
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 alpha2,6-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 alpha2,6-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 alpha2,6-sialoglycopolypeptide using ST6Gal1. The alpha2,6-sialoglycopolypeptide selectively inhibited hemagglutination induced by Sambucus nigra (SNA) lectin, showing about 780-fold higher affinity than the control fetuin. Asialoglycopolypeptide and gamma-polyglutamic acid did not affect SNA lectin-mediated hemagglutination.


CONCLUSIONS
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.
Ogata
Makoto
M
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan. w5613008@ipc.shizuoka.ac.jp
Nakajima
Makoto
M
Kato
Tatsuya
T
Obara
Takakiyo
T
Yagi
Hirokazu
H
Kato
Koichi
K
Usui
Taichi
T
Park
Enoch Y
EY
D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism; D012831:Bombyx / Q000378:metabolism* / Q000821:virology; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006385:Hemagglutination Inhibition Tests; D007814:Larva / Q000378:metabolism / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D009975:Orthomyxoviridae / Q000187:drug effects; D037121:Plant Lectins / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D054788:Ribosome Inactivating Proteins / Q000378:metabolism; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012799:Sialyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D000998:Antiviral Agents; D037121:Plant Lectins; D011994:Recombinant Proteins; C050600:Sambucus nigra lectins; D012795:Sialoglycoproteins; D012799:Sialyltransferases; D054788:Ribosome Inactivating Proteins; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase