Engineering a therapeutic lectin by uncoupling mitogenicity from antiviral activity.

A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code.

Synthesis and Thermodynamic Evaluation of Sialyl-Tn MUC1 Glycopeptides Binding to Macrophage Galactose-Type Lectin.

Interactions between the tumor-associated carbohydrate antigens of Mucin 1 (MUC1) and the carbohydrate-binding proteins, lectins, often lead to the creation of a pro-tumor microenvironment favoring tumor initiation, progression, metastasis, and immune evasion. Macrophage galactose binding lectin (MGL) is a C-type lectin receptor found on antigen-presenting cells that facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. Considering the crucial role of tumor-associated forms of MUC1 and MGL in tumor immunology, a thorough understanding of their binding interaction is essential for it to be exploited for cancer vaccine strategies. The synthesis of MUC1 glycopeptide models carrying a single or multiple Tn and/or sialyl-Tn antigen(s) is described. A novel approach for the sialyl-Tn threonine building block suitable for the solid phase peptide synthesis was developed. The thermodynamic profile of the binding interaction between the human MGL and MUC1 glycopeptide models was analyzed using isothermal titration calorimetry. The measured dissociation constants for the sialyl-Tn-bearing peptide epitopes were consistently lower compared to the Tn antigen and ranged from 10 µM for mono- to 100 nM for triglycosylated MUC1 peptide, respectively. All studied interactions, regardless of the glycan's site of attachment or density, exhibited enthalpy-driven thermodynamics.

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.

The glycobiology of the CD system: a dictionary for translating marker designations into glycan/lectin structure and function.

The profile of cell surface molecules, the biochemical platform for cellular communication, can be likened to a molecular fingerprint. Historically, raising monoclonal antibodies by immunization with cells has been instrumental in obtaining tools suited for phenotyping and functional analysis. Initially for leukocyte antigens, the resulting cluster of differentiation (CD) nomenclature has become a popular system for classification. Glycans presented on proteins or lipids and receptors for carbohydrate structures (lectins) are part of the CD list. Our review presents biochemical and biomedical highlights of the respective CD entries.

Bioactive cell-like hybrids coassembled from (glyco)dendrimersomes with bacterial membranes.

A library of amphiphilic Janus dendrimers including two that are fluorescent and one glycodendrimer presenting lactose were used to construct giant dendrimersomes and glycodendrimersomes. Coassembly with the components of bacterial membrane vesicles by a dehydration-rehydration process generated giant cell-like hybrid vesicles, whereas the injection of their ethanol solution into PBS produced monodisperse nanometer size assemblies. These hybrid vesicles contain transmembrane proteins including a small membrane protein, MgrB, tagged with a red fluorescent protein, lipopolysaccharides, and glycoproteins from the bacterium Escherichia coli. Incorporation of two colored fluorescent probes in each of the components allowed fluorescence microscopy to visualize and demonstrate coassembly and the incorporation of functional membrane channels. Importantly, the hybrid vesicles bind a human galectin, consistent with the display of sugar moieties from lipopolysaccharides or possibly glycosylated membrane proteins. The present coassembly method is likely to create cell-like hybrids from any biological membrane including human cells and thus may enable practical application in nanomedicine.

Onion-like glycodendrimersomes from sequence-defined Janus glycodendrimers and influence of architecture on reactivity to a lectin.

A library of eight amphiphilic Janus glycodendrimers (GDs) with d-mannose (Man) headgroups, a known routing signal for lectin-mediated transport processes, was constructed via an iterative modular methodology. Sequence-defined variations of the Janus GD modulate the surface density and sequence of Man after self-assembly into multilamellar glycodendrimersomes (GDSs). The spatial mode of Man presentation is decisive for formation of either unilamellar or onion-like GDS vesicles. Man presentation and Janus GD concentration determine GDS size and number of bilayers. Beyond vesicle architecture, Man topological display affects kinetics and plateau level of GDS aggregation by a tetravalent model lectin: the leguminous agglutinin Con A, which is structurally related to endogenous cargo transporters. The agglutination process was rapid, efficient, and readily reversible for onion-like GDSs, demonstrating their value as versatile tools to explore the nature of physiologically relevant glycan/lectin pairing.

