Jun Hirabayashi

Kagawa University, Takamatu, Kagawa, Japan

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Publications (269)989.01 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Previous studies indicated that sclerotes of the phytopathogenic Ascomycete Sclerotinia sclerotiorum contain a lectin that based on its molecular structure, specificity and N-terminal amino acid sequence could not be classified yet into any lectin family. Using a combination of molecular cloning, frontal affinity chromatography and molecular modelling the identity of the S. sclerotiorum agglutinin (SSA) was analyzed. Molecular cloning demonstrated that SSA shares no sequence similarity with any known fungal lectin or protein. The lectin is synthesized as a 153 amino acid polypeptide without signal peptide and undergoes apart from the removal of the N-terminal methionine no further processing. Frontal affinity chromatography revealed that the binding site of SSA primarily accommodates a non-reducing terminal GalNAc with a preference for the alpha- over the beta-anomer. SSA also strongly interacts with both glycolipid type glycans with terminal non-reducing Gal or GalNAc and galactosylated N-glycans. SSA shares a residual sequence similarity with part of the non-toxin haemagglutinin HA33/A from Clostridium botulinum. Molecular modeling using the three-dimensional structure of HA33/A as a template indicated that SSA can fold into a similar beta-trefoil domain. Though these results should be interpreted with care it is tempting to speculate that the Sclerotiniaceae lectins thus appear to be structurally related to the ricin-B superfamily. All evidence suggests that SSA represents a novel family of fungal lectins with a unique sequence and sugar-binding properties. Taking into account that orthologues of SSA are fairly common within the family Sclerotiniaceae but could not be identified in any other fungal species one can reasonably conclude that SSA-type lectins are confined to a small taxonomic group of the Ascomycota.
    No preview · Article · May 2007 · Glycoconjugate Journal
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    ABSTRACT: Pyridylamination is a versatile method for fluorescence labeling of oligosaccharides. The technique affords sensitive detection of saccharides with reducing termini and high-resolution separation by high-performance liquid chromatography. The conventional method, based on a liquid-phase reaction, has been extensively used in various aspects of glycobiology and glycotechnology. Unfortunately, the necessity for removing excess 2-aminopyridine makes the technique both laborious and time-consuming. Furthermore, removal of excess reagent can result in a significant loss of short saccharide components. In the present paper, we report an alternative methodology based on a "gas-phase" reaction, in which dried saccharides are reacted with vaporized 2-aminopyridine. The resultant Schiff base was also reduced in the gas phase within the same reaction microtube using a purpose-built device. The newly developed procedure was applied to both monosaccharide (GlcNAc) and oligosaccharides (isomalto-oligosaccharides) at quantitative yields with no requirement to remove excess reagent. The acid-labile sialyl linkages of alpha2-6-disialobiantennary oligosaccharides proved to be fully stable during the procedure. The developed method was also successfully applied to profiling N-linked oligosaccharides liberated from glycoproteins by hydrazinolysis and, thus, should contribute to various fields of glycomics.
    No preview · Article · May 2007 · Analytical Chemistry
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    ABSTRACT: The argasid tick Ornithodoros moubata is a vector of various viral and borrelian diseases in animals and humans. We report here molecular characterization and oligosaccharide-binding properties of a novel galectin (OmGalec) from this tick. OmGalec consisted of 333 amino acids with a predicted molecular weight of 37.4 kDa. Its amino acid sequence did not contain a signal peptide or transmembrane domain. It possessed tandem-repeated carbohydrate recognition domains, in which the typical motifs important for carbohydrate affinity were conserved. OmGalec was expressed both transcriptionally and translationally at all stages of the tick life cycle and in multiple organs and was abundant in hemocytes, midguts, and reproductive organs, which are of importance in immunity, interaction with pathogens, and development, respectively, suggesting that OmGalec is a multifunctional molecule. The oligosaccharide affinity profile analyzed by applying an automated frontal affinity chromatography system revealed that rOmGalec showed a general feature of the galectin family, i.e. significant affinity for lactosamine-type disaccharides, Galbeta1-3(4)Glc(NAc), via recognition of 4-OH and 6-OH of galactose and 3 (4)-OH of Glc(NAc). Its preference for type I saccharides and alpha1-3GalNAc-containing oligosaccharides might provide clues for identifying its ligands and its potential multiple functions. Our results may contribute to the elucidation of galectin functions in the development and immunity of arthropods and/or vector and pathogen interaction and provide valuable information for the development of novel tick control strategies.
