Mucosal galectin genes in all freshwater eels of the genus Anguilla

In this study, we determined the genomic DNA sequences of the mucosal galectin-encoding genes from all 19 species and subspecies of the genus Anguilla. The nucleotide sequences of the galectin genes were c. 2.3–2.5 kb long and the organisation of their four exons and three introns was conserved in all species. An unusual sequence was found in the fourth exon of Anguilla reinhardtii, resulting in a unique deduced amino-acid sequence at the C-terminus. All six amino-acid residues important for β-galactoside binding were conserved in three species, while one residue (R⁷³) was substituted to K⁷³ in the other 16 species–subspecies, including Anguilla marmorata. However, this substitution did not appear to affect the sugar-binding ability of galectins because the galectin of A. marmorata was previously shown to bind to lactose. We also discuss the molecular evolution of galectins among Anguilla spp. and the homologues previously identified in Conger myriaster.     

Emerging role of alpha2,6-sialic acid as a negative regulator of galectin binding and function

Galectins are β-galactoside-binding lectins that regulate diverse cell behaviors, including adhesion, migration, proliferation, and apoptosis. Galectins can be expressed both intracellularly and extracellularly, and extracellular galectins mediate their effects by associating with cell-surface oligosaccharides. Despite intensive current interest in galectins, strikingly few studies have focused on a key enzyme that acts to inhibit galectin signaling, namely β-galactoside α2,6-sialyltransferase (ST6Gal-I). ST6Gal-I adds an α2,6-linked sialic acid to the terminal galactose of N-linked glycans, and this modification blocks galectin binding to β-galactosides. This minireview summarizes the evidence suggesting that ST6Gal-I activity serves as an "off switch" for galectin function.     

Detoxification of ferulic acid by ectomycorrhizal fungi

The ectomycorrhizal fungi Laccaria amethystina and Lactarius deterrimus grown in liquid culture were used to study the fate of added ferulic acid. Laccaria amethystina degraded ferulic acid to the major metabolite vanillic acid. The intermediate vanillin was not detected. Lactarius deterrimus showed a completely different detoxification pattern. Two dimers and one trimer of ferulic acid could be identified as polymerization products of this fungus. A bioassay of the possible biological activities of ferulic acid and vanillic acid on these fungi revealed that vanillic acid was less toxic than ferulic acid for Laccaria amethystina but that both phenolic acids were toxic for Lactarius deterrimus. The results are discussed with respect to ectomycorrhizal fungal growth in the organic layer of forest soils and between living root cells of ectomycorrhizas.     

A tandem-repeat galectin from blood clam Tegillarca granosa and its induced mRNA expression response against bacterial challenge

Galectins are a family of β-galactoside-binding lectins which play crucial roles in innate immunity of vertebrates and invertebrates. In the present study, we have cloned and characterized the first galectin to be identified in Tegillarca granosa (designated Tg-GAL). The full-length cDNA of Tg-GAL was of 2,394 bp nucleotides, encoding a polypeptide of 354 amino acids. The amino acid sequence of T. granosa galectin (Tg-GAL) showed striking sequence similarity to invertebrate and vertebrate galectins in carbohydrate recognition domains (CRD) and contained amino acids that are crucial for binding β-galactoside sugars. Structurally, the Tg-GAL was a tandem repeat galectin containing two CRD connected by a unique peptide link. Quantitative real-time PCR was employed to investigate the tissue distribution of Tg-GAL mRNA and temporal expression in haemocytes of clams challenged with Vibrio parahaemolyticus, lipopolysaccharide (LPS) and peptidogylcan (PGN). The Tg-GAL mRAN expression was concentrated in hepatopancreas and mantle. The up-regulation of Tg-GAL after bacteria V. parahaemolyticus, LPS and PGN challenge showed that Tg-GAL might play a pivotal role in anti-bacterial immunity. Further study should investigate the effects of Tg-GAL absence by siRNA knockout.     

