[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
Applied and Environmental Microbiology 01/2006; 72(5):3515-3523. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glycans have important roles in living organisms with their structural diversity. Thus, glycomics, especially aspects involving the assignment of functional glycans in a high-throughput manner, has been an emerging field in the postproteomics era. To date, however, there has been no versatile method for glycan profiling. Here we describe a new microarray procedure based on an evanescent-field fluorescence-detection principle, which allows sensitive, real-time observation of multiple lectin-carbohydrate interactions under equilibrium conditions. The method allows quantitative detection of even weak lectin-carbohydrate interactions (dissociation constant, K(d) > 10(-6) M) as fluorescent signals for 39 immobilized lectins. We derived fully specific signal patterns for various Cy3-labeled glycoproteins, glycopeptides and tetramethylrhodamine (TMR)-labeled oligosaccharides. The obtained results were consistent with the previous reports of glycoprotein and lectin specificities. We investigated the latter aspects in detail by frontal affinity chromatography, another profiling method. Thus, the developed lectin microarray should contribute to creation of a new paradigm for glycomics.
[Show abstract][Hide abstract] ABSTRACT: An exo-beta-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-beta-1,3-galactanase indicated that the enzyme consists of an N-terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydrate-binding module family 6 (CBM6) in the C-terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS-PAGE. The enzyme showed reactivity only toward beta-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze beta-1,4-linked galacto-oligosaccharides, beta-1,6-linked galacto-oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze beta-1,3-galactosyl galactosaminide, beta-1,3-galactosyl glucosaminide, or beta-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal beta-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from beta-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl beta-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass beta-1,6-linked galactosyl side chains. The C-terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and beta-1,3-glucan, although it bound beta-1,3-galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a beta-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.
Journal of Biological Chemistry 08/2005; 280(27):25820-9. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Lectins belonging to the jacalin-related lectin family are distributed widely in the plant kingdom. Recently, two mannose-specific lectins having tandem repeat-type structures were discovered in Castanea crenata (angiosperm) and Cycas revoluta (gymnosperm). The occurrence of such similar molecules in taxonomically less related plants suggests their importance in the plant body. To obtain clues to understand their physiological roles, we performed detailed analysis of their sugar-binding specificity. For this purpose, we compared the dissociation constants (K(d)) of Castanea crenata agglutinin (CCA) and Cycas revoluta leaf lectin (CRLL) by using 102 pyridylaminated and 13 p-nitrophenyl oligosaccharides with a recently developed automated system for frontal affinity chromatography. As a result, we found that the basic carbohydrate-binding properties of CCA and CRLL were similar, but differed in their preference for larger N-linked glycans (e.g. Man7-9 glycans). While the affinity of CCA decreased with an increase in the number of extended alpha1-2 mannose residues, CRLL could recognize these Man7-9 glycans with much enhanced affinity. Notably, both lectins also preserved considerable affinity for mono-antennary, complex type N-linked glycans, though the specificity was much broader for CCA. The information obtained here should be helpful for understanding their functions in vivo as well as for development of useful probes for animal cells. This is the first systematic approach to elucidate the fine specificities of plant lectins by means of high-throughput, automated frontal affinity chromatography.