    Preview · Article · Apr 2007 · Glycobiology
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    ABSTRACT: Sialic acid (Sia) is a typical terminal sugar, which modifies various types of glycoconjugates commonly found in higher animals. Its regulatory roles in diverse biological phenomena are frequently triggered by interaction with Sia-binding lectins. When using natural Sia-binding lectins as probes, however, there have been practical problems concerning their repertoire and availability. Here, we show a rational creation of a Sia-binding lectin based on the strategy 'natural evolution-mimicry', where Sia-binding lectins are engineered by error-prone PCR from a Gal-binding lectin used as a scaffold protein. After selection with fetuin-agarose using a recently reinforced ribosome display system, one of the evolved mutants SRC showed substantial affinity for alpha2-6Sia, which the parental Gal-binding lectin EW29Ch lacked. SRC was found to have additional practical advantages in productivity and in preservation of affinity for Gal. Thus, the developed novel Sia-recognition protein will contribute as useful tools to sialoglycomics.
    No preview · Article · Apr 2007 · Journal of Biochemistry
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    ABSTRACT: The sugar-binding specificities of C-type lectins isolated from marine invertebrates were investigated by frontal affinity chromatography (FAC) using 100 oligosaccharides. The lectins included BRA-2 and BRA-3, multiple lectins from the hemolymph of the acorn barnacle, Megabalanus rosa, and BRL from the acorn barnacle, Balanus rostatus. The diverse sugar-binding specificities of the C-type lectins were determined by FAC analysis. BRA-2 recognized alpha2-6 sialylation but not alpha2-3 sialylation on glycans. On the other hand, BRA-3 showed high affinity for oligosaccharides with alpha-linked non-reducing terminal galactose, but not for sialylated forms, and BRL showed enhanced recognition activity towards Lewis(x) and Lewis(a) epitopes.
    No preview · Article · Mar 2007 · Bioscience Biotechnology and Biochemistry
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    ABSTRACT: Patients with metastatic cancer commonly have increased serum galectin-3 concentrations, but it is not known whether this has any functional implications for cancer progression. We report that MUC1, a large transmembrane mucin protein that is overexpressed and aberrantly glycosylated in epithelial cancer, is a natural ligand for galectin-3. Recombinant galectin-3 at concentrations (0.2-1.0 microg/ml) similar to those found in the sera of patients with metastatic cancer increased adhesion of MUC1-expressing human breast (ZR-75-1) and colon (HT29-5F7) cancer cells to human umbilical vein endothelial cells (HUVEC) by 111% (111 +/- 21%, mean +/- S.D.) and 93% (93 +/- 17%), respectively. Recombinant galectin-3 also increased adhesion to HUVEC of MUC1 transfected HCA1.7+ human breast epithelial cells that express MUC1 bearing the oncofetal Thomsen-Friedenreich antigen (Galbeta1,3 GalNAc-alpha (TF)) but did not affect adhesion of MUC1-negative HCA1.7-cells. MUC1-transfected, Ras-transformed, canine kidney epithelial-like (MDE9.2+) cells, bearing MUC1 that predominantly carries sialyl-TF, only demonstrated an adhesive response to galectin-3 after sialidase pretreatment. Furthermore, galectin-3-mediated adhesion of HCA1.7+ to HUVEC was reduced by O-glycanase pretreatment of the cells to remove TF. Recombinant galectin-3 caused focal disappearance of cell surface MUC1 in HCA1.7+ cells, suggesting clustering of MUC1. Co-incubation with antibodies against E-Selectin or CD44H, but not integrin-beta1, ICAM-1 or VCAM-1, largely abolished the epithelial cell adhesion to HUVEC induced by galectin-3. Thus, galectin-3, by interacting with cancer-associated MUC1 via TF, promotes cancer cell adhesion to endothelium by revealing epithelial adhesion molecules that are otherwise concealed by MUC1. This suggests a critical role for circulating galectin-3 in cancer metastasis and highlights the functional importance of altered cell surface glycosylation in cancer progression.