Structural basis for chitotetraose coordination by CGL3, a novel galectin-related protein from Coprinopsis cinerea

Recent advances in genome sequencing efforts have revealed an abundance of novel putative lectins. Among these, many galectin-related proteins, characterized by many conserved residues but intriguingly lacking critical amino acids, have been found in all corners of the eukaryotic superkingdom. Here we present a structural and biochemical analysis of one representative, the galectin-related lectin CGL3 found in the inky cap mushroom Coprinopsis cinerea. This protein contains all but one conserved residues known to be involved in β-galactoside binding in galectins. A Trp residue strictly conserved among galectins is changed to an Arg in CGL3 (R81). Accordingly, the galectin-related protein is not able to bind lactose. Screening of a glycan array revealed that CGL3 displays preference for oligomers of β1-4-linked N-acetyl-glucosamines (chitooligosaccharides) and GalNAcβ1-4GlcNAc (LacdiNAc). Carbohydrate-binding affinity of this novel lectin was quantified using isothermal titration calorimetry, and its mode of chitooligosaccharide coordination not involving any aromatic amino acid residues was studied by X-ray crystallography. Structural information was used to alter the carbohydrate-binding specificity and substrate affinity of CGL3. The importance of residue R81 in determining the carbohydrate-binding specificity was demonstrated by replacing this Arg with a Trp residue (R81W). This single-amino-acid change led to a lectin that failed to bind chitooligosaccharides but gained lactose binding. Our results demonstrate that, similar to the legume lectin fold, the galectin fold represents a conserved structural framework upon which dramatically altered specificities can be grafted by few alterations in the binding site and that, in consequence, many metazoan galectin-related proteins may represent lectins with novel carbohydrate-binding specificities.     

Conformational change of a unique sequence in a fungal galectin from Agrocybe cylindracea controls glycan ligand-binding specificity

A fungal galectin from Agrocybe cylindracea (ACG) exhibits broad binding specificity for β-galactose–containing glycans. We determined the crystal structures of wild-type ACG and the N46A mutant, with and without glycan ligands. From these structures and a saccharide-binding analysis of the N46A mutant, we revealed that a conformational change of a unique insertion sequence containing Asn46 provides two binding modes for ACG, and thereby confers broad binding specificity. We propose that the unique sequence provides these two distinct glycan-binding modes by an induced-fit mechanism.     

Binding of synthetic sulfated ligands by human splenic galectin 1, a β-galactoside-binding lectin

The carbohydrate-binding site of galectin 1, a vertebrate β-galactoside-binding lectin, has a pronounced specificity for the βGal(1→3)- and βGal(1→4)GlcNAc sequences. The binding inhibition study reported herein was carried out to determine whether sulfation of saccharides would influence their binding by galectin 1. The presence of 6′-OSO3- on LacNAc greatly reduces the inhibitory potency relative to LacNAc. 3′-OSO3-LacNAc, 3′-OSO3-Galβ(1→3)GlcNAcβ1-OBzl and 3-OSO3-Galβ1-OMe are more potent inhibitors than the non-sulfated parent compounds. Surprisingly, 2′-OSO3-LacNAc showed over 40 fold less inhibitory potency relative to LacNAc. Ovarian carcinoma A121 cells were shown to synthesize sulfated macromolecules that bind to galectin 1. Modulation in vivo of saccharide sulfation may lead to modulation of galectin 1 interaction with glycoconjugates; hence, sulfation could play a role in modulating lectin functions.     

