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Nozomu Nishi

Publications and source records attributed to Nozomu Nishi.

At least 19 recordsLinked to original sources
PubMed2007

Carbohydrate-recognition domains of galectin-9 are involved in intermolecular interaction with galectin-9 itself and other members of the galectin family.

Galectin-9 (Gal-9) is a tandem-repeat-type member of the galectin family associated with diverse biological processes, such as apoptosis, cell aggregation, and eosinophil chemoattraction. Although the detailed sugar-binding specificity of Gal-9 has been elucidated, molecular mechanisms that underlie these functions remain to be investigated. During the course of our binding study by affinity chromatography and surface plasmon resonance (SPR) analysis, we found that human Gal-9 interacts with immobilized Gal-9 in the protein-protein interaction mode. Interestingly, this intermolecular interaction strongly depended on the activity of the carbohydrate recognition domain (CRD), because the addition of potent saccharide inhibitors abolished the binding. The presence of multimers was also confirmed by Ferguson plot analysis of result of polyacrylamide gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Moreover, this intermolecular interaction was observed between Gal-9 and other galectin members, such as Gal-3 and Gal-8, but not Gal-1. Because such properties have not been reported yet, they may explain an unidentified mechanism underlying the diverse functions of Gal-9.

Chromatography, Affinity↗
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

Exosomes released by EBV-infected nasopharyngeal carcinoma cells convey the viral latent membrane protein 1 and the immunomodulatory protein galectin 9.

BACKGROUND: Nasopharyngeal carcinomas (NPC) are consistently associated with the Epstein-Barr virus (EBV). Their malignant epithelial cells contain the viral genome and express several antigenic viral proteins. However, the mechanisms of immune escape in NPCs are still poorly understood. EBV-transformed B-cells have been reported to release exosomes carrying the EBV-encoded latent membrane protein 1 (LMP1) which has T-cell inhibitory activity. Although this report suggested that NPC cells could also produce exosomes carrying immunosuppressive proteins, this hypothesis has remained so far untested. METHODS: Malignant epithelial cells derived from NPC xenografts--LMP1-positive (C15) or negative (C17)--were used to prepare conditioned culture medium. Various microparticles and vesicles released in the culture medium were collected and fractionated by differential centrifugation. Exosomes collected in the last centrifugation step were further purified by immunomagnetic capture on beads carrying antibody directed to HLA class II molecules. Purified exosomes were visualized by electron microscopy and analysed by western blotting. The T-cell inhibitory activities of recombinant LMP1 and galectin 9 were assessed on peripheral blood mononuclear cells activated by CD3/CD28 cross-linking. RESULTS: HLA-class II-positive exosomes purified from C15 and C17 cell supernatants were containing either LMP1 and galectin 9 (C15) or galectin 9 only (C17). Recombinant LMP1 induced a strong inhibition of T-cell proliferation (IC50 = 0.17 nM). In contrast recombinant galectin 9 had a weaker inhibitory effect (IC50 = 46 nM) with no synergy with LMP1. CONCLUSION: This study provides the proof of concept that NPC cells can release HLA class-II positive exosomes containing galectin 9 and/or LMP1. It confirms that the LMP1 molecule has intrinsic T-cell inhibitory activity. These findings will encourage investigations of tumor exosomes in the blood of NPC patients and assessment of their effects on various types of target cells.

Animals↗
PubMed2006

Soluble megalin is accumulated in the lumen of the rat endolymphatic sac.

