Kazuya Kabayama

Tokai University, Hiratuka, Kanagawa, Japan

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Publications (27)84.37 Total impact

  • e-Journal of Surface Science and Nanotechnology 01/2014; 12:400-403. DOI:10.1380/ejssnt.2014.400
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    ABSTRACT: Compared to other organs, the mouse thymus exhibits a high level of sialidase activity in both the soluble and crude membrane fractions, as measured at neutral pH using 4-methylumbelliferyl 5-acetyl neuraminic acid as a substrate. The main purpose of this study is to identify the sialidase with a high level of the activity at neutral pH in the crude membrane. Several parameters were analyzed using the soluble (S) fraction, N and D fractions that were obtained by NP-40 or DOC/NP-40 solubilization from the thymus crude membrane. The main sialidase activity in the N fraction exhibited almost the same isoelectric point to that of soluble Neu2 and 60% of the activity was removed from the membrane by three washes with 10 mM Tris-buffer, at pH 7.0. The N fraction preferentially hydrolyzed the sialic acid bond of glycoprotein and exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5. The same activity was observed in a plasma membrane-rich fraction. To date, the removal of sialic acid from fetuin at pH 7.0 was reported only with soluble Neu2 and the membrane fraction from Neu2-transfected COS cells. We analyzed the gene that controls the sialidase activity in the crude membrane fraction at pH 7.0 using SMXA recombinant mice and found that compared with other three genes, Neu2 presented the best correlation with the activity level. We suggest that Neu2 is most likely responsible for the main activity in the N fraction, due to its association with the membrane by an unknown mechanism.
    Microbiology and Immunology 06/2013; DOI:10.1111/1348-0421.12076 · 1.31 Impact Factor
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    ABSTRACT: Extraction of thin-layer chromatography (TLC) spot with organic solvents is one of the convenient methods for glycolipid isolation from crude samples, but other background contaminants are also co-extracted during this process. Calcium sulfate (gypsum), starch and synthetic polymers are commonly used as binders in TLC plates, and the co-extracted synthetic polymers usually interfere with further structural analyses, especially in analysis by mass spectrometry (MS). Several column chromatographic processes are therefore used to purify these extracts. However, the purification processes are time consuming and suffer from sample loss. In our previous study, we established a method for detergent removal from glycolipids in a detergent-resistant membrane microdomain (DRM). The procedure is based on selective detergent extraction, in which the sample is dried in a glass tube, followed by washing with non-polar organic solvents, 1,2-dichloroethane (DCE). In this study, we have attempted to apply this method for the background removal from gangliosides and glycosylinositolphosphoceramides extracted from high performance TLC (HPTLC) spots. After DCE washing, these glycolipids were detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, and it was found that DCE washing could effectively remove HPTLC backgrounds.
    Analytical methods 01/2013; 5(23):6617-6621. DOI:10.1039/C3AY41011K · 1.94 Impact Factor
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    ABSTRACT: Brine shrimp are primitive crustacean arthropodal model organisms, second to daphnia, which can survive in high-salinity environments. Their oviposited cysts, cuticle-covered diapausing eggs, are highly resistant to dryness. To elucidate specialties of brine shrimp, this study characterized glycosphingolipids, which are signal transduction-associated material. A group of novel and complex fucosyl glycosphingolipids were separated and identified from cysts of the brine shrimp Artemia franciscana by repeated lipid extraction, alkaline methanolysis, acid treatment, successive column chromatography, and post-source decay measurements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of the glycosphingolipids were elucidated by conventional structural characterization and mass spectrometry, and the compounds were identified as GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer. These compounds also contained a branching, non-arthro-series disaccharide with an α-GlcNAc terminus, similar to that found in a previously reported ceramide hexasaccharide (III3(GlcNAcα2Fucα)-At4Cer). The glycans within these complex GSLs are longer than reported glycans of the animal kingdom containing α-GlcNAc terminus. These complex GSLs as well as the longest GSL with ten sugar residues, ceramide decasaccharide (CDeS), contain the fucosylated LacdiNAc sequence reported to associate with parasitism/immunosuppression and the α-GlcNAc terminus reported to show a certain antibacterial effect in other reports. CDeS, the longest GSL of this species, was found in the highest amount, which indicates that CDeS may be functionally important. Electronic supplementary material The online version of this article (doi:10.1007/s10719-012-9436-8) contains supplementary material, which is available to authorized users.
