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ABSTRACT: Galectin-mediated ligation of glycoepitopes on T-cell activation markers induces an increase in the cytosolic calcium concentration ([Ca(2+)](i)) originating from a transient Ca(2+) release of internal stores as well as a sustained influx across the plasma membrane. In transiently transfected Jurkat T-lymphocytes, galectins [galectin-1 (gal-1), recombinant human galectin-1 (rec gal-1), nematode 32-kDa galectin (LEC-1), nematode 16-kDa galectin (LEC-6)] differentially stimulate the expression of the luciferase reporter gene constructs pNFAT-TA-Luc and pAP1(PMA)-TA-Luc, which are activated by the nuclear factor of activated T-cells (NFAT) or the transcription factor, activator protein 1 (AP-1), respectively. The galectin-stimulated expression of the reporter constructs is completely inhibited by lactose (30 mM) and asialofetuin (30 microM) carrying Galbeta1-4GlcNAc sequences. Preincubation of pNFAT-TA-Luc-transfected cells with cyclosporine A (0.1 microM) and FK506 (0.01 microM) abrogated the gal-1-induced expression of the reporter luciferase (Luc). Electrophoretic mobility shift assays (EMSAs) provided evidence for gal-1-stimulated increase in the binding of nuclear extracts to a synthetic oligonucleotide with an AP-1 consensus sequence.
Cellular Signalling 11/2002; 14(10):861-8. · 4.06 Impact Factor
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Jun Hirabayashi,
Tomomi Hashidate,
Yoichiro Arata,
Nozomu Nishi,
Takanori Nakamura,
Mitsuomi Hirashima,
Tadasu Urashima,
Toshihiko Oka,
Masamitsu Futai,
Werner E G Muller,
Fumio Yagi,
Ken-ichi Kasai
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ABSTRACT: Galectins are widely distributed sugar-binding proteins whose basic specificity for beta-galactosides is conserved by evolutionarily preserved carbohydrate-recognition domains (CRDs). Although they have long been believed to be involved in diverse biological phenomena critical for multicellular organisms, in only few a cases has it been proved that their in vivo functions are actually based on specific recognition of the complex carbohydrates expressed on cell surfaces. To obtain clues to understand the physiological roles of diverse members of the galectin family, detailed analysis of their sugar-binding specificity is necessary from a comparative viewpoint. For this purpose, we recently reinforced a conventional system for frontal affinity chromatography (FAC) [J. Chromatogr., B, Biomed. Sci. Appl. 771 (2002) 67-87]. By using this system, we quantitatively analyzed the interactions at 20 degrees C between 13 galectins including 16 CRDs originating from mammals, chick, nematode, sponge, and mushroom, with 41 pyridylaminated (PA) oligosaccharides. As a result, it was confirmed that galectins require three OH groups of N-acetyllactosamine, as had previously been denoted, i.e., 4-OH and 6-OH of Gal, and 3-OH of GlcNAc. As a matter of fact, no galectin could bind to glycolipid-type glycans (e.g., GM2, GA2, Gb3), complex-type N-glycans, of which both 6-OH groups are sialylated, nor Le-related antigens (e.g., Le(x), Le(a)). On the other hand, considerable diversity was observed for individual galectins in binding specificity in terms of (1) branching of N-glycans, (2) repeating of N-acetyllactosamine units, or (3) substitutions at 2-OH or 3-OH groups of nonreducing terminal Gal. Although most galectins showed moderately enhanced affinity for branched N-glycans or repeated N-acetyllactosamines, some of them had extremely enhanced affinity for either of these multivalent glycans. Some galectins also showed particular preference for alpha1-2Fuc-, alpha1-3Gal-, alpha1-3GalNAc-, or alpha2-3NeuAc-modified glycans. To summarize, galectins have evolved their sugar-binding specificity by enhancing affinity to either "branched", "repeated", or "substituted" glycans, while conserving their ability to recognize basic disaccharide units, Galbeta1-3/4GlcNAc. On these bases, they are considered to exert specialized functions in diverse biological phenomena, which may include formation of local cell-surface microdomains (raft) by sorting glycoconjugate members for each cell type.