    Full-text · Article · Feb 2007 · Journal of Biological Chemistry
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    Julie Nieminen · Atsushi Kuno · Jun Hirabayashi · Sachiko Sato
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    ABSTRACT: Galectin-3, a member of the galectin family of carbohydrate binding proteins, is widely expressed, particularly in cells involved in the immune response. Galectin-3 has also been indicated to play a role in various biological activities ranging from cell repression to cell activation and adhesion and has, thus, been recognized as an immunomodulator. Whereas those activities are likely to be associated with ligand cross-linking by this lectin, galectin-3, unlike other members of the galectin family, exists as a monomer. It has consequently been proposed that oligomerization of the N-terminal domains of galectin-3 molecules, after ligand binding by the C-terminal domain, is responsible for this cross-linking. The oligomerization status of galectin-3 could, thus, control the majority of its extracellular activities. However, little is known about the actual mode of action through which galectin-3 exerts its function. In this report we present data suggesting that oligomerization of galectin-3 molecules occurs on cell surfaces with physiological concentrations of the lectin. Using galectin-3 labeled at the C terminus with Alexa 488 or Alexa 555, the oligomerization between galectin-3 molecules on cell surfaces was detected using fluorescence resonance energy transfer. We observed this fluorescence resonance energy transfer signal in different biological settings representing the different modes of action of galectin-3 that we previously proposed; that is, ligand crosslinking leading to cell activation, cell-cell interaction/adhesion, and lattice formation. Furthermore, our data suggest that galectin-3 lattices are robust and could, thus, be involved, as previously proposed, in the restriction of receptor clustering.
    Preview · Article · Feb 2007 · Journal of Biological Chemistry
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    ABSTRACT: Podoplanin (Aggrus) is a mucin-type sialoglycoprotein that plays a key role in tumor cell-induced platelet aggregation. Podoplanin possesses a platelet aggregation-stimulating (PLAG) domain, and Thr52 in the PLAG domain of human podoplanin is important for its activity. Endogenous or recombinant human podoplanin were purified, and total glycosylation profiles were surveyed by lectin microarray. Analyses of glycopeptides produced by Edman degradation and mass spectrometry revealed that the disialyl-corel (NeuAc alpha2-3Gal beta l-3(NeuAc alpha2-6)GalNAc alpha l-O-Thr) structure was primarily attached to a glycosylation site at residue Thr52. Sialic acid-deficient podoplanin recovered its activity after additional sialylation. These results indicated that the sialylated Corel at Thr52 is critical for podoplanin-induced platelet aggregation.
    Full-text · Article · Feb 2007 · FEBS Letters
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    ABSTRACT: Frontal affinity chromatography using fluorescence detection (FAC-FD) is a versatile technique for the precise determination of dissociation constants (Kd) between glycan-binding proteins (lectins) and fluorescent-labeled glycans. A series of glycan-containing solutions is applied to a lectin-immobilized column, and the elution profile of each glycan (termed the 'elution front', V) is compared with that (V0) for an appropriate control. Here we describe our standard protocol using an automated FAC system (FAC-1), consisting of two isocratic pumps, an autosampler, a column oven and two miniature columns connected to a fluorescence detector. Analysis time for 100 sugar-protein interactions is approximately 10 h, using as little as 2.5 pmol of pyridylaminated (PA) oligosaccharide per analysis. Using FAC-FD, we have so far obtained quantitative interaction data of >100 lectins for >100 PA oligosaccharides.
    No preview · Article · Feb 2007 · Nature Protocol
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    ABSTRACT: Both saturated and unsaturated forms of isomeric unsulfated glycosaminoglycan (GAG) oligosaccharides, i.e., tetrasaccharides of chondroitin (CH) and hyaluronan (HA), were analyzed by electrospray ionization mass spectrometry/mass spectrometry. Although the only structural difference between them was the hydroxyl group at the C-4 position in N-acetylhexosamine (GalNAc or GlcNAc, respectively), given the same m/z value of precursor ions, these isomers in both their saturated and unsaturated forms could be separated by careful examination of diagnostic fragment ions in their product ion mass spectra when the relative abundances of these fragment ions were considered. In addition, the product ion mass spectrum of the unsaturated HA tetrasaccharide was compared with its linkage isomer, N-acetylheparosan tetrasaccharide. In this case, the isomers were more easily differentiated by comparing their characteristic spectral patterns. By adopting this approach, systematic differentiation of isomeric unsulfated GAG oligosaccharides should be achieved by means of fragmentation. It should also contribute widely to GAG-related biochemical and medicinal research in the future.