Galectin-1 Couples Glycobiology to Inflammation in Osteoarthritis through the Activation of an NF-κB-Regulated Gene Network.

Osteoarthritis is a degenerative joint disease that ranks among the leading causes of adult disability. Mechanisms underlying osteoarthritis pathogenesis are not yet fully elucidated, putting limits to current disease management and treatment. Based on the phenomenological evidence for dysregulation within the glycome of chondrocytes and the network of a family of adhesion/growth-regulatory lectins, that is, galectins, we tested the hypothesis that Galectin-1 is relevant for causing degeneration. Immunohistochemical analysis substantiated that Galectin-1 upregulation is associated with osteoarthritic cartilage and subchondral bone histopathology and severity of degeneration (p < 0.0001, n = 29 patients). In vitro, the lectin was secreted and it bound to osteoarthritic chondrocytes inhibitable by cognate sugar. Glycan-dependent Galectin-1 binding induced a set of disease markers, including matrix metalloproteinases and activated NF-κB, hereby switching on an inflammatory gene signature (p < 10(-16)). Inhibition of distinct components of the NF-κB pathway using dedicated inhibitors led to dose-dependent impairment of Galectin-1-mediated transcriptional activation. Enhanced secretion of effectors of degeneration such as three matrix metalloproteinases underscores the data's pathophysiological relevance. This study thus identifies Galectin-1 as a master regulator of clinically relevant inflammatory-response genes, working via NF-κB. Because inflammation is critical to cartilage degeneration in osteoarthritis, this report reveals an intimate relation of glycobiology to osteoarthritic cartilage degeneration.

Unraveling functional significance of natural variations of a human galectin by glycodendrimersomes with programmable glycan surface.

Surface-presented glycans (complex carbohydrates) are docking sites for adhesion/growth-regulatory galectins within cell-cell/matrix interactions. Alteration of the linker length in human galectin-8 and single-site mutation (F19Y) are used herein to illustrate the potential of glycodendrimersomes with programmable glycan displays as a model system to reveal the functional impact of natural sequence variations in trans recognition. Extension of the linker length slightly reduces lectin capacity as agglutinin and slows down aggregate formation at low ligand surface density. The mutant protein is considerably less active as agglutinin and less sensitive to low-level ligand presentation. The present results suggest that mimicking glycan complexity and microdomain occurrence on the glycodendrimersome surface can provide key insights into mechanisms to accomplish natural selectivity and specificity of lectins in structural and topological terms.

Galectin-related protein: An integral member of the network of chicken galectins: 2. From expression profiling to its immunocyto- and histochemical localization and application as tool for ligand detection.

BACKGROUND: Galectin-related protein (GRP), present in vertebrates, is special within this family of adhesion/growth-regulatory proteins due to its strong positive selection and loss of canonical lectin activity. METHODS: RT-PCR and Western blotting together with flow cytofluorimetry and immunocyto- and histochemistry monitor expression and localization of chicken GRP. The promoter sequence of the GRP gene is processed computationally to detect putative sites for binding transcription factors. The labeled protein is applied as probe to detect binding sites on cells and in sections, along with glycocompounds to test inhibition of the association. RESULTS: Expression of GRP in chicken is limited to bursa of Fabricius, immunohistochemically found in B cells, also in bursal epithelium and vessels. Presence in B cells is shared with only one canonical galectin, i.e. CG-8. Binding to a chicken lymphoma line was specific and saturable, not affected by lactose but completely blocked by heparin, as also seen in sections. CONCLUSIONS: Expression monitoring initiated for GRP reveals a distinct site of localization in chicken, much more restricted than for any of its canonical galectins.

Teaming up synthetic chemistry and histochemistry for activity screening in galectin-directed inhibitor design.