    Glycoconjugate Journal 08/2012; 30(3). DOI:10.1007/s10719-012-9436-8 · 1.95 Impact Factor
  • Yusuke Suzuki, Kazuya Kabayama
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    ABSTRACT: Although detergents are often essential in protocols, they are usually incompatible with further biochemical analysis. There are several methods for detergent removal, but the procedures are complicated or suffer from sample loss. Here, we describe a convenient and rapid method for detergent removal from sialic acid-containing glycosphingolipids (gangliosides) and neutral glycolipids in detergent-resistant membrane (DRM) microdomain. It is based on selective detergent extraction, in which the sample is dried on a glass tube, followed by washing with organic solvent. We investigated 18 organic solvents and used high performance thin-layer chromatography (HPTLC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) to confirm that dichloroethane (DCE) was the most suitable solvent and completely removed the nonionic detergent Triton X-100. Furthermore, DCE extraction effectively removed interference caused by other nonionic, zwitterionic, or ionic detergents in MALDI-QIT-TOF MS analysis.
    The Journal of Lipid Research 01/2012; 53(3):599-608. DOI:10.1194/jlr.D020545 · 4.73 Impact Factor
  • Kazuya Kabayama
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    ABSTRACT: Membrane lateral heterogeneity is accepted as a requirement for the function of biological membranes, and the notion of the "raft/microdomain" gives specificity to this concept. Recently, fluorescence-based techniques such as fluorescence recovery after photobleaching (FRAP), single particle tracking (SPT), and fluorescence correlation spectroscopy (FCS) have shown promise for application to the dynamics of membrane molecules in microdomains. We previously revealed, by performing live-cell FRAP and SPT studies, a mechanism of insulin resistance in which dissociation of the insulin receptor (IR)-caveolin-1 (Cav1) complex was caused by an interaction between the IRβ subunit and the ganglioside GM3 cluster, a glycolipid-enriched membrane microdomain. We hoped to demonstrate that an alteration in the lipid component of microdomains affects lateral diffusion of membrane receptors. We therefore established an experimental system for monitoring the membrane organization of receptors by analyzing their lateral diffusion parameters in the plasma membranes of living cells using FRAP and SPT. In this study, measurement of the lateral diffusion of the IR was performed by fitting analysis to fluorescence recovery curves and trace analysis to individual fluorescent spots, which provided the diffusion constant. The results show how fast IR molecules diffuse before and after a change in membrane environment, such as stimulation by cholesterol depression or treatment with a glycosphingolipid (GSL) inhibitor. Using these techniques, we have established a method for determining the diffusion constant for the lateral movement of IR-EGFP, expressed in CHO-K1 cells. We will use these techniques for the lateral diffusion analysis of membrane receptors under other assay conditions, such as use of GSL-deficient cells or pathologic samples.
    YAKUGAKU ZASSHI 01/2012; 132(4):417-23. DOI:10.4052/tigg.20.353 · 0.31 Impact Factor
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    ABSTRACT: Previously, we demonstrated that an inhibitor of ganglioside biosynthesis, d-PDMP, could restore impaired insulin signaling in tumor necrosis factor α (TNFα)-treated adipocytes by blocking the increase of GM3 ganglioside. Here, we analyzed the interaction between insulin receptor (IR) and GM3 in the plasma membranes using immunoelectron microscopy. In normal adipocytes, most GM3 molecules localized at planar and non-caveolar regions. Approximately 19% of IR molecules were detected in caveolar regions. The relative ratio of IRs associated with caveolae in TNFα-treated adipocytes was decreased to one-fifth of that in normal adipocytes, but this decrease was restored by d-PDMP. Thus, we could obtain direct evidence that insulin resistance is a membrane microdomain disorder caused by aberrant expression of ganglioside.
