Biomedical subjects

Hiroki Shoji

Publications and source records attributed to Hiroki Shoji.

11 recordsLinked to original sources

PubMed2006

Characterization of galectin-9-induced death of Jurkat T cells.

Galectin-9, a mammalian lectin with affinity for beta-galactosides, is known as an apoptosis inducer of activated T lymphocytes. In the present study, we examined the properties of galectin-9-mediated cell death of Jurkat T cells. Galectin-9NC (wild-type), consisting of two CRDs (N-terminal and C-terminal carbohydrate recognition domains), and derivatives of it, galectins-9-NN and -9-CC, induced Jurkat T-cell apoptosis. However, a single CRD (galectin-9NT or -CT) had no effect, suggesting the stable dimeric structure of two CRDs is required for the activity. The apoptosis was inhibited by pretreatment with an N-glycan synthesis inhibitor, indicating that the expression of N-glycans in the cells is essential for galectin-9-induced apoptosis. We previously showed that the apoptosis of MOLT-4 cell is mediated by galectin-9 via a Ca(2+)-calpain-caspase-1-dependent pathway. In Jurkat cells, the cell death by galectin-9, was insufficiently suppressed by caspase inhibitors, Ca(2+)-chelator or calpain inhibitor. Furthermore, we observed the loss of mitochondrial membrane potential and significant AIF release in galectin-9-treated cells. These findings suggest that caspase-dependent and-independent death pathways exist in Jurkat cells, and the main pathway might vary with the T-cell type.

Apoptosis↗

PubMed2006

Galectin-8 and galectin-9 are novel substrates for thrombin.

Galectin-8 and galectin-9, which each consist of two carbohydrate recognition domains (CRDs) joined by a linker peptide, belong to the tandem-repeat-type subclass of the galectin family. Alternative splicing leads to the formation of at least two and three distinct splice variants (isoforms) of galectin-8 and galectin-9, respectively, with tandem-repeat-type structures. The isoforms share identical CRDs and differ only in the linker region. In a search for differences in biological activity among the isoforms, we found that their isoforms with the longest linker peptide, that is, galectin-8L and galectin-9L (G8L and G9L), are highly susceptible to thrombin cleavage, whereas the predominant isoforms, galectin-8M and galectin-9M (G8M and G9M), and other members of human galectin family so far examined were resistant to thrombin. Amino acid sequence analysis of proteolytic fragments and site-directed mutagenesis showed that the thrombin cleavage sites (-IAPRT- and -PRPRG- for G8L and G9L, respectively) resided within the linker peptides. Although intact G8L stimulated neutrophil adhesion to substrate more efficiently than G8M, the activity of G8L but not that of G8M decreased on thrombin digestion. Similarly, thrombin treatment almost completely abolished eosinophil chemoattractant (ECA) activity of G9L. These observations suggest that G8L and G9L play unique roles in relation to coagulation and inflammation.

Amino Acid Sequence↗

PubMed2006

Analysis of the function of activin betaC subunit using recombinant protein.

Activins, TGF-beta superfamily members, have multiple functions in a variety of cells and tissues. Additional activin beta subunit genes, betaC and betaE, have been identified in humans and rodents. To explore the role of activin betaC subunit, we generated recombinant human activin C using Chinese hamster ovary cells. Recombinant activin C from the conditioned medium was purified by consecutive hydrophobic, size-exclusion, and high performance liquid chromatography. SDS-PAGE and Western blot analysis of the purified protein revealed that activin C formed disulfide bridges. However, activin C had no effect on the proliferation of cultured liver cells. Furthermore, there were no significant differences in erythroid differentiation and follicle stimulating hormone secretion in vitro. It was also shown that immunoreactive bands indicated the hetrodimer of activin betaC, and inhibin alpha subunits were detected in the conditioned medium from the activin C-producing cells, which were stably transfected with inhibin alpha subunit cDNA. This suggests that activin betaC subunit may have been present and that it may exert its effect as inhibin C.

Animals↗

PubMed2005

Development of highly stable galectins: truncation of the linker peptide confers protease-resistance on tandem-repeat type galectins.

