Evaluating the affinity and kinetics of small molecule glycomimetics for human and mouse galectin-3 using surface plasmon resonance.

Galectin-3 is a beta-galactoside-binding mammalian lectin that is one of a 15-member galectin family that can bind several cell surface glycoproteins via its carbohydrate recognition domain (CRD). As a result, it can influence a range of cellular processes including cell activation, adhesion and apoptosis. Galectin-3 has been implicated in various diseases, including fibrotic disorders and cancer, and is now being therapeutically targeted by both small and large molecules. Historically, the screening and triaging of small molecule glycomimetics that bind to the galectin-3 CRD has been completed in fluorescence polarisation (FP) assays to determine KD values. Surface plasmon resonance (SPR) has not been widely used for compound screening and in this study it was used to compare human and mouse galectin-3 affinity measures between FP and SPR, as well as investigate compound kinetics. The KD estimates for a set of compounds selected from mono- and di-saccharides with affinities across a 550-fold range, correlated well between FP and SPR assay formats for both human and mouse galectin-3. Increases in affinity for compounds binding to human galectin-3 were driven by changes in both kon and koff whilst for mouse galectin-3 this was primarily due to kon. The reduction in affinity observed between human to mouse galectin-3 was also comparable between assay formats. SPR has been shown to be a viable alternative to FP for early drug discovery screening and determining KD values. In addition, it can also provide early kinetic characterisation of small molecule galectin-3 glycomimetics with robust kon and koff values generated in a high throughput manner.

Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays.

Galectins are ß-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His6-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His6-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6-3-fold increase in binding efficiency for HSYGal-3 (His6-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His6-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His6-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.

Selectively Modified Lactose and N-Acetyllactosamine Analogs at Three Key Positions to Afford Effective Galectin-3 Ligands.

Galectins constitute a family of galactose-binding lectins overly expressed in the tumor microenvironment as well as in innate and adaptive immune cells, in inflammatory diseases. Lactose ((ß-D-galactopyranosyl)-(1→4)-ß-D-glucopyranose, Lac) and N-Acetyllactosamine (2-acetamido-2-deoxy-4-O-ß-D-galactopyranosyl-D-glucopyranose, LacNAc) have been widely exploited as ligands for a wide range of galectins, sometimes with modest selectivity. Even though several chemical modifications at single positions of the sugar rings have been applied to these ligands, very few examples combined the simultaneous modifications at key positions known to increase both affinity and selectivity. We report herein combined modifications at the anomeric position, C-2, and O-3' of each of the two sugars, resulting in a 3'-O-sulfated LacNAc analog having a Kd of 14.7 µM against human Gal-3 as measured by isothermal titration calorimetry (ITC). This represents a six-fold increase in affinity when compared to methyl ß-D-lactoside having a Kd of 91 µM. The three best compounds contained sulfate groups at the O-3' position of the galactoside moieties, which were perfectly in line with the observed highly cationic character of the human Gal-3 binding site shown by the co-crystal of one of the best candidates of the LacNAc series.

Investigation of anti-galectin-8 levels in patients with multiple sclerosis: A consort-clinical study.

BACKGROUND: Galectins are a family of endogenous mammalian lectins involved in pathogen recognition, killing, and facilitating the entry of microbial pathogens and parasites into the host. They are the intermediators that decipher glycan-containing information about the host immune cells and microbial structures to modulate signaling events that cause cellular proliferation, chemotaxis, cytokine secretion, and cell-to-cell communication. They have subgroups that take place in different roles in the immune system. The effect of galectin-8 on multiple sclerosis disease (MS) has been studied in the literature, but the results seemed unclear. In this study, we aimed to determine anti-galectin-8 (anti-Gal-8) levels in MS and their potential use as biomarkers. METHODS: In this experimental study, 45 MS patients diagnosed according to McDonald criteria were included in the patient group. The healthy control group contained 45 people without MS diagnosis and any risk factors. Demographic data, height, weight, body mass index, blood glucose, thyroid-stimulating hormone, alanine transaminase, aspartate transaminase, creatinine, low-density lipoprotein, anti-Gal-8 levels, the prevalence of hypertension, diabetes mellitus and coronary artery disease were recorded. In addition, the expanded disability status scale and disease duration were evaluated in the patient group. Data were presented as mean ±â€…standard deviations. RESULTS: The mean blood anti-galectin-8 value of the patient group was 4.84 ±â€…4.53 ng/mL, while it was 4.67 ±â€…3.40 ng/mL in the control group, and the difference in these values was found statistically insignificant (P > .05). Moreover, body mass index, glucose, alanine transaminase, aspartate transaminase, thyroid-stimulating hormone, and low-density lipoprotein levels were also statistically insignificant (P > .05). CONCLUSION: This study examined anti-Gal-8 levels in MS patients. The relationship between MS and galectin-8 and anti-Gal-8 levels in patients needs further clarification. As a result, the study's results could help elucidate the pathogenesis of MS and give more evidence for diagnosis.