    FEBS letters 12/2011; 586(2):191-5. DOI:10.1016/j.febslet.2011.12.019 · 3.34 Impact Factor
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    ABSTRACT: The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. In SAT-I null mice, hearing ability, assessed by brainstem auditory-evoked potentials (BAEP), was impaired at the onset of hearing and had been completely lost by 17 days after birth (P17), showing a deformity in hair cells in the organ of Corti. By 2 months of age, the organ of Corti had selectively and completely disappeared without effect on balance or motor function or in the histology of vestibule. Interestingly, spatiotemporal changes in localization of individual gangliosides, including GM3 and GT1b, were observed during the postnatal development and maturation of the normal inner ear. GM3 expressed in almost all regions of cochlea at P3, but at the onset of hearing it distinctly localized in stria vascularis, spiral ganglion, and the organ of Corti. In addition, SAT-I null mice maintain the function of stria vascularis, because normal potassium concentration and endocochlear potential of endolymph were observed even when they lost the BAEP completely. Thus, the defect of hearing ability of SAT-I null mice could be attributed to the functional disorganization of the organ of Corti, and the expression of gangliosides, especially GM3, during the early part of the functional maturation of the cochlea could be essential for the acquisition and maintenance of hearing function.
    Proceedings of the National Academy of Sciences 07/2009; 106(23):9483-8. DOI:10.1073/pnas.0903279106 · 9.81 Impact Factor
  • Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 09/2008; 53(12 Suppl):1552-7.
  • Jin-ichi Inokuchi, Kazuya Kabayama
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    ABSTRACT: Caveolae are a subset of membrane microdomains (lipid raft) particularly abundant in adipocytes. Critical dependence of the insulin metabolic signal transduction on caveolae/microdomains in adipocytes has been demonstrated. These microdomains can be biochemically isolated with their detergent insolubility and were designated as detergent resistant microdomains (DRM). Gangliosides are known as structurally and functionally important components in microdomains. We demonstrated that increased GM3 expression was accompanied in the state of insulin resistance in mouse 3T3-L1 adipocytes induced by TNFα and in the adipose tissues of obese/diabetic rodent models such as Zucker fa/fa rats and ob/ob mice (Tagami et al. 2002). We examined the effect of TNFα on the composition and function of DRM in adipocytes and demonstrated that increased GM3 levels result in the elimination of insulin receptor (IR) from the DRM while caveolin and flotillin remain in the DRM, leading to the inhibition of insulin’s metabolic signaling (Kabayama et al. 2005). These findings are further supported by the report that mice lacking GM3 synthase exhibit enhanced insulin signaling (Yamashita et al. 2003). Thus, we present a new pathological feature of insulin resistance in adipocytes induced by TNFα.
    12/2007: pages 333-336;
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    ABSTRACT: Expression of gangliosides and alterations in their composition have been observed during cell proliferation and differentiation and in certain cell cycle phases, brain development and cancer malignancy. To investigate the characteristics of GM3 synthase, SAT-I mRNA and ganglioside GM3 expression levels in lung cancer, we examined the expression levels of SAT-I mRNA as well as GM3 in 40 tumor tissues surgically removed from non-small cell lung cancer patients. Adenocarcinoma tissues expressed SAT-I mRNA levels that were significantly higher than those of squamous and other carcinomas (P < 0.0001). Moreover, the SAT-I mRNA levels were high in the bronchioalveolar carcinoma subtype and low in the solid and mucin subtypes of adenocarcinomas (P = 0.049, 0.049 and 0.013, respectively). To clarify the relationship between SAT-I mRNA and epidermal growth factor receptor (EGFR)-tyrosine kinase (TK) inhibitor sensitivity, we carried out drug sensitivity tests for the EGFR-TK inhibitors gefitinib and AG1478 using eight adenocarcinoma cell lines expressing no EGFR mutations. The IC(50) values for gefitinib and AG1478 decreased dramatically with increasing SAT-I mRNA levels (R(2) = 0.81 and 0.59, respectively), representing a wide range of drug sensitivities among adenocarcinoma cell lines. To explore a possible mechanism of how GM3 could enhance the sensitivity to EGFR-TK inhibitors, the SAT-I gene was introduced stably into a GM3-negative clone of murine 3LL lung cancer cells to produce GM3-reconstituted clones. We found an increase in EGFR protein levels and gefitinib sensitivity in GM3-reconstituted cells, suggesting the involvement of GM3 in the turnover of EGFR protein. Therefore, it is highly expected that, by measuring the expression levels of SAT-I mRNA in lung biopsy samples from non-small cell lung cancer patients, enhanced pathological identification and individualized chemotherapeutic strategies can be established for the appropriate use of EGFR-TK inhibitors.