The endolymphatic sac (ES) is believed to play an important role in maintaining homeostasis in the inner ear by the absorption and endocytosis of endolymph. Megalin is a 600-kDa multiligand endocytic receptor expressed in certain types of absorptive epithelia including kidney proximal tubules. We analyzed the immunoreactivity for megalin in rat ES by immunofluorescence, immunogold electron microscopy, and immunoblotting. With immunostaining, the luminal substances of the ES were strongly stained for megalin. Megalin was also localized in luminal macrophage-like cells and both types of epithelial cell (mitochondria-rich cells and ribosome-rich cells). In these cells, the megalin was localized in the lumen of endosomes, but was not membrane associated. This localization pattern indicates that the megalin in these cells is not a membrane receptor, but merely one of the constituents that are endocytosed from the lumen of the ES. Immunoblotting indicated that the megalin in the ES is a 210-kDa molecule lacking a cytoplasmic domain. This suggests that the megalin in the ES may be a soluble form, different from the 600-kDa membrane-bound receptor expressed in kidneys. Taken together, it is likely that the megalin in the ES lumen is a soluble component and may be endocytosed by the ES epithelial cells. Furthermore, we found that the tectorial membrane, an acellular structure in the cochlea, gave a strong megalin immunoreaction. Since the cochlea is connected to the ES, the megalin may be transported alone or with the components of the tectorial membrane from the cochlea to the ES lumen through longitudinal flow.

Animals↗
PubMed2006

Crystal structure of the galectin-9 N-terminal carbohydrate recognition domain from Mus musculus reveals the basic mechanism of carbohydrate recognition.

The galectins are a family of beta-galactoside-binding animal lectins with a conserved carbohydrate recognition domain (CRD). They have a high affinity for small beta-galactosides, but binding specificity for complex glycoconjugates varies considerably within the family. The ligand recognition is essential for their proper function, and the structures of several galectins have suggested their mechanism of carbohydrate binding. Galectin-9 has two tandem CRDs with a short linker, and we report the crystal structures of mouse galectin-9 N-terminal CRD (NCRD) in the absence and the presence of four ligand complexes. All structures form the same dimer, which is quite different from the canonical 2-fold symmetric dimer seen for galectin-1 and -2. The beta-galactoside recognition mechanism in the galectin-9 NCRD is highly conserved among other galectins. In the apo form structure, water molecules mimic the ligand hydrogen-bond network. The galectin-9 NCRD can bind both N-acetyllactosamine (Galbeta1-4GlcNAc) and T-antigen (Galbeta1-3GalNAc) with the proper location of Arg-64. Moreover, the structure of the N-acetyllactosamine dimer (Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc) complex shows a unique binding mode of galectin-9. Finally, surface plasmon resonance assay showed that the galectin-9 NCRD forms a homophilic dimer not only in the crystal but also in solution.

Amino Acid Sequence↗
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↗
PubMed2005

Galectin-9 as a prognostic factor with antimetastatic potential in breast cancer.

PURPOSE: Galectin-9, a member of the beta-galactoside-binding galectin family, induces aggregation of certain cell types. We assessed the contribution of galectin-9 to the aggregation of breast cancer cells as well as the relation between galectin-9 expression in tumor tissue and distant metastasis in patients with breast cancer. EXPERIMENTAL DESIGN: Subclones of MCF-7 breast cancer cells with high or low levels of galectin-9 expression were established and either cultured on plastic dishes or transplanted into nude mice. The tumors of 84 patients with breast cancer were tested for galectin-9 expression by immunohistochemistry. The patients were followed up for 14 years. RESULTS: MCF-7 subclones with a high level of galectin-9 expression formed tight clusters during proliferation in vitro, whereas a subclone (K10) with the lowest level of galectin-9 expression did not. However, K10 cells stably transfected with a galectin-9 expression vector aggregated in culture and in nude mice. Ectopic expression of galectin-9 also reduced MCF-7 cell adhesion to extracellular matrix proteins. Tumors of 42 of the 84 patients were galectin-9 positive, and those of 19 of the 21 patients with distant metastasis were galectin-9 negative. None of the 13 patients with galectin-9-positive tumors and lymph node metastasis up to level II manifested distant metastasis. The cumulative disease-free survival ratio for galectin-9-positive patients was more favorable than that for the galectin-9-negative group (P < 0.0001). Multivariate analysis revealed that galectin-9 status influenced distant metastasis independently of and to a greater extent than lymph node metastasis. CONCLUSIONS: Galectin-9 is a possible prognostic factor with antimetastatic potential in breast cancer.

