We have obtained the X-ray crystal structure of the galectin-8 N-terminal domain (galectin-8N) with a previously reported quinoline-galactoside ligand at a resolution of 1.6 Å. Based on this X-ray structure, a collection of galactosides derivatised at O3 with triazole, benzimidazole, benzothiazole, and benzoxazole moieties were designed and synthesised. This led to the discovery of a 3-O-(N-methylbenzimidazolylmethyl)-galactoside with a Kd of 1.8 μM for galectin-8N, the most potent selective synthetic galectin-8N ligand to date. Molecular dynamics simulations showed that benzimidazole-galactoside derivatives bind the non-conserved amino acid Gln47, accounting for the higher selectivity for galectin-8N. Galectin-8 is a carbohydrate-binding protein that plays a key role in pathological lymphangiogenesis, modulation of the immune system, and autophagy. Thus, the benzimidazole-derivatised galactosides represent promising compounds for studies of the pathological implications of galectin-8, as well as a starting point for the development of anti-tumour and anti-inflammatory therapeutics targeting galectin-8.

Pseudomonas aeruginosa is a widespread opportunistic pathogen that is capable of colonizing various human tissues and is resistant to many antibiotics. LecA is a galactose binding tetrameric lectin involved in adhesion, infection and biofilm formation. This study reports on the binding characteristics of mono- and divalent (chelating) ligands to LecA using different techniques. These techniques include affinity capillary electrophoresis, bio-layer interferometry, native mass spectrometry and a thermal shift assay. Aspects of focus include: affinity, selectivity, binding kinetics and residence time. The affinity of a divalent ligand was determined to be in the low-nanomolar range for all of the used techniques and with a ligand residence time of approximately 7 h, while no strong binding was seen to related lectin tetramers. Each of the used techniques provides a unique and complementary insight into the chelation based binding mode of the divalent ligand to the LecA tetramer.

Soybean meal (SBM) is an important protein source in animal feed. However, the levels of SBM inclusion are restricted in some animal species by the presence of antinutritional factors (ANFs), including non-starch polysaccharides (NSPs) and α-galactosides (GOSs). The aim of this study was to reduce the soybean meal NSPs and GOSs by solid-state fermentation (SSF) using a combination of cellulolytic bacteria isolated from different environments (termites, earthworms, corn silage and bovine ruminal content). To analyse the key enzymatic activities, the isolates were grown in minimal media containing NSPs extracted from SBM. The selected bacterial strains belonged to the genera Streptomyces, Cohnella and Cellulosimicrobium. SSF resulted in a reduction of nearly 24% in the total NSPs, 83% of stachyose and 69% of raffinose and an increase in the protein content. These results suggest that cellulolytic bacteria-based SSF processing facilitates SBM nutritional improvement. In addition, the use of fermented SBM in animal diets can be recommended.

Placental Protein 13 (PP13), an early biomarker of preeclampsia, is a placenta-specific galectin that binds beta-galactosides, building-blocks of ABO blood-group antigens, possibly affecting its bioavailability in blood.

Flavonoid biosynthesis profile was clarified by fruit bagging and re-exposure treatments in the green Chinese pear 'Zaosu' (Pyrus bretschneideri Rehd.) and its red mutant 'Red Zaosu'. Two distinct biosynthesis patterns of flavonoid 3-glycosides were found in 'Zaosu' pear. By comparison with 'Red Zaosu', the biosynthesis of flavonoid 3-galactosides and flavonoid 3-arabinosides were inhibited by bagging and these compounds only re-accumulated to a small degree in the fruit peel of 'Zaosu' after the bags were removed. In contrast, the biosynthesis of flavonoid 3-gluctosides and flavonoid 3-rutinosides was reduced by bagging and then increased when the fruits were re-exposed to sunlight. A combination of correlation, multicollinearity test and partial-correlation analyses among major flavonoid metabolites indicated that biosynthesis of each phenolic compound was independent in 'Zaosu' pear, except for the positive correlation between flavonoid 3-rutincosides and flavanols. In contrast with the green pear cultivar, almost all phenolic compounds in the red mutant had similar biosynthesis patterns except for arbutin. However, only the biosynthesis of flavonoid 3-galactosides was relatively independent and strongly affected the synthesis of the other phenolic compounds. Therefore, we propose a hypothesis that the strong accumulation of flavonoid 3-galactosides stimulated the biosynthesis of other flavonoid compounds in the red mutant and, therefore, caused systemic variation of flavonoid biosynthesis profiles between 'Zaosu' and its red mutant. This hypothesis had been further demonstrated by the enzyme activity of UFGT, and transcript levels of flavonoid biosynthetic genes and been well tested by a stepwise linear regression forecasting model. The gene that encodes flavonoid 3-galacosyltransferase was also identified and isolated from the pear genome.