A hallmark of endogenous lectins is their ability to select a few distinct glycoconjugates as counterreceptors for functional pairing from the natural abundance of cellular glycoproteins and glycolipids. As a consequence, assays to assess inhibition of lectin binding should necessarily come as close as possible to the physiological situation, to characterize an impact of a synthetic compound on biorelevant binding with pharmaceutical perspective. We here introduce in a proof-of-principle manner work with sections of paraffin-embedded tissue (jejunum, epididymis) and labeled adhesion/growth-regulatory galectins, harboring one (galectin-1 and galectin-3) or two (galectin-8) types of lectin domain. Six pairs of synthetic lactosides from tailoring of the headgroup (3'-O-sulfation) and the aglycone (ß-methyl to aromatic S- and O-linked extensions) as well as three bi- to tetravalent glycoclusters were used as test compounds. Varying extents of reduction in staining intensity by synthetic compounds relative to unsubstituted/free lactose proved the applicability and sensitivity of the method. Flanking cytofluorimetric assays on lectin binding to native cells gave similar grading, excluding a major impact of tissue fixation. The experiments revealed cell/tissue binding of galectin-8 preferentially via one domain, depending on the cell type so that the effect of an inhibitor in a certain context cannot be extrapolated to other cells/tissues. Moreover, the work with the other galectins attests that this assay enables comprehensive analysis of the galectin network in serial tissue sections to determine overlaps and regional differences in inhibitory profiles.

Direct Enzymatic Branch-End Extension of Glycocluster-Presented Glycans: An Effective Strategy for Programming Glycan Bioactivity.

The sequence of a glycan and its topology of presentation team up to determine the specificity and selectivity of recognition by saccharide receptors (lectins). Structure-activity analysis would be furthered if the glycan part of a glycocluster could be efficiently elaborated in situ while keeping all other parameters constant. By using a bacterial α2,6-sialyltransferase and a small library of bi- to tetravalent glycoclusters, we illustrate the complete conversion of scaffold-presented lactoside units into two different sialylated ligands based on N-acetyl/glycolyl-neuraminic acid incorporation. We assess the ensuing effect on their bioactivity for a plant toxin, and present an analysis of the noncovalent substrate binding contacts that the added sialic acid moiety makes to the lectin. Enzymatic diversification of a scaffold-presented glycan can thus be brought to completion in situ, offering a versatile perspective for rational glycocluster engineering.

Bivalent O-glycoside mimetics with S/disulfide/Se substitutions and aromatic core: Synthesis, molecular modeling and inhibitory activity on biomedically relevant lectins in assays of increasing physiological relevance.

The emerging significance of recognition of cellular glycans by lectins for diverse aspects of pathophysiology is a strong incentive for considering development of bioactive and non-hydrolyzable glycoside derivatives, for example by introducing S/Se atoms and the disulfide group instead of oxygen into the glycosidic linkage. We report the synthesis of 12 bivalent thio-, disulfido- and selenoglycosides attached to benzene/naphthalene cores. They present galactose, for blocking a plant toxin, or lactose, the canonical ligand of adhesion/growth-regulatory galectins. Modeling reveals unrestrained flexibility and inter-headgroup distances too small to bridge two sites in the same lectin. Inhibitory activity was first detected by solid-phase assays using a surface-presented glycoprotein, with relative activity enhancements per sugar unit relative to free cognate sugar up to nearly 10fold. Inhibitory activity was also seen on lectin binding to surfaces of human carcinoma cells. In order to proceed to characterize this capacity in the tissue context monitoring of lectin binding in the presence of inhibitors was extended to sections of three types of murine organs as models. This procedure proved to be well-suited to determine relative activity levels of the glycocompounds to block binding of the toxin and different human galectins to natural glycoconjugates at different sites in sections. The results on most effective inhibition by two naphthalene-based disulfides and a selenide raise the perspective for broad applicability of the histochemical assay in testing glycoclusters that target biomedically relevant lectins.

Studying the Structural Significance of Galectin Design by Playing a Modular Puzzle: Homodimer Generation from Human Tandem-Repeat-Type (Heterodimeric) Galectin-8 by Domain Shuffling.