    No preview · Article · Jan 2007 · Journal of the Mass Spectrometry Society of Japan
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    ABSTRACT: A novel lectin, PPL, was isolated from the mantle of penguin wing oyster (Pteria penguin) by affinity chromatography on mucin-Sepharose 4B and cation exchange chromatography on HiTrap SP. This lectin was estimated to be a 21-kDa monomer by gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted time of flight (MALDI-TOF) mass spectrometry. However, dynamic light scattering experiments revealed that a non-covalently linked dimer formed under high salt conditions (500 mM NaCl). Interestingly, PPL showed an increasing hemagglutinating activity with increasing salt concentration. The amino acid sequence of PPL was determined by direct protein sequence analysis and cDNA cloning. The 167-amino acid sequence included 24 lysine residues and had two tandemly repeated homologous domains (residues 20-78 and 107-165) with 44% internal homology. PPL showed sequence homology to L-rhamnose-binding lectins from fish eggs and a D-galactose-binding lectin from sea urchin eggs, with sequence identities in the range 37-48%. PPL agglutinated various animal erythrocytes independently of calcium ions. The minimum concentration of PPL needed to agglutinate rabbit erythrocytes was 0.5 micro g/ml, and the most effective saccharides to inhibit the hemagglutination were D-galactose, methyl-D-galactopyranoside and N-acetyl-D-lactosamine. Lactose also inhibited hemagglutination, but L-rhamnose did so only weakly despite the sequence homology with trout egg L-rhamnose-binding lectins. The carbohydrate-binding specificity of PPL was further examined by frontal affinity chromatography using 37 different pyridylaminated oligosaccharides. PPL was found to have strong binding affinity for various oligosaccharides that have Galbeta1-4Glu/GlcNAc, Galbeta1-3GalNAc/GlcNAc and Galalpha 1-4Gal moieties in their structure. PPL had a high thermal stability and retained 50% of its hemagglutinating activity after incubation at 70 degrees C for 100 min. It agglutinated some Gram-negative bacteria by recognizing lipopolysaccharides. Together, these results suggest that PPL is a new member of the trout egg lectin family which participates in the self-defense mechanism against bacteria and pathogens with a distinct carbohydrate-binding specificity. We conclude that the trout egg lectin family proteins, in particular their carbohydrate recognition domains, have acquired diverse carbohydrate-binding specificities during molecular evolution.
    Full-text · Article · Dec 2006 · Molecular Diversity
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    ABSTRACT: The mucin-type sialoglycoprotein, podoplanin (aggrus), is a platelet-aggregating factor on cancer cells. We previously described up-regulated expression of podoplanin in malignant astrocytic tumors including glioblastoma. Its expression was associated with tumor malignancy. In the present study, we investigated podoplanin expression and platelet-aggregating activities of glioblastoma cell lines. First, we established a highly reactive anti-podoplanin antibody, NZ-1, which inhibits podoplanin-induced platelet aggregation completely. Of 15 glioblastoma cell lines, LN319 highly expressed podoplanin and induced platelet aggregation. Glycan profiling using a lectin microarray showed that podoplanin on LN319 possesses sialic acid, which is important in podoplanin-induced platelet aggregation. Interestingly, NZ-1 neutralized platelet aggregation by LN319. These results suggest that podoplanin is a main reason for platelet aggregation induced by LN319. We infer that NZ-1 is useful to determine whether platelet aggregation is podoplanin-specific or not. Furthermore, podoplanin might become a therapeutic target of glioblastoma for antibody-based therapy.
    Full-text · Article · Dec 2006 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Alpha-L-arabinofuranosidase catalyses the hydrolysis of the alpha-1,2-, alpha-1,3-, and alpha-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 alpha-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-alpha-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 10(3) M(-1). These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.
    Preview · Article · Nov 2006 · Biochemical Journal
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    ABSTRACT: Structural glycomics plays a fundamental role in glycoscience and glycotechnology. In this paper, a novel strategy for the structural characterization of glycans is described, in which MS2 analysis involving a LIFT-TOF/TOF procedure is combined with frontal affinity chromatography (FAC). As model compounds, 20 neutral pyridylaminated (PA) oligosaccharides were chosen, which included four groups of structural isomers differing in sequence, linkage, position, or branching features. By depicting significant diagnostic ions on MS2, most of the analyzed oligosaccharides were successfully differentiated, while two pairs of linkage isomers, i.e., LNT/LNnT, and LNH/LNnH were not. For subsequent analysis by FAC, 14 lectins showing significant affinity to either LNT (type 1) or LNnT (type 2) were screened, and a galectin from the marine sponge Geodia cydonium (GC1) and a plant seed lectin from Ricinus communis (RCA-I) were used for determination of type 1 and 2 chains, respectively. With these specific probes, both of the isomeric pairs were unambiguously differentiated. Furthermore, a pair of triantennary, asparagine-linked oligosaccharide isomers could also be successfully differentiated. Thus, the combination of MS2 and FAC is a practical alternative for the structural characterization of complex glycans.