Solid-phase assays for study of carbohydrate specificity of galectins

We have recently shown that the carbohydrate-binding pattern of galectins in cells differs from that determined in artificial (non-cellular) test-systems. To understand the observed discrepancy, we compared several test-systems differing in the mode of galectin presentation on solid phase. The most representative system was an assay where the binding of galectin (human galectins-1 and -3 were studied) to asialofetuin immobilized on solid phase was inhibited by polyacrylamide glycoconjugates, Glyc-PAA. This approach permits us to range quantitatively glycans (Glyc) by their affinity to galectin, i.e. to study both high and low affinity ligands. Our attempts to imitate the cell system by solid-phase assay were not successful. In the cell system galectin binds glycoconjugates by one carbohydrate-recognizing domain (CRD), and after that the binding to the remaining non-bound CRD is studied by means of fluorescein-labeled Glyc-PAA. In an “imitation” variant when galectins are loaded on adsorbed asialofetuin or Glyc-PAA followed by revealing of binding by the second Glyc-PAA, the interaction was not observed or glycans were ordered poorly, unlike in the inhibitory assay. When galectins were adsorbed on corresponding antibodies (when all CRDs were free for recognition by carbohydrate), a good concentration dependence was observed and patterns of specificities were similar (though not identical) for the two methods; notably, this system does not reflect the situation in the cell. Besides the above-mentioned, other variants of solid-phase analysis of galectin specificity were tested. The results elucidate the mechanism and consequence of galectin CRD cis-masking on cell surface.     

Evidence from population genetics that the ectomycorrhizal basidiomycete Laccaria amethystina is an actual multihost symbiont

It is commonly assumed that ectomycorrhizal (ECM) fungi associated with temperate forest tree roots are not host-specific. Because this assumption relies on species delineations based on fruitbodies morphology or ribosomal DNA sequences, host-specific, cryptic biological species cannot be ruled out. To demonstrate that Laccaria amethystina has true generalist abilities, we sampled 510 fruitbodies on three French sites situated 150–450 km away from each other. At each site, populations from monospecific stands ( Abies alba , Castanea europea and Fagus sylvatica ) or mixed stands ( F. sylvatica  +  Quercus robur or Q. robur  + Carpinus betulus ) were sampled. Three different sets of markers were used for genotyping: (i) five microsatellite loci plus the ribosomal DNA intergenic spacer, (ii) the mitochondrial large ribosomal DNA subunit, and (iii) direct amplification of length polymorphism (DALP), a new method for fungi providing dominant markers. Evidence for allogamous populations (with possible inbreeding at local scale) and possibly for biparental mitochondrial inheritance was found. All markers congruently demonstrated that L. amethystina populations show little structure at this geographical scale, indicating high gene flow (as many as 50% of founding spores in all populations being of external origin). Our results also showed that host species contributed even less to population differentiation, and there was no evidence for cryptic biological species. This first in situ demonstration of a true multihost ability in an ECM species is discussed in terms of ecology and evolutionary biology.     

Engineering of the glycan-binding specificity of Agrocybe cylindracea galectin towards α(2,3)-linked sialic acid by saturation mutagenesis

Sialic acid represents a critical sugar component located at the outermost position of glycoconjugates, playing important roles in extensive biological processes. To date, however, there have been only few probes which show affinity to α(2,3)-linked sialic acid-containing glycoconjugates. Agrocybe cylindracea galectin is known to have a relatively high affinity towards Neu5Acα(2,3)Galβ(1,4)Glc (3'-sialyl lactose), but it significantly recognizes various β-galactosides, such as Galβ(1,4)GlcNAcβ (LacNAc) and Galβ(1,3)GalNAcα (T-antigen). To eliminate this background specificity, we focused an acidic amino acid residue (Glu86), which interacts with the glucose unit of 3'-sialyl lactose and substituted it with all other amino acids. Carbohydrate-binding specificity of the derived 14 mutants was analysed by surface plasmon resonance, and it was found that E86D mutant (Glu86 substituted with Asp) substantially lost the binding ability to LacNAc and T-antigen, while it retained the high affinity for 3'-sialyl lactose. Further, frontal affinity chromatography analysis using 132 pyridylaminated oligosaccharides confirmed that the E86D mutant had a strong preference for α(2,3)-disialo biantennary N-linked glycan. However, it showed the large decrease in the affinity for any of the asialo complex-type N-glycans and the glycolipid-type glycans. Thus, the developed mutant E86D will be of practical use in various fields relevant to cell biology and glycotechnology.     

