Frontal Affinity Chromatography as a Tool for Elucidation of Sugar Recognition Properties of Lectins

Hirabayashi, Jun; Arata, Yoichiro; Kasai, Ken‐ichi

doi:10.1016/s0076-6879(03)01025-5

Cited by 91 publications

(64 citation statements)

References 14 publications

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“…The method was essentially as described by Hirabayashi et al (17,18), using pyridylaminated (PA) oligosaccharides containing a terminal galactose residue as shown in Fig. 6B.…”

Section: Exo-␤-13-galactanase From Phanerochaete Chrysosporium 25822mentioning

confidence: 99%

An Exo-β-1,3-galactanase Having a Novel β-1,3-Galactan-bindingModule from Phanerochaetechrysosporium

Ichinose¹,

Yoshida

Kotake

et al. 2005

Journal of Biological Chemistry

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110

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An exo-␤-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-␤-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 ␤-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze ␤-1,4-linked galacto-oligosaccharides, ␤-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 ␤-1,3-galactosyl galactosaminide, ␤-1,3-galactosyl glucosaminide, or ␤-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal ␤-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from ␤-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl ␤-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass ␤-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 ␤-1,3-glucan, although it bound ␤-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 ␤-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.Arabinogalactan proteins (AGPs) 1 are a family of complex proteoglycans widely distributed in plants. They are found in the extracellular matrix associated with the plasma membrane and cell wall (1, 2). Although their precise functions are unknown, AGPs have been implicated in diverse developmental roles, including differentiation, cell-cell recognition, and embryogenesis (3-5). AGPs are characterized by large amounts of carbohydrate components rich in galactose (sugars in the present study are D series unless otherwise designated) and Larabinose, and protein components rich in hydroxyproline, serine, threonine, alanine, and glycine (1). The carbohydrate moieties of AGPs have a common structure consisting of a ␤-1,3-galactosyl backbone to which side chains of ␤-1,6-galactosyl residues are attached through O-6. L-Arabinose and lesser amounts of other auxiliary sugars, such as glucuronic acid, L-rhamnose, and L-fucose, are attached to t...

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“…The method was essentially as described by Hirabayashi et al (17,18), using pyridylaminated (PA) oligosaccharides containing a terminal galactose residue as shown in Fig. 6B.…”

Section: Exo-␤-13-galactanase From Phanerochaete Chrysosporium 25822mentioning

confidence: 99%

An Exo-β-1,3-galactanase Having a Novel β-1,3-Galactan-bindingModule from Phanerochaetechrysosporium

Ichinose¹,

Yoshida

Kotake

et al. 2005

Journal of Biological Chemistry

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110

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“…All of the Cys mutants were adsorbed onto the asialofetuin-immobilized affinity column and were eluted with lactose, suggesting that they retained their ability to bind to the N-acetlyllactosamine structure abundant in immobilized asialofetuin. Furthermore, the expressed mutants were subjected to frontal affinity chromatography 6,9,20) to determine whether they maintained a sugar-binding profile similar to that of wild-type hGal-1. All five (S7C, A51C, K63C, A121C, and D123C) mutant recombinant proteins showed sugar binding profiles similar to those of wild-type hGal-1 (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Crosslinking of Cys-Mutated Human Galectin-1 to the Model Glycoprotein Ligands Asialofetuin and Laminin by Using a Photoactivatable Bifunctional Reagent

