Breaking barriers: How modified citrus pectin inhibits galectin-8.

Inhibition of galectin-3-mediated interactions by modified citrus pectin (MCP) could affect several rate-limiting steps in cancer metastasis, but the ability of MCP to antagonize galectin-8 function remains unknown. We hypothesized that MCP could bind to galectin-8 in addition to galectin-3. In this study, a combination of gradual ethanol precipitation and DEAE-Sepharose Fast Flow chromatography was used to isolate several fractions from MCP. The ability of these fractions to antagonize galectin-8 function was studied as well as the primary structure and initial structure-function relationship of the major active component MCP-30-3. The results showed that MCP-30-3 (168 kDa) was composed of Gal (13.8%), GalA (63.1%), GlcA (13.0%), and Glc (10.1%). MCP-30-3 could specifically bind to galectin-8, with an MIC value of 0.04 mg mL-1. After MCP-30-3 was hydrolyzed by ß-galactosidase or pectinase, its binding activity was significantly reduced. These results provide new insights into the interaction between MCP structure and galectin function, as well as the potential utility in the development of functional foods.

Endocytic Roles of Glycans on Proteins and Lipids.

Most cell surface proteins are decorated by glycans, and the plasma membrane is rich in glycosylated lipids. The mechanisms by which the enormous complexity of these glycan structures on proteins and lipids is exploited to control glycoprotein activity by setting their cell surface residence time and the ways by which they are taken up into cells are still under active investigation. Here, two mechanisms are presented, termed galectin lattices and glycolipid-lectin (GL-Lect)-driven endocytosis, which are among the most prominent to establish a link between glycan information and endocytosis. Types of glycans on glycoproteins and glycolipids are reviewed from the angle of their interaction with glycan-binding proteins that are at the heart of galectin lattices and GL-Lect-driven endocytosis. Examples are given to show how these mechanisms affect cellular functions ranging from cell migration and signaling to vascularization and immune modulation. Finally, outstanding challenges on the link between glycosylation and endocytosis are discussed.

Placental galectins: a subfamily of galectins lose the ability to bind ß-galactosides with new structural features†.

Galectins are a phylogenetically conserved family of soluble ß-galactoside binding proteins. There are 16 different of galectins, each with a specific function determined by its distinct distribution and spatial structure. Galectin-13, galectin-14, and galectin-16 are distinct from other galectin members in that they are primarily found in placental tissue. These galectins, also referred to as placental galectins, play critical roles in regulating pregnancy-associated processes, such as placenta formation and maternal immune tolerance to the embedded embryo. The unique structural characteristics and the inability to bind lactose of placental galectins have recently received significant attention. This review primarily examines the novel structural features of placental galectins, which distinguish them from the classic galectins. Furthermore, it explores the correlation between these structural features and the loss of ß-galactoside binding ability. In addition, the newly discovered functions of placental galectins in recent years are also summarized in our review. A detailed understanding of the roles of placental galectins may contribute to the discovery of new mechanisms causing numerous pregnancy diseases and enable the development of new diagnostic and therapeutic strategies for the treatment of these diseases, ultimately benefiting the health of mothers and offspring.

Identification of Galectin 9 and its antibacterial function in yellow drum (Nibea albiflora).

Galectins are a family of evolutionarily conserved lectins that contain carbohydrate recognition domains (CRDs) specifically recognizing ß-galactoside. Galectin-9 plays a crucial role in various biological processes during pathogenic infections. In a previous study, galectin-9 was identified as a candidate gene for resistance to Vibrio harveyi disease in yellow drum using a genome-wide association study (GWAS) analysis. In this study, a galectin-9 gene was identified from Nibea albiflora and named YdGal-9. The mRNA transcripts of YdGal-9 were distributed in all the detected tissues and the highest level was found in the kidney. The subcellular localization of YdGal-9-EGFP proteins was observed in both nucleus and cytoplasm in the kidney cells of N. albiflora. The expression of YdGal-9 in the brain increased significantly after infection with Vibrio harveyi. The red blood cells from rabbits, Larimichthys crocea, and N. albiflora were agglutinated by the purified recombinant YdGal-9 proteins. The results of the agglutination activity of deletion mutants of YdGal-9 proved that the conserved sugar binding motifs (H-NPR and WG-EE-) were critical for YdGal-9's agglutination activity. In addition, YdGal-9 killed some gram-negative bacteria by inducing cell wall destruction including Pseudomonas plecoglossicida, Aeromonas hydrophila, Escherichia coli, V. parahemolyticus, V. harveyi, and V. alginolyticus. Taken together, these results suggested that the YdGal-9 protein of N. albiflora played a vital role in fighting bacterial infections.

