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.

Comparative lectinology: Delineating glycan-specificity profiles of the chicken galectins using neoglycoconjugates in a cell assay.

A major aspect of carbohydrate-dependent galectin functionality is their cross-linking capacity. Using a cell surface as biorelevant platform for galectin binding and a panel of 40 glycans as sensor part of a fluorescent polyacrylamide neoglycopolymer for profiling galectin reactivity, properties of related proteins can be comparatively analyzed. The group of the chicken galectins (CGs) is an especially suited system toward this end due to its relatively small size, compared with mammalian galectins. The experiments reveal particularly strong reactivity toward N-acetyllactosamine repeats for all tested CGs and shared reactivity of CG-1A and CG-2 to histo-blood group ABH determinants. In cross-species comparison, CG-1B's properties closely resembled those of human galectin-1, as was the case for the galectin-2 (but not galectin-3) ortholog pair. Although binding-site architectures are rather similar, reactivity patterns can well differ.

Mimicking biological membranes with programmable glycan ligands self-assembled from amphiphilic Janus glycodendrimers.

An accelerated modular synthesis produced 18 amphiphilic Janus glycodendrimers with three different topologies formed from either two or one carbohydrate head groups or a mixed constellation with a noncarbohydrate hydrophilic arm. By simple injection of their THF solutions into water or buffer, all of the Janus compounds self-assembled into uniform, stable, and soft unilamellar vesicles, denoted glycodendrimersomes. The mixed constellation topology glycodendrimersomes were demonstrated to be most efficient in binding plant, bacterial, and human lectins. This evidence with biomedically relevant receptors offers a promising perspective for the application of such glycodendrimersomes in targeted drug delivery, vaccines, and other areas of nanomedicine.

Modular synthesis of amphiphilic Janus glycodendrimers and their self-assembly into glycodendrimersomes and other complex architectures with bioactivity to biomedically relevant lectins.

The modular synthesis of 7 libraries containing 51 self-assembling amphiphilic Janus dendrimers with the monosaccharides D-mannose and D-galactose and the disaccharide D-lactose in their hydrophilic part is reported. These unprecedented sugar-containing dendrimers are named amphiphilic Janus glycodendrimers. Their self-assembly by simple injection of THF or ethanol solution into water or buffer and by hydration was analyzed by a combination of methods including dynamic light scattering, confocal microscopy, cryogenic transmission electron microscopy, Fourier transform analysis, and micropipet-aspiration experiments to assess mechanical properties. These libraries revealed a diversity of hard and soft assemblies, including unilamellar spherical, polygonal, and tubular vesicles denoted glycodendrimersomes, aggregates of Janus glycodendrimers and rodlike micelles named glycodendrimer aggregates and glycodendrimermicelles, cubosomes denoted glycodendrimercubosomes, and solid lamellae. These assemblies are stable over time in water and in buffer, exhibit narrow molecular-weight distribution, and display dimensions that are programmable by the concentration of the solution from which they are injected. This study elaborated the molecular principles leading to single-type soft glycodendrimersomes assembled from amphiphilic Janus glycodendrimers. The multivalency of glycodendrimersomes with different sizes and their ligand bioactivity were demonstrated by selective agglutination with a diversity of sugar-binding protein receptors such as the plant lectins concanavalin A and the highly toxic mistletoe Viscum album L. agglutinin, the bacterial lectin PA-IL from Pseudomonas aeruginosa, and, of special biomedical relevance, human adhesion/growth-regulatory galectin-3 and galectin-4. These results demonstrated the candidacy of glycodendrimersomes as new mimics of biological membranes with programmable glycan ligand presentations, as supramolecular lectin blockers, vaccines, and targeted delivery devices.

Down-regulation of the epidermal growth factor receptor by altering N-glycosylation: emerging role of ß1,4-galactosyltransferases.

BACKGROUND/AIM: Blocking N-glycosylation of the epidermal growth factor receptor (EGFR) by tunicamycin inhibits its cellular accumulation. Due to the toxic potential of this drug, finding less drastic routes to reduce EGFR expression is desirable. MATERIALS AND METHODS: Four glycosylation mutants of Chinese hamster ovary (CHO) cells with defects in N-glycan processing and branch-end maturation were tested for EGFR gene expression, production, functionality and routing after transfection with a vector encoding for human EGFR. RESULTS: Lack of conversion of paucimannosidic to hybrid/complex-type N-glycans and drastic reductions in sialylation/galactosylation did not lead to major effects. In contrast, EGFR expression in a mutant with reduced presence of ß1,4-galactosyltransferases-I-VI was markedly reduced. Misrouting or defects in transfection/transcription were excluded. CONCLUSION: ß1,4-Galactosyl-transferases warrant for further attention as effector(s) in order to attenuate EGFR-dependent signaling.