Biochimica et Biophysica Acta 10/2002; 1572(2-3):232-54. · 4.66 Impact Factor
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ABSTRACT: Galectins, a family of soluble beta-galactosyl-binding lectins, are believed to mediate cell-cell and cell-extracellular matrix interactions during development, inflammation, apoptosis, and tumor metastasis. However, neither the detailed mechanisms of their function(s) nor the identities of their natural ligands have been unequivocally elucidated. Of the several galectins present in the nematode Caenorhabditis elegans, the 16-kDa "proto" type and the 32-kDa "tandem-repeat" type are the best characterized so far, but their carbohydrate specificities have not been examined in detail. Here, we report the carbohydrate-binding specificity of the recombinant C. elegans 16-kDa galectin and the structural analysis of its binding site by homology modeling. Our results indicate that unlike the galectins characterized so far, the C. elegans 16-kDa galectin interacts with most blood group precursor oligosaccharides (type 1, Galbeta1,3GlcNAc, and type 2, Galbeta1,4GlcNAc; Talpha, Galbeta1,3GalNAcalpha; Tbeta, Galbeta1,3GalNAcbeta) and gangliosides containing the Tbeta structure. Homology modeling of the C. elegans 16-kDa galectin CRD revealed that a shorter loop containing residues 66-69, which enables interactions of Glu(67) with both axial and equatorial -OH at C-3 of GlcNAc (in Galbeta1,4GlcNAc) or at C-4 of GalNAc (in Galbeta1,3GalNAc), provides the structural basis for this novel carbohydrate specificity.
Glycobiology 09/2002; 12(8):451-61. · 3.58 Impact Factor
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ABSTRACT: Protein glycosylation is a central issue for post-genomic (proteomic) sciences. We have taken a systematic approach for analyzing soluble glycoproteins produced in the nematode Caenorhabditis elegans. The approach aims at assigning (i) genes that encode glycoproteins, (ii) sites where glycosylation occurs, and (iii) types of attached glycan structures. A soluble extract of C. elegans, as a starting material, was applied first to a concanavalin A (ConA) column (specific for high-mannose type N-glycans), and then the flow-through fraction was applied to a galectin LEC-6 (GaL6) column (specific for complex-type N-glycans). The adsorbed glycoproteins were digested with lysylendopeptidase, and the resultant glycopeptides were selectively recaptured with the same lectin columns. The glycopeptides were separated by reversed-phase chromatography and then subjected to sequence determination. As a result, 44 and 23 glycopeptides captured by the ConA and GaL6 columns, respectively, were successfully analyzed and assigned to 32 and 16 corresponding genes, respectively. For these glycopeptides, 49 N-glycosylation sites were experimentally confirmed, whereas 21 sites remained as potential sites. Of the identified genes, about 80% had apparent homologues in other species, as represented by typical secreted proteins. However, the two sets of genes assigned for the ConA and GaL6-recognized glycopeptides showed only 1 overlap with each other. Proof of the practical applicability of the glyco-catch method to a model organism, C. elegans, directs us to explore more complex multicellular organisms.