Galectin-9 and galectin-8, members of beta-galactoside-binding animal lectin family, are promising agents for the treatment of immune-related and neoplastic diseases. The proteins consist of two carbohydrate recognition domains joined by a linker peptide, which is highly susceptible to proteolysis. To increase protease resistance, we prepared mutant proteins by serial truncation of the linker peptide. As a result, mutant forms lacking the entire linker peptide were found to be highly stable against proteolysis and retained their biological activities. These mutant proteins might be useful tools for analyzing the biological functions and evaluating the therapeutic potential of galectin-9 and galectin-8.

Base Sequence↗

PubMed2005

Xenopus galectin-VIIa binds N-glycans of members of the cortical granule lectin family (xCGL and xCGL2).

We have identified members of the Xenopus cortical granule lectin (xCGL) family as candidate target glycoproteins of Xenopus galectin-VIIa (xgalectin-VIIa) in Xenopus embryos. In addition to the original xCGL, we also identified a novel member of the xCGL family, xCGL2. Expression of the mRNAs of xCGL and xCGL2, as well as that of xgalectin-VIIa, was observed throughout early embryogenesis. Two and three potential N-glycosylation sites were deduced from the amino acid sequences of xCGL and xCGL2, respectively, and xgalectin-VIIa recognizes N-glycans linked to a common site in xCGL and xCGL2 and also recognizes N-glycans linked to a site specific to xCGL2. However, interaction between xgalectin-Ia and xCGLs was not detectable. We also obtained consistent results on surface plasmon resonance analysis involving xCGLs as ligands and xgalectins as analytes. The Kd value of the interaction between xgalectin-VIIa and xCGLs was calculated to be 35.9 nM. The structures of the N-glycans of xCGLs, which were recognized by xgalectin-VIIa, were analyzed by the two-dimensional sugar map method, and three kinds of N-acetyllactosamine type, biantennary N-glycans were identified as the major neutral N-glycans. The binding specificity of oligosaccharides for xgalectin-VIIa was analyzed by frontal affinity chromatography (FAC). The oligosaccharide specificity pattern of xgalectin-VIIa was similar to that of the human homolog galectin-3, and it was also shown that the N-acetyllactosamine type, biantennary N-glycans exhibit high affinity for xgalectin-VIIa (Kd = 11 microM). These results suggest that xgalectin-VIIa interacts with xCGLs through binding to N-acetyllactosamine type N-glycans and that this interaction might make it possible to organize a lectin network involving members of different lectin families.

Amino Acid Sequence↗

PubMed2003

Galectin-8 modulates neutrophil function via interaction with integrin alphaM.

The members of the galectin family are associated with diverse cellular events, including immune response. We investigated the effects of galectin-8 on neutrophil function. Human galectin-8 induced firm and reversible adhesion of peripheral blood neutrophils but not eosinophils to a plastic surface in a lactose-sensitive manner. Other human galectins, galectins-1, -3, and -9, showed low or negligible effects on neutrophil adhesion. Confocal microscopy revealed actin bundle formation in the presence of galectin-8. Cytochalasins inhibited both actin assembly and cell adhesion induced by galectin-8. Affinity purification of galectin-interacting proteins from solubilized neutrophil membrane revealed that N-terminal carbohydrate recognition domain (CRD) of galectin-8 bound promatrix metalloproteinase-9 (proMMP-9), and C-terminal CRD bound integrin alphaM/CD11b and proMMP-9. A mutant galectin-8 lacking the carbohydrate-binding activity of N-terminal CRD (galectin-8R69H) retained adhesion-inducing activity, but inactivation of C-terminal CRD (galectin-8R233H) abolished the activity. MMP-3-mediated processing of proMMP-9 was accelerated by galectin-8, and this effect was inhibited by lactose. Galectins-1 and -3 did not affect the processing. Superoxide production, an essential event in bactericidal function of neutrophils, was stimulated by galectin-8 to an extent comparable to that induced by fMLP. Galectin-8R69H but not galectin-8R233H could stimulate superoxide production. Taken together, these results suggest that galectin-8 is a novel factor that modulates the neutrophil function related to transendothelial migration and microbial killing.