Tissue lectins are emerging (patho)physiological effectors with broad significance. The capacity of adhesion/growth-regulatory galectins to form functional complexes with distinct cellular glycoconjugates is based on molecular selection of matching partners. Engineering of variants by changing the topological display of carbohydrate recognition domains (CRDs) provides tools to understand the inherent specificity of the functional pairing. We here illustrate its practical implementation in the case of human tandem-repeat-type galectin-8 (Gal-8). It is termed Gal-8 (NC) due to presence of two different CRDs at the N- and C-terminal positions. Gal-8N exhibits exceptionally high affinity for 3'-sialylated/sulfated ß-galactosides. This protein is turned into a new homodimer, i.e., Gal-8 (NN), by engineering. The product maintained activity for lactose-inhibitable binding of glycans and glycoproteins. Preferential association with 3'-sialylated/sulfated (and 6-sulfated) ß-galactosides was seen by glycan-array analysis when compared to the wild-type protein, which also strongly bound to ABH-type epitopes. Agglutination of erythrocytes documented functional bivalency. This result substantiates the potential for comparative functional studies between the variant and natural Gal-8 (NC)/Gal-8N.

Detection of Proteome Changes in Human Colon Cancer Induced by Cell Surface Binding of Growth-Inhibitory Human Galectin-4 Using Quantitative SILAC-Based Proteomics.

Endogenous lectins have the capacity to translate glycan-encoded information on the cell surface into effects on cell growth. As test cases to examine changes in protein presence associated with tumor growth inhibition, we applied SILAC-based proteomics on human colon carcinoma cells treated with galectin-4 (Gal-4). The five tested lines-LS 180, Vaco 432, Colo 205, CX 1, and HCT 116-responded with differentiation and reduced proliferation to Gal-4 binding. In proteomic analysis (mass spectral data deposited with PRIDE, PXD003489), 2654 proteins were quantified, of which 190 were down-regulated and 115 were up-regulated (>2-fold). 1D annotation analysis of the results indicated down-regulation of DNA replication-associated processes, while protein presence for secretory and transport functions appeared increased. The strongest induction was found for CALB2 (calretinin; ∼24-fold), TGM2 (protein-glutamine γ-glutamyltransferase 2; ∼11-fold), S100A3 (∼10-fold), and GSN (gelsolin; 9.5-fold), and the most pronounced decreases were seen for CDKN2A (tumor suppressor ARF; ∼6-fold), EPCAM (epithelial cell adhesion molecule; ∼6-fold), UBE2C (ubiquitin-conjugating enzyme E2 C; ∼5-fold), KIF2C (kinesin-like protein KIF2C; 5-fold), and LMNB1 (lamin-B1; ∼5-fold). The presence of the common proliferation marker Ki-67 was diminished about 4-fold. By tracing significant alterations of protein expression likely relevant for the observed phenotypic effects, the capacity of a galectin to affect the proteome of human colon cancer cells at multiple sites is revealed.

Galectin-8 enhances adhesion of multiple myeloma cells to vascular endothelium and is an adverse prognostic factor.

Multiple myeloma is characterized by abnormal infiltration of malignant plasma cells into bone marrow. Testing the hypothesis that bivalent galectin-8 (Gal-8) may influence homing of myeloma cells to vascular endothelium as a key prerequisite for infiltration, we analyzed the two Gal-8 splice variants (Gal-8S, Gal-8L). They differ in the length of their linker peptide connecting the two lectin domains. Both Gal-8 isoforms bind to cells of the myeloma lines Gal-8+ MOLP-8 and Gal-8- LP-1 in a glycan-inhibitable manner. Both Gal-8 isoforms led to enhanced adhesion of myeloma cells to vascular endothelium under dynamic shear stress conditions, Gal-8L (by more than 40-fold) even stronger than Gal-8S. Additional treatment of endothelial cells with tumour necrosis factor prior to the dynamic shear stress assay entailed an almost 100-fold enhanced adhesion of myeloma cells without addition of Gal-8 variants and a further 1.5-1.7-fold enhancement by addition of Gal-8 variants. We also found that elevated expression of Gal-8 in native multiple myeloma cells is an adverse prognostic factor for overall and event-free survival using patients' gene expression profile data of the total therapy 2 and 3 myeloma studies. Also, elevated concentrations of Gal-8 were detected (45%, 19/42 patients) in sera of multiple myeloma patients compared to those of healthy, age-matched donors. Both experimental and clinical data strongly point to the significance of Gal-8 for multiple myeloma development.