[Show abstract][Hide abstract] ABSTRACT: The establishment of HIV type 1 (HIV-1) infection is initiated by the stable attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between virus-encoded gp120 and cell surface CD4, a number of distinct interactions influence binding of HIV-1 to host cells. In this study, we report that galectin-1, a dimeric beta-galactoside-binding protein, promotes infection with R5, X4, and R5X4 variants. Galectin-1 acts as a soluble adhesion molecule by facilitating attachment of HIV-1 to the cell surface. This postulate is based on experiments where galectin-1 rendered HIV-1 particles more refractory to various agents that block HIV-1 adsorption and coreceptor binding (i.e., a blocking anti-CD4, soluble CD4, human anti-HIV-1 polyclonal Abs; stromal cell-derived factor-1alpha; RANTES). Experiments performed with the fusion inhibitor T-20 confirmed that galectin-1 is primarily affecting HIV-1 attachment. The relevance of the present findings for the pathogenesis of HIV-1 infection is provided by the fact that galectin-1 is abundantly expressed in the thymus and lymph nodes, organs that represent major reservoirs for HIV-1. Moreover, galectin-1 is secreted by activated CD8(+) T lymphocytes, which are found in high numbers in HIV-1-positive patients. Therefore, it is proposed that galectin-1, which is released in an exocrine fashion at HIV-1 replication sites, can cross-link HIV-1 and target cells and promote a firmer adhesion of the virus to the cell surface, thereby augmenting the efficiency of the infection process. Overall, our findings suggest that galectin-1 might affect the pathogenesis of HIV-1 infection.
The Journal of Immunology 05/2005; 174(7):4120-6. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Electrospray ionization combined with ion trap mass spectrometry (ESI-ITMS) is a powerful tool for structural analysis of complex carbohydrates. Although its application to sulfated glycans has been limited so far, it should provide critical information, such as sulfate positions, on their structures. In this work, MS(n) spectra of nine monosulfated monosaccharides, consisting of five hexoses and four N-acetylhexosamines, were measured in negative ion mode to find basic fragmentation rules for sulfated sugars. Two pairs of positional isomers with respect to sulfation, i.e., Gal4S and Gal6S, and GalNAc4S and GalNAc6S, showed characteristic fragmentation patterns in MS(3), and could be discriminated from one another by the appearance of particular diagnostic fragment ions that characterize individual isomers. It was also demonstrated that, even if a mixture of these positional isomers was analyzed, the proportion of each species could be estimated through analysis of the abundance ratios of the diagnostic ions. However, 3-O-sulfated saccharides (Glc3S and GlcNAc3S) gave a single abundant diagnostic ion in MS(2) corresponding to the hydrogensulfate ion, [OSO(3)H](-), and this characteristic clearly differentiated them from their positional isomers. In contrast, 6-O-sulfated diastereomers consisting of two groups, Glc6S, Man6S, Gal6S, and GlcNAc6S, GalNAc6S, could not be discriminated by the types of fragment ions; however, the abundance ratios of particular fragment ions differed significantly between Glc(NAc)6S and Gal(NAc)6S. Since ESI-ITMS yielded large quantities of useful information on structures of monosulfated hexoses and N-acetylhexosamines in an extremely simple and reproducible manner, establishment of a comprehensive strategy based on ESI-ITMS(n) appears to be a promising technique for structural elucidation of sulfated complex carbohydrates.
Rapid Communications in Mass Spectrometry 02/2005; 19(13):1788-96. · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The galactose-binding lectin EW29 from the earthworm Lumbricus terrestris is composed of two homologous domains, both of which are members of the R-type lectin family. The truncated mutant rC-half comprising the C-terminal domain was crystallized by the hanging-drop vapour-diffusion method. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 61.2, c = 175.6 A, and diffracted to beyond 1.9 A resolution. Matthews coefficient calculations suggested that this crystal contained two molecules per asymmetric unit.
[Show abstract][Hide abstract] ABSTRACT: Human galectin-1 is a potent multifunctional effector that participates in specific protein-carbohydrate and protein-protein (lipid) interactions. By determining its X-ray structure, we provide the basis to define the structure of its ligand-binding pocket and to perform rational drug design. We have also analysed whether single-site mutations introduced at some distance from the carbohydrate recognition domain can affect the lectin fold and influence sugar binding. Both the substitutions introduced in the C2S and R111H mutants altered the presentation of the loop, harbouring Asp123 in the common "jelly-roll" fold. The orientation of the side-chain was inverted 180 degrees and the positions of two key residues in the sugar-binding site of the R111H mutant were notably shifted, i.e. His52 and Trp68. Titration calorimetry was used to define the decrease in ligand affinity in both mutants and a significant increase in the entropic penalty was found to outweigh a slight enhancement of the enthalpic contribution. The position of the SH-groups in the galectin appeared to considerably restrict the potential to form intramolecular disulphide bridges and was assumed to be the reason for the unstable lectin activity in the absence of reducing agent. However, this offers no obvious explanation for the improved stability of the C2S mutant under oxidative conditions. The noted long-range effects in single-site mutants are relevant for the functional divergence of closely related galectins and in more general terms, the functionality definition of distinct amino acids.