Linker remodels human Galectin-8 structure and regulates its hemagglutination and pro-apoptotic activity.

Numerous articles have reported the involvement of linker in regulating bioactivity of tandem-repeat galectins. We hypothesize that linker interacts with N/C-CRDs to regulate the bioactivity of tandem-repeat galectins. To further investigate structural molecular mechanism of linker in regulating bioactivity of Gal-8, Gal-8LC was crystallized. Gal-8LC structure revealed formation of ß-strand S1 by Asn174 to Pro176 from linker. S1-strand interacts with C-terminal of C-CRD via hydrogen bond interactions, mutually influencing their spatial structures. Our Gal-8 NL structure have demonstrated that linker region from Ser154 to Gln158 interacts with the N-terminal of Gal-8. Ser154 to Gln158 and Asn174 to Pro176 are likely involved in regulation of Gal-8's biological activity. Our preliminary experiment results revealed different hemagglutination and pro-apoptotic activities between full-length and truncated forms of Gal-8, indicating involvement of linker in regulating these activities. We generated several mutant and truncated forms of Gal-8 (Gal-8 M3, Gal-8 M5, Gal-8TL1, Gal-8TL2, Gal-8LC-M3 and Gal-8_177-317). Ser154 to Gln158 and Asn174 to Pro176 were found to be involved in regulating hemagglutination and pro-apoptotic activities of Gal-8. Ser154 to Gln158 and Asn174 to Pro176 are critical functional regulatory regions within linker. Our study holds significant importance in providing a profound understanding of how linker regulates biological activity of Gal-8.

Oligosaccharide Ligands of Galectin-4 and Its Subunits: Multivalency Scores Highly.

Galectins are carbohydrate-binding lectins that modulate the proliferation, apoptosis, adhesion, or migration of cells by cross-linking glycans on cell membranes or extracellular matrix components. Galectin-4 (Gal-4) is a tandem-repeat-type galectin expressed mainly in the epithelial cells of the gastrointestinal tract. It consists of an N- and a C-terminal carbohydrate-binding domain (CRD), each with distinct binding affinities, interconnected with a peptide linker. Compared to other more abundant galectins, the knowledge of the pathophysiology of Gal-4 is sparse. Its altered expression in tumor tissue is associated with, for example, colon, colorectal, and liver cancers, and it increases in tumor progression, and metastasis. There is also very limited information on the preferences of Gal-4 for its carbohydrate ligands, particularly with respect to Gal-4 subunits. Similarly, there is virtually no information on the interaction of Gal-4 with multivalent ligands. This work shows the expression and purification of Gal-4 and its subunits and presents a structure-affinity relationship study with a library of oligosaccharide ligands. Furthermore, the influence of multivalency is demonstrated in the interaction with a model lactosyl-decorated synthetic glycoconjugate. The present data may be used in biomedical research for the design of efficient ligands of Gal-4 with diagnostic or therapeutic potential.

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.

Lactose and Galactose Promote the Crystallization of Human Galectin-10.