Journal of Biochemistry 08/2002; 132(1):103-14. · 2.37 Impact Factor
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ABSTRACT: Progress in genome projects has provided us with fundamentals on genetic information; however, the functions of a large number of genes remain to be elucidated. To understand the in vivo functions of eukaryotic genes, it is essential to grasp the features of their post-translational modifications. Among them, protein glycosylation is a central issue to be discussed, considering the predominant roles of glycoproteins in cell-cell and cell-substratum recognition events in multicellular organisms. In this context, it is necessary to establish a core strategy for analyzing glycosylated proteins under the concept of the "glycome" [Trends Glycosci. Glycotechnol. 12 (2000) 1]. Though the term glycome should be defined, in analogy to the genome and proteome, as "a whole set of glycans produced in a single organism", here we propose a glycome project specifically focusing on glycoproteins. Principal objectives in the project are to identify: (1) which genes encode glycoproteins (i.e. genome information); (2) which sites among potential glycosylation sites are actually glycosylated (i.e. glycosylation site information); (3) what are the structures of glycans (i.e. structural information); and (4) what are the effects (functions) of glycosylation (functional information). For these purposes, two affinity technologies have been introduced. One is named the "glyco-catch method" to identify genes encoding glycoproteins [Proteomics 1 (2001) 295], and the other is the recently reinforced "frontal affinity chromatography" [J. Chromatogr. A 890 (2000) 261]. By the former method, genes that encode glycoproteins as well as glycosylation sites are systematically identified by the efficient combination of conventional lectin-affinity chromatography and contemporary in silico database searching. The following three actions have been devised for rapid and systematic characterization of glycans: (1) mass spectrometry to acquire exact mass information; (2) 2-D/3-D mapping to obtain refined chemical information; and (3) reinforced frontal affinity chromatography to determine affinity constants (K(a)-values) for a set of lectins. Pyridylaminated glycans are used throughout the characterization processes. In this review, the concept and strategy of glycomic approaches are described referring to the on-going glycome project focused on the nematode Caenorhabditis elegans.
Journal of Chromatography B 06/2002; 771(1-2):67-87. · 2.89 Impact Factor
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Miki Sato,
Nozomu Nishi,
Hiroki Shoji,
Masako Seki,
Tomomi Hashidate, Jun Hirabayashi,
Ken-ichi Kasai Ki,
Yuiro Hata,
Shigehiko Suzuki,
Mitsuomi Hirashima,
Takanori Nakamura
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ABSTRACT: 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.
Glycobiology 04/2002; 12(3):191-7. · 3.58 Impact Factor
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ABSTRACT: The affinity constants of recombinant human galectin-1 and galectin-3 for sugars were determined by capillary affinophoresis. The monoliganded affinophore contains p-aminophenyl-beta-lactoside as an affinity ligand in the matrix of succinylglutathione and has three negative charges. An analysis of the mobility change of the lectins caused by the affinophore and its inhibition by neutral sugars allowed, for the first time, a determination of the affinity constants between the binding sites of the lectins and sugars. The relative magnitude of the affinity constants for each of the sugars in terms of dissociation constants found to be consistent with previously reported data on the concentrations of sugars that caused a 50% inhibition (I50) in the binding assay of the lectin to oligosaccharide-immobilized agarose beads but the absolute values of the dissociation constants were considerably smaller than the I50 values. Capillary affinophoresis indicated microheterogeneity of the lectin preparations and enabled the separate analysis of the affinity of each component simultaneously showing the advantage in using a separation method for analysis of bioaffinity.
Journal of Chromatography B 03/2002; 768(1):199-210. · 2.89 Impact Factor
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ABSTRACT: The 32-kDa galectin (LEC-1 or N32) of the nematode Caenorhabditis elegans is the first example of a tandem repeat-type galectin and is composed of two domains, each of which is homologous to typical
vertebrate 14-kDa-type galectins. To elucidate the biological meaning of this unique structure containing two probable sugar
binding sites in one molecule, we analyzed in detail the sugar binding properties of the two domains by using a newly improved
frontal affinity chromatography system. The whole molecule (LEC-1), the N-terminal lectin domain (Nh), and the C-terminal
lectin domain (Ch) were expressed in Escherichia coli, purified, and immobilized on HiTrap gel agarose columns, and the extent of retardation of various sugars by the columns
was measured. To raise the sensitivity of the system, we used 35 different fluorescence-labeled oligosaccharides (pyridylaminated
(PA) sugars). All immobilized proteins showed affinity for N-acetyllactosamine-containingN-linked complex-type sugar chains, and the binding was stronger for more branched sugars. Ch showed 2–5-fold stronger binding
toward all complex-type sugars compared with Nh. Both Nh and Ch preferred Galβ1–3GlcNAc to Galβ1–4GlcNAc. Because the Fucα1–2Galβ1–3GlcNAc
(H antigen) structure was found to interact with all immobilized protein columns significantly, theK
d value of pentasaccharide Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc-PA for each column was determined by analyzing the concentration
dependence. Obtained values for immobilized LEC-1, Nh, and Ch were 6.0 × 10−
5, 1.3 × 10−
4, and 6.5 × 10−
5 m, respectively. The most significant difference between Nh and Ch was in their affinity for GalNAcα1–3(Fucα1–2)Galβ1–3GlcNAcβ1–3Galβ1–4Glc-PA,
which contains the blood group A antigen; the K
d value for immobilized Nh was 4.8 × 10−
5
m, and that for Ch was 8.1 × 10−
4 m. The present results clearly indicate that the two sugar binding sites of LEC-1 have different sugar binding properties.