Comparative study of the glycan specificities of cell-bound human tandem-repeat-type galectin-4, -8 and -9.

Adhesion/growth-regulatory galectins (gals) exert their functionality by the cis/trans-cross-linking of distinct glycans after initial one-point binding. In order to define the specificity of ensuing association events leading to cross-linking, we recently established a cell-based assay using fluorescent glycoconjugates as flow cytometry probes and tested it on two human gals (gal-1 and -3). Here we present a systematic study of tandem-repeat-type gal-4, -8 and -9 loaded on Raji cells resulting in the following key insights: (i) all three gals bound to oligolactosamines; (ii) binding to ligands with Galß1-3GlcNAc or Galß1-3GalNAc as basic motifs was commonly better than that to canonical Galß1-4GlcNAc; (iii) all three gals bound to 3'-O-sulfated and 3'-sialylated disaccharides mentioned above better than that to parental neutral forms and (iv) histo-blood group ABH antigens were the highest affinity ligands in both the cell and the solid-phase assay. Fine specificity differences were revealed as follows: (i) gal-8 and -9, but not gal-4, bound to disaccharide Galß1-3GlcNAc; (ii) increase in binding due to negatively charged substituents was marked only in the case of gal-4 and (iii) gal-4 and -8 bound preferably to histo-blood group A glycans, whereas gal-9 targeted B-type glycans. Experiments with single carbohydrate recognition domains (CRDs) of gal-4 showed that the C-CRD preferably bound to ABH glycans, whereas the N-CRD associated with oligolactosamines. In summary, the comparative analysis disclosed the characteristic profiles of glycan reactivity for the accessible CRD of cell-bound gals. These results indicate the distinct sets of functionality for these three members of the same subgroup of human gals.

Galectin-4, a novel neuronal regulator of myelination.

Myelination of axons by oligodendrocytes (OLGs) is essential for proper saltatory nerve conduction, i.e., rapid transmission of nerve impulses. Among others, extracellular matrix (ECM) molecules, neuronal signaling, and axonal adhesion regulate the biogenesis and maintenance of myelin membranes, driven by polarized transport of myelin-specific proteins and lipids. Galectin-4, a tandem-repeat-type lectin with affinity to sulfatide and nonsialylated termini of N-glycans, has the ability to regulate adhesion of cells to ECM components and is also involved in polarized membrane trafficking. We, therefore, anticipated that galectin-4 might play a role in myelination. Here, we show that in developing postnatal rat brains galectin-4 expression is downregulated just before the onset of myelination. Intriguingly, when immature OLGs were treated with galectin-4, OLG maturation was retarded, while a subset of the immature OLGs reverted to a morphologically less complex progenitor stage, displaying concomitantly an increase in proliferation. Similarly, myelination was inhibited when galectin-4 or anti-galectin-4 antibodies were added to co-cultures of dorsal root ganglion neurons and OLGs. Neurons and OLGs were identified as a possible source of galectin-4, both in vitro and in vivo. In culture, neurons but not OLGs released galectin-4. Interestingly, in co-cultures, a reduced release of endogenous galectin-4 correlated with the onset of myelination. Moreover, galectin-4-reactive sites are transiently expressed on processes of premyelinating primary OLGs, but not on neurons. Taken together, these results identify neuronal galectin-4 as a candidate for a soluble regulator of OLG differentiation and, hence, myelination. © 2012 Wiley Periodicals, Inc.

Nitric oxide changes distinct aspects of the glycophenotype of human neuroblastoma NB69 cells.