Tamura

Watanabe

Igarashi

et al. 2014

Biological & Pharmaceutical Bulletin

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Galectins are a group of animal lectins characterized by their specificity for β-galactosides. In our previous study, we showed that a human galectin-1 (hGal-1) mutant, in which a cysteine residue was introduced at Lys 28 , forms a covalently cross-linked complex with the model glycoprotein ligands asialofetuin and laminin by using the photoactivatable sulfhydryl reagent benzophenone-4-maleimide (BPM). In the present study, we used several hGal-1 mutants in which single cysteine residues were introduced at different positions and examined their ability to form a covalent complex with asialofetuin or laminin by using BPM. We found that the efficiency of formation of the cross-linked products differed depending on the positions of the cysteine introduced and also on the ligand used for crosslinking. Therefore, by using different cysteine hGal-1 mutants, the chances of isolating different ligands for hGal-1 should increase depending on the systems and cells used.Key words galectin; ligand; crosslink; maleimide; benzophenone Galectins comprise a group of animal lectins that specifically bind to β-galactosides and are characterized by an evolutionarily conserved sequence motif at their carbohydratebinding site. [1][2][3] Galectins are involved in a wide variety of biological phenomena, including development, cell differentiation, tumor metastasis, apoptosis, RNA splicing, and regulation of immune function. 4,5) Interactions between lectins and their carbohydrate ligands play important roles in various biological systems. Although the basic sugar structure recognized by mammalian galectins, such as human galectin-1 (hGal-1), is the N-acetyllactosamine disaccharide unit (Galβ1 4GlcNAc), the sugar-binding specificity of each galectin could be enhanced by branched, repeated, or substituted glycans, 6,7) suggesting that galectins could interact with a variety of endogenous ligands possessing different carbohydrate structures. However, because the binding ability of galectins is insufficient for some sugar structures, it may not be possible to isolate some endogenous ligand glycoconjugates by conventional methods such as affinity chromatography.We previously prepared several mutants of LEC-1 (a galectin from the nematode Caenorhabditis elegans), which has two lectin domains tandemly repeated in a single polypeptide chain, 8,9) in which a cysteine residue was introduced near the sugar-binding site.10,11) Cross-linked products were formed using a photoactivatable bifunctional reagent, benzophenone-4-maleimide (BPM), [12][13][14] when the mutant Q38C was incubated with a model glycoprotein ligand, asialofetuin. 9) BPM 12) reacts with the sulfhydryl groups of cysteine residues via its maleimide moiety. Upon ultraviolet irradiation, a reactive species that can form covalent bonds with neighboring groups is generated. We used the same method to form a cross-linked product between hGal-1 and N-acetyllactosmine-containing glycoproteins via BPM. 15) Lys 28 in hGal-1, at a location that corresponds to Gln 38 of LEC-1, was selected f...

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“…To investigate the detailed sugar-binding specificities of lectins, the frontal affinity chromatography (FAC) system was recently reinforced by an efficient combination of the advantages of high-performance liquid chromatography (HPLC) and a large repertoire of pyridylaminated (PA)-oligosaccharides. 19,20) In this study, we applied the automated FAC system to explore the detailed sugar-binding specificities of C-type lectins isolated from acorn barnacles. The FAC system indicated that the marine invertebrate C-type lectins BRAs and BRL had diverse binding specificities toward oligosaccharides.…”

Section: )mentioning

confidence: 99%

“…19,20) For this purpose, various concentrations ([A] 0 ) of pNP-glycosides of 6 0 SiaLac (Neu5Ac2-6Gal1-4Glc) for the BRA-2 column, Gal-for the BRA-3 column, and LacNAc-(Gal1-4GlcNAc-) for the BRL column dissolved in the running buffer were injected into the column. The elution front relative to that of pNP-Man-(the control saccharide), viz., V-V 0 , was determined.…”

Section: )mentioning

confidence: 99%

Diverse Sugar-Binding Specificities of Marine Invertebrate C-Type Lectins

Matsubara¹,

Nakamura‐Tsuruta²,

Hirabayashi³

et al. 2007

Bioscience, Biotechnology, and Biochemistry

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Frontal Affinity Chromatography as a Tool for Elucidation of Sugar Recognition Properties of Lectins

Cited by 91 publications

References 14 publications

An Exo-β-1,3-galactanase Having a Novel β-1,3-Galactan-bindingModule from Phanerochaetechrysosporium

An Exo-β-1,3-galactanase Having a Novel β-1,3-Galactan-bindingModule from Phanerochaetechrysosporium

Crosslinking of Cys-Mutated Human Galectin-1 to the Model Glycoprotein Ligands Asialofetuin and Laminin by Using a Photoactivatable Bifunctional Reagent

Diverse Sugar-Binding Specificities of Marine Invertebrate C-Type Lectins

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