Targeting disordered-structured domain interactions in Galectin-3 based on NMR and enhanced MD.

Intrinsically disordered regions (IDRs) are common and important functional domains in many proteins. However, IDRs are difficult to target for drug development due to the lack of defined structures that would facilitate the identification of possible drug-binding pockets. Galectin-3 is a carbohydrate-binding protein of which overexpression has been implicated in a wide variety of disorders, including cancer and inflammation. Apart from its carbohydrate-recognition/binding domain (CRD), Galectin-3 also contains a functionally important disordered N-terminal domain (NTD) that contacts the C-terminal domain (CTD) and could be a target for drug development. To overcome challenges involved in inhibitor design due to lack of structure and the highly dynamic nature of the NTD, we used a protocol combining nuclear magnetic resonance data from recombinant Galectin-3 with accelerated molecular dynamics (MD) simulations. This approach identified a pocket in the CTD with which the NTD makes frequent contact. In accordance with this model, mutation of residues L131 and L203 in this pocket caused loss of Galectin-3 agglutination ability, signifying the functional relevance of the cavity. In silico screening was used to design candidate inhibitory peptides targeting the newly discovered cavity, and experimental testing of only three of these yielded one peptide that inhibits the agglutination promoted by wild-type Galectin-3. NMR experiments further confirmed that this peptide indeed binds to a cavity in the CTD, not within the actual CRD. Our results show that it is possible to apply a combination of MD simulations and NMR experiments to precisely predict the binding interface of a disordered domain with a structured domain, and furthermore use this predicted interface for designing inhibitors. This procedure can potentially be extended to many other targets in which similar IDR interactions play a vital functional role.

Galectin-3 Decreases 4-1BBL Bioactivity by Crosslinking Soluble and Membrane Expressed 4-1BB.

4-1BB is a T cell costimulatory receptor and a member of the tumor necrosis factor receptor superfamily. Here, we show that Galectin-3 (Gal-3) decreases the cellular response to its ligand (4-1BBL). Gal-3 binds to both soluble 4-1BB (s4-1BB) and membrane-bound 4-1BB (mem4-1BB), without blocking co-binding of 4-1BBL. In plasma, we detected complexes composed of 4-1BB and Gal-3 larger than 100 nm in size; these complexes were reduced in synovial fluid from rheumatoid arthritis. Both activated 4-1BB+ T cells and 4-1BB-transfected HEK293 cells depleted these complexes from plasma, followed by increased expression of 4-1BB and Gal-3 on the cell surface. The increase was accompanied by a 4-fold decrease in TNFα production by the 4-1BBhighGal-3+ T cells, after exposure to 4-1BB/Gal-3 complexes. In RA patients, complexes containing 4-1BB/Gal-3 were dramatically reduced in both plasma and SF compared with healthy plasma. These results support that Gal-3 binds to 4-1BB without blocking the co-binding of 4-1BBL. Instead, Gal-3 leads to formation of large soluble 4-1BB/Gal-3 complexes that attach to mem4-1BB on the cell surfaces, resulting in suppression of 4-1BBL's bioactivity.

Novel Galectin-3 Roles in Neurogenesis, Inflammation and Neurological Diseases.