    No preview · Article · Oct 2006 · Journal of Biochemistry
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    ABSTRACT: beta1,4-N-acetylgalactosaminyltransferase III (beta4GalNAc-T3), which was recently cloned and identified, exhibits GalNAc transferase activity toward a GlcNAcbeta residue with beta1,4-linkage, forming the N,N'-diacetyllactosediamine, GalNAcbeta1,4GlcNAc (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 beta4GalNAc-T3 expression and its product LacdiNAc, we examined the exact localization of beta4GalNAc-T3 in human gastric mucosa using a newly developed antibody, monoclonal antibody (mAb) K1356. This antibody specifically detected the enzyme that transfected the beta4GalNAc-T3 gene into MKN45 cells, and the terminal betaGalNAc epitope yielded on the cell surface was recognized by a lectin, Wisteria floribunda agglutinin (WFA). beta4GalNAc-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, beta4GalNAc-T3 was observed in the basolateral position of the nucleus, where no WFA reactivity was detected. The anti-Tn (GalNAcalpha-O-Ser/Thr) antibody staining did not overlap with the WFA staining. By measuring the binding activity of WFA using automated frontal affinity chromatography (FAC), we found WFA to bind most strongly LacdiNAc among the sugar chains examined. Neither beta4GalNAc-T3 nor WFA-positive staining was detected in intestinal metaplastic cells. These results suggest that the supra-nuclear expression of beta4GalNAc-T3 is essential for the formation of LacdiNAc on the surface mucous cells and that LacdiNAc and beta4GalNAc-T3 are novel differentiation markers of surface mucous cells in the gastric mucosa.
    Preview · Article · Oct 2006 · Glycobiology
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    ABSTRACT: Lectin-based structural glycomics requires a search for useful lectins and their biochemical characterization to profile complex features of glycans. In this paper, two GlcNAc-binding lectins are reported with their detailed oligosaccharide specificity. One is a classic plant lectin, Griffonia simplicifolia lectin-II (GSL-II), and the other is a novel fungal lectin, Boletopsis leucomelas lectin (BLL). Their sugar-binding specificity was analyzed by frontal affinity chromatography using 146 glycans (125 pyridylaminated and 21 p-nitrophenyl saccharides). As a result, it was found that both GSL-II and BLL showed significant affinity toward complex-type N-glycans, which are either partially or completely agalactosylated. However, their branch-specific features differed significantly: GSL-II strongly bound to agalacto-type, tri- or tetra-antennary N-glycans with its primary recognition of a GlcNAc residue transferred by GlcNAc-transferase IV, while BLL preferred N-glycans with fewer branches. In fact, the presence of a GlcNAc residue transferred by GlcNAc-transferase V abolishes the binding of BLL. Thus, GSL-II and BLL forms a pair of complementally probes to profile a series of agalacto-type N-glycans.
    No preview · Article · Sep 2006 · Journal of Biochemistry
  • Toshikazu Minamisawa · Jun Hirabayashi
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    ABSTRACT: Structural complexity and heterogeneity of glycosaminoglycans (GAGs) have troubled biochemists in the research field for many years, thereby the progress of its functional research has long been delayed. Recently, rapid progress in mass spectrometry (MS), especially a tandem MS that can perform MS/MS (MS n) experiments, has lead to its increasing use for structural studies of GAGs. By using MS, though still under research, it is becoming possible to obtain information on not only molecular weights of GAG oligosaccharides but also position of sulfation, epimerization of a uronic acid, and more detail on the oligosaccharide sequence. Further refinement of MS methodology is expected to accelerate biochemical research of GAGs as well as their medical applications.
