Method for Preparing Recombinant Galectin-2 Protein without Escherichia coli-Specific Post-translational Modifications.

Galectin-2 (Gal-2) is an animal lectin with specificity for ß-galactosides. It is predominantly expressed and suggested to play a protective function in the gastrointestinal tract; therefore, it can be used as a protein drug. Recombinant proteins have been expressed using Escherichia coli and used to study the function of Gal-2. The recombinant human Gal-2 (hGal-2) protein purified via affinity chromatography after being expressed in E. coli was not completely homogeneous. Mass spectrometry confirmed that some recombinant Gal-2 were phosphogluconoylated. In contrast, the recombinant mouse Gal-2 (mGal-2) protein purified using affinity chromatography after being expressed in E. coli contained a different form of Gal-2 with a larger molecular weight. This was due to mistranslating the original mGal-2 stop codon TGA to tryptophan (TGG). In this report, to obtain a homogeneous Gal-2 protein for further studies, we attempted the following methods: for hGal-2, 1) replacement of the lysine (Lys) residues, which was easily phosphogluconoylated with arginine (Arg) residues, and 2) addition of histidine (His)-tag on the N-terminus of the recombinant protein and cleavage with protease after expression; for mGal-2, 3) changing the stop codon from TGA to TAA, which is commonly used in E. coli. We obtained an almost homogeneous recombinant Gal-2 protein (human and mouse). These results have important implications for using Gal-2 as a protein drug.

Expression, S-Nitrosylation, and Measurement of S-Nitrosylation Ratio of Recombinant Galectin-2.

S-nitrosylation, which involves the coupling of an NO group to the reactive thiol of Cys residue(s) in a polypeptide, is an important posttranslational modification detected in a variety of proteins. Here, we present the S-nitrosylation of recombinant galectin-2 (Gal-2) using S-nitrosocysteine and the measurement of the molecular ratio of S-nitrosylation of Cys residues in the Gal-2 protein.

Potential Interaction between Galectin-2 and MUC5AC in Mouse Gastric Mucus.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. Of these, galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract. In the current study, we used a mouse gastric mucous fraction to investigate whether Gal-2 is secreted from epithelial cells and identify its potential ligands in gastric mucus. Gal-2 was detected in the mouse gastric mucous fraction and could be eluted from it by the addition of lactose. Affinity chromatography using recombinant mouse galectin-2 (mGal-2)-immobilized adsorbent and subsequent LC-MS/MS identified MUC5AC, one of the major gastric mucin glycoproteins, as a potential ligand of mGal-2. Furthermore, MUC5AC was detected in the mouse gastric mucous fraction by Western blotting, and recombinant mGal-2 was adsorbed to this fraction in a carbohydrate-dependent manner. These results suggested that Gal-2 and MUC5AC in mouse gastric mucus interact in a ß-galactoside-dependent manner, resulting in a stronger barrier structure protecting the mucosal surface.

Galectin-2 suppresses nematode development by binding to the invertebrate-specific galactoseß1-4fucose glyco-epitope.

Galactoseß1-4Fucose (GalFuc) is a unique disaccharide found in invertebrates including nematodes. A fungal galectin CGL2 suppresses nematode development by recognizing the galactoseß1-4fucose epitope. The Caenorhabditis elegans galectin LEC-6 recognizes it as an endogenous ligand and the Glu67 residue of LEC-6 is responsible for this interaction. We found that mammalian galectin-2 (Gal-2) also has a comparable glutamate residue, Glu52. In the present study, we investigated the potential nematode-suppressing activity of Gal-2 using C. elegans as a model and focusing on Gal-2 binding to the GalFuc epitope. Gal-2 suppressed C. elegans development whereas its E52D mutant (Glu52 substituted by Asp), galectin-1 and galectin-3 had little effect on C. elegans growth. Lectin-staining using fluorescently-labeled Gal-2 revealed that, like CGL2, it specifically binds to the C. elegans intestine. Natural C. elegans glycoconjugates were specifically bound by immobilized Gal-2. Western blotting with anti-GalFuc antibody showed that the bound glycoconjugates had the GalFuc epitope. Frontal affinity chromatography with pyridylamine-labeled C. elegans N-glycans disclosed that Gal-2 (but not its E52D mutant) recognizes the GalFuc epitope. Gal-2 also binds to the GalFuc-bearing glycoconjugates of Ascaris and the GalFuc epitope is present in the parasitic nematodes Nippostrongylus brasiliensis and Brugia pahangi. These results indicate that Gal-2 suppresses C. elegans development by binding to its GalFuc epitope. The findings also imply that Gal-2 may prevent infestations of various parasitic nematodes bearing the GalFuc epitope.