Homology model and targeted mutagenesis identify critical residues for arachidonic acid inhibition of Kv4 channels

Polyunsaturated fatty acids such as arachidonic acid (AA) exhibit inhibitory modulation of Kv4 potassium channels. Molecular docking approaches using a Kv4.2 homology model predicted a membrane-embedded binding pocket for AA comprised of the S4-S5 linker on one subunit and several hydrophobic residues within S3, S5 and S6 from an adjacent subunit. The pocket is conserved among Kv4 channels. We tested the hypothesis that modulatory effects of AA on Kv4.2/KChIP channels require access to this site. Targeted mutation of a polar residue (K318) and a nonpolar residue (G314) within the S4-S5 linker as well as a nonpolar residue in S3 (V261) significantly impaired the effects of AA on K⁺ currents in Xenopus oocytes. These residues may be important in stabilizing (K318) or regulating access to (V261, G314) the negatively charged carboxylate moiety on the fatty acid. Structural specificity was supported by the lack of disruption of AA effects observed with mutations at residues located near, but not within the predicted binding pocket. Furthermore, we found that the crystal structure of the related Kv1.2/2.1 chimera lacks the structural features present in the proposed AA docking site of Kv4.2 and the Kv1.2/2.1 K⁺ currents were unaffected by AA. We simulated the mutagenic substitutions in our Kv4.2 model to demonstrate how specific mutations may disrupt the putative AA binding pocket. We conclude that AA inhibits Kv4 channel currents and facilitates current decay by binding within a hydrophobic pocket in the channel in which K318 within the S4-S5 linker is a critical residue for AA interaction.     

Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions

Galectins are a family of beta-galactose binding lectins associated with functions such as immunological and malignant events. To study the binding affinity of galectins for natural and artificial saccharides and glycoconjugates we have developed an assay using fluorescence polarization. A collection of fluorescein-conjugated saccharides was synthesized and used as probes with galectins-1 and -3 and the two carbohydrate recognition domains of galectin-4. Direct binding of a fixed probe amount with different amounts of each galectin defined specificity and selectivity and permitted selection of the optimal probe for inhibition studies. Then fixed amounts of galectin and selected probe were used to screen the inhibitory potency of a library of nonfluorescent compounds. As the assay is in solution and does not require separation of free and bound probe, it is simple and rapid and can easily be applied to different unlabeled galectins. As all interaction components are known, K(d) values for galectin-inhibitor interaction can be directly calculated without approximation other than the assumption of a simple one-site competition.     

Theoretical investigation on the glycan‐binding specificity of Agrocybe cylindracea galectin using molecular modeling and molecular dynamics simulation studies

Galectins are β‐galactoside binding proteins which have the ability to serve as potent antitumor, cancer biomarker, and induce tumor cell apoptosis. Agrocybe cylindracea galectin (ACG) is a fungal galectin which specifically recognizes α(2,3)‐linked sialyllactose at the cell surface that plays extensive roles in the biological recognition processes. To investigate the change in glycan‐binding specificity upon mutations, single point and double point site‐directed in silico mutations are performed at the binding pocket of ACG. Molecular dynamics (MD) simulation studies are carried out for the wild‐type (ACG) and single point (ACG1) and double point (ACG2) mutated ACGs to investigate the dynamics of substituted mutants and their interactions with the receptor sialyllactose. Plausible binding modes are proposed for galectin–sialylglycan complexes based on the analysis of hydrogen bonding interactions, total pair‐wise interaction energy between the interacting binding site residues and sialyllactose and binding free energy of the complexes using molecular mechanics–Poisson–Boltzmann surface area. Our result shows that high contribution to the binding in different modes is due to the direct and water‐mediated hydrogen bonds. The binding specificity of double point mutant Y59R/N140Q of ACG2 is found to be high, and it has 26 direct and water‐mediated hydrogen bonds with a relatively low‐binding free energy of −47.52 ± 5.2 kcal/mol. We also observe that the substituted mutant Arg59 is crucial for glycan‐binding and for the preference of α(2,3)‐linked sialyllactose at the binding pocket of ACG2 galectin. When compared with the wild‐type and single point mutant, the double point mutant exhibits enhanced affinity towards α(2,3)‐linked sialyllactose, which can be effectively used as a model for biological cell marker in cancer therapeutics. Copyright © 2015 John Wiley & Sons, Ltd.     

