Taiichi Usui

Chemoenzymatic synthesis, characterization, and application of glycopolymers carrying lactosamine repeats as entry inhibitors against influenza virus infection

To control interspecies transmission of influenza viruses, it is essential to elucidate the molecular mechanisms of the interaction of influenza viruses with sialo-glycoconjugate receptors expressed on different host cells. Competitive inhibitors containing mimetic receptor carbohydrates that prevent virus entry may be useful tools to address such issues. We chemoenzymatically synthesized and characterized the glycopolymers that were carrying terminal 2,6-sialic acid on lactosamine repeats as influenza virus inhibitors. In vitro and in vivo infection experiments using these glycopolymers demonstrated marked differences in inhibitory activity against different species of viruses. Human viruses, including clinically isolated strains, were consistently inhibited by glycopolymers carrying lactosamine repeats with higher activity than those containing a single lactosamine. A swine virus also showed the same recognition properties as those from human hosts. In contrast, avian and equine viruses were not inhibited by any of the glycopolymers examined carrying single, tandem, or triplet lactosamine repeats. Hemagglutination inhibition and solid-phase binding analyses indicated that binding affinity of glycopolymers with influenza viruses contributes dominantly to the inhibitory activity against viral infection. Sequence analysis and molecular modeling of human viruses indicated that specific amino acid substitutions on hemagglutinin may affect binding affinity of glycopolymers carrying lactosamine repeats with viruses. In conclusion, glycopolymers carrying lactosamine repeats of different lengths are useful to define molecular mechanisms of virus recognition. The core carbohydrate portion as well as sialyl linkages on the receptor glycoconjugate may affect host cell recognition of human and swine viruses.

Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli

A soluble and active form of recombinant human ST6Gal I was expressed in Escherichia coli. The gene encoding the soluble form of ST6Gal I lacking the membrane and cytosolic regions was introduced into a bacterial expression vector, pMAL-p2X, fused in frame with a maltose-binding protein (MBP) tag. Low-temperature cultivation at 13∘C during IPTG-induction significantly improved both solubility and MBP-tagging of the recombinant enzyme expressed in bacteria. The supernatant prepared by disruption of the cells demonstrated sialic acid transfer activity to both an oligosaccharide and a glycoprotein, asialofetuin, indicating that the enzyme expressed in bacteria is soluble and active. The MBP-tagged enzyme was efficiently purified by a combination of cation-exchange column and amylase-conjugated agarose column chromatography. The purified recombinant enzyme exerted enzymatic activity even in the absence of detergents in the reaction mixture. Acceptor substrate specificity of the enzyme was marginally different from that of rat liver ST6Gal I. These observations suggest that membrane and cytosolic regions of ST6Gal I may affect the properties of the enzyme. The purified recombinant enzyme was applied to convert desialylated fetuin to resialylated fetuin. Lectin blotting demonstrated that resialylated fetuin possesses a single Neu5Ac α 2-6 residue. The resialylated fetuin efficiently blocked hemagglutination induced by influenza virus strain A/Memphis/1/71 (H3N2), indicating that resialylated carbohydrate chains on the protein are so active as to competitively inhibit virus-receptor interaction. In conclusion, soluble recombinant ST6Gal I obtained using our bacterial expression system is a valuable tool to investigate the molecular mechanisms of biological and pathological interactions mediated via carbohydrates. Published in 2005.