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Tadashi Tai

Publications and source records attributed to Tadashi Tai.

13 recordsLinked to original sources
PubMed2006

Identification of N-glycan-binding proteins for E3 ubiquitin ligases.

N-glycans serve as a degradation signal by the SCF(Fbx2) ubiquitin ligase complex in the cytosol. Fbx2, an F-box protein, binds specifically to proteins attached with N-linked high-mannose type oligosaccharides, and subsequently contributes to ubiquitination of glycoproteins. Pre-integrin beta1 is identified as one of the Fbx2 targets. These two proteins bind in the cytosol after inhibition of the proteasome. These results indicate that SCF(Fbx2) ubiquitinates N-linked glycoproteins, which are translocated from the endoplasmic reticulum to the cytosol by the quality control mechanism. This chapter describes methods, including a binding protein assay for N-glycans, a ubiquitination assay for N-linked glycoproteins with SCF(Fbx2) ubiquitin ligase complex, an overlay assay for the detection of Fbx2 binding proteins, and a pull-down assay for the interaction between Fbx2 and N-linked glycoproteins, used to identify N-glycan-binding proteins for E3 ubiquitin ligases.

Animals↗
PubMed2005

Production of a recombinant single-chain variable-fragment (scFv) antibody against sulfoglycolipid.

Mammalian sulfoglycolipids are comprised of two major classes of compounds, sulfatide (SO(3)-3Gal-ceramide) and seminolipid (SO(3)-3Gal-alkylacylglycerol). Sulfatide is present in relatively high levels in myelin, and seminolipid is present in testis. The sulfation of these sulfoglycolipids is catalyzed by a common enzyme, cerebroside sulfotransferase (CST). Disruption of the Cst gene in mice revealed that sulfatide and seminolipid are essential for, respectively, myelin formation and spermatogenesis. The present study describes the generation of a recombinant single-chain variable fragment (scFv) antibody against sulfoglycolipid, for use in the functional analysis of sulfoglycolipids in living cells. A positive hybridoma producing anti-sulfoglycolipid IgG3, referred to as DI8, was initially obtained by immunizing CST-null mice with an isolated sulfatide. The DI8 monoclonal antibody was found to bind specifically to sulfoglycolipids with the terminal 3-O-sulfated galactose structure, as evidenced by ELISA and thin-layer chromatogram-immunostaining. The antibody stained seminolipid on the cell surface of spermatogenic cells of wild-type testis, but it did not react with any cells in the seminiferous tubules of CST-null testis. Total RNA was extracted from this hybridoma, and cDNAs that encode the variable regions of the heavy and light chains of IgG3 were obtained by RT-PCR. These DNA fragments were linked through a DNA linker coding (Gly(4)Ser)(3), and the recombinant scFv fragment was then inserted into a phagemid vector pCANTAB 5E. The scFv antibody that was displayed at the tip of the M13 phage in the form of a g3p fusion protein bound to sulfatide. Furthermore, a soluble form of the scFv antibody was also found to bind to the sulfoglycolipids in ELISA.

Amino Acid Sequence↗
PubMed2004

Galactosylceramide expression factor-1 induces myogenesis in MDCK and C3H10T1/2 cells.

We previously reported that galactosylceramide expression factor-1 (GEF-1), a rat homolog of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs/Hgs), induces galactosylceramide and/or sulfatide expression and morphological changes in epithelial cells. Here, we show that GEF-1 induces myogenesis in MDCK and C3H10T1/2 cells. GEF-1 overexpression in MDCK cells (MDCK/GEF-1) appeared to promote trans-differentiation to myoblasts that expressed MyoD and myosin heavy chain (MHC). MDCK/GEF-1 cells also expressed several DNA-binding proteins (MyoD and MEF-2) that are essential for myogenesis. These results suggest that GEF-1 induces MDCK cells to enter an early stage of myogenesis. Subsequently, we tested whether GEF-1 could induce myogenesis in C3H10T1/2 mouse fibroblasts, which have the potential to differentiate into myoblast-like cells. Indeed, GEF-1 induced morphological changes that were consistent with myoblast-like cells, and both MyoD and MHC were expressed. Our results suggest that GEF-1 may induce MDCK and C3H10T1/2 cells to trans-differentiate into myoblast-like cells.