It is an open question whether the presence of nitric oxide (NO) affects the cell glycophenotype. A panel of six plant lectins was used in this study to monitor distinct aspects of cell surface glycosylation under nitrosative stress. We determined that treating human neuroblastoma NB69 cells with the long-lived NO donor 2,2'-(hydroxynitrosohydrazono)bis-ethanimine (DETA/NO) and monitoring the non-apoptotic adherent cell population significantly increases the presentation of N-glycans as detected by concanavalin A. Examining fine-structural features, bisected N-glycans and branch-end tailoring including α2,6-sialylation were found to be enhanced. Confocal fluorescence microscopy and cell permeabilization experiments pointed to a major effect of NO on the extent of cell surface N-glycan presentation. We also show that NO increases the level of protein O-GlcNAcylation, a multifunctional post-translational modification. Our results thus establish the first evidence for NO as modulator of distinct aspects of cell glycosylation.

Multivalent binding of carbohydrates by the human alpha-defensin, HD5.

Four of the six human alpha-defensins (human neutrophil peptides 1-3 and human alpha-defensin 5; HD5) have a lectin-like ability to bind glycosylated proteins. Using HD5 as a model, we applied surface plasmon resonance techniques to gain insights into this property. HD5 bound natural glycoproteins > neoglycoproteins based on BSA > nonglycosylated BSA >> free sugars. The affinity of HD5 for simple sugars covalently bound to BSA was orders of magnitude greater than its affinity for the same sugars in solution. The affinity of HD5 for protein-bound carbohydrates resulted from multivalent interactions which may also involve noncarbohydrate residues of the proteins. HD5 showed concentration-dependent self-association that began at submicromolar concentrations and proceeded to dimer and tetramer formation at concentrations below 5 microM. The (R9A, R28A) and (R13A, R32A) analogs of HD5 showed greatly reduced self-association as well as minimal binding to BSA and to BSA-affixed sugars. From this and other evidence, we conclude that the extensive binding of HD5 to (neo)glycoproteins results from multivalent nonspecific interactions of individual HD5 molecules with carbohydrate and noncarbohydrate moieties of the target molecule and that the primary binding events are magnified and enhanced by subsequent in situ assembly and oligomerization of HD5. Self-association and multivalent binding may play integral roles in the ability of HD5 to protect against infections caused by viruses and other infectious agents.

N-acetyl-seryl-aspartyl-lysyl-proline prevents cardiac remodeling and dysfunction induced by galectin-3, a mammalian adhesion/growth-regulatory lectin.

Galectin-3 (Gal-3) is secreted by activated macrophages. In hypertension, Gal-3 is a marker for hypertrophic hearts prone to develop heart failure. Gal-3 infused in pericardial sac leads to cardiac inflammation, remodeling, and dysfunction. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), a naturally occurring tetrapeptide, prevents and reverses inflammation and collagen deposition in the heart in hypertension and heart failure postmyocardial infarction. In the present study, we hypothesize that Ac-SDKP prevents Gal-3-induced cardiac inflammation, remodeling, and dysfunction, and these effects are mediated by the transforming growth factor (TGF)-beta/Smad3 signaling pathway. Adult male rats were divided into four groups and received the following intrapericardial infusion for 4 wk: 1) vehicle (saline, n = 8); 2) Ac-SDKP (800 microg x kg(-1) x day(-1), n = 8); 3) Gal-3 (12 microg/day, n = 7); and 4) Ac-SDKP + Gal-3 (n = 7). Left ventricular ejection fraction, cardiac output, and transmitral velocity were measured by echocardiography; inflammatory cell infiltration, cardiomyocyte hypertrophy, and collagen deposition in the heart by histological and immunohistochemical staining; and TGF-beta expression and Smad3 phosphorylation by Western blot. We found that, in the left ventricle, Gal-3 1) enhanced macrophage and mast cell infiltration, increased cardiac interstitial and perivascular fibrosis, and causes cardiac hypertrophy; 2) increased TGF-beta expression and Smad3 phosphorylation; and 3) decreased negative change in pressure over time response to isoproterenol challenge, ratio of early left ventricular filling phase to atrial contraction phase, and left ventricular ejection fraction. Ac-SDKP partially or completely prevented these effects. We conclude that Ac-SDKP prevents Gal-3-induced cardiac inflammation, fibrosis, hypertrophy, and dysfunction, possibly via inhibition of the TGF-beta/Smad3 signaling pathway.

Versatile strategy for the synthesis of biotin-labelled glycans, their immobilization to establish a bioactive surface and interaction studies with a lectin on a biochip.