Intra- and intermolecular interactions of human galectin-3: assessment by full-assignment-based NMR.

Galectin-3 is an adhesion/growth-regulatory protein with a modular design comprising an N-terminal tail (NT, residues 1-111) and the conserved carbohydrate recognition domain (CRD, residues 112-250). The chimera-type galectin interacts with both glycan and peptide motifs. Complete (13)C/(15)N-assignment of the human protein makes NMR-based analysis of its structure beyond the CRD possible. Using two synthetic NT polypeptides covering residues 1-50 and 51-107, evidence for transient secondary structure was found with helical conformation from residues 5 to 15 as well as proline-mediated, multi-turn structure from residues 18 to 32 and around PGAYP repeats. Intramolecular interactions occur between the CRD F-face (the 5-stranded ß-sheet behind the canonical carbohydrate-binding 6-stranded ß-sheet of the S-face) and NT in full-length galectin-3, with the sequence P(23)GAW(26)…P(37)GASYPGAY(45) defining the primary binding epitope within the NT. Work with designed peptides indicates that the PGAX motif is crucial for self-interactions between NT/CRD. Phosphorylation at position Ser6 (and Ser12) (a physiological modification) and the influence of ligand binding have minimal effect on this interaction. Finally, galectin-3 molecules can interact weakly with each other via the F-faces of their CRDs, an interaction that appears to be assisted by their NTs. Overall, our results add insight to defining binding sites on galectin-3 beyond the canonical contact area for ß-galactosides.

Functional interplay between ganglioside GM1 and cross-linking galectin-1 induces axon-like neuritogenesis via integrin-based signaling and TRPC5-dependent Ca²âº influx.

Axon-like neuritogenesis in neuroblastoma (NG108-15) cells and primary cerebellar granular neurons is furthered by the presence of ganglioside GM1. We describe here that galectin-1 (Gal-1), a homobivalent endogenous lectin, is an effector by cross-linking the ganglioside and its associated glycoprotein α5 ß1 -integrin. The thereby triggered signaling cascade involves autophosphorylation of focal adhesion kinase and activation of phospholipase Cγ and phosphoinositide-3 kinase. This leads to a transient increase in the intracellular Ca(2+) concentration by opening of TRPC5 channels, which belong to the signal transduction-gated cation channels. Controls with GM1-defective cells (NG-CR72 and neurons from ganglio-series KO mice) were retarded in axonal growth, underscoring the relevance of GM1 as functional counterreceptor for Gal-1. The lectin's presence was detected in the NG108-15 cells, suggesting an autocrine mechanism of action, and in astrocytes in situ. Gal-1, as cross-linking lectin, can thus translate metabolic conversion of ganglioside GD1a to GM1 by neuraminidase action into axon growth. Galectin-1 (Gal-1) was shown an effector of axonogenesis in cerebellar granule neurons (CGNs) and NG108-15 cells by cross-linking GM1 ganglioside and its associated glycoprotein α5 ß1 -integrin. The resulting signaling led to a transient increase in intracellular Ca(2+) by opening TRPC5 channels. CGNs deficient in GM1 showed retarded axonogenesis, underscoring the relevance of GM1 as functional counterreceptor for Gal-1 in this process. This Gal-1/GM1-induced signaling was manifest only at the earliest, initiating stage of axon development.

Merging carbohydrate chemistry with lectin histochemistry to study inhibition of lectin binding by glycoclusters in the natural tissue context.