Galectins are proteins that bind β-galactosides such as N-acetyllactosamine present in N-linked and O-linked glycoproteins and that seem to be implicated in inflammatory and immune responses as well as fibrotic mechanisms. This preliminary study investigated serum galectins as clinical biomarkers in lung transplant patients with chronic lung allograft dysfunction (CLAD), phenotype bronchiolitis obliterans syndrome (BOS).

Tissue lectins are emerging (patho)physiological effectors with broad significance. The capacity of adhesion/growth-regulatory galectins to form functional complexes with distinct cellular glycoconjugates is based on molecular selection of matching partners. Engineering of variants by changing the topological display of carbohydrate recognition domains (CRDs) provides tools to understand the inherent specificity of the functional pairing. We here illustrate its practical implementation in the case of human tandem-repeat-type galectin-8 (Gal-8). It is termed Gal-8 (NC) due to presence of two different CRDs at the N- and C-terminal positions. Gal-8N exhibits exceptionally high affinity for 3'-sialylated/sulfated β-galactosides. This protein is turned into a new homodimer, i.e., Gal-8 (NN), by engineering. The product maintained activity for lactose-inhibitable binding of glycans and glycoproteins. Preferential association with 3'-sialylated/sulfated (and 6-sulfated) β-galactosides was seen by glycan-array analysis when compared to the wild-type protein, which also strongly bound to ABH-type epitopes. Agglutination of erythrocytes documented functional bivalency. This result substantiates the potential for comparative functional studies between the variant and natural Gal-8 (NC)/Gal-8N.

Trichoderma reesei colonizes predecayed wood in nature and metabolizes cellulose and hemicellulose from the plant biomass. The respective enzymes are industrially produced for application in the biofuel and biorefinery industry. However, these enzymes are also induced in the presence of lactose (1,4-0-ß-d-galactopyranosyl-d-glucose), a waste product from cheese manufacture or whey processing industries. In fact, lactose is the only soluble carbon source that induces these enzymes in T. reesei on an industrial level but the reason for this unique phenomenon is not understood. To answer this question, we used systems analysis of the T. reesei transcriptome during utilization of lactose. We found that the respective CAZome encoded all glycosyl hydrolases necessary for cellulose degradation and particularly for the attack of monocotyledon xyloglucan, from which ß-galactosides could be released that may act as the inducers of T. reesei's cellulases and hemicellulases. In addition, lactose also induces a high number of putative transporters of the major facilitator superfamily. Deletion of fourteen of them identified one gene that is essential for lactose utilization and lactose uptake, and for cellulase induction by lactose (but not sophorose) in pregrown mycelia of T. reesei. These data shed new light on the mechanism by which T. reesei metabolizes lactose and offers strategies for its improvement. They also illuminate the key role of ß-D-galactosides in habitat specificity of this fungus.

Galectins form a large family of beta-galactoside-binding proteins in metazoa and fungi. This report presents a comparative study of the functions of potential galectin genes found in the genome database of Caenorhabditis elegans. We isolated full-length cDNAs of eight potential galectin genes (lec-2-5 and 8-11) from a lambdaZAP cDNA library. Among them, lec-2-5 were found to encode 31-35-kDa polypeptides containing two carbohydrate-recognition domains similar to the previously characterized lec-1, whereas lec-8-11 were found to encode 16-27-kDa polypeptides containing a single carbohydrate-recognition domain and a C-terminal tail of unknown function. Recombinant proteins corresponding to lec-1-4, -6, and 8-10 were expressed in Escherichia coli, and their sugar-binding properties were assessed. Analysis using affinity adsorbents with various beta-galactosides, i.e., N-acetyllactosamine (Galbeta1-4GlcNAc), lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), and asialofetuin, demonstrated that LEC-1-4, -6, and -10 have a significant affinity for beta-galactosides, while the others have a relatively lower affinity. These results indicate that the integrity of key amino acid residues responsible for recognition of lactose (Galbeta1-4Glc) or N-acetyllactosamine in vertebrate galectins is also required in C. elegans galectins. However, analysis of their fine oligosaccharide-binding properties by frontal affinity chromatography suggests their divergence towards more specialized functions.