The emerging role of glycans as versatile biochemical signals in diverse aspects of cellular sociology calls for establishment of sensitive methods to monitor carbohydrate recognition by receptors such as lectins. Most of these techniques involve the immobilization of one of the binding partners on a surface, e.g. atomic force microscopy, glycan array and Surface Plasmon Resonance (SPR), hereby simulating cell surface presentation. Here, we report the synthesis of fluorescent glycoconjugates, with a functionalization strategy which avoids the frequently occurring ring opening at the reducing end for further immobilization on a surface or derivatization with biotin. In order to improve the versatility of these derivatized glycans for biological studies, a new approach for the synthesis of biotinylated and fluorescent glycans has also been realized. Finally, to illustrate their usefulness the neoglycoconjugates were immobilized on different surfaces, and the interaction analysis with a model lectin, the toxin from mistletoe, proved them to act as potent ligands, underscoring the merit of the presented synthetic approach.

Galectin-loaded cells as a platform for the profiling of lectin specificity by fluorescent neoglycoconjugates: a case study on galectins-1 and -3 and the impact of assay setting.

The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end, it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells. The extent of surface binding of fluorescent neoglycoconjugates depended on the lectin presence and the type of lectin, the nature of the probes' carbohydrate headgroup and the density of unsubstituted beta-galactosides on the cell surface. Using the most frequently studied galectins-1 and -3, application of this assay led to rather equal binding levels for linear and branched oligomers of N-acetyllactosamine. A clear preference of galectin-3 for alpha1-3-linked galactosylated lactosamine was noted. In parallel, a panel of 24 neoglycoconjugates was tested as inhibitors of galectin binding from solution to N-glycans of surface-immobilized asialofetuin. These two assays differ in presentation of the galectin and ligand, facilitating identification of assay-dependent properties. Under the condition of the cell assay, selectivity among oligosaccharides for the lectins was higher, and extraordinary affinity of galectin-1 to 3'-O-sulfated probes in a solid-phase assay was lost in the cell assay. Having introduced and validated a cell assay, the comprehensive profiling of ligand binding to cell-surface-presented galectins is made possible.

AB-type lectin (toxin/agglutinin) from mistletoe: differences in affinity of the two galactoside-binding Trp/Tyr-sites and regulation of their functionality by monomer/dimer equilibrium.

Viscumin of mistletoe (Viscum album L.) has a concentration-dependent activity profile unique to plant AB-toxins. It starts with lectin-dependent mitogenicity and then covers toxicity and cell agglutination, associated with shifts in the monomer/dimer equilibrium. Each lectin subunit harbors two sections for ligand contact. In the dimer, the B-chain sites in subdomain 2 gamma (designated as the Tyr-sites) appear fully accessible, whereas Trp-sites in subdomain 1 alpha are close to the dimer interface. It is unclear whether both types of sites operate similarly in binding glycoligands in solution. By systematically covering a broad range of lactose/lectin ratio in isothermal titration calorimetry, we obtained evidence for two sites showing dissimilar binding affinity. Intriguingly, the site with higher affinity was only partially occupied. To assign the observed properties to the Trp/Tyr-sites, we next performed chemically induced dynamic nuclear polarization measurements of Trp and Tyr accessibility. A Tyr signal, but not distinct Trp peaks, was recorded when testing the dimer. Lactose-quenchable Trp peaks became visible on the destabilization of the dimer by citraconylation, intimating Trp involvement in ligand contact in the monomer. Fittingly, Tyr acetylation but not mild Trp oxidation reduced the dimer hemagglutination activity and the extent of binding to asialofetuin-Sepharose 4B. Altogether, the results attribute lectin activity in the dimer primarily to Tyr-sites. Full access to Trp-sites is gained on dimer dissociation. Thus, the monomer/dimer equilibrium of viscumin regulates the operativity of these sites. Their structural divergence affords the possibility for differences in ligand selection when comparing monomers (Tyr- and Trp-sites) with dimers (primarily Tyr-sites).

A proteomic approach to the identification of new tPA receptors in pancreatic cancer cells.

We have developed a strategy to identify putative tissue-type plasminogen activator (tPA)receptors present in pancreatic cancer cells by affinity capture with tPA-Sepharose followed by 2-DE and MALDI-MS PMF. Proteins pulled down from either total lysates or raft membrane fractions were characterized and compared with those from a total lysate of an endothelial cell line (HUVEC) to identify pancreas-restricted tPA receptors. A total of 31 proteins were found by this approach, including annexin A2, already described as a tPA receptor in pancreas and endothelial cells, other proteins acting as tPA receptors (i.e., enolase, cytokeratins 8 and 18) in other tissues, and additional proteins not previously identified as candidate tPA receptors. Confirmation of the results was performed for some of these proteins using immunoblotting. These studies are the basis for further functional analyses on the role of these proteins in the biological effects of tPA.