Actins↗

PubMed2003

Characterization of the Xenopus galectin family. Three structurally different types as in mammals and regulated expression during embryogenesis.

We have isolated six novel galectin cDNAs from a Xenopus laevis kidney cDNA library. The newly identified X. laevis galectins (xgalectins) comprise one proto type (xgalectin-Vb), one chimera type (xgalectin-VIIa), and four tandem repeat types (xgalectin-IIb, -IIIb, -VIa, and -VIIIa). Thus, together with those mentioned in our previous work (Shoji, H., Nishi, N., Hirashima, M., and Nakamura, T. (2002) Glycobiology 12, 163-172), the 12 xgalectins are classified into three types based on their domain structures, as in mammals. The xgalectins whose counterparts in other species have not been identified (xgalectin-IVa, -Vb, and -VIa) were confirmed to possess lactose-binding activity by expression of their recombinant forms. This shows that they truly function as animal lectins. The protein purification study revealed that the major xgalectins in kidney are xgalectin-Ib, -IIa, -IIb, -IIIa, and -VIIa. The mRNAs of xgalectin-IIb, -IIIb, -Vb, and -VIa were localized to specific adult tissues, whereas those of xgalectin-VIIa and -VIIIa were broadly distributed. The temporal expression patterns of the mRNAs of the 12 xgalectins during embryogenesis were analyzed and categorized into three groups: 1) mRNA observed to exist throughout embryogenesis, i.e. maternal mRNA also exists (xgalectin-Ia, -IIa, -IIIa, -IIIb, -Va, -VIIa, and -VIIIa); 2) mRNA observed from the gastrula stage (xgalectin-VIa); and 3) mRNA observed from the tail bud or the tadpole stage (xgalectin-Ib, -IIb, -IVa, and -Vb). The mRNA of the most abundant xgalectin in embryos, xgalectin-VIIa, was localized to the surface layer of embryos, the epidermis, the cement gland, and various placodes. Xgalectin-VIIa protein was also observed to exist throughout embryogenesis by Western blot analysis with specific antiserum. These results show that the expression of each member is spatiotemporally regulated from eggs to adulthood, suggesting that galectins play multiple roles not only in adults, but also in development.

Amino Acid Sequence↗

PubMed2002

Possible role of galectin-9 in cell aggregation and apoptosis of human melanoma cell lines and its clinical significance.

Galectin-9 expression was examined in 6 human melanoma cell lines. Among them, MM-BP proliferated with colony formation, but MM-RU failed. RT-PCR analysis revealed evident expression of galectin-9 mRNA in MM-BP but not in MM-RU. MM-BP expressed galectin-9 protein both on the surface and in the cytoplasm, whereas MM-RU expressed it only weakly in the cytoplasm. Exogenous galectin-9 induced in vitro both cell aggregation and apoptosis of MM-RU proliferating without colony formation. Association of galectin-9 expression in melanoma cells with prognosis of the patients bearing melanocytic tumors was further examined. Galectin-9 protein was strongly and homogeneously expressed in melanocytic nevi, but down-regulated in melanoma cells especially in metastatic lesions. High galectin-9 expression was inversely correlated with the progression of this disease, suggesting that high galectin-9 expression in primary melanoma lesions links to a better prognosis.

Annexin A5↗

PubMed2002

Purification and cDNA cloning of Xenopus liver galectins and their expression.