Journal of Biological Chemistry 02/2001; 276(5):3068-3077. · 4.77 Impact Factor
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ABSTRACT: Galectins are a family of soluble β-galactoside-binding lectins distributed in both vertebrates and invertebrates and, more
recently, found also in fungus. The 32-kDa galectin isolated from the nematode Caenorhabditis elegans(Hirabayashi, J., Satoh, M., and Kasai, K. (1992) J. Biol. Chem. 267, 15485–15490) was the first “tandem repeat-type” galectin, containing two homologous carbohydrate-binding sites. Here,
we report the structure of the nematode 32-kDa galectin gene. Physical mapping by yeast artificial chromosome polytene filter
hybridization revealed that the 32-kDa galectin gene is located on chromosome II. Analysis of the transcript (1.4 kilobases)
showed the presence at its 5′-end of a 22-nucleotide trans-spliced leader sequence (SL1). The entire genomic structure spanning >5 kilobase pairs (kbp), including the 5′-noncoding
region, two intervening sequences (introns 1 and 2), and the 3′-noncoding region, was completely determined by the combination
of genomic polymerase chain reaction and conventional colony hybridization. Intron 1 was relatively long (2.4 kbp) and was
found to be inserted after the ninth codon (TAC) from the initiation codon. This position proved to be almost homologous to
the conserved first intron insertion position in the vertebrate galectin genes (i.e. genes of mammalian galectin-1, -2, and -3 and chick 14-kDa galectin). On the other hand, intron 2 was much shorter (0.6 kbp),
and it was inserted into the central region of the second carbohydrate-binding site. Although such an insertion pattern has
never been observed in the vertebrate galectin genes, it seems to be common in C. elegans tandem repeat-type galectin genes, as predicted by the C. elegans genome project (Coulson, A., and the C. elegans Genome Consortium (1996) Biochem. Soc. Trans. 24, 289–291). Based on extensive sequence comparison, the origin and molecular evolution of the tandem repeat-type galectins
are discussed.
Journal of Biological Chemistry 10/1997; 272(42):26669-26677. · 4.77 Impact Factor
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ABSTRACT: The localization of the 32-kDa galectin (-galactoside-binding lectin) of the nematodeCaenorhabditis elegans, which is the first lectin to be found in a nematode, was examined immunohistochemically using an anti-lectin antiserum. The lectin was found to be localized most abundantly in the adult cuticle and also in the terminal bulb of the pharynx. However, it was difficult to locate the galectin in larval animals, though immunochemical experiments suggested its presence. These results suggest that one of the fundamental roles of the galectin may be as a component of the durable outer barrier, as in the case of the morphogenesis of chick embryonic skin.
The Histochemical Journal 02/1996; 28(3):201-207.
