Tadanobu Takahashi

Role of sulfatide in normal and pathological cells and tissues

Sulfatide is 3-O-sulfogalactosylceramide that is synthesized by two transferases (ceramide galactosyltransferase and cerebroside sulfotransferase) from ceramide and is specifically degraded by a sulfatase (arylsulfatase A). Sulfatide is a multifunctional molecule for various biological fields including the nervous system, insulin secretion, immune system, hemostasis/thrombosis, bacterial infection, and virus infection. Therefore, abnormal metabolism or expression change of sulfatide could cause various diseases. Here, we discuss the important biological roles of sulfatide in the nervous system, insulin secretion, immune system, hemostasis/thrombosis, cancer, and microbial infections including human immunodeficiency virus and influenza A virus. Our review will be helpful to achieve a comprehensive understanding of sulfatide, which serves as a fundamental target of prevention of and therapy for nervous disorders, diabetes mellitus, immunological diseases, cancer, and infectious diseases

Edible bird's nest extract inhibits influenza virus infection

Abstract Edible birds 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.

Sialidase Activity of Influenza A Virus in an Endocytic Pathway Enhances Viral Replication

ABSTRACT N2 neuraminidase (NA) genes of the 1957 and 1968 pandemic influenza virus strains possessed avian-like low-pH stability of sialidase activity, unlike most epidemic strains. We generated four reverse-genetics viruses from a genetic background of A/WSN/33 (H1N1) that included parental N2 NAs of 1968 pandemic (H3N2) and epidemic (H2N2) strains or their counterpart N2 NAs in which the low-pH stability of the sialidase activity was changed by substitutions of one or two amino acid residues. We found that the transfectant viruses bearing low-pH-stable sialidase (WSN/Stable-NAs) showed 25- to 80-times-greater ability to replicate in Madin-Darby canine kidney (MDCK) cells than did the transfectant viruses bearing low-pH-unstable sialidase (WSN/Unstable-NAs). Enzymatic activities of WSN/Stable-NAs were detected in endosomes of MDCK cells after 90 min of virus internalization by in situ fluorescent detection with 5-bromo-4-chloro-indole-3-yl-α-N-acetylneuraminic acid and Fast Red Violet LB. Inhibition of sialidase activity of WSN/Stable-NAs on the endocytic pathway by pretreatment with 4-guanidino-2,4-dideoxy-N-acetylneuraminic acid (zanamivir) resulted in a significant decrease in progeny viruses. In contrast, the enzymatic activities of WSN/Unstable-NAs, the replication of which had no effect on pretreatment with zanamivir, were undetectable in cells under the same conditions. Hemadsorption assays of transfectant-virus-infected cells revealed that the low-pH stability of the sialidase had no effect on the process of removal of sialic acid from hemagglutinin in the Golgi regions. Moreover, high titers of viruses were recovered from the lungs of mice infected with WSN/Stable-NAs on day 3 after intranasal inoculation, but WSN/Unstable-NAs were cleared from the lungs of the mice. These results indicate that sialidase activity in late endosome/lysosome traffic enhances influenza A virus replication.

Sulfatide Is Required for Efficient Replication of Influenza A Virus

ABSTRACT Sulfatide is abundantly expressed in various mammalian organs, including the intestines and trachea, in which influenza A viruses (IAVs) replicate. However, the function of sulfatide in IAV infection remains unknown. Sulfatide is synthesized by two transferases, ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST), and is degraded by arylsulfatase A (ASA). In this study, we demonstrated that sulfatide enhanced IAV replication through efficient translocation of the newly synthesized IAV nucleoprotein (NP) from the nucleus to the cytoplasm, by using genetically produced cells in which sulfatide expression was down-regulated by RNA interference against CST mRNA or overexpression of the ASA gene and in which sulfatide expression was up-regulated by overexpression of both the CST and CGT genes. Treatment of IAV-infected cells with an antisulfatide monoclonal antibody (MAb) or an anti-hemagglutinin (HA) MAb, which blocks the binding of IAV and sulfatide, resulted in a significant reduction in IAV replication and accumulation of the viral NP in the nucleus. Furthermore, antisulfatide MAb protected mice against lethal challenge with pathogenic influenza A/WSN/33 (H1N1) virus. These results indicate that association of sulfatide with HA delivered to the cell surface induces translocation of the newly synthesized IAV ribonucleoprotein complexes from the nucleus to the cytoplasm. Our findings provide new insights into IAV replication and suggest new therapeutic strategies.