Galectin-3 (Gal-3) is an evolutionarily conserved and multifunctional protein that drives inflammation in disease. Gal-3's role in the central nervous system has been less studied than in the immune system. However, recent studies show it exacerbates Alzheimer's disease and is upregulated in a large variety of brain injuries, while loss of Gal-3 function can diminish symptoms of neurodegenerative diseases such as Alzheimer's. Several novel molecular pathways for Gal-3 were recently uncovered. It is a natural ligand for TREM2 (triggering receptor expressed on myeloid cells), TLR4 (Toll-like receptor 4), and IR (insulin receptor). Gal-3 regulates a number of pathways including stimulation of bone morphogenetic protein (BMP) signaling and modulating Wnt signalling in a context-dependent manner. Gal-3 typically acts in pathology but is now known to affect subventricular zone (SVZ) neurogenesis and gliogenesis in the healthy brain. Despite its myriad interactors, Gal-3 has surprisingly specific and important functions in regulating SVZ neurogenesis in disease. Gal-1, a similar lectin often co-expressed with Gal-3, also has profound effects on brain pathology and adult neurogenesis. Remarkably, Gal-3's carbohydrate recognition domain bears structural similarity to the SARS-CoV-2 virus spike protein necessary for cell entry. Gal-3 can be targeted pharmacologically and is a valid target for several diseases involving brain inflammation. The wealth of molecular pathways now known further suggest its modulation could be therapeutically useful.

Galectin-3 N-terminal tail prolines modulate cell activity and glycan-mediated oligomerization/phase separation.

Galectin-3 (Gal-3) has a long, aperiodic, and dynamic proline-rich N-terminal tail (NT). The functional role of the NT with its numerous prolines has remained enigmatic since its discovery. To provide some resolution to this puzzle, we individually mutated all 14 NT prolines over the first 68 residues and assessed their effects on various Gal-3-mediated functions. Our findings show that mutation of any single proline (especially P37A, P55A, P60A, P64A/H, and P67A) dramatically and differentially inhibits Gal-3-mediated cellular activities (i.e., cell migration, activation, endocytosis, and hemagglutination). For mechanistic insight, we investigated the role of prolines in mediating Gal-3 oligomerization, a fundamental process required for these cell activities. We showed that Gal-3 oligomerization triggered by binding to glycoproteins is a dynamic process analogous to liquid-liquid phase separation (LLPS). The composition of these heterooligomers is dependent on the concentration of Gal-3 as well as on the concentration and type of glycoprotein. LLPS-like Gal-3 oligomerization/condensation was also observed on the plasma membrane and disrupted endomembranes. Molecular- and cell-based assays indicate that glycan binding-triggered Gal-3 LLPS (or LLPS-like) is driven mainly by dynamic intermolecular interactions between the Gal-3 NT and the carbohydrate recognition domain (CRD) F-face, although NT-NT interactions appear to contribute to a lesser extent. Mutation of each proline within the NT differentially controls NT-CRD interactions, consequently affecting glycan binding, LLPS, and cellular activities. Our results unveil the role of proline polymorphisms (e.g., at P64) associated with many diseases and suggest that the function of glycosylated cell surface receptors is dynamically regulated by Gal-3.

Degraded Arabinogalactans and Their Binding Properties to Cancer-Associated Human Galectins.

Galectins represent ß-galactoside-binding proteins with numerous functions. Due to their role in tumor progression, human galectins-1, -3 and -7 (Gal-1, -3 and -7) are potential targets for cancer therapy. As plant derived glycans might act as galectin inhibitors, we prepared galactans by partial degradation of plant arabinogalactan-proteins. Besides commercially purchased galectins, we produced Gal-1 and -7 in a cell free system and tested binding capacities of the galectins to the galactans by biolayer-interferometry. Results for commercial and cell-free expressed galectins were comparable confirming functionality of the cell-free produced galectins. Our results revealed that galactans from Echinacea purpurea bind to Gal-1 and -7 with KD values of 1-2 µM and to Gal-3 slightly stronger with KD values between 0.36 and 0.70 µM depending on the sensor type. Galactans from the seagrass Zostera marina with higher branching of the galactan and higher content of uronic acids showed stronger binding to Gal-3 (0.08-0.28 µM) compared to galactan from Echinacea. The results contribute to knowledge on interactions between plant polysaccharides and galectins. Arabinogalactan-proteins have been identified as a new source for production of galactans with possible capability to act as galectin inhibitors.