Adult↗
PubMed2005

36-kDa microfibril-associated glycoprotein (MAGP-36) is an elastin-binding protein increased in chick aortae during development and growth.

MAGP-36 was discovered in porcine aorta in 1989 and is thought to be one of the microfibril-associated proteins. MAGP-36 has been localized on the surface of elastic fibers or laminae in immunohistochemical studies. However, its functional role in the aorta is obscure. Herein, we report on the binding activity of MAGP-36 to components of the aortic wall and its accumulation pattern in the aorta during development and growth. In vitro, MAGP-36 bound to elastin and collagen in a Ca(2+)-dependent manner, and mediated the adhesion of human aortic smooth muscle cells. This cell adhesion mostly depended on the RGD-containing domain of MAGP-36. We examined the accumulation of MAGP-36 with quantitative Western blot analysis and immunoelectron microscopy in chick aortae during development and growth. The amount of MAGP-36 increased on the surface of elastic fibers or laminae between days 14 and 34 after the start of incubation, and reached a plateau at about 53 days. This accumulation of MAGP-36 roughly correlated with an increase in blood pressure for this period. Thus, MAGP-36 might be a bridging protein that connects elastin to other components of the aortic wall and might play a role in maintaining the integrity of the aortic structure under arterial pressure.

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↗
PubMed2005

In nasopharyngeal carcinoma cells, Epstein-Barr virus LMP1 interacts with galectin 9 in membrane raft elements resistant to simvastatin.

Nasopharyngeal carcinomas (NPC) are etiologically related to the Epstein-Barr virus (EBV), and malignant NPC cells have consistent although heterogeneous expression of the EBV latent membrane protein 1 (LMP1). LMP1 trafficking and signaling require its incorporation into membrane rafts. Conversely, raft environment is likely to modulate LMP1 activity. In order to investigate NPC-specific raft partners of LMP1, rafts derived from the C15 NPC xenograft were submitted to preparative immunoprecipitation of LMP1 combined with mass spectrometry analysis of coimmunoprecipitated proteins. Through this procedure, galectin 9, a beta-galactoside binding lectin and Hodgkin tumor antigen, was identified as a novel LMP1 partner. LMP1 interaction with galectin 9 was confirmed by coimmunoprecipitation and Western blotting in whole-cell extracts of NPC and EBV-transformed B cells (lymphoblastoid cell lines [LCLs]). Using mutant proteins expressed in HeLa cells, LMP1 was shown to bind galectin 9 in a TRAF3-independent manner. Galectin 9 is abundant in NPC biopsies as well as in LCLs, whereas it is absent in Burkitt lymphoma cells. In subsequent experiments, NPC cells were treated with Simvastatin, a drug reported to dissociate LMP1 from membrane rafts in EBV-transformed B cells. We found no significant effects of Simvastatin on the distribution of LMP1 and galectin 9 in NPC cell rafts. However, Simvastatin was highly cytotoxic for NPC cells, regardless of the presence or absence of LMP1. This suggests that Simvastatin is a potentially useful agent for the treatment of NPCs although it has distinct mechanisms of action in NPC and LCL cells.

Blotting, Western↗
PubMed2004

Immunocytochemical analysis of cubilin-mediated endocytosis of high density lipoproteins (HDL) in epithelial cells of the rat visceral yolk sac.

