Galectin-9 as a Potential Modulator of Lymphocyte Adhesion to Endothelium via Binding to Blood Group H Glycan.

The recruitment of leukocytes from blood is one of the most important cellular processes in response to tissue damage and inflammation. This multi-step process includes rolling leukocytes and their adhesion to endothelial cells (EC), culminating in crossing the EC barrier to reach the inflamed tissue. Galectin-8 and galectin-9 expressed on the immune system cells are part of this process and can induce cell adhesion via binding to oligolactosamine glycans. Similarly, these galectins have an order of magnitude higher affinity towards glycans of the ABH blood group system, widely represented on ECs. However, the roles of gal-8 and gal-9 as mediators of adhesion to endothelial ABH antigens are practically unknown. In this work, we investigated whether H antigen-gal-9-mediated adhesion occurred between Jurkat cells (of lymphocytic origin and known to have gal-9) and EA.hy 926 cells (immortalized endothelial cells and known to have blood group H antigen). Baseline experiments showed that Jurkat cells adhered to EA.hy 926 cells; however when these EA.hy 926 cells were defucosylated (despite the unmasking of lactosamine chains), adherence was abolished. Restoration of fucosylation by insertion of synthetic glycolipids in the form of H (type 2) trisaccharide Fucα1-2Galß1-4GlcNAc restored adhesion. The degree of lymphocyte adhesion to native and the "H-restored" (glycolipid-loaded) EA.hy 926 cells was comparable. If this gal-9/H (type 2) interaction is similar to processes that occur in vivo, this suggests that only the short (trisaccharide) H glycan on ECs is required.

Altering the Modular Architecture of Galectins Affects its Binding with Synthetic α-Dystroglycan O-Mannosylated Core M1 Glycoconjugates In situ.

The multifunctionality of galectins helps regulate a broad range of fundamental cellular processes via cis-binding and trans-bridging activities and has gained widespread attention with respect to the importance of the natural specificity/selectivity of this lectin family to its glycoconjugate receptors. Combining galectin (Gal)-1, -3, -4, and -9 variant test panels, achieved via rational protein engineering, and a synthetic α-dystroglycan (DG) O-Mannosylated core M1 glycopeptide library, a detailed comparative analysis was performed, utilizing microarray experiments to delineate the design-functionality relationships within this lectin family. Enhancement of prototype Gal-1 and chimera-type Gal-3 cis-binding toward the prepared ligands is possible by transforming these lectins into tandem-repeat type and prototypes, respectively. Furthermore, Gal-1 variants demonstrated improved trans-bridging capabilities between core M1 α-DG glycopeptides and laminins in microarray, suggesting the possible translational applications of these galectin variants in the treatment of some forms of α-dystroglycanopathy.

The sweet side of wound healing: galectins as promising therapeutic targets in hemostasis, inflammation, proliferation, and maturation/remodeling.

INTRODUCTION: Understanding the molecular and cellular processes involved in skin wound healing may pave the way for the development of innovative approaches to transforming the identified natural effectors into therapeutic tools. Based on the extensive involvement of the ga(lactoside-binding)lectin family in (patho)physiological processes, it has been well established that galectins are involved in a wide range of cell-cell and cell-matrix interactions. AREAS COVERED: In the present paper, we provide an overview of the biological role of galectins in repair and regeneration, focusing on four main phases (hemostasis, inflammation, proliferation, and maturation/remodeling) of skin repair using basic wound models (open excision vs. sutured incision). EXPERT OPINION: The reported data make a strong case for directing further efforts to treat excisional and incisional wounds differently. Functions of galectins essentially result from their modular presentation. In fact, Gal-1 seems to play a role in the early phases of healing (anti-inflammatory) and wound contraction, Gal-3 accelerates re-epithelization and increases tensile strength (scar inductor). Galectins have also become subject of redesigning by engineering to optimize the activity. Clinically relevant, these new tools derived from the carbohydrate recognition domain platform may also prove helpful for other purposes, such as potent antibacterial agglutinins and opsonins.

Targeting osteoarthritis-associated galectins and an induced effector class by a ditopic bifunctional reagent: Impact of its glycan part on binding measured in the tissue context.