Carbohydrate-induced modulation of cell membrane. VIII. Agglutination with mammalian lectin galectin-1 increases osmofragility and membrane fluidity of trypsinized erythrocytes.

Interaction of lectins with cell surface determinants may alter membrane properties. Using trypsinized rabbit erythrocytes as model we tested the capacity of an endogenous lectin in this respect. Galectin-1 is a member of an adhesion/growth-regulatory family known to interact for example with ganglioside GM(1) and also the hydrophobic tail of oncogenic H-Ras. Assays on membrane fluidity and osmofragility detect galectin-1's capacity to increase the parameters. Moreover, it increases susceptibility of erythrocytes to radical damage. These observations indicate the potential of this endogenous lectin to affect membrane properties beyond the immediate interaction with cell surface epitopes.

Galectins bind to the multivalent glycoprotein asialofetuin with enhanced affinities and a gradient of decreasing binding constants.

Our previous isothermal titration microcalorimetry (ITC) studies of the binding of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) showed negative binding cooperativity that was due to the carbohydrate ligands and not the proteins [Dam, T. K., et al. (2002) Biochemistry 41, 1351-1358]. The negative cooperativity was associated with the decreasing functional valence of the carbohydrates upon progressive binding of their epitopes. The present study also shows negative cooperativity in the ITC binding data of asialofetuin (ASF), a glycoprotein that possesses nine LacNAc epitopes, to galectin-1, -2, -3, -4, -5, and -7, and truncated, monomer versions of galectin-3 and -5, which are members of a family of animal lectins. Although the observed K(a) values for binding of ASF to the galectins and two truncated forms are only 50-80-fold greater than that of LacNAc, analysis of the data in terms of the relationship between the observed macroscopic free energy of binding and the decreasing microscopic free energies of binding of the epitopes shows that the first LacNAc epitope of ASF binds with approximately 6000-fold higher affinity than the last epitope. Thus, the microscopic binding constants of the galectins for the first epitope(s) of ASF are in the nanomolar range, with a gradient of decreasing binding constants of the remaining epitopes. The results indicate that the above galectins bind with fractional, high affinities to multivalent glycoproteins such as ASF, independent of the quaternary structures of the galectins. These findings have important implications for the binding of galectins to multivalent carbohydrate receptors.

Monomer/dimer equilibrium of the AB-type lectin from mistletoe enables combination of toxin/agglutinin activities in one protein: analysis of native and citraconylated proteins by ultracentrifugation/gel filtration and cell biological consequences of dimer destabilization.

The biological activity of a lectin is influenced by its quaternary structure. Viscumin is special among the family members of toxic AB-type plant lectins, because it triggers mitogenicity, toxicity, and agglutination. Its activity profile is dependent on the concentration, motivating a thorough inspection of the status of quaternary structure. Over a broad range of protein concentrations (0.01-25 mg/mL), viscumin occurs as a dimer. At high concentrations, the solutions exhibited nonideality, self-association, and polydispersity in sedimentation equilibrium and velocity experiments caused by irreversible aggregation. Calculation of viscumin's overall shape based on sedimentation velocity data resulted in an elongated dimer form resembling that of crystallized agglutinin. Appearance of monomers was restricted to concentrations in the submicrogram/mL level, as demonstrated by fast protein liquid chromatography gel-filtration analysis. To shift the equilibrium to the monomer for comparative cell biological assays, we performed chemical modification under conditions protecting the lectin activity. Citraconylation was effective to destabilize the dimer. Binding studies by fluorescence-activated cell scan analysis revealed a reduction in cell association upon modification and a tendency for increased sensitivity towards haptenic inhibitors at microg/mL concentrations. Nonetheless, growth inhibition continued to be potent for the ricin-like monomer despite reduced extent of binding. Occurrence of a concentration-dependent monomer/dimer equilibrium appears to achieve the same objectives as the development of two separate protein entities in Ricinus communis, an alternative strategy to emergence of a monomeric toxin, and cell cross-linking dimeric agglutinin.