Adenosine Triphosphatases↗
PubMed2004

Synaptic function of cholinergic-specific Chol-1alpha ganglioside.

The function of a cholinergic-specific ganglioside, Chol-1alpha, was investigated. The release of acetylcholine from synaptosomes was inhibited by anti-Chol-1alpha monoclonal antibody but not by monoclonal antibodies against other brain gangliosides tested. Chol-1alpha ganglioside stimulated the high-affinity choline uptake by synaptosomes and consequently enhanced acetylcholine synthesis, resulting in an increased release of acetylcholine from synaptosomes. The memory and learning abilities of rats given anti-Chol-1alpha antibody were remarkably suppressed. These in vitro and in vivo studies suggest that Chol-1alpha ganglioside plays a pivotal role in cholinergic synaptic transmission and participates in cognitive function.

Animals↗
PubMed2003

Fbs2 is a new member of the E3 ubiquitin ligase family that recognizes sugar chains.

F-box proteins are substrate recognition components of Skp1-Cullin1-F-box protein-Roc1 (SCF) E3 ubiquitin-protein ligases. We reported previously that Fbs1 (F-box protein that recognizes sugar chains; equivalent to Fbx2 or NFB42) binds specifically to proteins attached with high mannose oligosaccharides and subsequently contributes to elimination of N-glycoproteins in cytosol (Yoshida, Y., Chiba, T., Tokunaga, F., Kawasaki, H., Iwai, K., Suzuki, T., Ito, Y., Matsuoka, K., Yoshida, M., Tanaka, K., and Tai, T. (2002) Nature 418, 438-442). Here we report the identification of another F-box protein that recognizes N-glycan, Fbs2 (called Fbx6b or FBG2 previously). Although the expression of Fbs1 was restricted to the adult brain and testis, the Fbs2 transcript was widely expressed. The Fbs2 protein forms an SCFFbs2 ubiquitinligase complex that targets sugar chains in N-glycoproteins for ubiquitylation. Only glycoproteins bound to concanavalin A lectin and not to wheat germ agglutinin or Ricinus communis agglutinin interacted with Fbs2 in various tissues and cell lines. Pull-down analysis using various oligosaccharides revealed that Man3-9GlcNAc2 glycans were required for efficient Fbs2 binding, whereas modifications of mannose residues by other sugars or deletion of inner GlcNAc reduced Fbs2 binding. Fbs2 interacted with N-glycans of T-cell receptor alpha-subunit (TCRalpha), a typical substrate of the endoplasmic reticulum-associated degradation (ERAD) pathway, and the forced expression of mutant Fbs2DeltaF, which lacks the F-box domain essential for forming the SCF complex, and decrease of endogenous Fbs2 by small interfering RNA led to inhibition of TCRalpha degradation in cells. Thus, Fbs2 is a novel member of F-box protein family that recognizes N-glycans and plays a role in ERAD.

Animals↗
PubMed2003

Expansion of natural killer cells in mice transgenic for IgM antibody to ganglioside GD2: demonstration of prolonged survival after challenge with syngeneic tumor cells.

IgM antibodies to gangliosides, sialic acid-containing glycosphingolipids, have been shown to mediate anti-tumor effects in cancer patients with melanoma and neuroblastoma and to correlate with survival. Mechanisms by which the antibodies induce tumor suppression, however, have not been systematically studied. To investigate this point, we produced and characterized C57BL/6 mice transgenic for IgM antibody to ganglioside GD2. The transgenic (TG) mice showed high IgM, but not IgG antibody titers against GD2 in their sera. No significant clinical symptoms were observed. When EL4 cells, syngeneic T lymphoma that express ganglioside GD2, were injected into TG mice, prolonged survival was observed. Complement-dependent cytotoxicity (CDC) of EL4 cells was mediated with TG mice sera. Neither antibody-dependent cellular cytotoxicity with their sera nor cytotoxic T lymphocyte activity to EL4 cells was shown in TG mice. Spleen lymphocytes from TG mice had increased numbers of natural killer (NK) cells, but not T cells, B cells, or macrophages compared with wild-type mice. Depletion of NK cells with anti-asialo GM1 rabbit serum reduced or abrogated the observed anti-tumor effects, suggesting that NK cells play a major role in tumor eradication or suppression. NK cell activity in TG mice was much higher than wild-type mice. Moreover, TG mice showed prolonged survival after injection with syngeneic B16 melanoma cells, which express GM3, but not GD2 or GD3. Taking these results together, our studies demonstrate that the TG mice have significant anti-tumor characteristics, probably due to CDC and NK cell expansion and activation with anti-ganglioside GD2 antibody.

