Substrate Specificity of Equine and Human Influenza A Virus Sialidase to Molecular Species of Sialic Acid.

Most equine influenza A viruses (IAVs) show strong binding to glycoconjugates containing N-glycolylneuraminic acid (Neu5Gc) as well as N-acetylneuraminic acid (Neu5Ac). Therefore, the progeny of equine IAV is thought to be released from the infected cell surface through removal of sialic acids by the viral sialidase. In the present study, equine IAV sialidases showed significantly lower substrate affinity than that of human IAV sialidases to artificial and natural Neu5Gc-conjugated substrates. The substrate specificity of equine IAV sialidases is in disagreement with their binding specificity to molecular species of sialic acid. The results suggest that substrate specificity of equine IAV sialidase for Neu5Ac, rather than for Neu5Gc, is important for an advantage at the early infection stage and the process of progeny virus release from the surface of infected cells.

Production and Purification of Secretory Simian Cytidine Monophosphate-N-acetylneuraminic Acid Hydroxylase Using Baculovirus-Protein Expression System.

Cytidine monophosphate (CMP) N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) is an essential enzyme for N-glycolylneuraminic acid (Neu5Gc) synthesis. In humans, Neu5Gc cannot be synthesized because of a deletion in the CMAH gene. Since Neu5Gc research has not been actively performed in comparison with Neu5Ac research, little is known about the function of Neu5Gc. Possible reasons are that CMAH for controlling Neu5Gc synthesis is not understood well at the molecular level, that commercial Neu5Gc is expensive, and that addition of exogenous Neu5Gc to glycoconjugates is not a general method because of the difficulty in obtaining CMAH. One solution to these problems is to achieve large-scale production of CMAH with enzymatic activity. To produce and purify CMAH as simply as possible, we generated simian CMAH as a secretory protein with a histidine tag using a baculovirus protein expression system. After culture of baculovirus-infected cells in serum-free medium, secretory simian CMAH (approximately 180 µg) was highly purified from the supernatant (150 mL) of cell culture. HPLC analysis showed conversion of CMP-Neu5Ac to CMP-Neu5Gc by the secretory CMAH. We succeeded in producing secretory CMAH with enzymatic activity that is easy to purify. In addition, peptide-N-glycosidase F treatment of CMAH indicated that secretory CMAH was a glycoprotein with N-glycan. It will also contribute to research on Neu5Gc function by easy-to-use methods for controlling Neu5Gc synthesis, for exogenous addition of Neu5Gc to glycoconjugates and by application to industrial Neu5Gc synthesis.

N-glycolylneuraminic acid on human epithelial cells prevents entry of influenza A viruses that possess N-glycolylneuraminic acid binding ability.

UNLABELLED: Some animal influenza A viruses (IAVs) bind not only to N-acetylneuraminic acid (Neu5Ac) but also to N-glycolylneuraminic acid (Neu5Gc), which has been discussed as a virus receptor. Human cells cannot synthesize Neu5Gc due to dysfunction of the CMP-Neu5Ac hydroxylase (CMAH) gene, which converts CMP-Neu5Ac to CMP-Neu5Gc. However, exogenous Neu5Gc from Neu5Gc-rich dietary sources is able to be metabolically incorporated into surfaces of tissue cells and may be related to enhancement of the infectivity and severity of IAV. Here, we investigated the receptor function of Neu5Gc on IAV infection in Neu5Gc-expressing cells by transfection of the monkey CMAH gene into human cells or by incubation with human cells in the presence of N-glycolylmannosamine. Expression of Neu5Gc on human cells clearly suppressed infectivity of IAVs that possess Neu5Gc binding ability. Furthermore, there was no difference in infectivity of a transfectant virus that included the wild-type HA gene from A/Memphis/1/1971 (H3N2), which shows no Neu5Gc binding, between parent MCF7 cells and cells stably expressing the monkey CMAH gene (CMAH-MCF7 cells). On the other hand, cell entry of the transfectant virus that included the Neu5Gc-binding HA gene with a single mutation to Tyr at position Thr155 was arrested at the stage of internalization from the plasma membrane of the CMAH-MCF7 cells. These results indicate that expression of Neu5Gc on the surface of human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to work as a decoy receptor of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. IMPORTANCE: Influenza A viruses (IAVs) bind to the host cell surfaces through sialic acids at the terminal of glycoconjugates. For IAV binding to sialic acids, some IAVs bind not only to N-acetylneuraminic acid (Neu5Ac) as a receptor but also to N-glycolylneuraminic acid (Neu5Gc). Neu5Gc has been discussed as a receptor of human and animal IAVs. Our results showed that Neu5Gc expression on human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to be a "decoy receptor" of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. Human cells cannot synthesize Neu5Gc because of dysfunction of the CMP-N-acetylneuraminic acid hydroxylase gene but can exogenously and metabolically incorporate Neu5Gc from dietary sources. The expression of Neu5Gc on human epithelial cells by taking in exogenous Neu5Gc from Neu5Gc-rich dietary sources may be related to restriction of the infection of IAVs that have acquired Neu5Gc binding ability.

Binding kinetics of sulfatide with influenza A virus hemagglutinin.

Association of a sulfated galactosyl ceramide, sulfatide, with the viral envelope glycoprotein hemagglutinin (HA) delivered to the cell surface is required for influenza A virus (IAV) replication through efficient translocation of the newly synthesized viral nucleoprotein from the nucleus to the cytoplasm. To determine whether the ectodomain of HA can bind to sulfatide, a secreted-type HA (sHA), in which the transmembrane region and cytoplasmic tail were deleted, was generated by using a baculovirus expression system. The receptor binding ability and antigenic structure of sHA were evaluated by a hemagglutination assay, solid-phase binding assay and hemagglutination inhibition assay. sHA showed subtype-specific antigenicity and binding ability to both sulfatide and gangliosides. Kinetics of sHA binding to sulfatide and GD1a was demonstrated by quartz crystal microbalance (QCM) analysis. QCM analysis showed that the sHA bound with the association rate constant (k on) of 1.41 × 10(4) M(-1) sec(-1), dissociation rate constant (k off) of 2.03 × 10(-4) sec(-1) and K d of 1.44 × 10(-8) M to sulfatide immobilized on a sensor chip. The k off values of sHA were similar for sulfatide and GD1a, whereas the k on value of sHA binding to sulfatide was 2.56-times lower than that of sHA binding to GD1a. The results indicate that sulfatide directly binds to the ectodomain of HA with high affinity.