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ABSTRACT: The localization of an endogenous 14-kDa -galactoside-binding lectin (galectin) and its pattern of gene expression were examined in normal human skin by light- and electron microscopy. Under the light microscope, immunostaining of 14-kDa galectin was observed in the cell membrane of cells in the basal and spinous layers of the epidermis. Galectin was also found in the Langerhans cells, as shown by double labeling using anti-14-kDa galectin and anti-CD1a antibodies. In the dermis, immunostaining for the 14-kDa galectin was positive in the extracellular matrix and fibroblasts. At the electron-microscopic level of resolution, galectin was located primarily along the plasma membrane of keratinocytes, and in both the cytoplasm and nucleus of Langerhans cells in the epidermis, whereas in the dermis it was detected in the extracellular matrix and in both the nucleus and cytoplasm of fibroblasts. The gene expression of 14-kDa galectin was visualized by the HRP-staining method following in situ hybridization techniques. The expression was detected in the cytoplasm of cells in the basal and spinous layers of the epidermis; whereas, in the dermis, it was detected in the cytoplasm of fibroblasts. Moreover, SDS-polyacrylamide gel electrophoresis and lectin-blot analysis revealed that this galectin bound to glycoproteins of approximately 17, 62, and 72 kDa in the epidermis and to those of 29, 54, and 220 kDa in the dermis. The present study indicates that 1) normal human skin produces the -galactoside-binding 14-kDa galectin, and 2) this galectin is located in both the epidermis, particularly in the keratinocytes and Langerhans cells, and in the dermis. These results suggest that galectin is important for cell-cell contact and/or adhesion in the epidermis and for cell-extracellular matrix interaction in the dermis.
Cell and Tissue Research 03/1995; 280(1):1-10. · 3.11 Impact Factor
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ABSTRACT: To identify critical amino acid residues for carbohydrate binding of galectins (soluble -galactoside-binding lectins found in the animal kingdom), site-directed mutagenesis was performed on human galectin-1. On the basis of the previous results (Hirabayashi and Kasai (1992)J Biol Chem
266:23648-53), more systematic mutagenesis experiments were performed in order to confirm the concept that conserved hydrophilic residues play a central role. When a homologous substitution was made for highly conserved His44, Arg48 or Asn61, the resultant mutant (H44Q, R48H or N61D, respectively) almost completely lacked carbohydrate-binding ability, as found previously for Asn46, Glu71 and Arg73 mutants. This suggests these six hydrophilic residues are essential. On the other hand, when less conserved Lys63, Arg111 or Asp125 were substituted, the resultant mutant (K63H, R111H or D125E, respectively) retained almost the same affinities to asialofetuin and lactose as the wild-type galectin. Therefore, none of these residues is directly involved in the binding. These results, together with the previous observation that the above six essential residues are all encoded in the largest exon of the gene and are located close to each other in the central, most hydrophilic region of the protein, suggest that the residues form a carbohydrate-binding site of galectin.
Glycoconjugate Journal 09/1994; 11(5):437-442. · 2.12 Impact Factor
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ABSTRACT: The polylactosamine structure is a fundamental structure of carbohydrate chains and carries a lot of biofunctional carbohydrate epitopes. To investigate the biological function of polylactosamine chains, here we generated and analyzed knockout mice lacking the gene B3gnt2, which encodes a major polylactosamine synthase. In β1,3-N-acetylglucosaminyltransferase (B3gnt2) B3gnt2-deficient (B3gnt2−/−) mice, the number of polylactosamine structures was markedly lower than in wild-type mice. Flow cytometry, LEL lectin-blotting, and glycan analysis by metabolic labeling demonstrated that the amount of polylactosamine chains on N-glycans was greatly reduced in the tissues of B3gnt2−/− mice. We examined whether immunological abnormalities were present in B3gnt2−/− mice. We screened polylactosamine-carrying molecules of wild-type mice by lectin microarray analysis and found that polylactosamine was present on CD28 and CD19, two established immune co-stimulatory molecules. Polylactosamine levels on these molecules were lower in B3gnt2−/− mice than in wild-type mice. B3gnt2−/− T cells were more sensitive to the induction of intracellular Ca2+ flux on stimulation with anti-CD3ε/CD28 antibodies and proliferated more strongly than wild-type T cells. B3gnt2−/− B cells also showed hyperproliferation on BCR stimulation. These results showed that hyperactivation of lymphocytes occurred due to a lack of polylactosamine on receptor molecules in B3gnt2−/− mice. This finding indicates that polylactosamine has an important role in immunological biofunctions. We can therefore attempt to identify the in vivo biological function of glycans using glycogene-deficient mice.
