Molecular Species Of Sialic Acid Research Articles

Role of Glycoconjugates and Mammalian Sialidases Involved in Viral Infection and Neural Function

Glycoconjugates are present in various organisms, ranging from animals to viruses. Glycoconjugates are involved in several biologically significant functions, including viral infection and neurotransmission. However, the role of glycoconjugates in virus replication and neural function remains unknown. We discovered that the influenza A virus (IAV) binds to sulfatide, which lacks a sialic acid residue, and that delivering sulfatide combined with newly synthesized IAV hemagglutinin (HA) to the target plasma membrane induces the translocation of viral ribonucleoprotein complexes from the nucleus to the cytoplasm. Molecular species of sialic acid are largely classified as N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). We discovered that two amino acids in IAV H3 HA play a critical role in the recognition of Neu5Ac and Neu5Gc. Also, we observed that human parainfluenza virus types 1 (hPIV-1) and 3 (hPIV-3) bind to different forms of gangliosides. These viruses preferentially recognized oligosaccharides containing branched N-acetyllactosaminoglycans with terminal Neu5Acα2-3Gal. Neu5Gcα2-3Gal- and NeuAcα2-6Gal-containing receptors were identified by hPIV-3, but not by hPIV-1. We constructed a novel sialidase fluorescent substrate, 2-benzothiazol-2-yl-phenol derivative-based N-acetylneuraminic acid (BTP3-Neu5Ac), which detects sialidase activity in living mammalian tissues and virus-infected cells expressing viral neuraminidase. We discovered that neural activity-dependent desialylation by sialidase contributes to rat hippocampal memory processing. Using BTP3-Neu5Ac, we developed a rapid and sensitive approach for detecting and isolating drug-resistant influenza viruses. This review summarizes the role of sialylglycoconjugates and sulfatide in virus replication as well as mammalian sialidases involved in neural function and insulin secretion.

Functional analysis of glyco-molecules that bind with influenza virus.

Influenza A virus (IAV) recognizes terminal sialic acid of sialoglyco-conjugates on host cells through the viral envelope glycoprotein hemagglutinin (HA), followed by initiation of entry into the cells. Molecular species of sialic acid are largely divided into two moieties: N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). A receptor for IAV infection generally means Neu5Ac. Almost all equine IAVs and some human, swine, and duck IAVs bind not only to Neu5Ac but also to Neu5Gc. In nonhuman animals, Neu5Gc has been detected in swine and equine tracheas and the duck colon, which are the main replication sites of mammalian and avian IAVs. Therefore, Neu5Gc in these sites has been suggested to be a functional receptor for IAV infection. Humans cannot synthesize Neu5Gc due to a genetic defect of the Neu5Gc-synthesizing enzyme. We evaluated the receptor function of Neu5Gc in IAV infection in human cells. Our results indicated that Neu5Gc expression on the surface of human cells is not a functional receptor for IAV infection and that it has a negative effect on infectivity of IAV possessing Neu5Gc binding ability. IAV also binds to non-sialo 3-O-sulfated galactosylceramide (sulfatide). Sulfatide has been suggested to be a functional receptor for IAV infection. However, we have shown that sulfatide is not a functional receptor for IAV infection and that the binding of HA with sulfatide enhances progeny virus production. It is expected that functions of these glyco-molecules can be used in prevention and development of new drugs against IAV.

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.

Preferential Accumulation of 14C-N-Glycolylneuraminic Acid over 14C-N-Acetylneuraminic Acid in the Rat Brain after Tail Vein Injection.

The two main molecular species of sialic acid existing in nature are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Neu5Ac is abundant in mammalian brains and plays crucial roles in many neural functions. In contrast, Neu5Gc is present only at a trace level in vertebrate brains. The brain-specific suppression of Neu5Gc synthesis, which is a common feature in mammals, suggests that Neu5Gc has toxicity against brain functions. However, in vivo kinetics of Neu5Gc in the whole body, especially in the brain, has not been studied in sufficient detail. To determine the in vivo kinetics of Neu5Gc, 14C-Neu5Gc was enzymatically synthesized and injected into rat tail veins. Although most of 14C-Neu5Gc was excreted in urine, a small amount of 14C-Neu5Gc was detected in the brain. Brain autoradiography indicated that 14C-Neu5Gc was accumulated predominantly in the hippocampus. 14C-Neu5Gc transferred into the brain was incorporated into gangliosides including GM1, GD1a, GD1b, GT1b and GQ1b. Reduction of 14C-Neu5Gc after intracerebroventricular infusion was slower than that of 14C-Neu5Ac in the brain and hippocampus. The results suggest that Neu5Gc is transferred from blood into the brain across the blood brain barrier and accumulates in the brain more preferentially than does Neu5Ac.