Penicillin-binding protein 3 of Streptococcus pneumoniae and its application in screening of β-lactams in milk

The soluble form of penicillin-binding protein 3 (sPBP3^∗) from Streptococcus pneumoniae was expressed in Escherichia coli as a six-histidine fusion protein. The protein was purified and used to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP–Amp) to the sPBP3^∗ by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins) could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from 74.06 to 106.31% in skimmed milk and from 63.97 to 107.26% in whole milk, with coefficients of variation (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on sPBP3^∗ exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk.     

Extensive gene flow over Europe and possible speciation over Eurasia in the ectomycorrhizal basidiomycete Laccaria amethystina complex

Biogeographical patterns and large-scale genetic structure have been little studied in ectomycorrhizal (EM) fungi, despite the ecological and economic importance of EM symbioses. We coupled population genetics and phylogenetic approaches to understand spatial structure in fungal populations on a continental scale. Using nine microsatellite markers, we characterized gene flow among 16 populations of the widespread EM basidiomycete Laccaria amethystina over Europe (i.e. over 2900 km). We also widened our scope to two additional populations from Japan (10(4) km away) and compared them with European populations through microsatellite markers and multilocus phylogenies, using three nuclear genes (NAR, G6PD and ribosomal DNA) and two mitochondrial ribosomal genes. European L. amethystina populations displayed limited differentiation (average F(ST) = 0.041) and very weak isolation by distance (IBD). This panmictic European pattern may result from effective aerial dispersal of spores, high genetic diversity in populations and mutualistic interactions with multiple hosts that all facilitate migration. The multilocus phylogeny based on nuclear genes confirmed that Japanese and European specimens were closely related but clustered on a geographical basis. By using microsatellite markers, we found that Japanese populations were strongly differentiated from the European populations (F(ST) = 0.416), more than expected by extrapolating the European pattern of IBD. Population structure analyses clearly separated the populations into two clusters, i.e. European and Japanese clusters. We discuss the possibility of IBD in a continuous population (considering some evidence for a ring species over the Northern Hemisphere) vs. an allopatric speciation over Eurasia, making L. amethystina a promising model of intercontinental species for future studies.     

Site directed mutagenesis of Histidine residues in Anthrax toxin lethal factor binding domain reduces toxicity

Anthrax lethal toxin is a mixture of protective antigen (PA, 735 AA) and lethal factor (LF, 776 AA). Earlier studies have shown that 254 residues of lethal factor are sufficient for PA binding to cause internalization (Arora N and Leppla SH, J Biol Chem 268: 3334-3342, 1993). The present study was undertaken to determine residues which are important for binding of LF to PA. LF modification with diethyl pyrocarbonate (DEPC, modifies histidine residue primarily) results in the loss of binding and toxicity in mammalian cells. There are nine histidine residues in the binding domain. To locate the important residue(s), site-directed mutagenesis of these histidines were performed by recombinant methods. Replacement of His42 with Gly42 destablizes the protein and hence it could not be purified. His35 when mutagenized to Gly35 (mLF-DTA) diminishes the toxicity by 20 fold. Time dependent studies show that binding of mLF-DTA was reduced at shorter incubations and longer incubations taper off this difference. Gel shift assay suggested 8-10% less binding of mLF-DTA as compared to LF-DTA. In conclusion His35 is important for binding and His42 is critical and confers proper conformation for LF binding to PA.     