    Cancer Science 11/2007; 98(10):1625-32. DOI:10.1111/j.1349-7006.2007.00578.x · 3.48 Impact Factor
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    ABSTRACT: Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling. We previously demonstrated that, in adipocytes in a state of TNFalpha-induced insulin resistance, the inhibition of insulin metabolic signaling and the elimination of insulin receptors (IR) from the caveolae microdomains were associated with an accumulation of the ganglioside GM3. To gain insight into molecular mechanisms behind interactions of IR, caveolin-1 (Cav1), and GM3 in adipocytes, we have performed immunoprecipitations, cross-linking studies of IR and GM3, and live cell studies using total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching techniques. We found that (i) IR form complexes with Cav1 and GM3 independently; (ii) in GM3-enriched membranes the mobility of IR is increased by dissociation of the IR-Cav1 interaction; and (iii) the lysine residue localized just above the transmembrane domain of the IR beta-subunit is essential for the interaction of IR with GM3. Because insulin metabolic signal transduction in adipocytes is known to be critically dependent on caveolae, we propose a pathological feature of insulin resistance in adipocytes caused by dissociation of the IR-Cav1 complex by the interactions of IR with GM3 in microdomains.
    Proceedings of the National Academy of Sciences 09/2007; 104(34):13678-83. DOI:10.1073/pnas.0703650104 · 9.81 Impact Factor
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    ABSTRACT: Ganglioside GM3, one of the sialic acid containing glycosphingolipids, is known to form clusters in lipid microdomains, which serve as platforms for effective signal transduction. In an attempt to clarify the GM3 cluster effect, we enzymatically synthesized GM3 mimetic polymer (GM3-p), with an acrylamide backbone from LacCer mimetic polymer (LacCer-p). Interestingly, GM3-p, but not LacCer-p, reversibly inhibited proliferation of NIH3T3 cells, which are normally resistant to exogenously added GM3. Moreover, we found that the introduction of carbonic acid into the acrylamide chain aided well-oriented cluster formation and enhanced the inhibitory effect of GM3-p. Since sialyllactosyl polymer and GM4 mimetic polymer, but not GM2 mimetic polymer, also inhibited cell proliferation, sialic acid-galactose units must be essential for the biological activity of GM3-p. These results suggest that the formation of sialic acid-galactose clusters is necessary for the suppressive effect of GM3-p. GM3-p treatment did not affect the serum-dependent activation of ERK1/2 or c-fos expression, but caused a reduction in the gene and/or protein expression of cyclin D1, cyclin E, cyclin-dependent kinase (cdk)4, and cdk2, which are involved in the cell cycle. Therefore, GM3-p inhibits cell proliferation by reducing cyclin D1-cdk4 and cyclin E-cdk2 complexes without affecting growth factor signaling from serum to c-fos.
    Glycobiology 07/2007; 17(6):568-77. DOI:10.1093/glycob/cwm020 · 3.75 Impact Factor
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    ABSTRACT: The ganglioside patterns have been shown to dramatically change during cell proliferation and differentiation and in certain cell-cycle phases, brain development, and cancer malignancy. To investigate the significance of the ganglioside GM3 in cancer malignancy, we established GM3-reconstituted cells by transfecting the cDNA of GM3 synthase into a GM3-deficient subclone of the 3LL Lewis lung carcinoma cell line (Uemura, S. (2003) Glycobiology, 13, 207-216). The GM3-reconstituted cells were resistant to apoptosis induced by etoposide and doxorubicin. There were no changes in the expression levels of topoisomerase IIalpha or P-glycoprotein, or in the uptake of doxorubicin between the GM3-reconstituted cells and the mock-transfected cells. To understand the mechanism of the etoposide-resistant phenotype acquired in the GM3-reconstituted cells, we investigated their apoptotic signaling. Although no difference was observed in the phosphorylation of p53 at serine-15-residue site by etoposide between the GM3-reconstituted cells and mock-transfected cells, the activation of both caspase-3 and caspase-9 was specifically inhibited in the former. We found that the anti-apoptotic protein B-cell leukemia/lymphoma 2 (Bcl-2) was increased in the GM3-reconstituted cells. Moreover, wild-type 3LL Lewis lung carcinoma cells, which have an abundance of GM3, exhibited no DNA fragmentation following etoposide treatment and expressed higher levels of the Bcl-2 protein compared with the J5 subclone. Thus, these results support the conclusion that endogenously produced GM3 is involved in malignant phenotypes, including anticancer drug resistance through up-regulating the Bcl-2 protein in this lung cancer cell line.