Characterisation and functional comparison of single-CRD and multidomain containing galectins CgGal-2 and CgGal-3 from oyster Crassostrea gigas.

Galectins are ß-galactoside binding lectins that play crucial roles in innate immunity in vertebrates and invertebrates through their conserved carbohydrate-recognition domains (CRDs). In the present study, single- and four-CRD-containing galectins were identified in oyster Crassostrea gigas (designated CgGal-2 and CgGal-3). The open reading frames (ORFs) of CgGal-2 and CgGal-3 encode polypeptides of 200 and 555 amino acids, respectively. All CRDs of CgGal-3 include two consensus motifs essential for ligand-binding, and a novel motif is present in CgGal-2. Pathogen-associated molecular pattern (PAMP) profiles were determined for recombinant rCgGal-2 and rCgGal-3, and rCgGal-2 displayed low binding affinity for PAMPs, while rCgGal-3 bound various PAMPs including glucan, lipopolysaccharide (LPS), and peptidoglycan (PGN) with relatively high affinity. Furthermore, rCgGal-2 and rCgGal-3 exhibited different microbe binding profiles; rCgGal-2 bound to Gram-negative bacteria (Escherichia coli and Vibrio vulnificus) and fungi (Saccharomyces cerevisiae and Pichia pastoris), while rCgGal-3 bound to these microbes but also to Gram-positive bacteria (Micrococcus luteus). In addition, rCgGal-3 possessed microbial agglutinating activity and coagulation activity against fungi and erythrocytes, respectively, but rCgGal-2 lacked any agglutinating activity. Carbohydrate binding specificity analysis showed that rCgGal-3 specifically bound D-galactose. Furthermore, rCgGal-2 and rCgGal-3 functioned as opsonin participating in the clearance against invaders in C. gigas. Thus, CgGal-2 with one CRD and CgGal-3 with four CRDs are new members of the galectin family involved in immune responses against bacterial infection. Differences in the organisation and amino acid sequences of CRDs may affect their specificity and affinity for nonself substances.

Structural mechanisms for the S-nitrosylation-derived protection of mouse galectin-2 from oxidation-induced inactivation revealed by NMR.

Galectin-2 (Gal-2) is a lectin thought to play protective roles in the gastrointestinal tract. Oxidation of mouse Gal-2 (mGal-2) by hydrogen peroxide (H2 O2 ) results in the loss of sugar-binding activity, whereas S-nitrosylation of mGal-2, which does not change its sugar-binding profile, has been shown to protect the protein from H2 O2 -induced inactivation. One of the two cysteine residues, C57, has been identified as being responsible for controlling H2 O2 -induced inactivation; however, the underlying molecular mechanism has not been elucidated. We performed structural analyses of mGal-2 using nuclear magnetic resonance (NMR) and found that residues near C57 experienced significant chemical shift changes following S-nitrosylation, and that S-nitrosylation slowed the H2 O2 -induced aggregation of mGal-2. We also revealed that S-nitrosylation improves the thermal stability of mGal-2 and that the solvent accessibility and/or local dynamics of residues near C57 and the local dynamics of the core-forming residues in mGal-2 are reduced by S-nitrosylation. Structural models of Gal-2 indicated that C57 is located in a hydrophobic pocket that can be plugged by S-nitrosylation, which was supported by the NMR experiments. Based on these results, we propose two structural mechanisms by which S-nitrosylation protects mGal-2 from H2 O2 -induced aggregation without changing its sugar-binding profile: (a) stabilization of the hydrophobic pocket around C57 that prevents oxidation-induced destabilization of the pocket, and (b) prevention of oxidation of C57 during the transiently unfolded state of the protein, in which the residue is exposed to H2 O2 . DATABASE: Nuclear magnetic resonance assignments for non-S-nitrosylated mGal-2 and S-nitrosylated mGal-2 have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu/) under ID code 27237 for non-S-nitrosylated mGal-2 and ID code 27238 for S-nitrosylated mGal-2.