Recognition of glycans by lectins leads to cell adhesion and growth regulation. The specificity and selectivity of this process are determined by carbohydrate structure (sequence and shape) and topology of its presentation. The synthesis of (neo)glycoconjugates with bi- to oligo-valency (glycoclusters) affords tools to delineate structure-activity relationships by blocking lectin binding to an artificial matrix, often a glycoprotein, or cultured cell lines. The drawback of these assays is that glycan presentation is different from that in tissues. In order to approach the natural context, we here introduce lectin histochemistry on fixed tissue sections to determine the susceptibility of binding of two plant lectins, i.e., GSA-II and WGA, to a series of 10 glycoclusters. Besides valency, this panel covers changes in the anomeric position (α/ß) and the atom at the glycosidic linkage (O/S). Flanked by cell and solid-phase assays with human tumor lines and two mucins, respectively, staining (intensity and profile) was analyzed in sections of murine jejunum, stomach and epididymis as a function of glycocluster presence. The marked and differential sensitivity of signal generation to structural aspects of the glycoclusters proves the applicability of this method. This enables comparisons between data sets obtained by using (neo)glycoconjugates, cells and the tissue context as platforms. The special advantage of processing tissue sections is the monitoring of interference with lectin association at sites that are relevant for functionality. Testing glycoclusters in lectin histochemistry will especially be attractive in cases of multi-target recognition (glycans, proteins and lipids) by a tissue lectin.

From lectin structure to functional glycomics: principles of the sugar code.

Lectins are carbohydrate-binding proteins which lack enzymatic activity on their ligand and are distinct from antibodies and free mono- and oligosaccharide sensor/transport proteins. Emerging insights into the functional dimension of lectin binding to cellular glycans have strongly contributed to the shaping of the 'sugar code'. Fittingly, over a dozen folds and a broad spectrum of binding site architecture, ranging from shallow grooves to deep pockets, have developed sugar-binding capacity. A central question is how the exquisite target specificity of endogenous lectins for certain cellular glycans can be explained. In this regard, affinity regulation is first systematically dissected into six levels. Experimentally, the strategic combination of methods to monitor distinct aspects of the lectin-glycan interplay offers a promising perspective to answer this question.

Thermodynamic Switch in Binding of Adhesion/Growth Regulatory Human Galectin-3 to Tumor-Associated TF Antigen (CD176) and MUC1 Glycopeptides.

A shift to short-chain glycans is an observed change in mucin-type O-glycosylation in premalignant and malignant epithelia. Given the evidence that human galectin-3 can interact with mucins and also weakly with free tumor-associated Thomsen-Friedenreich (TF) antigen (CD176), the study of its interaction with MUC1 (glyco)peptides is of biomedical relevance. Glycosylated MUC1 fragments that carry the TF antigen attached through either Thr or Ser side chains were synthesized using standard Fmoc-based automated solid-phase peptide chemistry. The dissociation constants (Kd) for interaction of galectin-3 and the glycosylated MUC1 fragments measured by isothermal titration calorimetry decreased up to 10 times in comparison to that of the free TF disaccharide. No binding was observed for the nonglycosylated control version of the MUC1 peptide. The most notable feature of the binding of MUC1 glycopeptides to galectin-3 was a shift from a favorable enthalpy to an entropy-driven binding process. The comparatively diminished enthalpy contribution to the free energy (ΔG) was compensated by a considerable gain in the entropic term. (1)H-(15)N heteronuclear single-quantum coherence spectroscopy nuclear magnetic resonance data reveal contact at the canonical site mainly by the glycan moiety of the MUC1 glycopeptide. Ligand-dependent differences in binding affinities were also confirmed by a novel assay for screening of low-affinity glycan-lectin interactions based on AlphaScreen technology. Another key finding is that the glycosylated MUC1 peptides exhibited activity in a concentration-dependent manner in cell-based assays revealing selectivity among human galectins. Thus, the presentation of this tumor-associated carbohydrate ligand by the natural peptide scaffold enhances its affinity, highlighting the significance of model studies of human lectins with synthetic glycopeptides.