Methods in Enzymology.
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ABSTRACT: Frontal affinity chromatography is a method for quantitative analysis of biomolecular interactions. We reinforced it by incorporating various merits of a contemporary liquid chromatography system. As a model study, the interaction between an immobilized Caenorhabditis elegans galectin (LEC-6) and fluorescently labeled oligosaccharides (pyridylaminated sugars) was analyzed. LEC-6 was coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (100 μm diameter), and packed into a miniature column (e.g., 10×4.0 mm, 0.126 ml). Twelve pyridylaminated oligosaccharides were applied to the column through a 2-ml sample loop, and their elution patterns were monitored by fluorescence. The volume of the elution front (V) determined graphically for each sample was compared with that obtained in the presence of an excess amount of hapten saccharide, lactose (V0); and the dissociation constant, Kd, was calculated according to the literature [K. Kasai, Y. Oda, M. Nishikawa, S. Ishii, J. Chromatogr. 376 (1986) 33]. This system also proved to be useful for an inverse confirmation; that is, application of galectins to an immobilized glycan column (in the present case, asialofetuin was immobilized on Sepharose 4 Fast Flow), and the elution profiles were monitored by fluorescence based on tryptophan. The relative affinity of various galectins for asialofetuin could be easily compared in terms of the extent of retardation. The newly constructed system proved to be extremely versatile. It enabled rapid (analysis time 12 min/cycle) and sensitive (20 nM for pyridylaminated derivatives, and 1 μg/ml for protein) analyses of lectin–carbohydrate interactions. It should become a powerful tool for elucidation of biomolecular interactions, in particular for functional analysis of a large number of proteins that should be the essential issues of post-genome projects.
Journal of Chromatography A.
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ABSTRACT: A complementary DNA clone preferentially expressed in the gastrointestinal tract was obtained from a rat stomach library. The protein coded by the clone had a single carbohydrate recognition domain having conserved motifs for β-galactoside binding and showed 67% amino acid identity with human galectin-2. The recombinant protein synthesized inEscherichia colicould bind to an asialofetuin column and was eluted with β-galactopyranoside. From these observations, we named the protein rat galectin-2 coded by the cDNA. The rat galectin-2 was predominantly expressed in the epithelial cells of stomach. Thus this protein may form a mucin layer cross-linking with the β-galactoside moiety of glycoproteins.