Identification of amino acid residues of influenza A virus H3 HA contributing to the recognition of molecular species of sialic acid

To identify a determinant of human H3 hemagglutinin (HA) amino acid residues linked to the recognition of molecular species of sialic acid, we generated six mutant viruses possessing either the wild-type HA gene from A/Memphis/1/71 (H3N2) or a genetically single-mutated HA gene at position 137, 144, 155, 158 or 193 from a genetic backbone of A/WSN/33 (H1N1) by reverse genetics. We evaluated the binding ability with four types of synthetic sialylglycolipids. The results indicate that the amino acid substitutions Thr155 to Tyr and Glu158 to Gly in H3 HA facilitate virus binding to N-glycolylneuraminic acid.

Edible bird's nest extract inhibits influenza virus infection

Edible bird's nest (EBN) is the nest of the swift that is made from its saliva. Although EBN has been widely used for enhancing immunocompetence, its antiviral efficacy has not been studied in detail. We found that EBN extract could strongly inhibit infection with influenza viruses in a host range-independent manner when it was hydrolyzed with Pancreatin F. Western blotting assay showed that the EBN extract bound to influenza virus. Furthermore, EBN extract could neutralize the infection of MDCK cells with influenza viruses and inhibit hemagglutination of influenza viruses to erythrocytes, but it could not inhibit the activity of influenza virus sialidase. Fluorometric HPLC indicated that the major molecular species of sialic acid in EBN is N-acetylneuraminic acid. The results suggest that EBN is a safe and valid natural source for the prevention of influenza viruses.

Host mediated variation and receptor binding specificity of influenza viruses.

There are at least two possible mechanisms for the host range variation of influenza A viruses. First mechanism is the pressure of the antibody, this results has already reported by us, in 1989(12). In this case, Ser 205 which is located in antigenic site D in the hemagglutinin trimer. When Ser 205 is substituted to Tyr, receptor binding specificity become 2-6 predominant rather than 2-3 predominant. Ser205 is located far away from the receptor binding pocket in the subunit itself, however, very close to the pocket in the next subunit. When the Ser 205 is substituted to bulky amino acid Tyr, the three dimensional structure of the receptor binding pocket in next subunit may be influenced to become 2-6 binding property rather than 2-3 binding. Second mechanism is the selective pressure for the appearance of host cell variant with altered receptor binding specificities. In this case, amino acid 226 located in receptor binding pocket is clitical for the recognition of 2-6 and 2-3 linkages. Only single amino acid alteration of Leu 226 to Gln made remarkable change of receptor binding specificity 2-6 to 2-3. In this study, we also identified amino acid 155 Thr, 158 Glu, and 228 Ser of the hemagglutinin which may be clitical for the recognition of the molecular species of sialic acid, such as Neu5Ac and Neu5Gc. Very recently, we reported the amino acid residues contributing to the substrate specificity of the influenza A virus sialidase. If these amino acid alteration occurres in the host animal bodies in nature, the virus aquires the new receptor binding specificity which allows the infection to the different animal species, such as bird to human population. This process may be one of the important mechanism for the host mediated variation, and the influenza virus transmission between different hosts, and also emerging of new subtypes of influenza viruses in human population.

Swine influenza virus strains recognize sialylsugar chains containing the molecular species of sialic acid predominantly present in the swine tracheal epithelium

We determined the ratio of N-glycolylneuraminic acid (Neu5Gc) to N-acetylneuraminic acid (Neu5Ac) in swine respiratory epithelia by fluorometric high-performance liquid chromatography, and examined the binding specificity of swine influenza virus strains for gangliosides containing different molecular species of sialic acid (Neu5Ac and Neu5Gc), and for bovine erythrocyte sialoglycoprotein 2 (GP-2) containing Neu5Gc as its predominate sialic acid (96% of total sialic acids). The presence of Neu5Gc, which had not been detected in human tracheal epithelia, and Neu5Ac in swine tracheal epithelia was observed in a 1:1 ratio. The swine influenza virus H1 and H3 isolates tested, except for A/swine/Iowa/15/30 (H1N1), displayed a marked binding ability for sialylsugar chains containing Neu5Gc compared with that of the human influenza virus strains. These results suggest that swine influenza viruses recognize sialylsugar chains containing the molecular species of sialic acid present predominantly in the swine tracheal epithelium. © 1997 Federation of European Biochemical Societies.

Structural determination of gangliosides that bind to influenza A, B, and C viruses by an improved binding assay: Strain-specific receptor epitopes in sialo-sugar chains