Galectin-3 in T cell-mediated immunopathology and autoimmunity.

Galectin-3 (Gal-3) is the only member of galectin family able to form pentamers and heterodimers with chemokines. Its presence in various cells and tissues suggests variety of regulatory functions in physiological conditions, but increasing body of evidence indicates involvement of Gal-3 in pathological cascades of many diseases. Gal-3 exerts different, sometimes opposite, effects in various disorders or in different phases of the same disease. These differences in action of Gal-3 are related to the localization of Gal-3 in the cell, types of receptors through which it acts, or the types of cells that secrete it. As a regulator of immune response and T-cell activity, Gal-3 appears to have important role in development of autoimmunity mediated by T cells. Absence of Gal-3 in C57Bl6 mice favors Th2 mediated inflammatory myocarditis but attenuate fibrosis. Recent data also indicate Gal-3 involvement in development atherosclerosis. In pathogenesis of diabetes type 1 and autoimmune components of diabetes type 2 Gal-3 may have detrimental or protective role depending on its intracellular or extracellular localization. Gal-3 mediates autoimmune hepatic damage through activation of T-cells or natural killer T cells. Gal-3 is an important mediator in neurodevelopment, neuropathology and behavior due to its expression both in neurons and glial cells. All together, assessing the role of Gal-3 in immunopathology and autoimmunity it could be concluded that it is an important participant in pathogenesis, as well as promising monitoring marker and therapeutic target.

Topsy-turvy binding of negatively charged homogalacturonan oligosaccharides to galectin-3.

Galectin-3 is crucial to many physiological and pathological processes. The generally accepted dogma is that galectins function extracellularly by binding specifically to ß(1→4)-galactoside epitopes on cell surface glycoconjugates. Here, we used crystallography and NMR spectroscopy to demonstrate that negatively charged homogalacturonans (HG, linear polysaccharides of α(1→4)-linked-D-galacturonate (GalA)) bind to the galectin-3 carbohydrate recognition domain. The HG carboxylates at the C6 positions in GalA rings mandate that this saccharide bind galectin-3 in an unconventional, "topsy-turvy" orientation that is flipped by about 180o relative to that of the canonical ß-galactoside lactose. In this binding mode, the reducing end GalA ß-anomer of HGs takes the position of the nonreducing end galactose residue in lactose. This novel orientation maintains interactions with the conserved tryptophan and seven of the most crucial lactose-binding residues, albeit with different H-bonding interactions. Nevertheless, the HG molecular orientation and new interactions have essentially the same thermodynamic binding parameters as lactose. Overall, our study provides structural details for a new type of galectin-sugar interaction that broadens glycospace for ligand binding to Gal-3 and suggests how the lectin may recognize other negatively charged polysaccharides like glycoaminoglycans (e.g. heparan sulfate) on the cell surface. This discovery impacts on our understanding of galectin-mediated biological function.

Automated orientation of water molecules in neutron crystallographic structures of proteins.

The structure and function of proteins are strongly affected by the surrounding solvent water, for example through hydrogen bonds and the hydrophobic effect. These interactions depend not only on the position, but also on the orientation, of the water molecules around the protein. Therefore, it is often vital to know the detailed orientations of the surrounding ordered water molecules. Such information can be obtained by neutron crystallography. However, it is tedious and time-consuming to determine the correct orientation of every water molecule in a structure (there are typically several hundred of them), which is presently performed by manual evaluation. Here, a method has been developed that reliably automates the orientation of a water molecules in a simple and relatively fast way. Firstly, a quantitative quality measure, the real-space correlation coefficient, was selected, together with a threshold that allows the identification of water molecules that are oriented. Secondly, the refinement procedure was optimized by varying the refinement method and parameters, thus finding settings that yielded the best results in terms of time and performance. It turned out to be favourable to employ only the neutron data and a fixed protein structure when reorienting the water molecules. Thirdly, a method has been developed that identifies and reorients inadequately oriented water molecules systematically and automatically. The method has been tested on three proteins, galectin-3C, rubredoxin and inorganic pyrophosphatase, and it is shown that it yields improved orientations of the water molecules for all three proteins in a shorter time than manual model building. It also led to an increased number of hydrogen bonds involving water molecules for all proteins.