Antiviral effect of strictinin on influenza virus replication

Strictinin, which is a member of the ellagitanin family of hydrolyzable tannins, prevented replication of human, duck and swine influenza A viruses (IAVs) in vitro at non-toxic concentrations. The addition of strictinin at the same time as IAV inoculation to MDCK cells inhibited viral replication in a dose-dependent manner. Strictinin showed 50% inhibitory concentrations for IAVs from 0.09±0.021 to 0.28±0.037μM (mean±S.E.M.) by the focus-forming assay. Treatment of MDCK cells with strictinin before and after viral inoculation resulted in no significant antiviral activity. Further studies showed that strictinin inhibited IAV-induced hemifusion. However, strictinin exhibited no inhibitory effect against receptor binding, sialidase activity. Strictinin also showed an antiviral effect on influenza B virus and human parainfluenza virus type-1 in vitro. The results indicate that strictinin is a useful antiviral agent.

Down-regulation of TRPM6-mediated magnesium influx by cyclosporin A

Transient receptor potential melastatin 6 (TRPM6) is distributed along the apical membrane of the renal tubular cells and is involved in the reabsorption of magnesium. In this study, we show that TRPM6 expression is suppressed by cyclosporin A (CsA) via a down-regulation of c-Fos expression. TRPM6 was expressed in NRK-52E, but not in Madin-Darby canine kidney cells. In contrast, its homolog, TRPM7, was equally expressed in both cells. In NRK-52E cells, CsA dose-dependently decreased TRPM6 expression without affecting TRPM7 expression. Magnesium load measurements revealed the rise in the intracellular free magnesium concentration ([Mg2+]i) to be inhibited by CsA. The transfection of TRPM6 siRNA decreased TRPM6 expression without affecting TRPM7 expression and inhibited the elevation of [Mg2+]i. CsA did not affect the intracellular distribution of nuclear factor of activated T cells (NFATc). Furthermore, TRPM6 expression was not changed by a NFATc inhibitor. Next, we examined the effect of CsA on the transcription factors c-Fos and c-Jun. CsA decreased c-Fos expression without affecting c-Jun expression. The transfection of c-Fos siRNA suppressed TRPM6 expression without affecting TRPM7 expression. We suggest that CsA decreases TRPM6 expression mediated by inhibition of c-Fos transcription, resulting in a decrease of renal Mg2+ reabsorption.

The expression of sialylated high-antennary N-glycans in edible bird’s nest

Edible birds nest (EBN) is the nest made from the saliva of Collocalia swift. Recently, we have found that EBN extract could strongly inhibit infection of influenza viruses in a host-range-independent manner [Guo, C. T.; Takahashi, T.; Bukawa, W.; Takahashi, N.; Yagi, H.; Kato, K.; Hidari, K. I.; Miyamoto, D.; Suzuki, T.; Suzuki, Y. Antiviral Res.2006, 70, 140-146]. Although this antiviral activity might be attributed to O- or N-glycoconjugates, no N-glycan structures have so far been described for EBN. Here, we report the N-glycosylation profile of EBN, in which a tri-antennary N-glycan bearing the alpha2,3-N-acetylneuraminic acid residues is displayed as a major component. We suggest that the sialylated high-antennary N-glycans of EBN contribute to the inhibition of influenza viral infection.