Journal of Molecular Biology 11/2004; 343(4):957-70. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Brain-derived neurotrophic factor (BDNF) is a neuroprotective polypeptide that is thought to be responsible for neuron proliferation, differentiation, and survival. An agent that enhances production of BDNF is expected to be useful for the treatment of neurodegenerative diseases. Here we report that galectin-1, a member of the family of beta-galactoside binding proteins, induces astrocyte differentiation and strongly inhibits astrocyte proliferation, and then the differentiated astrocytes greatly enhance their production of BDNF. Induction of astrocyte differentiation and BDNF production by an endogenous mammalian lectin may be a new mechanism for preventing neuronal loss after injury.
[Show abstract][Hide abstract] ABSTRACT: Structural glycomics (SG) plays a fundamental part of concurrent glycobiology aiming at comprehensive elucidation of glycan functions ( i.e. , functional glycomics) in the context of post-genome sciences. The SG project started in April 2003 and will continue for 3 years in the framework of NEDO (New Energy and Industrial Technology Organization) under the METI (the Ministry of Economy, Trade, and Industry), Japan. The main purpose of the project is the development of high-throughput and robust machines, which should greatly contribute to the structural analysis of complex glycans. In this chapter, 2 major research items, i.e. , (1) glycoproteomics, which enables comprehensive analysis of glycoproteins, and (2) "glycan profiling" by means of lectins, are described. For the latter, frontal affinity chromatography has been adopted as a starting tool for comprehensive analysis of the interaction of 100 lectins and 100 oligosaccharides under the concept of "hect-by-hect," which refers to 100 x 100.
[Show abstract][Hide abstract] ABSTRACT: Galectin-1 (Gal-1), the prototype of a family of beta-galactoside-binding proteins, has been shown to attenuate experimental acute and chronic inflammation. In view of the fact that endothelial cells (ECs), but not human polymorphonuclear leukocytes (PMNs), expressed Gal-1 we tested here the hypothesis that the protein could modulate leukocyte-EC interaction in inflammatory settings. In vitro, human recombinant (hr) Gal-1 inhibited PMN chemotaxis and trans-endothelial migration. These actions were specific as they were absent if Gal-1 was boiled or blocked by neutralizing antiserum. In vivo, hrGal-1 (optimum effect at 0.3 micro g equivalent to 20 pmol) inhibited interleukin-1beta-induced PMN recruitment into the mouse peritoneal cavity. Intravital microscopy analysis showed that leukocyte flux, but not their rolling velocity, was decreased by an anti-inflammatory dose of hrGal-1. Binding of biotinylated Gal-1 to resting and postadherent human PMNs occurred at concentrations inhibitory in the chemotaxis and transmigration assays. In addition, the pattern of Gal-1 binding was differentially modulated by PMN or EC activation. In conclusion, these data suggest the existence of a previously unrecognized function of Gal-1, that is inhibition of leukocyte rolling and extravasation in experimental inflammation. It is possible that endogenous Gal-1 may be part of a novel anti-inflammatory loop in which the endothelium is the source of the protein and the migrating PMNs the target for its anti-inflammatory action.
American Journal Of Pathology 11/2003; 163(4):1505-15. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Biological Chemistry 07/2003; 278(25):22223-30. · 4.65 Impact Factor