Pairing glycans with tissue lectins controls multiple effector pathways in (patho)physiology. A clinically relevant example is the prodegradative activity of galectins-1 and -3 (Gal-1 and -3) in the progression of osteoarthritis (OA) via matrix metalloproteinases (MMPs), especially MMP-13. The design of heterobifunctional inhibitors that can block galectin binding and MMPs both directly and by preventing their galectin-dependent induction selectively offers a perspective to dissect the roles of lectins and proteolytic enzymes. We describe the synthesis of such a reagent with a bivalent galectin ligand connected to an MMP inhibitor and of two tetravalent glycoclusters with a subtle change in headgroup presentation for further elucidation of influence on ligand binding. Testing was performed on clinical material with mixtures of galectins as occurring in vivo, using sections of fixed tissue. Two-colour fluorescence microscopy monitored binding to the cellular glycome after optimization of experimental parameters. In the presence of the inhibitor, galectin binding to OA specimens was significantly reduced. These results open the perspective to examine the inhibitory capacity of custom-made ditopic compounds on binding of lectins in mixtures using sections of clinical material with known impact of galectins and MMPs on disease progression.

Combining Recombinase-Mediated Cassette Exchange Strategy with Quantitative Proteomic and Phosphoproteomic Analyses to Inspect Intracellular Functions of the Tumor Suppressor Galectin-4 in Colorectal Cancer Cells.

Galectin-4 (Gal4) has been suggested to function as a tumor suppressor in colorectal cancer (CRC). In order to systematically explore its function in CRC, we established a CRC cell line where Gal4 expression can be regulated via the doxycycline (dox)-inducible expression of a single copy wildtype LGALS4 transgene generated by recombinase-mediated cassette exchange (RMCE). Using this model and applying in-depth proteomic and phosphoproteomic analyses, we systematically screened for intracellular changes induced by Gal4 expression. Overall, 3083 cellular proteins and 2071 phosphosites were identified and quantified, of which 1603 could be matched and normalized to their protein expression levels. A bioinformatic analysis revealed that most of the regulated proteins and phosphosites can be localized in the nucleus and are categorized as nucleic acid-binding proteins. The top candidates whose expression was modulated by Gal4 are PURB, MAPKAPK3, BTF3 and BCAR1, while the prime candidates with altered phosphorylation included ZBTB7A, FOXK1, PURB and CK2beta. In order to validate the (phospho)proteomic data, we confirmed these candidates by a radiometric metabolic-labelling and immunoprecipitation strategy. All candidates exert functions in the transcriptional or translational control, indicating that Gal4 might be involved in these processes by affecting the expression or activity of these proteins.

What Happens If a Human Galectin Enters the Endoplasmic Reticulum?

Mammalian galectins have no signal peptide, and it is not known what would happen if a galectin is directed to take the classical export route. The corresponding engineering of galectin-specific cDNA will answer questions on the fate of a signal peptide-bearing protein variant after its entry into the endoplasmic reticulum (ER). Affinity chromatography and mass-spectrometric analysis of occupancy of potential N-glycosylation sites for the galectin, binding and functional assays with cells as well as subcellular fractionation by density gradient ultracentrifugation and immunocytochemical colocalization with ER/Golgi markers report on aspects of the consequences of letting a galectin enter new territory. Applying these methods will help to clarify why galectins are leaderless and thus produced by free ribosomes.

Exploring the Galectin Network by Light and Fluorescence Microscopy.

Dynamic changes of a cell's glycophenotype are increasingly interpreted as shifts in the capacity to interact with tissue (endogenous) lectins. The status of glycan branching or chain length (e.g., core 1 vs core 2 mucin-type O-glycans and polyLacNAc additions) as well as of sialylation/sulfation has been delineated to convey signals. They are "read" by galectins, for example regulating lattice formation on the membrane and cell growth. Owing to the discovery of the possibility that these effectors act in networks physiologically resulting in functional antagonism or cooperation, their detection and distribution profiling need to be expanded from an individual (single) protein to the-at best-entire family. How to work with non-cross-reactive antibodies and with the labeled tissue-derived proteins (used as probes) is exemplarily documented for chicken and human galectins including typical activity and specificity controls. This description intends to inspire the systematic (network) study of members of a lectin family and also the application of tissue proteins beyond a single lectin category in lectin histochemistry.

Examining Galectin Gene Regulation by Reporter Assays.

Matching their role as potent and versatile effectors in cellular homeostasis and disease processes, galectins are subject to a fine-tuned transcriptional regulation of gene expression. It can apparently even involve coregulation with certain elements of the enzymatic machinery for glycan biosynthesis/remodeling and/or functional carriers of galectin-binding glycans such as the α5ß1-integrin. All this suggests not yet fully known combinatorial processes to reach the desired outcome. Identification of transcription start point(s), cloning of upstream promoter region, and the design of plasmids for luciferase-based reporter assays establish the platform to initiate a systematic search of regulatory sequences. Their elucidation is also a step toward rationally manipulating expression of galectin genes in pathogenesis.