Inhibition of human parainfluenza virus type 1 sialidase by analogs of 2-deoxy-2,3-didehydro- N-acetylneuraminic acid

Eleven novel analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) modified at the C-4 and C-9 positions were designed and tested for their ability to inhibit sialidase of human parainfluenza virus type 1 (hPIV-1). The analogs modified by the cyanomethyl, amidinomethyl, and thiocarbamoylmethyl groups at the C-4 position exhibited potent inhibition against hPIV-1 sialidase compared with Neu5Ac2en. The most effective compound was thiocarbamoylmethyl analog (4-O-thiocarbamoylmethyl-Neu5Ac2en). The activity of 4-O-thiocarbamoylmethyl-Neu5Ac2en causing 50% enzyme inhibition at a concentration of approximately 1.0×10−5M was 30-fold larger than Neu5Ac2en. While, the analogs of Neu5Ac2en modified by the azido and N-acetyl groups at the C-9 showed a decrease in inhibition of sialidase compared with the 9-hydroxy analogs. In addition, 4-O-thiocarbamoylmethyl-Neu5Ac2en strongly inhibited hPIV-1 infections of Lewis lung carcinoma-monkey kidney cells in comparison with Neu5Ac2en. The present findings would provide useful information for the development of anti-human parainfluenza virus compounds.

A molecular mechanism for the low-pH stability of sialidase activity of influenza A virus N2 neuraminidases1

Four human pandemic influenza A virus strains isolated in 1957 and 1968, but not most of the epidemic strains isolated after 1968, possess sialidase activity under low‐pH conditions. Here, we used cell‐expressed neuraminidases (NAs) to determine the region of the N2 NA that is associated with low‐pH stability of sialidase activity. We found that consensus amino acid regions responsible for low‐pH stability did not exist in pandemic NAs but that two amino acid substitutions in the low‐pH‐stable A/Hong Kong/1/68 (H3N2) NA and a single substitution in the low‐pH‐unstable A/Texas/68 (H2N2) NA resulted in significant change in low‐pH stability.

Claudin-16 Is Directly Phosphorylated by Protein Kinase A Independently of a Vasodilator-Stimulated Phosphoprotein-Mediated Pathway

Claudin‐16 (CLDN‐16) is involved in the paracellular reabsorption of Mg2+ in the thick ascending limb of Henle. The tight junctional localization and Mg2+ transport of CLDN‐16 are regulated by cAMP/PKA‐dependent phosphorylation. Here, we examined whether PKA phosphorylates CLDN‐16 in a direct or indirect manner. CLDN‐16 was stably expressed in Madin‐Darby canine kidney (MDCK) cells using a Tet‐OFF system. The phosphorylation of CLDN‐16 is upregulated by fetal calf serum (FCS). This phosphorylation was completely inhibited by a PKA inhibitor, N‐[2‐(p‐bromocinnamylamino)ethyl]‐5‐isoquinolinesulfonamide dihydrochloride. Without FCS, dibutyryl cAMP (DBcAMP) increased the phosphoserine level of CLDN‐16 in a concentration‐dependent manner. The phosphorylated CLDN‐16 elicited increases of transepithelial electrical resistance (TER) and transepithelial transport of Mg2+. Vasodilator‐stimulated phosphoprotein (VASP) was also phosphorylated in the presence of FCS or DBcAMP. In the glutathione‐S‐transferase (GST) pull down assay, a cytosolic carboxyl domain of CLDN‐16 was associated with PKA, but not with VASP. Furthermore, PKA was immunoprecipitated with CLDN‐16 in MDCK cells, but VASP was not. In cells expressing a dephosphorylated mutant (Ser160Ala) of VASP, CLDN‐16 was phosphorylated by DBcAMP and was associated with ZO‐1, a tight junctional‐scaffolding protein, without integral cell–cell junctions. We suggest that PKA directly phosphorylates CLDN‐16, resulting in the localization to tight junctions (TJs) and the maintenance of Mg2+ reabsorption. J. Cell. Physiol. 214:221–229, 2008.