    No preview · Article · Sep 2006 · Trends in Glycoscience and Glycotechnology
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    ABSTRACT: Agaricus bisporus agglutinin (ABA) is known as a useful lectin to detect T-antigen (Core1) disaccharide (Galbeta1-3GalNAcalpha) and related O-linked glycans. However, a recent X-ray crystallographic study revealed the presence of another intrinsic sugar-binding site, i.e., for GlcNAc. To confirm this possibility, detailed analysis was performed using two advanced methods: lectin microarray and frontal affinity chromatography (FAC). In the lectin microarray, intense signals were observed on ABA spots for both N-glycanase-treated and O-glycanase/beta1-4galactosidase-treated Cy3-labeled asialofetuin. This indicates substantial affinity for both O-linked and agalactosylated (GlcNAc-exposed) N-linked glycans. A further approach by FAC using 20 pNP and 130 PA-oligosaccharides demonstrated that ABA bound to Core1 (K(d) = 3.4 x 10(-6) M) and Core2 (1.9 x 10(-5) M) but not to Core3 and Core6 O-linked glycans. It also showed substantial affinity to mono-, bi-, and tri-antennary agalactosylated complex-type N-linked glycans (K(d) > 1.8 x 10(-5) M). These results establish ABA as a lectin having dual sugar-binding sites with distinct specificity, i.e., for Gal-exposed O-linked glycans and GlcNAc-exposed N-linked glycans.
    No preview · Article · Aug 2006 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Intraocular inflammatory diseases are a common cause of severe visual impairment and blindness. In this study, we investigated the immunoregulatory role of galectin-1 (Gal-1), an endogenous lectin found at sites of T cell activation and immune privilege, in experimental autoimmune uveitis (EAU), a Th1-mediated model of retinal disease. Treatment with rGal-1 either early or late during the course of interphotoreceptor retinoid-binding protein-induced EAU was sufficient to suppress ocular pathology, inhibit leukocyte infiltration, and counteract pathogenic Th1 cells. Administration of rGal-1 at the early or late phases of EAU ameliorated disease by skewing the uveitogenic response toward nonpathogenic Th2 or T regulatory-mediated anti-inflammatory responses. Consistently, adoptive transfer of CD4(+) regulatory T cells obtained from rGal-1-treated mice prevented the development of active EAU in syngeneic recipients. In addition, increased levels of apoptosis were detected in lymph nodes from mice treated with rGal-1 during the efferent phase of the disease. Our results underscore the ability of Gal-1 to counteract Th1-mediated responses through different, but potentially overlapping anti-inflammatory mechanisms and suggest a possible therapeutic use of this protein for the treatment of human uveitic diseases of autoimmune etiology.
    Full-text · Article · Jun 2006 · The Journal of Immunology
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    ABSTRACT: A gene encoding an exo-beta-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-beta-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50 degrees C and catalyzes hydrolysis only of beta-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from beta-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a beta-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-beta-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including beta-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a beta-1,3-galactobiose, beta-1,4-galactosyl glucose, or beta-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward beta-1,3-galactan and alpha-l-arabinofuranosidase-treated AGP.
    Full-text · Article · May 2006 · Applied and Environmental Microbiology

Publication Stats

10k Citations
989.01 Total Impact Points

Institutions

  • 2005-2015
    • Kagawa University
      • • Life Science Research Center
      • • Division of Glyco-Bioindustry and Functional Glycomics
      Takamatu, Kagawa, Japan
  • 2003-2015
    • National Institute of Advanced Industrial Science and Technology
      • • Research Center for Stem Cell Engineering
      • • Research Center for Medical Glycoscience
      Tsukuba, Ibaraki, Japan
  • 2014
    • Kitasato University
      • Department of Marine Biosciences
      Edo, Tōkyō, Japan
  • 2011-2013
    • Japan Advanced Institute of Science and Technology
      KMQ, Ishikawa, Japan
    • The University of Tokyo
      Tōkyō, Japan
  • 2008
    • Ludwig-Maximilians-University of Munich
      • Faculty of Veterinary Medicine
      München, Bavaria, Germany
  • 1986-2008
    • Teikyo University
      • • Department of Biochemistry
      • • Faculty of Pharmaceutical Sciences
      Edo, Tokyo, Japan
  • 2006
    • Advance Institute of Science and Technology
      Dehra, Uttarakhand, India
    • Saitama University
      • Faculty of Science
      Saitama, Saitama, Japan
  • 2001
    • Kanazawa Medical University
      • Department of Pathology
      Kanazawa, Ishikawa, Japan
  • 1998
    • Kyorin University
      • Department of Anatomy
      Edo, Tōkyō, Japan