The human gastrointestinal tract can be positively modulated by dietary supplementation of probiotic bacteria in combination with prebiotic carbohydrates. Here differential transcriptomics and functional genomics were used to identify genes in Lactobacillus acidophilus NCFM involved in the uptake and catabolism of 11 potential prebiotic compounds consisting of α- and β-linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems (PTS), galactoside pentose hexuronide (GPH) permease, and ATP-binding cassette (ABC) transporters. PTS systems were upregulated primarily by di- and tri-saccharides such as cellobiose, isomaltose, isomaltulose, panose and gentiobiose, while ABC transporters were upregulated by raffinose, Polydextrose, and stachyose. A single GPH transporter was induced by lactitol and galactooligosaccharides (GOS). The various transporters were associated with a number of glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65, involved in the catabolism of various α- and β-linked glucosides and galactosides. Further subfamily specialization was also observed for different PTS-associated GH1 6-phospho-β-glucosidases implicated in the catabolism of gentiobiose and cellobiose. These findings highlight the broad oligosaccharide metabolic repertoire of L. acidophilus NCFM and establish a platform for selection and screening of both probiotic bacteria and prebiotic compounds that may positively influence the gastrointestinal microbiota.

α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae.

Galectin-3 (Gal-3), the only chimeric β-galactosides-binding lectin, consists of Gal-3N (N-terminal regulatory peptide) and Gal-3C (C-terminal carbohydrate-recognition domain). Interestingly, Gal-3C could specifically inhibit endogenous full-length Gal-3 to exhibit anti-tumor activity. Here, we aimed to further improve the anti-tumor activity of Gal-3C via developing novel fusion proteins.

During pregnancy, placental protein-13 (galectin-13) is highly expressed in the placenta and fetal tissue, and less so in maternal serum that is related to pre-eclampsia. To understand galectin-13 function at the molecular level, we solved its crystal structure and discovered that its dimer is stabilized by two disulfide bridges between Cys136 and Cys138 and six hydrogen bonds involving Val135, Val137, and Gln139. Native PAGE and gel filtration demonstrate that this is not a crystallization artifact because dimers also form in solution. Our biochemical studies indicate that galectin-13 ligand binding specificity is different from that of other galectins in that it does not bind β-galactosides. This is partly explained by the presence of Arg53 rather than His53 at the bottom of the carbohydrate binding site in a position that is crucial for interactions with β-galactosides. Mutating Arg53 to histidine does not re-establish normal β-galactoside binding, but rather traps cryoprotectant glycerol molecules within the ligand binding site in crystals of the R53H mutant. Moreover, unlike most other galectins, we also found that GFP-tagged galectin-13 is localized within the nucleus of HeLa and 293 T cells. Overall, galectin-13 appears to be a new type of prototype galectin with distinct properties.

Chickpea grains may represent an alternative to soybean meals and energetic concentrates in animal feeding, as their nutritional value can help to increase the sustainability of livestock systems. Unfortunately, the presence of bioactive compounds with anti-nutritional effects can prevent its direct use, especially in mono-gastrics. It is known that the synthesis of these compounds depends on genetic expression, which is also influenced by growth conditions. The objective of this two-year study was to assess the effect of sowing date (winter versus spring) and seeding rate (70 versus 110 seeds m-2) on the accumulation of soluble carbohydrates, α-galactosides, trypsin inhibitors, and inositol phosphates in the grains of two Kabuli cultivars, in the Mediterranean climate. The results showed that seeds collected from winter sowing contained more trypsin inhibitors than those seeded in spring (+ 4%, on average), reaching values between 16.1 and 18.6 TIU mg protein-1. The seeding rate affects only the α-galactosides content, which increases (+9%) at lower densities (70 seeds m-2). These findings suggest that agronomic management can be used to modulate the content of some anti-nutritional factors in the seeds, even though the genetic characteristics and phenotypic expression, in relation to the climatic conditions, seem to deeply affect the content of all the bioactive compounds investigated.

Generally, extruded gluten-free foods are mostly phytochemically deficient. In this study inositol phosphates, α-galactosides, lectins, protease inhibitors, and phenols, their antioxidant activity and sensorial analysis of some rice/bean/whole carob fruit flour blends were determined in unprocessed (controls) and extruded formulations. The fortification of rice-based extrudates with both legumes has a positive influence on both their bioactive compound content and their acceptability by consumers. The extruded formulations contained around twice as much (p < 0.05) total α-galactosides than their unprocessed counterparts. Extrusion significantly reduced the phytic acid content (10%) and significantly increased the less phosphorylated forms (16%-70%). After extrusion, the lectins and protease inhibitors were eliminated. The different phenolic compounds mostly increased (11%-36%), notably in the formulations with carob fruit. The antioxidant activity and the different groups of phenols showed a positive correlation in the extrudates. All the experimental extrudates had higher amounts of bioactive compounds than the commercial extruded rice. Considering the amount of phytochemicals determined in the novel gluten-free extrudates and the scores of sensorial analysis, formulations containing 20%-40% bean and 5% carob fruit could be adequate in promoting health-related functions, helping to increase pulse consumption, and allowing the food industry to satisfy consumers' requirement for functional foods.