Archives of Biochemistry and Biophysics 361(2):195-201. · 2.93 Impact Factor
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Yuzuru Ikehara,
Takashi Sato,
Toru Niwa,
Sachiko Nakamura,
Masanori Gotoh,
Sanae Kabata Ikehara,
Katsue Kiyohara,
Chihiro Aoki,
Toshie Iwai,
Hayao Nakanishi, Jun Hirabayashi,
Masae Tatematsu,
Hisashi Narimatsu
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ABSTRACT: β1,4- N -acetylgalactosaminyltransferase III (β4GalNAc-T3), which was recently cloned and identified, exhibits GalNAc transferase activity toward a GlcNAcβ residue with β1,4-linkage, forming the N,N ’-diacetyllactosediamine, GalNAcβ1,4GlcNAc (LacdiNAc or LDN).Though LacdiNAc has not been found in the gastric mucosa, a large amount of transcript was detected in our previous study. To increase our knowledge of β4GalNAc-T3 expression and its product LacdiNAc, we examined the exact localization of β4GalNAc-T3 in human gastric mucosa using a newly developed antibody, mAb K1356. This antibody specifically detected the enzyme that trasfected the β4GalNAc-T3 gene into MKN45 cells, and the terminal βGalNAc epitope yielded on the cell surface was recognized by a lectin, Wisteria floribunda agglutinin (WFA). β4GalNAc-T3 was localized in the supra-nuclear region of surface mucous cells in gastric mucosa, and WFA positively stained the mucins secreted by the cells. In contrast, in the cells of the glandular compartment in the fundic glands and a few cells in the pyloric glands, β4GalNAc-T3 was observed in the basolateral position of nucleus, where no WFA reactivity was detected. The anti-Tn (GalNAcα -O- Ser/Thr) antibody staining did not overlap with the WFA staining. From measuring the binding activity of WFA using automated frontal affinity chromatography, WFA was found to bind most strongly LacdiNAc among the sugar chains examined. Neither β4GalNAc-T3 nor WFA-positive staining was detected in intestinal metaplastic cells. These results suggest that the supra-nuclear expression of β4GalNAc-T3 is essential for the formation of LacdiNAc on the surface mucous cells, and that LacdiNAc and β4GalNAc-T3 are novel differentiation markers of surface mucous cells in the gastric mucosa.
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ABSTRACT: Monoclonal antibodies against an endogenous β-galactoside-binding lectin (monomer molecular weight 14,000, 14K lectin) of chick embryo were prepared and characterized. The inhibitory activities against hemagglutination, antigenic determinants and binding specificities were examined. Monoclonal antibody S1A4-5 strongly inhibited the hemagglutination activity of this lectin. This antibody did not bind to any cyanogen bromide (BrCN) fragment of the lectin. Another monoclonal antibody, S1A4-3, bound to one of the BrCN fragments (residues 34–66). However, this antibody inhibited hemagglutination only weakly. The bindings to isolectins of β-galactoside-binding lectin, namely 14K lectin, 16K lectin (monomer molecular weight 16,000) and a third species which is assumed to be a hybrid molecule consisting of 14K and 16K lectin subunits, were examined. The antibody SIA4-5 bound to 14K lectin but not to 16K lectin. In the case of the third species, intermediate binding was observed.
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ABSTRACT: Two carbohydrate-binding proteins (subunit molecular masses, 32 and 16 kDa, respectively) were isolated for the first time from a nematode, Caenorhabditis elegans . They were specifically extracted with lactose and adsorbed on asialofetuin-Sepharose in the absence of a metal ion. Although these two proteins were co-eluted from a gel filtration column at a position corresponding to an apparent molecular size of 30 kDa under non-denaturing conditions, they could be separated by reversed-phase chromatography. The 32 kDa protein, the main component, was further characterized. Together with its solubility, saccharide specificity and metal independence, some other structural properties, including its amino acid composition, UV spectrum, and partial amino acid sequence, strongly suggested that the 32 kDa protein is a member of a class of soluble β-galactoside-binding lectins which had previously been only found in vertebrates.
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ABSTRACT: A β-galactoside-binding lectin was extracted from human placenta homogenate with lactose solution and purified to apparent homogeneity by affinity chromatography on asialofetuin-Sepharose. The apparent subunit molecular weight of the lectin was 13,800 and its isoelectric point was about 5. Several saccharides containing D-galactose inhibited the hemagglutinating activity. The lectin resembles other vertebrate β-galactoside-binding lectins in various biochemical characteristics.
Biochemical and Biophysical Research Communications.
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ABSTRACT: The complete amino acid sequence of a β-galactoside-binding lectin from human placenta was determined at protein level. The lectin consists of 134 amino acids and its N-terminal alanine is blocked with acetate. The lectin shows about 50% similarity with chick 14K lectin, which was the first vertebrate β-galactoside-binding lectin completely sequenced. Only 14 residues proved to be different from those of rat lung lectin, the sole mammalian lectin of which the complete sequence has been reported.