    Glycobiology 08/2006; 16(7):641-50. DOI:10.1093/glycob/cwj103 · 3.75 Impact Factor
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    ABSTRACT: Lipid rafts, specialized membrane microdomains enriched in sphingolipids and cholesterol, have been shown to function as signaling platforms in T cells. Surface raft expression is known to be increased in human T cells upon activation, and this increased raft expression may account for efficient signaling capability and decreased dependency for co-stimulation in effector and/or activated T cells. However, raft-mediated signaling ability in activated T cells remains to be clarified. In this study, we analyzed the structure and function of lipid rafts in human activated T cells. We demonstrated that raft protein constituents are dramatically changed after activation along with an increase in lipid contents. T cells stimulated with anti-CD3 plus anti-CD28 antibodies showed an increase not only in surface monosialoganglioside GM1 expression but also in total amounts of raft-associated lipids such as sphingomyelin, cholesterol and glycosphingolipids. Raft proteins increased after activation include Csk, Csk-binding protein and Fyn, the molecules known to be involved in negative regulation of T cell activation. Consistent with the increase in expression of these proteins, TCR-mediated Ca(2+) response, a response dependent on raft integrity, was clearly inhibited in activated T cells. Thus, the structure and function of lipid rafts in human activated T cells seem to be quite distinct from those in naive T cells. Further, human activated T cells are relatively resistant to signaling, at least transiently, by TCR re-stimulation even though their raft expression is increased.
    International Immunology 07/2005; 17(6):749-58. DOI:10.1093/intimm/dxh257 · 3.18 Impact Factor
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    ABSTRACT: Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling, but their role in the pathogenesis of insulin resistance has not been investigated. Detergent-resistant membrane microdomains (DRMs), isolated in the low-density fractions, are highly enriched in cholesterol, glycosphingolipids and various signaling molecules. Tumor necrosis factor alpha (TNFalpha) induces insulin resistance in type 2 diabetes, but its mechanism of action is not fully understood. In other studies we have found a selective increase in the acidic glycosphingolipid ganglioside GM3 in 3T3-L1 adipocytes treated with TNFalpha, suggesting a specific function for GM3. In the DRMs from TNFalpha-treated 3T3-L1 adipocytes, GM3 levels were doubled compared with results in normal adipocytes. Additionally, insulin receptor (IR) accumulations in the DRMs were diminished, whereas caveolin and flotillin levels were unchanged. Furthermore, insulin-dependent IR internalization and intracellular movement of the IR substrate 1(IRS-1) were both greatly suppressed in the treated cells, leading to an uncoupling of IR-IRS-1 signaling. GM3 depletion was able to counteract the TNFalpha-induced inhibitions of IR internalization and accumulation into DRMs. Together, these findings provide compelling evidence that in insulin resistance the insulin metabolic signaling defect can be attributed to a loss of IRs in the microdomains due to an accumulation of GM3.
    Glycobiology 02/2005; 15(1):21-9. DOI:10.1093/glycob/cwh135 · 3.75 Impact Factor
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    ABSTRACT: Tenascin-X (TNX) is a member of the tenascin family of glycoproteins of the extracellular matrix. Here, we observed abnormalities in the skin of TNX-deficient mice in comparison with that of wild-type mice. Histological analysis with Oil Red O staining demonstrated that there was considerable accumulation of lipid in the skin of TNX-deficient (TNX-/-) mice. By thin-layer chromatography of total lipids, it was found that the level of triglyceride was significantly increased in TNX-/- mice. The mRNA levels of most of the lipogenic enzyme genes examined were remarkably increased in TNX-/- mice. By gas chromatography-mass spectrometry analysis of triglyceride-associated fatty acids in the skin, saturated fatty acid palmitoic acid was decreased, whereas unsaturated fatty acids palmitoleic acid and oleic acid were increased in TNX-/- mice compared with those in wild-type mice. Conversely, fibroblast cell lines transfected with TNX showed a significant decrease in the amount of triglyceride. An increase in the saturated fatty acid stearic acid and decreases in the unsaturated fatty acids palmitoleic acid, oleic acid and linoleic acid, compared to those in mock-transfected cells were also caused by over-expression of TNX. These results indicate that TNX is involved in the regulation of triglyceride synthesis and the regulation of composition of triglyceride-associated fatty acids.