Animals↗
PubMed2002

E3 ubiquitin ligase that recognizes sugar chains.

N-glycosylation of proteins in the endoplasmic reticulum (ER) has a central role in protein quality control. Here we report that N-glycan serves as a signal for degradation by the Skp1-Cullin1-Fbx2-Roc1 (SCF(Fbx2)) ubiquitin ligase complex. The F-box protein Fbx2 (ref. 4) binds specifically to proteins attached to N-linked high-mannose oligosaccharides and subsequently contributes to ubiquitination of N-glycosylated proteins. Pre-integrin beta 1 is a target of Fbx2; these two proteins interact in the cytosol after inhibition of the proteasome. In addition, expression of the mutant Fbx2 Delta F, which lacks the F-box domain that is essential for forming the SCF complex, appreciably blocks degradation of typical substrates of the ER-associated degradation pathway. Our results indicate that SCF(Fbx2) ubiquitinates N-glycosylated proteins that are translocated from the ER to the cytosol by the quality control mechanism.

Animals↗
PubMed2002

Molecular cloning and characterization of galactosylceramide expression factor-1 (GEF-1).

A rat brain cDNA clone has been isolated using a eukaryotic cell transient expression system with anti-galactosylceramide (GalCer) monoclonal antibody (MAb), that induces GalCer expression in COS-7 cells. The protein was designated as GalCer expression factor-1 (GEF-1). The deduced amino acid sequences revealed a strikingly high homology to a mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), but no homology to UDP-galactose: ceramide galactosyltransferase. COS-7 cells transfected with the cDNA clone showed dramatic morphological changes and cell growth suppression. Overexpression of GEF-1 in MDCK (MDCK/GEF-1) cells showed GalCer-derived sulfatide expression as well as morphological changes, but not cell growth suppression. The enzyme activity and the mRNA level of CGT increased significantly in MDCK/GEF-1 cells compared with control cells. Taking these results together, it is suggested that GEF-1 may play an important role in regulating GalCer and sulfatide expression in the epithelial cells as well as in the brain.

Adenosine Triphosphatases↗
PubMed2002

Association of GM4 ganglioside with the membrane surrounding lipid droplets in shark liver.

By TLC, GM4 was found to be the major ganglioside in the liver of six shark species examined: Odontaspis taurus, Negaprion brevirostris, Sphyrna lewini, Mustelus griseus, Mustelus manazo, and Prionace glauca. A detailed analysis of the glycosphingolipids (GSLs) in the liver of O. taurus (sand tiger shark) showed that it contained approximately 110 nmol of lipid-bound sialic acid per gram of wet tissue, of which 80% was GM4. By extracting the liver of O. taurus with chloroform/methanol, followed by chromatographic separation of GSLs using DEAE-Sephadex A-25 and Iatrobeads columns, we have isolated GM4 in pure form with a yield of approximately 5 mg per 100 g of wet tissue. The structures of both the sugar chain and the ceramide moiety of this GM4 were analyzed by chemical analysis, mass spectrometry, and NMR spectroscopy. Similar to GM4 isolated from other sources, 92% of fatty acids in the ceramide of this GM4 were 2-hydroxylated. However, unlike the long-chain bases found in other GSLs, the total long-chain bases in this GM4 were found to contain 43% octadecasphingenine and 50% nonadecasphingenine. Immunohistochemical analysis using a monoclonal antibody against GM4 revealed that the hepatocytes of both M. griseus (spotless smooth hound) and M. manazo (smooth hound) were filled with lipid droplets and GM4 was primarily associated with the membrane structure surrounding lipid droplets.

Animals↗