Cubilin was recently shown to function as an endocytic receptor for high density lipoprotein (HDL) holoparticles and apolipoprotein A-I (apo A-I), the main protein constituent of HDL. In the present study, we analyzed the distribution and intracellular trafficking of cubilin and HDL in rat visceral yolk sac epithelial cells. After epithelial cells were loaded with apolipoprotein E-free HDL for 30 min in vitro, double immunofluorescence showed that the apical cytoplasm of the cells was strongly stained with anti-cubilin antibodies and anti-apo A-I/HDL. Furthermore, double immunogold electron-microscopic observations revealed the distinct localization of cubilin and HDL in endocytic vacuoles. In early endosomes, both were colocalized on the membrane. Although, in late endosomes, cubilin was also localized on the membrane, HDL was mainly located in the matrix. Both were found in the matrix in lysosomes. In addition, cubilin was markedly localized in apical tubules (ATs), which are generally accepted as being receptor recycling compartments. Thus, HDL is internalized through cubilin-mediated endocytosis and is finally transported to lysosomes. By contrast, cubilin is mainly translocated to ATs for recycling, although some of the cubilin is degraded in lysosomes. Quantitative analysis further revealed that cubilin was not concentrated on the membranes of ATs, although it accumulated in the AT area. Some HDL were also observed in the AT area. These findings suggest that the translocation of cubilin and HDL to ATs from early endosomes occurs through a simple sorting mechanism based on the geometry of these compartments and the bulk membrane and volume flow.

Animals↗
PubMed2004

Double-stranded RNA enhances the expression of galectin-9 in vascular endothelial cells.

Treatment of cells with double-stranded RNA (dsRNA) in vitro mimics viral infection and regulates expression of various genes. We addressed the mechanisms of leucocyte traffic across the vascular endothelium induced by dsRNA. The present study focused on the expression of galectin-9, which is one of key molecules in the regulation of the interaction between vascular wall and white blood cells. Human umbilical vein endothelial cells (HUVEC) in culture were treated with polyinosinic-polycytidylic acid (poly IC), and expression of mRNA and protein of galectin-9 was analysed by reverse transcription polymerase-chain reaction (RT-PCR) and Western blotting. Poly IC enhanced the expression of galectin-9 mRNA and protein in concentration- and time-dependent manners. This effect of poly IC was almost completely suppressed by the pretreatment with 2-aminopurine, an inhibitor of dsRNA-dependent kinase. Poly IC treatment of HUVEC also enhanced the adherence of EoL-1 cells to the cells, which was inhibited by co-treatment with lactose. We conclude that poly IC upregulates galectin-9 expression in the vascular endothelium and this may explain part of the mechanism for leucocyte traffic through the vascular wall.

Base 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

Specific recognition of Leishmania major poly-beta-galactosyl epitopes by galectin-9: possible implication of galectin-9 in interaction between L. major and host cells.

Leishmania parasites are the causative agents of leishmaniasis, manifesting itself in a species-specific manner. The glycan epitopes on the parasite are suggested to be involved in the Leishmania pathogenesis. One of such established species-unique glycan structures is the poly-beta-galactosyl epitope (Galbeta1-3)n found on L. major, which can develop cutaneous infections with strong inflammatory responses. Interestingly, the polygalactosyl epitope is also suggested to be involved in the development of the parasites in its host vector, sand fly. Thus, the recognition of the galactosyl epitope by lectins expressed in host or sand fly should be implicated in the species-specific manifestations of leishmaniasis and in the parasite life cycle, respectively. We recently reported that one host beta-galactoside-binding protein, galectin-3, can distinguish L. major from the other species through its binding to the poly-beta-galactosyl epitope, proposing a role for galectin-3 as an immunomodulator that could influence the L. major-specific immune responses in leishmaniasis. Here we report that galectin-9 can also recognize L. major by binding to the L. major-specific polygalactosyl epitope. Frontal affinity analysis with different lengths of poly-beta-galactosyllactose revealed that the galectin-9 affinity for polygalactose was enhanced in proportion to the number of Galbeta1-3 units present. Even though both galectins have comparable affinities toward the polygalactosyl epitopes, only galectin-9 can promote the interaction between L. major and macrophages, suggesting distinctive roles for the galectins in the L. major-specific development of leishmaniasis in the host.

Animals↗
PubMed2003

Galectin-9 induces apoptosis through the calcium-calpain-caspase-1 pathway.

Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that beta-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4(+) and CD8(+) T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca(2+)) influx, and that either the intracellular Ca(2+) chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca(2+)-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.

Adjuvants, Immunologic↗
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↗