What is the Sugar Code?

A code is defined by the nature of the symbols, which are used to generate information-storing combinations (e. g. oligo- and polymers). Like nucleic acids and proteins, oligo- and polysaccharides are ubiquitous, and they are a biochemical platform for establishing molecular messages. Of note, the letters of the sugar code system (third alphabet of life) excel in coding capacity by making an unsurpassed versatility for isomer (code word) formation possible by variability in anomery and linkage position of the glycosidic bond, ring size and branching. The enzymatic machinery for glycan biosynthesis (writers) realizes this enormous potential for building a large vocabulary. It includes possibilities for dynamic editing/erasing as known from nucleic acids and proteins. Matching the glycome diversity, a large panel of sugar receptors (lectins) has developed based on more than a dozen folds. Lectins 'read' the glycan-encoded information. Hydrogen/coordination bonding and ionic pairing together with stacking and C-H/π-interactions as well as modes of spatial glycan presentation underlie the selectivity and specificity of glycan-lectin recognition. Modular design of lectins together with glycan display and the nature of the cognate glycoconjugate account for the large number of post-binding events. They give an entry to the glycan vocabulary its functional, often context-dependent meaning(s), hereby building the dictionary of the sugar code.

Structural Characterization of Rat Galectin-5, an N-Tailed Monomeric Proto-Type-like Galectin.

Galectins are multi-purpose effectors acting via interactions with distinct counterreceptors based on protein-glycan/protein recognition. These processes are emerging to involve several regions on the protein so that the availability of a detailed structural characterization of a full-length galectin is essential. We report here the first crystallographic information on the N-terminal extension of the carbohydrate recognition domain of rat galectin-5, which is precisely described as an N-tailed proto-type-like galectin. In the ligand-free protein, the three amino-acid stretch from Ser2 to Ser5 is revealed to form an extra ß-strand (F0), and the residues from Thr6 to Asn12 are part of a loop protruding from strands S1 and F0. In the ligand-bound structure, amino acids Ser2-Tyr10 switch position and are aligned to the edge of the ß-sandwich. Interestingly, the signal profile in our glycan array screening shows the sugar-binding site to preferentially accommodate the histo-blood-group B (type 2) tetrasaccharide and N-acetyllactosamine-based di- and oligomers. The crystal structures revealed the characteristically preformed structural organization around the central Trp77 of the CRD with involvement of the sequence signature's amino acids in binding. Ligand binding was also characterized calorimetrically. The presented data shows that the N-terminal extension can adopt an ordered structure and shapes the hypothesis that a ligand-induced shift in the equilibrium between flexible and ordered conformers potentially acts as a molecular switch, enabling new contacts in this region.

Imitating evolution's tinkering by protein engineering reveals extension of human galectin-7 activity.

Wild-type lectins have distinct types of modular design. As a step to explain the physiological importance of their special status, hypothesis-driven protein engineering is used to generate variants. Concerning adhesion/growth-regulatory galectins, non-covalently associated homodimers are commonly encountered in vertebrates. The homodimeric galectin-7 (Gal-7) is a multifunctional context-dependent modulator. Since the possibility of conversion from the homodimer to hybrids with other galectin domains, i.e. from Gal-1 and Gal-3, has recently been discovered, we designed Gal-7-based constructs, i.e. stable (covalently linked) homo- and heterodimers. They were produced and purified by affinity chromatography, and the sugar-binding activity of each lectin unit proven by calorimetry. Inspection of profiles of binding of labeled galectins to an array-like platform with various cell types, i.e. sections of murine epididymis and jejunum, and impact on neuroblastoma cell proliferation revealed no major difference between natural and artificial (stable) homodimers. When analyzing heterodimers, acquisition of altered properties was seen. Remarkably, binding properties and activity as effector can depend on the order of arrangement of lectin domains (from N- to C-termini) and on the linker length. After dissociation of the homodimer, the Gal-7 domain can build new functionally active hybrids with other partners. This study provides a clear direction for research on defining the full range of Gal-7 functionality and offers the perspective of testing applications for engineered heterodimers.

What Cyto- and Histochemistry Can Do to Crack the Sugar Code.