Galectin-3 marks activated macrophages in failure-prone hypertrophied hearts and contributes to cardiac dysfunction.

BACKGROUND: Inflammatory mechanisms have been proposed to be important in heart failure (HF), and cytokines have been implicated to add to the progression of HF. However, it is unclear whether such mechanisms are already activated when hypertrophied hearts still appear well-compensated and whether such early mechanisms contribute to the development of HF. METHODS AND RESULTS: In a comprehensive microarray study, galectin-3 emerged as the most robustly overexpressed gene in failing versus functionally compensated hearts from homozygous transgenic TGRmRen2-27 (Ren-2) rats. Myocardial biopsies obtained at an early stage of hypertrophy before apparent HF showed that expression of galectin-3 was increased specifically in the rats that later rapidly developed HF. Galectin-3 colocalized with activated myocardial macrophages. We found galectin-3-binding sites in rat cardiac fibroblasts and the extracellular matrix. Recombinant galectin-3 induced cardiac fibroblast proliferation, collagen production, and cyclin D1 expression. A 4-week continuous infusion of low-dose galectin-3 into the pericardial sac of healthy Sprague-Dawley rats led to left ventricular dysfunction, with a 3-fold differential increase of collagen I over collagen III. Myocardial galectin-3 expression was increased in aortic stenosis patients with depressed ejection fraction. CONCLUSIONS: This study shows that an early increase in galectin-3 expression identifies failure-prone hypertrophied hearts. Galectin-3, a macrophage-derived mediator, induces cardiac fibroblast proliferation, collagen deposition, and ventricular dysfunction. This implies that HF therapy aimed at inflammatory responses may need to be targeted at the early stages of HF and probably needs to antagonize multiple inflammatory mediators, including galectin-3.

Thermodynamic binding studies of bivalent oligosaccharides to galectin-1, galectin-3, and the carbohydrate recognition domain of galectin-3.

Galectins are a growing family of animal lectins with common consensus sequences that bind beta-Gal and LacNAc residues. There are at present 14 members of the galectin family; however, certain galectins possess different structures as well as biological properties. Galectin-1 is a dimer of two homologous carbohydrate recognition domains (CRDs) and possesses apoptotic and proinvasive activities. Galectin-3 consists of a C-terminal CRD and an N-terminal nonlectin domain implicated in the oligomerization of the protein and is often associated with antiapoptotic activity. Because many cellular oligosaccharide receptors are multivalent, it is important to characterize the interactions of multivalent carbohydrates with galectins-1 and -3. In the present study, binding of bovine heart galectin-1 and recombinant murine galectin-3 to a series of synthetic analogs containing two LacNAc residues separated by a varying number of methylene groups, as well as biantennary analogs possessing two LacNAc residues, were examined using isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements. The thermodynamics of binding of the multivalent carbohydrates to the C-terminal CRD domain of galectin-3 was also investigated. ITC results showed that each bivalent analog bound by both LacNAc residues to the two galectins. However, galectin-1 shows a lack of enhanced affinity for the bivalent straight chain and branched chain analogs, whereas galectin-3 shows enhanced affinity for only lacto-N-hexaose, a naturally occurring branched chain carbohydrate. The CRD domain of galectin-3 was shown to possess similar thermodynamic binding properties as the intact molecule. The results of this study have important implications for the design of carbohydrate inhibitors of the two galectins.

Quaternary solution structures of galectins-1, -3, and -7.

Galectins are a growing family of animal lectins with functions in growth regulation and cell adhesion that bind beta-Gal residues in oligosaccharides. Evidence indicates that some of the biological properties of galectins are due to their cross-linking activities with multivalent glycoconjugate receptors. Therefore determination of the quaternary solution structures of these proteins is important in understanding their structure-function properties. The present study reports analytical sedimentation velocity and equilibrium data for galectins-1, -3, and -7 in the absence and presence of bound LacNAc, the natural ligand epitope. Galectin-1 from bovine heart and recombinant human galectin-7 were found to be stable dimers by both methods. In contrast, recombinant murine galectin-3, as well as its proteolytical derived C-terminal domain, are predominantly monomeric. The presence of LacNAc at concentrations sufficient to fully saturate the proteins had no significant effect on either the weight average molecular weight determined by sedimentation equilibrium or the hydrodynamic properties determined from sedimentation velocity experiments. These results show that binding of a monovalent ligand does not affect oligomerization of these galectins.