Synthesis of tetravalent thio- and selenogalactopyranoside-containing glycoclusters using azide-alkyne click strategy is presented. Prepared compounds are potential ligands of Pseudomonas aeruginosa lectin PA-IL. P. aeruginosa is an opportunistic human pathogen associated with cystic fibrosis, and PA-IL is one of its virulence factors. The interactions of PA-IL and tetravalent glycoconjugates were investigated using hemagglutination inhibition assay and compared with mono- and divalent galactosides (propargyl 1-thio- and 1-seleno-β-d-galactopyranoside, digalactosyl diselenide and digalactosyl disulfide). The lectin-carbohydrate interactions were also studied by saturation transfer difference NMR technique. Both thio- and seleno-tetravalent glycoconjugates were able to inhibit PA-IL significantly better than simple d-galactose or their intermediate compounds from the synthesis.

The spore-forming lactic acid bacterium Bacillus coagulans MA-13 has been isolated from canned beans manufacturing and successfully employed for the sustainable production of lactic acid from lignocellulosic biomass. Among lactic acid bacteria, B. coagulans strains are generally recognized as safe (GRAS) for human consumption. Low-cost microbial production of industrially valuable products such as lactic acid and various enzymes devoted to the hydrolysis of oligosaccharides and lactose, is of great importance to the food industry. Specifically, α- and β-galactosidases are attractive for their ability to hydrolyze not-digestible galactosides present in the food matrix as well as in the human gastrointestinal tract.

Lactose permease (LacY) from Escherichia coli belongs to the major facilitator superfamily. It facilitates the co-transport of β-galactosides, including lactose, into cells by using a proton gradient towards the cell. We now show that LacY is capable of scrambling glycerophospholipids across a membrane. We found that purified LacY reconstituted into liposomes at various protein to lipid ratios catalyzed the rapid translocation of fluorescently labeled and radiolabeled glycerophospholipids across the proteoliposome membrane bilayer. The use of LacY mutant proteins unable to transport lactose revealed that glycerophospholipid scrambling was independent of H+/lactose transport activity. Unexpectedly, in a LacY double mutant locked into an occluded conformation glycerophospholipid, scrambling activity was largely inhibited. The corresponding single mutants revealed the importance of amino acids G46 and G262 for glycerophospholipid scrambling of LacY.

Wheat pasta has a compact structure built by a gluten network entrapping starch granules resulting in a low glycemic index, but is nevertheless unsuitable for gluten-intolerant people. High protein gluten-free legume flours, rich in fibers, resistant starch and minerals are thus a good alternative for gluten-free pasta production. In this study, gluten-free pasta was produced exclusively from faba, lentil or black-gram flours. The relationship between their structure, their cooking and Rheological properties and their in-vitro starch digestion was analyzed and compared to cereal gluten-free commercial pasta. Trypsin inhibitory activity, phytic acid and α-galactosides were determined in flours and in cooked pasta. All legume pasta were rich in protein, resistant starch and fibers. They had a thick but weak protein network, which is built during the pasta cooking step. This particular structure altered pasta springiness and increased cooking losses. Black-gram pasta, which is especially rich in soluble fibers, differed from faba and lentil pasta, with high springiness (0.85 vs. 0.75) and less loss during cooking. In comparison to a commercial cereal gluten-free pasta, all the legume pasta lost less material during cooking but was less cohesive and springy. Interestingly, due to their particular composition and structure, lentil and faba pasta released their starch more slowly than the commercial gluten-free pasta during the in-vitro digestion process. Anti-nutritional factors in legumes, such as trypsin inhibitory activity and α-galactosides were reduced by up to 82% and 73%, respectively, by pasta processing and cooking. However, these processing steps had a minor effect on phytic acid. This study demonstrates the advantages of using legumes for the production of gluten-free pasta with a low glycemic index and high nutritional quality.

Copper radical alcohol oxidases belonging to auxiliary activity family 5, subfamily 2 (AA5_2) catalyze the oxidation of galactose and galactosides, as well as aliphatic alcohols. Despite their broad applied potential, so far very few AA5_2 members have been biochemically characterized. We report the recombinant production and biochemical characterization of an AA5_2 oxidase from Penicillium rubens Wisconsin 54-1255 (PruAA5_2A), which groups within an unmapped clade phylogenetically distant from those comprising AA5_2 members characterized to date. PruAA5_2 preferentially oxidized raffinose over galactose; however, its catalytic efficiency was 6.5 times higher on glycolaldehyde dimer compared to raffinose. Deep sequence analysis of characterized AA5_2 members highlighted amino acid pairs correlated to substrate range and conserved within the family. Moreover, PruAA5_2 activity spans substrate preferences previously reported for AA5 subfamily 1 and 2 members, identifying possible functional overlap across the AA5 family.