Galectin-10 (Gal-10) forms Charcot-Leyden crystals (CLCs), which play a key role in the symptoms of asthma and allergies and some other diseases. Gal-10 has a carbohydrate-binding site; however, neither the Gal-10 dimer nor the CLCs can bind sugars. To investigate the monomer-dimer equilibrium of Gal-10, high-performance size-exclusion chromatography (SEC) was employed to separate serial dilutions of Gal-10 with and without carbohydrates. We found that both the dimerization and crystallization of Gal-10 were promoted by lactose/galactose binding. A peak position shift for the monomer was observed after treatment with either lactose or galactose, implying that the polarity of the monomer was reduced by lactose/galactose binding. Further experiments indicated that alkaline conditions of pH 8.8 mimicked the lactose/galactose-binding environment, and the time interval between monomers and dimers in the chromatogram decreased from 0.8 min to 0.4 min. Subsequently, the electrostatic potential of the Gal-10 monomers was computed. After lactose/galactose binding, the top side of the monomer shifted from negatively charged to electrically neutral, allowing it to interact with the carbohydrate-binding site of the opposing subunit during dimerization. Since lactose/galactose promotes the crystallization of Gal-10, our findings implied that dairy-free diets (free of lactose/galactose) might be beneficial to patients with CLC-related diseases.

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.

The role of galectins in immunity and infection.

The galectin family consists of carbohydrate (glycan) binding proteins that are expressed by a wide variety of cells and bind to galactose-containing glycans. Galectins can be located in the nucleus or the cytoplasm, or can be secreted into the extracellular space. They can modulate innate and adaptive immune cells by binding to glycans on the surface of immune cells or intracellularly via carbohydrate-dependent or carbohydrate-independent interactions. Galectins expressed by immune cells can also participate in host responses to infection by directly binding to microorganisms or by modulating antimicrobial functions such as autophagy. Here we explore the diverse ways in which galectins have been shown to impact immunity and discuss the opportunities and challenges in the field.

Glycocalix[4]arenes and their affinity to a library of galectins: the linker matters.

Galectins are lectins that bind ß-galactosides. They are involved in important extra- and intracellular biological processes such as apoptosis, and regulation of the immune system or the cell cycle. High-affinity ligands of galectins may introduce new therapeutic approaches or become new tools for biomedical research. One way of increasing the low affinity of ß-galactoside ligands to galectins is their multivalent presentation, e.g., using calixarenes. We report on the synthesis of glycocalix[4]arenes in cone, partial cone, 1,2-alternate, and 1,3-alternate conformations carrying a lactosyl ligand on three different linkers. The affinity of the prepared compounds to a library of human galectins was determined using competitive ELISA assay and biolayer interferometry. Structure-affinity relationships regarding the influence of the linker and the core structure were formulated. Substantial differences were found between various linker lengths and the position of the triazole unit. The formation of supramolecular clusters was detected by atomic force microscopy. The present work gives a systematic insight into prospective galectin ligands based on the calix[4]arene core.

Structural and functional analysis of a tandem repeat galacturonic acid-binding lectin from the sea hare Aplysia californica.

A d-galacturonic acid-specific lectin, named AcL, was purified from the sea hare Aplysia californica by galactose-agarose affinity chromatography. AcL has a molecular mass of 27.5 kDa determined by MALDI-TOF mass spectrometry. This lectin shows a good affinity for d-galacturonic acid and a lower affinity for galactosides: raffinose, melibiose, α and ß-lactose, and d-galactose. We determined the amino acid sequence of AcL by trypsin digestion and subsequent peptide analysis by mass spectrometry, resulting in a 238 amino acid protein with a theoretical molecular mass of 26.4 kDa. The difference between the theoretical and experimental values can be attributed to post-translational modifications. Thiol-disulfide quantification discerned five disulfide bonds and three free cysteines. The structure of Acl is mainly comprised of beta sheets, determined by circular dichroism, and predicted with AlphaFold. Theoretical models depict three nearly identical tandem domains consisting of two beta sheets each. From docking analysis, we identified AcL glycan-binding sites as multiple conserved motifs in each domain. Furthermore, phylogenetic analysis based on its structure and sequence showed that AcL and its closest homologues (GalULs) form a clear monophyletic group, distinct from other glycan-binding proteins with a jelly-roll fold: lectins of types F and H. GalULs possess four conserved sequence regions that distinguish them and are either ligand-binding motifs or stabilizing network hubs. We suggest that this new family should be referred to as GalUL or D-type, following the traditional naming of lectins; D standing for depilans, the epithet for the species (Aplysia depilans) from which a lectin of this family was first isolated and described.