Cloning and characterisation of tandem-repeat type galectin in rainbow trout (Oncorhynchus mykiss)

Fish beta-galactoside binding lectin (galectin) cDNA was cloned from the cDNA library of rainbow trout (Oncorhynchus mykiss) head kidney. The clone contained a single open reading frame encoding 341 amino acids (aa) (38 kDa protein), including the initiator methionine. Significant sequence homology to mammalian galectin-9 (40-55% identity) was observed. Its amino acid sequence showed two distinct N- and C-terminal domains (148 and 130 aa, respectively) connected by a peptide linker (63 aa). The galectin contains two consensus WG-E-R/K motifs thought to play an essential role in sugar-binding, indicating that this lectin is a member of the tandem-repeat type galectins which have not been identified in fish. The 1.6 kDa mRNA of the lectin was found by Northern blot analyses to be widely expressed in the spleen, head kidney, thymus, peritoneal exudate cells, ovary, gills and heart. Southern blot analyses with the probe for C-terminal of the lectin showed the existence of two hybridising genes. These results suggest that rainbow trout has at least one tandem-repeat type galectin as well as proto-type galectin.     

Analysis of the carbohydrate binding specificity of the mushroom Pleurotus ostreatus lectin by surface plasmon resonance

The sugar binding specificity of the mushroom Pleurotus ostreatus lectin (POL) was analyzed by surface plasmon resonance. The lectin was immobilized to a sensor chip, and asialo-bovine submaxillary mucin (asialo-BSM), one of the most potent inhibitors in the hemagglutination inhibition assay, tightly bound to the lectin. The binding specificity of various mono- or oligosaccharides to the lectin was evaluated by the coinjection method. The dissociation of asialo-BSM was promoted by injection of some haptenic saccharides. For the most part, the order of acceleration ability of the sugars to the dissociation in the coinjection experiment agreed with that of the inhibitory potency of each sugar evaluated by the hemagglutination inhibition assay. In conclusion, POL recognized a galactosyl residue, and the specificity was increased by substitution at the C-2 position of the galactosyl residue with a fucosyl or acetylamino group. This method using the coinjection method proved useful in analysis of carbohydrate-lectin binding specificity.     

Mycorrhizal synthesis of four ectomycorrhizal fungi in potted <Emphasis Type="Italic">Populus maximowiczii</Emphasis> seedlings

Ectomycorrhizal (ECM) syntheses between four ECM fungi, Laccaria amethystina, Hebeloma mesophaeum, Thelephora terrestris, and Tomentella sp., and Populus maximowiczii seedlings that are known to form ECM at a denuded area of Mt. Usu were performed in volcanic debris in a controlled growth chamber. The percentage of ECM colonization and seedling growth were determined 3 months after inoculation. Seedlings were successfully colonized by the inoculated ECM fungi with low contamination ratios. Seedling height and biomass were larger in the inoculated seedlings than in the control, although the effects of inoculation on seedling growth varied with the ECM fungus.     

Modulation of the carbohydrate-binding specificity of two Xenopus proto-type galectins by site-directed mutagenesis

The galectin family is a representative soluble lectin group, which is responsible for the modulation of various cell functions. Although the carbohydrate-binding specificity of galectins has been well-studied, the relationship between protein structure and specificity remains to be elucidated. We previously reported the characteristics of a Xenopus laevis skin galectin, xgalectin-Va, which had diverged from galectin-1. The carbohydrate selectivity of xgalectin-Va was different from that of human galectin-1 and xgalectin-Ib (a Xenopus laevis galectin-1 homolog). In this study, we clarified the key residues for this selectivity by site-directed mutagenesis. Substitution of two amino acids of xgalectin-Va, Val56Gly/Lys76Arg, greatly enhanced the binding ability to N-acetyllactosamine and conferred significant T-cell growth inhibition activity, although the wild type had no activity. These two residues, Gly54 and Arg74 in galectin-1, would cooperatively contribute to the N-acetyllactosamine recognition. The loop region between the S4 and S5 β-strands was involved in the binding to the TF-antigen disaccharide. The loop substitution successfully changed the carbohydrate selectivity of xgalectin-Va and xgalectin-Ib.