We have characterized galectin family proteins in adult tissues of Xenopus laevis and purified 14-kDa and 36-kDa proteins from the liver. The liver galectins showed comparable hemagglutination activities to those of mammalian galectins. Furthermore, we isolated five galectin cDNAs from a Xenopus liver library. These cDNAs revealed that X. laevis galectins (xgalectins) form a family consisting of at least proto and tandem repeat types based on their domain structures, like the mammalian galectin family. Two proto-type xgalectins, -Ia and -Ib, exhibited a high sequence identity (91%) with each other at the amino acid level and were most similar (49-50% identity) to human galectin-1. From their sequence similarity and ubiquitous tissue distributions, xgalectins-Ia and -Ib both seemed to be Xenopus homologues of mammalian galectin-1. Three tandem repeat-type xgalectins were newly identified. Two of them, xgalectins-IIa and -IIIa, seemed to be homologous to human galectins-4 and -9, respectively, judging from their high sequence similarities (42-50% identity). However, xgalectin-IVa seemed to be a novel type. Distributions of mRNAs of xgalectins were analyzed by northern hybridization. In addition to adult tissues, either of three tandem repeat-type xgalectins were expressed in whole embryos. Moreover, amino acid sequence analysis of liver proteins indicated that xgalectins-Ia, -IIa, and -IIIa are produced as abundant galectins in the adult liver.

Amino Acid Sequence↗

PubMed2002

Functional analysis of the carbohydrate recognition domains and a linker peptide of galectin-9 as to eosinophil chemoattractant activity.

Human galectin-9 is a beta-galactoside-binding protein consisting of two carbohydrate recognition domains (CRDs) and a linker peptide. We have shown that galectin-9 represents a novel class of eosinophil chemoattractants (ECAs) produced by activated T cells. A previous study demonstrated that the carbohydrate binding activity of galectin-9 is indispensable for eosinophil chemoattraction and that the N- and C-terminal CRDs exhibit comparable ECA activity, which is substantially lower than that of full-length galectin-9. In this study, we investigated the roles of the two CRDs in ECA activity in conjunction with the sugar-binding properties of the CRDs. In addition, to address the significance of the linker peptide structure, we compare the three isoforms of galectin-9, which only differ in the linker peptide region, in terms of ECA activity. Recombinant proteins consisting of two N-terminal CRDs (galectin-9NN), two C-terminal CRDs (galectin-9CC), and three isoforms of galectin-9 (galectin-9S, -9M, and -9L) were generated. All the recombinant proteins had hemagglutination activity comparable to that of the predominant wild-type galectin-9 (galectin-9M). Galectin-9NN and galectin-9CC induced eosinophil chemotaxis in a manner indistinguishable from the case of galectin-9M. Although the isoform of galectin-9 with the longest linker peptide, galectin-9L, exhibited limited solubility, the three isoforms showed comparable ECA activity over the concentration range tested. The interactions between N- and C-terminal CRDs and glycoprotein glycans and glycolipid glycans were examined using frontal affinity chromatography. Both CRDs exhibited high affinity for branched complex type sugar chain, especially for tri- and tetraantennary N-linked glycans with N-acetyllactosamine units, and the oligosaccharides inhibited the ECA activity at low concentrations. These results suggest that the N- and C-terminal CRDs of galectin-9 interact with the same or a closely related ligand on the eosinophil membrane when acting as an ECA and that ECA activity does not depend on a specific structure of the linker peptide.

Base Sequence↗

PubMed2002

Quantification of galectin-7 and its localization in adult mouse tissues.

We developed a method to quantify galectin-7 extracted from adult mouse tissues by Western blot analysis. More than 0.5 ng of galectin-7 per mg of tissue was detectable by this method. The amounts of galectin-7 in tissues were determined as follows: skin, 62 +/- 3 ng/mg; esophagus, 23 +/- 8 ng/mg; stomach, 18 +/- 6 ng/mg; anus, 13 +/- 1 ng/mg; and tongue, 12 +/- 2 ng/mg. This indicates that galectin-7 production coincides with the degree of stratification of the epithelia. Interestingly, we also detected significant amounts of galectin-7, 5.9 plus minus 1.4 and 2.7 +/- 0.6 ng/mg, in the trachea and ovaries, respectively. Moreover, we found that galectin-7 is localized in the pseudostratified epithelium of the trachea and stromal epithelium of the ovaries by immunohistochemistry. Thus, galectin-7 protein might be produced primarily in stratified epithelia, but also in some wet epithelia, and plays a unique role in cell-mucus contact, or the growth of ovarian follicles.

Animals↗