As letters form the vocabulary of a language, biochemical 'symbols' (the building blocks of oligo- and polymers) make writing molecular messages possible. Compared to nucleotides and amino acids, sugars have chemical properties that facilitate to reach an unsurpassed level of oligomer diversity. These glycans are a part of the ubiquitous cellular glycoconjugates. Cyto- and histochemically, the glycans' structural complexity is mapped by glycophenotyping of cells and tissues using receptors ('readers', thus called lectins), hereby revealing its dynamic spatiotemporal regulation: these data support the concept of a sugar code. When proceeding from work with plant (haem)agglutinins as such tools to the discovery of endogenous (tissue) lectins, it became clear that a broad panel of biological meanings can indeed be derived from the sugar-based vocabulary (the natural glycome incl. post-synthetic modifications) by glycan-lectin recognition in situ. As consequence, the immunocyto- and histochemical analysis of lectin expression is building a solid basis for the steps toward tracking down functional correlations, for example in processes leading to cell adhesion, apoptosis, autophagy or growth regulation as well as targeted delivery of glycoproteins. Introduction of labeled tissue lectins to glycan profiling assists this endeavor by detecting counterreceptor(s) in situ. Combining these tools and their applications strategically will help to take the trip toward the following long-range aim: to compile a dictionary for the glycan vocabulary that translates each message (oligosaccharide) into its bioresponse(s), that is to crack the sugar code.

Characterizing ligand-induced conformational changes in clinically relevant galectin-1 by HN/H2O (D2O) exchange.

Glycans of cellular glycoconjugates serve as biochemical signals for a multitude of (patho)physiological processes via binding to their receptors (e.g. lectins). In the case of human adhesion/growth-regulatory galectin-1 (Gal-1), small angle neutron scattering and fluorescence correlation spectroscopy have revealed a significant decrease of its gyration radius and increase of its diffusion coefficient upon binding lactose, posing the pertinent question on the nature and region(s) involved in the underlying structural alterations. Requiring neither a neutron source nor labeling, diffusion measurements by 1H NMR spectroscopy are shown here to be sufficiently sensitive to detect this ligand-induced change. In order to figure out which region(s) of Gal-1 is (are) affected at the level of peptides, we first explored the use of H/D exchange mass spectrometry (HDX MS). Hereby, we found a reduction in proton exchange kinetics beyond the lactose-binding site. The measurement of fast HN/H2O exchange by phase-modulated NMR clean chemical exchange (CLEANEX) NMR on 15N-labeled Gal-1 then increased the spatial resolution to the level of individual amino acids. The mapped regions with increased protection from HN/H2O (D2O) exchange that include the reduction of solvent exposure around the interface can underlie the protein's compaction. These structural changes have potential to modulate this galectin's role in lattice formation on the cell surface and its interaction(s) with protein(s) at the F-face.

Calorimetric Analysis of the Interplay between Synthetic Tn Antigen-Presenting MUC1 Glycopeptides and Human Macrophage Galactose-Type Lectin.

Human macrophage galactose-type lectin (hMGL, HML, CD301, CLEC10A), a C-type lectin expressed by dendritic cells and macrophages, is a receptor for N-acetylgalactosamine α-linked to serine/threonine residues (Tn antigen, CD175) and its α2,6-sialylated derivative (sTn, CD175s). Because these two epitopes are among malignant cell glycan displays, particularly when presented by mucin-1 (MUC1), assessing the influence of the site and frequency of glycosylation on lectin recognition will identify determinants governing this interplay. Thus, chemical synthesis of the tandem-repeat O-glycan acceptor region of MUC1 and site-specific threonine glycosylation in all permutations were carried out. Isothermal titration calorimetry (ITC) analysis of the binding of hMGL to this library of MUC1 glycopeptides revealed an enthalpy-driven process and an affinity enhancement of an order of magnitude with an increasing glycan count from 6-8 µM for monoglycosylated peptides to 0.6 µM for triglycosylated peptide. ITC measurements performed in D2O permitted further exploration of the solvation dynamics during binding. A shift in enthalpy-entropy compensation and contact position-specific effects with the likely involvement of the peptide surroundings were detected. KinITC analysis revealed a prolonged lifetime of the lectin-glycan complex with increasing glycan valency and with a change in the solvent to D2O.

Probing sulfatide-tissue lectin recognition with functionalized glycodendrimersomes.