    Genes to Cells 09/2004; 9(8):737-48. DOI:10.1111/j.1356-9597.2004.00755.x · 2.86 Impact Factor
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    ABSTRACT: Valienamine analogues having alpha- and beta-galactose-type structures were synthesized by racemic modification from (1SR,2RS,3SR)-6-methylenecyclohex-4-ene-1,2,3-triol. Four N-alkyl derivatives of the beta-anomer were readily prepared selectively by treatment of key intermediate 2,6-di-O-acetyl-3,4-O-isopropylidene-5a-carba-alpha- and beta-l-arabino-hex-5(5a)-enopyranosyl bromides with alkyl amines. All compounds were assayed for inhibitory activity against six glycosidases, and the N-dodecyl derivative was shown to be a very strong inhibitor of beta-galactosidase (IC(50) 0.01 microM, bovine liver).
    Bioorganic & Medicinal Chemistry 04/2004; 12(5):995-1002. DOI:10.1016/j.bmc.2003.12.016 · 2.95 Impact Factor
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    ABSTRACT: To elucidate the biological significance of the lactosylceramide (LacCer) branching in glycosphingolipid (GSL) biosynthesis, we established ganglioside GM3- and lactosylsulfatide SM3-reconstituted cells by introducing the GM3 synthase gene and the sulfotransferase gene, respectively. In SM3-expressing cells, the reduction of beta1 integrin mRNA expression, the reduced adhesivity to fibronectin and laminin, and the suppression of anchorage-independent growth (tumorigenic potential) were observed. On the other hand, in GM3-expressing cells, anchorage-independent growth was promoted and the expression of PDGF alpha receptor mRNA was specifically reduced. Interestingly enough, no change in anchorage-dependent growth was observed in these cells, and tumorigenic signals were controlled selectively in both positive and negative directions. Thus, the spatio-temporal, gene expression control mechanism by individual GSL molecules accumulating in the cell membrane microdomain (raft) has been proven.
    Glycoconjugate Journal 02/2004; 20(3):169-78. DOI:10.1023/B:GLYC.0000024248.62242.1f · 1.95 Impact Factor
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    ABSTRACT: Lipid rafts are highly enriched in cholesterol and sphingolipids. In contrast to many reports that verify the importance of cholesterol among raft lipid components, studies that address the role of sphingolipids in raft organization and function are scarce. Here, we investigate the role of glycosphingolipids (GSLs) in raft structure and raft-mediated signal transduction in T lymphocytes by the usage of a specific GSL synthesis inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Surface GM1 expression and the expression of GSLs in rafts were profoundly reduced by D-PDMP treatment, whereas the expression of other lipid and protein constituents, such as cholesterol, sphingomyelin, Lck, and linker for activation of T cells, was not affected. T cell receptor-mediated signal transduction induced by antigen stimulation or by antibody cross-linking was normal in D-PDMP-treated T cells. In contrast, the signal through glycosylphosphatidylinositol (GPI)-anchored proteins was clearly augmented by D-PDMP treatment. Moreover, GPI-anchored proteins became more susceptible to phosphatidylinositol-specific phospholipase C cleavage in D-PDMP-treated cells, demonstrating that GSL depletion from rafts primarily influences the expression state and function of GPI-anchored proteins. Finally, by comparing the effect of D-PDMP with that of methyl-beta-cyclodextrin, we identified that compared with cholesterol depletion, GSL depletion has the opposite effect on the phosphatidylinositol-specific phospholipase C sensitivity and signaling ability of GPI-anchored proteins. These results indicate a specific role of GSLs in T cell membrane rafts that is dispensable for T cell receptor signaling but is important for the signal via GPI-anchored proteins.
    Journal of Biological Chemistry 01/2004; 278(51):51920-7. DOI:10.1074/jbc.M307674200 · 4.60 Impact Factor

Publication Stats

639 Citations
84.37 Total Impact Points


  • 2011–2013
    • Tokai University
      • Institute of Glycoscience
      Hiratuka, Kanagawa, Japan
  • 2007–2009
    • Tohoku Pharmaceutical University
  • 2000–2007
    • Hokkaido University
      • • Faculty of Pharmaceutical Sciences
      • • Graduate School of Pharmaceutical Sciences
      Sapporo, Hokkaidō, Japan
  • 1998–2004
    • Keio University
      • • Department of Biosciences and Informatics
      • • Department of Applied Chemistry
      Edo, Tōkyō, Japan