Identification of Galectin-2-Mucin Interaction and Possible Formation of a High Molecular Weight Lattice.

Galectins comprise a group of animal lectins characterized by their specificity for ß-galactosides. Galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract and has been identified as one of the main gastric mucosal proteins that are proposed to have a protective role in the stomach. As Gal-2 is known to form homodimers in solution, this may result in crosslinking of macromolecules with the sugar structures recognized by Gal-2. In this study, we report that Gal-2 could interact with mucin, an important component of gastric mucosa, in a ß-galactoside-dependent manner. Furthermore, Gal-2 and mucin could form an insoluble precipitate, potentially through the crosslinking of mucins via Gal-2 and the formation of a lattice, resulting in a large insoluble complex. Therefore, we suggest that Gal-2 plays a role in the gastric mucosa by strengthening the barrier structure through crosslinking the mucins on the mucosal surface.

Human galectin-2 interacts with carbohydrates and peptides non-classically: new insight from X-ray crystallography and hemagglutination.

Galectin-2 (Gal-2) plays a role in cancer, myocardial infarction, immune response, and gastrointestinal tract diseases. The only reported crystal structure of Gal-2 shows that it is a dimer in which the monomer subunits have almost identical structures, each binding with one molecule of lactose. In this study, we crystallized Gal-2 under new conditions that produced three crystal structures. In each Gal-2 dimer structure, lactose was shown to be bound to only one of the carbohydrate recognition domain subunits. In solution studies, the thermal shift assay demonstrated that inequivalent monomer subunits in the Gal-2 dimer become equivalent upon ligand binding. In addition, galectin-mediated erythrocyte agglutination assays using lactose and larger complex polysaccharides as inhibitors showed the structural differences between Gal-1 and Gal-2. Overall, our results reveal some novel aspects to the structural differentiation in Gal-2 and expand the potential for different types of molecular interactions that may be specific to this lectin.

Identification of the cysteine residue responsible for oxidative inactivation of mouse galectin-2.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. Mouse galectin-2 (mGal-2) is predominantly expressed in the gastrointestinal tract and has been identified as one of the main gastric mucosal proteins that are uniquely sensitive to S-nitrosylation. We have previously reported that oxidation of mGal-2 by hydrogen peroxide (H2O2) resulted in the loss of sugar-binding ability, whereas pre-treatment of mGal-2 with S-nitrosocysteine prevented H2O2-induced inactivation. In this study, we used point-mutated recombinant mGal-2 proteins to study which of the two highly conserved Cys residues in mGal-2 must be S-nitrosylated for protection against oxidative inactivation. Mutation of Cys57 to a Met residue (C57M) did not result in lectin inactivation following H2O2 treatment, whereas Cys75 mutation to Ser (C75S) led to significantly reduced lectin activity, as is the case for wild-type mGal-2. However, pre-treatment of the C75S mutant with S-nitrosocysteine protected the protein from H2O2-induced inactivation. Therefore, Cys57 is suggested to be responsible for oxidative inactivation of the mGal-2 protein, and protection of the sulfhydryl group of the Cys57 in mGal-2 by S-nitrosylation is likely important for maintaining mGal-2 protein function in an oxidative environment such as the gastrointestinal tract.

S-nitrosylation of mouse galectin-2 prevents oxidative inactivation by hydrogen peroxide.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. Galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract. A proteomic analysis identified Gal-2 as a protein that was S-nitrosylated when mouse gastric mucosal lysates were reacted with S-nitrosoglutathione, a physiologically relevant S-nitrosylating agent. In the present study, recombinant mouse (m)Gal-2 was S-nitrosylated using nitrosocysteine (CysNO), which had no effect on the sugar-binding specificity and dimerization capacity of the protein. On the other hand, mGal-2 oxidation by H2O2 resulted in the loss of sugar-binding ability, while S-nitrosylation prevented H2O2-inducted inactivation, presumably by protecting the Cys residue(s) in the protein. These results suggest that S-nitrosylation by nitric oxides protect Gal-2 from oxidative stress in the gastrointestinal tract.