The small 3-O-sulfated galactose head group of sulfatides, an abundant glycosphingolipid class, poses the (sphinx-like) riddle on involvement of glycan bridging by tissue lectins (sugar code). First, synthesis of head group derivatives for functionalization of amphiphilic dendrimers is performed. Aggregation of resulting (biomimetic) vesicles, alone or in combination with lactose, demonstrates bridging by a tissue lectin (galectin-4). Physiologically, this can stabilize glycolipid-rich microdomains (rafts) and associate sulfatide-rich regions with specific glycoproteins. Further testing documents importance of heterobivalency and linker length. Structurally, sulfatide recognition by galectin-8 is shown to involve sphingosine's OH group as substitute for the 3'-hydroxyl of glucose of lactose. These discoveries underscore functionality of this small determinant on biomembranes intracellularly and on the cell surface. Moreover, they provide a role model to examine counterreceptor capacity of more complex glycans of glycosphingolipids and to start their bottom-up glycotope surface programming.

From examining the relationship between (corona)viral adhesins and galectins to glyco-perspectives.

Glycan-lectin recognition is vital to processes that impact human health, including viral infections. Proceeding from crystallographical evidence of case studies on adeno-, corona-, and rotaviral spike proteins, the relationship of these adhesins to mammalian galectins was examined by computational similarity assessments. Intrafamily diversity among human galectins was in the range of that to these viral surface proteins. Our findings are offered to inspire the consideration of lectin-based approaches to thwart infection by present and future viral threats, also mentioning possible implications for vaccine development.

Glycobiology of developing chicken kidney: Profiling the galectin family and selected ß-galactosides.

The concept of the sugar code interprets the cellular glycophenotype as a rich source of information read by glycan-lectin recognition in situ. This study's aim is the comprehensive characterization of galectin expression by immunohistochemistry during chicken nephrogenesis along with mapping binding sites by (ga)lectin histochemistry. Light and two-color fluorescence microscopy were used. First, six plant/fungal lectins that are specific for galectin-binding parts of N- and O-glycans were applied. The spatiotemporally regulated distributions of these glycans in meso- and metanephros equip cells with potential binding partners for the galectins. Complete galectin profiling from HH Stage 20 (about 70-72 hr) onward revealed cell-, galectin-, and stage-dependent expression patterns. Representatives of all three types of modular architecture of the galectin family are detectable, and overlaps of signal distribution in light and two-color fluorescence microscopy illustrate a possibility for functional cooperation among them. Performing systematic galectin histochemistry facilitated comparisons between staining profiles of plant lectins and galectins. They revealed several cases for differences so that tissue lectins appear to be selective among the ß-galactosides. Notably, selectivity is also disclosed in intrafamily comparison. Thus, combining experimental series with plant and tissue lectins is a means to characterize target populations of glycans presented by cellular glycoconjugates for individual galectins. Our results document the presence and sophisticated level of elaboration among ß-galactosides and among the members of the family of galectins during organogenesis, using chicken galectins and kidney as model. Thus, they provide a clear guideline for functional assays using supramolecular tools, cells, and organ cultures.

Pro4 prolyl peptide bond isomerization in human galectin-7 modulates the monomer-dimer equilibrum to affect function.

Human galectin-7 (Gal-7; also termed p53-induced gene 1 product) is a multifunctional effector by productive pairing with distinct glycoconjugates and protein counter-receptors in the cytoplasm and nucleus, as well as on the cell surface. Its structural analysis by NMR spectroscopy detected doubling of a set of particular resonances, an indicator of Gal-7 existing in two conformational states in slow exchange on the chemical shift time scale. Structural positioning of this set of amino acids around the P4 residue and loss of this phenomenon in the bioactive P4L mutant indicated cis-trans isomerization at this site. Respective resonance assignments confirmed our proposal of two Gal-7 conformers. Mapping hydrogen bonds and considering van der Waals interactions in molecular dynamics simulations revealed a structural difference for the N-terminal peptide, with the trans-state being more exposed to solvent and more mobile than the cis-state. Affinity for lactose or glycan-inhibitable neuroblastoma cell surface contact formation was not affected, because both conformers associated with an overall increase in order parameters (S2). At low µM concentrations, homodimer dissociation is more favored for the cis-state of the protein than its trans-state. These findings give direction to mapping binding sites for protein counter-receptors of Gal-7, such as Bcl-2, JNK1, p53 or Smad3, and to run functional assays at low concentration to test the hypothesis that this isomerization process provides a (patho)physiologically important molecular switch for Gal-7.

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

In the original publication of the article, the "Result" section has been published.