A Rapid and Facile Purification Method for Glycan-Binding Proteins and Glycoproteins.

Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time-saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of crude extracts containing the target proteins from soybean flour Alternate Protocol 1: Preparation of crude extracts from Jack bean meal Alternate Protocol 2: Preparation of crude extracts from the corms of Colocasia esculenta, Xanthosoma sagittifolium, and from the bulbs of Allium sativum Alternate Protocol 3: Preparation of Escherichia coli cell lysates containing human galectin-3 Alternate Protocol 4: Preparation of crude extracts from chicken egg whites (source of ovalbumin) Basic Protocol 2: Preparation of 2% (v/v) red blood cell suspension Basic Protocol 3: Detection of lectin activity of the crude extracts Basic Protocol 4: Identification of multivalent inhibitors as target capturing agents by hemagglutination inhibition assays Basic Protocol 5: Testing the capturing abilities of target capturing agents by precipitation/turbidity assays Basic Protocol 6: Capturing of targets (lectins and glycoproteins) in the crude extracts by target capturing agents and separation of the target-TCA complex from other components of the crude extracts Basic Protocol 7: Releasing the captured targets (lectins and glycoproteins) by dissolving the complex Basic Protocol 8: Separation of the targets (lectins and glycoproteins) from their respective target capturing agents Basic Protocol 9: Verification of the purity of the isolated targets (lectins or glycoproteins).

Analysis of the water-soluble polysaccharides from Camellia japonica pollen and their inhibitory effects on galectin-3 function.

Pollen has been defined as dietary supplement used to supplement the diet in many countries, but the primary structure and activity of Camellia japonica pollen polysaccharide remain unclear. In this study, the water-soluble polysaccharide extracted from Camellia japonica pollen (WCPP) was fractionated into one neutral fraction (WCPP-N) and two acidic fractions (WCPP-A1 and WCPP-A2) by DEAE-cellulose column, and WCPP-A2 was further fractionated into two homogeneous sub-fractions (WCPP-A2a and WCPP-A2b) by Sepharose CL-6B column. Monosaccharide composition results showed that WCPP-N might mainly contain starch-like glucan as well as some arabinogalactan, while WCPP-A1, WCPP-A2 and its sub-fractions might mainly composed of rhamnogalacturonan I (RG-I) pectic polysaccharide domain backbone with some different types of side chains, including arabinan, galactan, and/or arabinogalactan. The primary structure analysis of WCPP-A2a by NMR spectra analysis suggested that WCPP-A2a was an RG-I-like pectic polysaccharide, branched at the O-4 of Rha residues in the backbone, with α-(1 â†’ 3,5)-L-arabinan as well as type-II arabinogalactan side chain to which were attached. The results of galectin-3-mediated hemagglutination assay indicated that WCPP-A2a exhibited the strongest inhibitory effect on galectin-3 with MIC value around 0.27 µg/mL. These results suggested the potential use of Camellia japonica pollen polysaccharide as a galectin3 inhibitor in functional foods.

Regioselective 3-O-Substitution of Unprotected Thiodigalactosides: Direct Route to Galectin Inhibitors.

The synthesis of tailored bioactive carbohydrates usually comprises challenging (de)protection steps, which lowers synthetic yields and increases time demands. We present here a regioselective single-step introduction of benzylic substituents at 3-hydroxy groups of ß-d-galactopyranosyl-(1→1)-thio-ß-d-galactopyranoside (TDG) employing dibutyltin oxide in good yields. These glycomimetics act as inhibitors of galectins-human lectins, which are biomedically attractive targets for therapeutic inhibition in, for example, cancerogenesis. The affinity of the prepared glycomimetics to galectin-1 and galectin-3 was studied in enzyme-linked immunosorbent (ELISA)-type assays and their potential to inhibit galectin binding on the cell surface was shown. We used our original in vivo biotinylated galectin constructs for easy detection by flow cytometry. The results of the biological experiments were compared with data from molecular modeling with both galectins. The present work reveals a facile and elegant synthetic route for the preparation of TDG-derived glycomimetics that exhibit differing selectivity and affinity to galectins depending on the choice of 3-O-substitution.