An improved binding assay for detection of ganglioside receptors for influenza A, B, and C viruses was developed. In this system, the virions bound to gangliosides that were developed on a silica gel thin-layer plate were detected by mouse monoclonal antibody against viral hemagglutinin and peroxidase-conjugated anti-mouse immunoglobrin. No hydrolysis of the gangliosides by viral receptor-destroying enzyme was detected in the present condition. The reactivity of the viruses to gangliosides depended on the amount of developed gangliosides (10 pmols-10 nmols), the molecular species of sialic acid, and their sugar sequences. Human influenza A (PR/8/34), B (Lee/40), and C (Ann Arbor/1/50) viruses bound different receptor epitopes of sialo-sugar chains of gangliosides. The A/PR/8 virus bound most effectively to Neu5Ac-containing lacto-series gangliosides carrying type I and type II sugar chains, followed by ganglio-series and hematoside-series gangliosides. The A/PR/8 virus weakly bound to Neu5Ac α2,6lactotetraosylceramide [IV 6(Neu5Ac)Lc4Cer] and Neu5Ac α2,6paragloboside [IV 6(Neu5Ac)nLc4Cer] carrying Neu5Ac α2,6Gal sequence, although thoir Neu5Ac α2,3Gal derivatives were the most potent gangliosides tested. B/Lee/40 bound restrictively to IV 6(Neu5Ac)Lc4Cer and IV 6(Neu5Ac)nLc4Cer, which carry Neu5Ac α2,6Gal sequence, and type I and type II lacto-series sugar chain, respectively. C/Ann Arbor/1/50 reacted only with 9-O-Ac-Neu5Ac-carrying sugar chains in all the gangliosides tested. This method also allowed the microanalysis of receptor gangliosides of unknown samples. ESK cells, sensitive to the influenza A viruses infection, expressed several kinds of receptor active gangliosides, while those from ESK-R cells, resistant to the virus infection, were undetectable.

Characterization of gangliosides of porcine erythrocyte membranes: occurrence of ganglioside GD3 as major ganglioside.

Four major ganglioside species were isolated from porcine erythrocyte membranes by DEAE-Sephadex and Iatrobeads column chromatography. Treatment of the lipids with graded neuraminidase and beta-galactosidase, gas chromatographic analysis of their carbohydrates, sphingosine bases and molecular species of sialic acid revealed that the structure of these gangliosides were GM3(NeuAc), GM3(NeuGc), GD3(NeuAc) and GD3(NeuGc), each of which was 16 +/- 2 micrograms, 304 +/- 42 micrograms, 30 +/- 3 micrograms and 240 +/- 26 micrograms, respectively, per gram of the dry erythrocyte stroma. The amount of GM3 and GD3 accounted for more than 95% of total gangliosides of the erythrocytes. Porcine erythrocytes may provide a good source for large scale preparation of ganglioside GD3 which recently was identified as a human melanoma-associated antigen.

Characterization of 2,7-anhydro-N-acetylneuraminic acid in human wet cerumen.

A molecular species of sialic acid was isolated in a free form from cerumen of the wet type, but not of the dry type, by an ion-exchange column chromatography and preparative high-performance liquid chromatography. Structural analysis of this sialic acid was performed by gas-liquid chromatography/mass spectrometry with chemical ionization (CI) and electron ionization (EI). In the CI mass spectra, the protonated molecular ion of the trimethylsilyl derivative was observed at m/z 580. and that of the methyl ester-trimethylsilyl derivative was at m/z 522. In the EI mass spectrum, the methyl ester-trimethylsilyl derivative gave characteristic ions at m/z 506, 462, 418, 416, 328, 316, 238, 228, 205, 186, and 173. This mass spectrum was identical with that of 2,7-anhydro-N-acetylneuraminic acid, which was reported by Lifely and Cottee (Carbohydr. Res. 107, 187-197, 1982) as the mass spectrum of a by-product prepared from N-acetylneuraminic acid by methanolysis. These results indicate that the compound in the wet cerumen is 2,7-anhydro-N-acetylneuraminic acid. Since this sialic acid species could not be detected in cerumens of the dry type, its formation in the wet type may be controlled by an autosomal dominant gene.

N-Acetylneuraminyllactosylceramide, GM3-NeuAc, a new influenza A virus receptor which mediates the adsorption-fusion process of viral infection. Binding specificity of influenza virus A/Aichi/2/68 (H3N2) to membrane-associated GM3 with different molecular species of sialic acid.

Agglutinates of native chicken erythrocytes caused by influenza virus A/Aichi/2/68 (H3N2) at 4 degrees C were potently fused and lysed at low pH (optimum pH 5.3) at 37 degrees C. Exogenous gangliosides GM3 (Sia alpha 2-3Gal beta 1-4Glc beta 1-ceramide) and GM2 (GalNAc beta 1-4(Sia alpha 2-3)-Gal beta 1-4Glc beta 1-ceramide) were integrated into the membranes of chicken asialoerythrocytes within 5-min incubation at 37 degrees C. We found that the incorporation of ganglioside GM3 containing N-acetylneuraminic acid into asialoerythrocytes restored the biological responsiveness to the virus as established by agglutination at 4 degrees C and fusion and hemolysis at 37 degrees C at pH 5.3. Biological responsiveness of GM3-NeuAc-erythrocytes to the virus was considerably higher than that of GM3-NeuGc-erythrocytes under the same experimental conditions. Treatment of the GM3-NeuAc-erythrocytes with neuraminidase again resulted in the complete abolishment of the response to the virus. Erythrocytes containing GM2-NeuAc showed no detectable biological responses toward the virus. The above results indicate that the hemagglutinin of influenza virus A/Aichi/2/68 (H3N2) recognizes the sialyloligosaccharide chain of ganglioside GM3 as its receptor which mediates the adsorption and fusion process on the virus entry into the host cells and has more preferential specificity for binding to N-acetylneuraminic acid-containing GM3 than that to N-glycolyl type in the target cell membranes.