Proximity labeling technologies to illuminate glycan-protein interactions.

Glycosylation is a ubiquitous post-translational modification read by glycan-binding proteins (GBP) to encode important functions, but a robust understanding of these interactions and their consequences can be challenging to uncover. Glycan-GBP interactions are transient and weak, making them difficult to capture, and glycosylation is dynamic and heterogenous, necessitating study in native cellular environments to identify endogenous ligands. Proximity labeling, an experimental innovation that labels biomolecules close to a protein of interest, has recently emerged as a powerful strategy to overcome these limitations, allowing interactors to be tagged in cells for subsequent enrichment and identification by mass spectrometry-based proteomics. We will describe this nascent technique and discuss its applications in the last five years with different GBP classes, including Siglecs, galectins, and non-human lectins.

The interplay of galectins-1, -3, and -9 in the immune-inflammatory response underlying cardiovascular and metabolic disease.

Galectins are ß-galactoside-binding proteins that bind and crosslink molecules via their sugar moieties, forming signaling and adhesion networks involved in cellular communication, differentiation, migration, and survival. Galectins are expressed ubiquitously across immune cells, and their function varies with their tissue-specific and subcellular location. Particularly galectin-1, -3, and -9 are highly expressed by inflammatory cells and are involved in the modulation of several innate and adaptive immune responses. Modulation in the expression of these proteins accompany major processes in cardiovascular diseases and metabolic disorders, such as atherosclerosis, thrombosis, obesity, and diabetes, making them attractive therapeutic targets. In this review we consider the broad cellular activities ascribed to galectin-1, -3, and -9, highlighting those linked to the progression of different inflammatory driven pathologies in the context of cardiovascular and metabolic disease, to better understand their mechanism of action and provide new insights into the design of novel therapeutic strategies.

Structural and evolutionary insights into the multidomain galectin from the red abalone Haliotis rufescens with specificity for sulfated glycans.

Galectins are an evolutionarily ancient family of lectins characterized by their affinity for ß-galactosides and a conserved binding site in the carbohydrate recognition domain (CRD). These lectins are involved in multiple physiological functions, including the recognition of glycans on the surface of viruses and bacteria. This feature supports their role in innate immune responses in marine mollusks. Here, we identified and characterized a galectin, from the mollusk Haliotis rufescens (named HrGal), with four CRDs that belong to the tandem-repeat type. HrGal was purified by affinity chromatography in a galactose-agarose resin and exhibited a molecular mass of 64.11 kDa determined by MALDI-TOF mass spectrometry. The identity of HrGal was verified by sequencing, confirming that it is a 555 amino acid protein with a mass of 63.86 kDa. This protein corresponds to a galectin reported in GenBank with accession number AHX26603. HrGal is stable in the presence of urea, reducing agents, and ions such as Cu2+ and Zn2+. The recombinant galectin (rHrGal) was purified from inclusion bodies in the presence of these ions. A theoretical model obtained with the AlphaFold server exhibits four non-identical CRDs, with a ß sandwich folding and the representative motifs for binding ß-galactosides. This allows us to classify HrGal within the tandem repeat galectin family. On the basis of a phylogenetic analysis, we found that the mollusk sequences form a monophyletic group of tetradomain galectins unrelated to vertebrate galectins. HrGal showed specificity for galactosides and glucosides but only the sulfated sugars heparin and ι-carrageenan inhibited its hemagglutinating activity with a minimum inhibitory concentration of 4 mM and 6.25 X 10-5% respectively. The position of the sulfate groups seemed crucial for binding, both by carrageenans and heparin.

Galectins: Important Regulators in Normal and Pathologic Pregnancies.