Fine-tuning of prototype chicken galectins: structure of CG-2 and structure-activity correlations.

The comparatively small number of members of the family of adhesion/growth-regulatory galectins in chicken predestines this system as an attractive model to study the divergence of these lectins after gene duplication. Expression profiling of the three homodimeric (prototype) chicken galectins (CG-1A, CG-1B and CG-2) has raised evidence of distinct functionalities, explaining the interest in a detailed crystallographic analysis of CG-2. As revealed here, marked differences are found in the ligand-binding site and in the contact pattern within the homodimer interface, underlying a characteristic orientation of the two subunits. Notably, a distinctive trimer of dimers that is unique in all galectin crystal structures reported to date forms the core unit of the crystallographic assembly. Combination with spectroscopic and thermodynamic measurements, and comparisons with CG-1A and CG-1B, identify differential changes in the circular-dichroism spectra in the presence of lactose, reflecting the far-reaching impact of the ligand on hydrodynamic behaviour, and inter-galectin differences in both the entropy and the enthalpy of binding. This structural information is a salient step to complete the analysis of the full set of galectins from this model organism.

Single-site mutational engineering and following monoPEGylation of the human lectin galectin-2: effects on ligand binding, functional aspects, and clearance from serum.

The emerging insights into the physiological significance of endogenous lectins prompted us to characterize the effect of monosubstitution with poly(ethylene glycol) (PEG; 5 kDa) on a human lectin. As role model, we used a member of the galectin family, that is, galectin-2, the Cys57Met (single-site) mutant and its monoPEGylated derivative. The activities of these three proteins were comparatively studied by biochemical, cell biological, and histochemical methods, using surface-immobilized glycoproteins, different types of cells presenting gangliosides or (glyco)proteins as counterreceptors in vitro and tissue sections. PEGylation led to decreases in affinity/signal intensity with context dependence. The introduction of the mutation, too, can influence reactivity. Assays on haemagglutination and inhibition of cell proliferation underscored that mutational engineering and substitution can (but must not necessarily) affect this protein's activity. Serum clearance in rats was markedly retarded by PEGylation. Overall, the bulky substitution, spatially comparable to N-glycans, can markedly reduce binding of the galectin to physiological binding sites.

Analysis of monoPEGylated human galectin-2 by small-angle X-ray and neutron scattering: concentration dependence of PEG conformation in the conjugate.

Protein conjugation with polyethylene glycol (PEG) is a valuable means for improving stability, solubility, and bioavailability of pharmaceutical proteins. Using human galectin-2 (hGal-2) and 5 kDa PEG as a model system we first produced a PEG-hGal-2 conjugate exclusively at the Cys75 residue, resulting in two monosubstituted subunits per hGal-2 homodimer. Small angle X-ray and neutron scattering (SAXS and SANS) were combined to provide complementary structural information about the PEG-hGal-2 conjugate, wherein signal generation in SAXS depends mainly on the protein while SANS data presents signals from both the protein and PEG moieties. SAXS data gave a constant radius of gyration (R(g) = 21.5 Å) for the conjugate at different concentrations and provided no evidence for an alteration of homodimeric structure or hGal-2 ellipsoidal shape upon PEGylation. In contrast, SANS data revealed a concentration dependence of R(g) for the conjugate, with the value decreasing from 31.5 Å at 2 mg/mL to 26 Å at 14 mg/mL (based on hGal-2 concentration). Scattering data have been successfully described by the model of the ellipsoidal homogeneous core (hGal-2) attached with polymer chains (PEG) at the surface. Evidently, the PEG conformation of the conjugate strongly depends on conjugate concentration and PEG's radius of gyration decreases from 24.5 to 15 Å. An excluded volume effect, arising from steric clashes between PEG molecules at high concentration, was quantified by estimating the second virial coefficient, A(2), of PEGylated hGal-2 from the SANS data. A positive value of A(2) (6.0 ± 0.4 × 10(-4) cm(3) mol g(-2)) indicates repulsive interactions between molecules, which are expected to protect the PEGylated protein against aggregation.