Galectin-3: Roles in Neurodevelopment, Neuroinflammation, and Behavior.

: There is a plethora of evidence to suggest that Galectin-3 plays an important role in normal functions of mammalian cells, as well as in different pathogenic conditions. This review highlights recent data published by researchers, including our own team, on roles of Galectin-3 in the nervous system. Here, we discuss the roles of Galectin-3 in brain development, its roles in glial cells, as well as the interactions of glial cells with other neural and invading cells in pathological conditions. Galectin-3 plays an important role in the pathogenesis of neuroinflammatory and neurodegenerative disorders, such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, and Huntington's disease. On the other hand, there is also evidence of the protective role of Galectin-3 due to its anti-apoptotic effect in target cells. Interestingly, genetic deletion of Galectin-3 affects behavioral patterns in maturing and adult mice. The results reviewed in this paper and recent development of highly specific inhibitors suggests that Galectin-3 may be an important therapeutic target in pathological conditions including the disorders of the central nervous system.

Influence of protein (human galectin-3) design on aspects of lectin activity.

The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure-activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms.

Characterization and functional study of Galectin3 from Japanese flounder (Paralichthys olivaceus).

Galectins belong to the ß-galactoside binding protein family and participate in both innate and acquired immunity. In this study, we described the molecular characteristics of Galectin3 gene from Japanese flounder (Paralichthys olivaceus), designed as PoGalectin3. Its open reading frame was 1128 bp, encoding a protein composed of 375 amino acids. PoGalectin3 belongs to chimeric galactose agglutinin, which contains a C-terminal carbohydrate recognition domain (CRD) (L250-P372), and its N-terminal is rich in proline (P) and glycine (G). Multiple sequence alignment and phylogenetic tree showed that PoGalectin3 was conservative in different aquatic animals. Tissue distribution confirmed that PoGalectin3 showed significantly highest expression in brain, moderate expression in liver, intestine and muscle. PoGalectin3 was significantly increased post infection with Edwardsiella tarda from intestine tissue of P. olivaceus. In order to investigate the binding ability of PoGalectin3 to pathogen-associated molecular patterns, the recombinant PoGalectin3 protein (rPoGalectin3) was successfully expressed and purified, and an Enzyme linked immunosorbent assay (ELISA) experiment was performed. ELISA refers to the qualitative and quantitative detection method of immune response by combining soluble antigen or antibody with solid-phase carrier. It was confirmed that rPoGalectin3 exhibited high affinity to lipopolysaccharide and peptidoglycan. The rPoGalectin3 also exhibited a concentration dependent binding capacity with Gram-positive bacteria (Bacillus pumilus, Bacillus subtilis, Bacillus cereus) and Gram-negative bacteria (Aeromonas salmonicida, E. tarda, Vibrio vulnificus). In addition, the results of microbial agglutination experiment showed that rPoGalectin3 could agglutinate Gram-positive bacteria (B. pumilus, B. subtilis) and Gram-negative bacteria (A. salmonicida, E. tarda) in the presence of Ca2+. In conclusion, this research laid an important foundation for the specific function analysis of PoGalectin3, which provide theoretical basis for the prevention and control of aquatic diseases.

Galectin-3 Is a Potential Mediator for Atherosclerosis.

Atherosclerosis is a multifactorial chronic inflammatory arterial disease forming the pathological basis of many cardiovascular diseases such as coronary heart disease, heart failure, and stroke. Numerous studies have implicated inflammation as a key player in the initiation and progression of atherosclerosis. Galectin-3 (Gal-3) is a 30 kDa ß-galactose, highly conserved and widely distributed intracellularly and extracellularly. Gal-3 has been demonstrated in recent years to be a novel inflammatory factor participating in the process of intravascular inflammation, lipid endocytosis, macrophage activation, cellular proliferation, monocyte chemotaxis, and cell adhesion. This review focuses on the role of Gal-3 in atherosclerosis and the mechanism involved and several classical Gal-3 agonists and antagonists in the current studies.