Galectins (Gal) are characterized by their affinity for galactoside structures on glycoconjugates. This relationship is mediated by carbohydrate recognition domains, which are multifunctional regulators of basic cellular biological processes with high structural similarity among family members. They participate in both innate and adaptive immune responses, as well as in reproductive immunology. Recently, the discovery that galectins are highly expressed at the maternal-fetal interface has garnerd the interest of experts in human reproduction. Galectins are involved in a variety of functions such as maternal-fetal immune tolerance, angiogenesis, trophoblast invasion and placental development and are considered to be important mediators of successful embryo implantation and during pregnancy. Dysregulation of these galectins is associated with abnormal and pathological pregnancies (e.g., preeclampsia, gestational diabetes mellitus, fetal growth restriction, preterm birth). Our work reviews the regulatory mechanisms of galectins in normal and pathological pregnancies and has implications for clinicians in the prevention, diagnosis and treatment of pregnancy-related diseases.

Purification of Recombinant Galectins from Different Species Using Distinct Affinity Chromatography Methods.

Galectins are lectins having the capacity to recognize ß-galactose-containing glycan structures and are widely distributed among various taxa. However, the exact physiological and biochemical functions mediated by galectins that necessitate their wide occurrence among diverse species have not yet been delineated in a precise manner. Purification of recombinant galectins in active form is a fundamental requirement to elucidate their biological function. In this chapter, we are describing methods to recombinantly express and purify galectins using three different methods of affinity purification, i.e., lactosyl-Sepharose chromatography for fungal galectin Coprinopsis cinerea galectin 2 (CGL2), nickel-chromatography for histidine-tagged human galectin-7, and glutathione-Sepharose chromatography for Glutathione S-transferase-tagged (GST-tagged) human galectin-7. Step-by-step instructions are provided for obtaining the above-mentioned recombinant galectins that retain carbohydrate-binding activity and are suitable for conducting biochemical experiments.

Investigation of the Molecular Details of the Interactions of Selenoglycosides and Human Galectin-3.

Human galectin-3 (hGal-3) is involved in a variety of biological processes and is implicated in wide range of diseases. As a result, targeting hGal-3 for clinical applications has become an intense area of research. As a step towards the development of novel hGal-3 inhibitors, we describe a study of the binding of two Se-containing hGal-3 inhibitors, specifically that of di(ß-D-galactopyranosyl)selenide (SeDG), in which two galactose rings are linked by one Se atom and a di(ß-D-galactopyranosyl)diselenide (DSeDG) analogue with a diseleno bond between the two sugar units. The binding affinities of these derivatives to hGal-3 were determined by 15N-1H HSQC NMR spectroscopy and fluorescence anisotropy titrations in solution, indicating a slight decrease in the strength of interaction for SeDG compared to thiodigalactoside (TDG), a well-known inhibitor of hGal-3, while DSeDG displayed a much weaker interaction strength. NMR and FA measurements showed that both seleno derivatives bind to the canonical S face site of hGal-3 and stack against the conserved W181 residue also confirmed by X-ray crystallography, revealing canonical properties of the interaction. The interaction with DSeDG revealed two distinct binding modes in the crystal structure which are in fast exchange on the NMR time scale in solution, explaining a weaker interaction with hGal-3 than SeDG. Using molecular dynamics simulations, we have found that energetic contributions to the binding enthalpies mainly differ in the electrostatic interactions and in polar solvation terms and are responsible for weaker binding of DSeDG compared to SeDG. Selenium-containing carbohydrate inhibitors of hGal-3 showing canonical binding modes offer the potential of becoming novel hydrolytically stable scaffolds for a new class of hGal-3 inhibitors.

Methods of in vitro study of galectin-glycomaterial interaction.

Galectins are a family of carbohydrate-binding lectins modulating cell events such as cell proliferation, apoptosis, adhesion or migration by cross-linking the glycan structures of cell membranes and/or extracellular matrix components. In a diseased organism, galectins are upregulated and trigger the progression of diseases such as inflammation, cancerogenesis, fibrosis, cardiovascular and metabolic disorders. Targeting galectins with glycomaterials for the aims of diagnostics or therapy is, therefore, a focus of biotechnological and biomedicinal research, and already led to candidates for clinical trials. Testing and evaluation of galectin-glycomaterial interactions require informative and versatile analytical methods at several levels of knowledge, from basic inter-molecular interaction to complex cell-based assays. This review aims to classify and characterize a selection of the most promising methods to identify the prospective glycomaterials for translating galectin targeting from the molecular level to the level of tailored in vivo assays.