Caenorhabditis elegans N-glycan core beta-galactoside confers sensitivity towards nematotoxic fungal galectin CGL2.

The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galbeta1,4Fucalpha1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galbeta1,4Fucalpha1,6GlcNAc trisaccharide at 1.5 A resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms.

Proteomic approach for identification of protein S-nitrosation in mouse gastric mucosa treated with S-nitrosoglutathione.

Nitric oxide, endogenously generated and exogenously supplied to the stomach, plays an important role in gastric physiological and pathological processes, including epithelial secretion, barrier function, immune response, and carcinogenesis. One of the mechanisms by which NO and related nitroso-compounds transmit their signals is S-nitrosation-mediated protein modification. To screen and identify gastric mucosal proteins that are uniquely sensitive to S-nitrosation, we reacted mouse gastric mucosal lysates with S-nitrosoglutathione, a physiologically relevant nitrosating agent, then performed proteomic analysis including the biotin-switch assay. The results showed that more than sixty protein spots on 2-DE were detected, and thirty-two of these were identified by MALDI-TOF MS and PMF. Eight of these identified proteins were unique S-nitrosated proteins not previously reported. Using Western blot technique, we further confirmed S-nitrosation especially in four proteins such as carbonic anhydrase-2, nucleoside diphosphate kinase B, Raf kinase inhibitor protein, and galectin-2, all known to be closely related to gastric mucosal protection and integrity, cell migration, and tumor metastasis. In addition, ex vivo study closer to in vivo situation also demonstrated these four proteins significantly S-nitrosated with S-nitrosoglutathione in mouse gastric mucosa. These findings will provide useful information regarding the linkage of protein S-nitrosation to gastric physiology and pathophysiology.

Single-site Cys-substituting mutation of human lectin galectin-2: modulating solubility in recombinant production, reducing long-term aggregation, and enabling site-specific monoPEGylation.

The effector capacity of endogenous lectins on cell adhesion/growth prompts studies to turn them into pharmaceutically stable forms. Using human galectin-2 as a proof-of-principle model, we first introduced mutations at the site of one of the two Cys residues, that is, C57A, C57M, and C57S. Only the C57M variant was expressed in bacteria in soluble form in high yield. No notable aggregation of the modified homodimeric lectin occurred during 3 weeks of storage. This mutational process also facilitated the site-directed introduction of poly(ethylene glycol) into the remaining sulfhydryl group (Cys75). Product analysis revealed rather complete conjugation with one chain per subunit in the homodimer. We note that neither the secondary structure alteration nor the absence of binding ability to a glycoprotein (asialofetuin) was observed. The results thus document the feasibility of tailoring a human galectin for enhanced stability to aggregation as well as monoPEGylation, which enables further testing of biological properties including functionality as growth regulator and the rate of serum clearance.

Structural basis for chitotetraose coordination by CGL3, a novel galectin-related protein from Coprinopsis cinerea.

Recent advances in genome sequencing efforts have revealed an abundance of novel putative lectins. Among these, many galectin-related proteins, characterized by many conserved residues but intriguingly lacking critical amino acids, have been found in all corners of the eukaryotic superkingdom. Here we present a structural and biochemical analysis of one representative, the galectin-related lectin CGL3 found in the inky cap mushroom Coprinopsis cinerea. This protein contains all but one conserved residues known to be involved in beta-galactoside binding in galectins. A Trp residue strictly conserved among galectins is changed to an Arg in CGL3 (R81). Accordingly, the galectin-related protein is not able to bind lactose. Screening of a glycan array revealed that CGL3 displays preference for oligomers of beta1-4-linked N-acetyl-glucosamines (chitooligosaccharides) and GalNAc beta 1-4GlcNAc (LacdiNAc). Carbohydrate-binding affinity of this novel lectin was quantified using isothermal titration calorimetry, and its mode of chitooligosaccharide coordination not involving any aromatic amino acid residues was studied by X-ray crystallography. Structural information was used to alter the carbohydrate-binding specificity and substrate affinity of CGL3. The importance of residue R81 in determining the carbohydrate-binding specificity was demonstrated by replacing this Arg with a Trp residue (R81W). This single-amino-acid change led to a lectin that failed to bind chitooligosaccharides but gained lactose binding. Our results demonstrate that, similar to the legume lectin fold, the galectin fold represents a conserved structural framework upon which dramatically altered specificities can be grafted by few alterations in the binding site and that, in consequence, many metazoan galectin-related proteins may represent lectins with novel carbohydrate-binding specificities.

Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens.

Human galectins have functionally divergent roles, although most of the members of the galectin family bind weakly to the simple disaccharide lactose (Galbeta1-4Glc). To assess the specificity of galectin-glycan interactions in more detail, we explored the binding of several important galectins (Gal-1, Gal-2, and Gal-3) using a dose-response approach toward a glycan microarray containing hundreds of structurally diverse glycans, and we compared these results to binding determinants on cells. All three galectins exhibited differences in glycan binding characteristics. On both the microarray and on cells, Gal-2 and Gal-3 exhibited higher binding than Gal-1 to fucose-containing A and B blood group antigens. Gal-2 exhibited significantly reduced binding to all sialylated glycans, whereas Gal-1 bound alpha2-3- but not alpha2-6-sialylated glycans, and Gal-3 bound to some glycans terminating in either alpha2-3- or alpha2-6-sialic acid. The effects of sialylation on Gal-1, Gal-2, and Gal-3 binding to cells also reflected differences in cellular sensitivity to Gal-1-, Gal-2-, and Gal-3-induced phosphatidylserine exposure. Each galectin exhibited higher binding for glycans with poly-N-acetyllactosamine (poly(LacNAc)) sequences (Galbeta1-4GlcNAc)(n) when compared with N-acetyllactosamine (LacNAc) glycans (Galbeta1-4GlcNAc). However, only Gal-3 bound internal LacNAc within poly(LacNAc). These results demonstrate that each of these galectins mechanistically differ in their binding to glycans on the microarrays and that these differences are reflected in the determinants required for cell binding and signaling. The specific glycan recognition by each galectin underscores the basis for differences in their biological activities.

Murine homodimeric adhesion/growth-regulatory galectins-1, -2 and -7: comparative profiling of gene/ promoter sequences by database mining, of expression by RT-PCR/immunohistochemistry and of contact sites for carbohydrate ligands by computational chemistry.

Following the detection of individual members of the family of galectins it is an obvious challenge to define the extent of functional overlap/divergence among these proteins. As a step to address this issue a comparative profiling has been started in the mouse as a model organism, combining sequence analysis, expression patterns and structural features in the cases of the homodimeric galectins-1, -2 and -7. Close relationship was apparent at the level of global gene organization. Scrutiny of the proximal promoter regions for putative transcription-factor-binding sites by two search algorithms uncovered qualitative and quantitative differences with potential to influence the combinatorial functionality of regulatory sequences. RT-PCR mapping with samples from an array of 17 organs revealed significant differences, separating rather ubiquitous gene expression of galectin-1 from the more restricted individual patterns of galectins-2 and -7. Using specific antisera obtained by affinity depletion including stringent controls to ascertain lack of cross-reactivity these results were corroborated at the level of galectin localization in fixed tissue sections. Nuclear presence was seen in the case of galectin-1. In addition to nonidentical expression profiles the mapping of the carbohydrate recognition domains of galectins-1 and -7 by homology modelling and docking of naturally occurring complex tetra- and pentasaccharides disclosed a series of sequence deviations which may underlie disparate affinities for cell surface glycans/glycomimetic peptides. In view of applicability the presented data can serve as useful reference to delineate changes with respect to disease and in genetically engineered models. To enable more general conclusions on the galectin network it is warranted to further pursue this combined approach within this lectin family.