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Inflammatory chemo- and cytokines and matrix-degrading proteases underlie the progression of osteoarthritis (OA). Aiming to define upstream regulators for these disease markers, we pursued initial evidence for an upregulation of members of the adhesion/growth-regulatory galectin family. Immunohistochemical localization of galectin-3 (Gal-3) in sections of human cartilage with increasing levels of degeneration revealed a linear correlation reaching a chondrocyte positivity of 60%. Presence in situ was cytoplasmic, the lectin was secreted from OA chondrocytes in culture and binding of Gal-3 yielded lactose-inhibitable surface staining. Exposure of cells to the lectin led to enhanced gene expression and secretion of functional disease markers. Genome-wide transcriptomic analysis broadened this result to reveal a pro-degradative/inflammatory gene signature under the control of NF-κB. Fittingly, targeting this route of activation by inhibitors impaired the unfavourable response to Gal-3 binding, as also seen by shortening the lectin's collagen-like repeat region. Gal-3's activation profile overlaps with that of homodimeric galectin-1 (Gal-1) and also has distinctive (supplementing) features. Tested at subsaturating concentrations in a mixture, we found cooperation between the two galectins, apparently able to team up to promote OA pathogenesis. In summary, our results suggest that a network of endogenous lectins is relevant for initiating this process cascade.

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., (Copyright © 2016 by The American Association of Immunologists, Inc.)

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 β1 -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 β1 -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., (© 2015 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Lectins translate information encoded in glycan chains of cellular glycoconjugates into bioeffects. The topological presentation of contact sites for cognate sugar binding is a crucial factor toward this end. To dissect the significance of such phylogenetically conserved properties, the design and engineering of non-natural variants are attractive approaches. Here, a homodimeric human lectin, i.e. adhesion/growth-regulatory galectin-1, is converted into a tandem-repeat display by introducing the 33-amino-acid linker of another family member (i.e. galectin-8). The yield of variant was reduced by about a third. This protein had ∼10-fold higher activity in hemagglutination. Nearly complete sequence determination by mass-spectrometric in-source decay and fingerprinting excluded the presence of any modifications. When (1)H-(15)N heteronuclear single-quantum coherence data on the (15)N-labeled variant and wild-type protein were compared, changes in chemical shifts, signal intensities and resonance multiplicities revealed reduction of stability of interfacial contacts between the lectin domains and an increase in inter-domain flexibility. When both binding sites in the variant were loaded with ligand, association of the two carbohydrate recognition domains was enhanced, corroborated by gel filtration. Dynamic changes in the spatial presentation of the two lectin domains in the context of a tandem-repeat display can alter counterreceptor targeting relative to the fixed positions found in the proto-type galectin homodimer., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The emerging significance of lectins for pathophysiological processes provides incentive for the design of potent inhibitors. To this end, systematic assessment of contributions to affinity and selectivity by distinct types of synthetic tailoring of glycosides is a salient step, here taken for the aglyconic modifications of two disaccharide core structures. Firstly we report the synthesis of seven N-linked-lactosides and of eight O-linked N-acetyllactosamines, each substituted with a 1,2,3-triazole unit, prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC). The totally regioselective β-D-(1 → 4) galactosylation of a 6-O-TBDPSi-protected N-acetylglucosamine acceptor provided efficient access to the N-acetyllactosamine precursor. The resulting compounds were then systematically tested for lectin reactivity in two binding assays of increasing biorelevance (inhibition of lectin binding to a surface-presented glycoprotein and to cell surfaces). As well as a plant toxin, we also screened the relative inhibitory potential with adhesion/growth-regulatory galectins (total of eight proteins). This type of modification yielded up to 2.5-fold enhancement for prototype proteins, with further increases for galectins-3 and -4. Moreover, the availability of (15)N-labeled proteins and full assignments enabled (1)H, (15)N HSQC-based measurements for hu- man galectins-1, -3, and -7 against p-nitrophenyl lactopyranoside, a frequently tested standard inhibitor containing an aromatic aglycone. The measurements confirmed the highest affinity against galectin-3 and detected chemical shift differences in its hydrophobic core upon ligand binding, besides common alterations around the canonical contact site for the lactoside residue. What can be accomplished in terms of affinity/selectivity by this type of core extension having been determined, the applied combined strategy should be instrumental for proceeding with defining structure-activity correlations at other bioinspired sites in glycans and beyond the tested lectin types., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Many human proteins have a modular design with receptor and structural domains. Using adhesion/growth-regulatory galectin-3 as model, we describe an interdisciplinary strategy to define the functional significance of its tail established by nine non-triple helical collagen-like repeats (I-IX) and the N-terminal peptide. Genetic engineering with sophisticated mass spectrometric product analysis provided the tools for biotesting, i.e. eight protein variants with different degrees of tail truncation. Evidently,various aspects of galectin-3 activity (cis binding and cell bridging) are affected by tail shortening in a different manner. Thus, this combined approach reveals an unsuspected complexity of structure-function relationship, encouraging further application beyond this chimera-type galectin., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Background/aim: Stromal cells in the tumor microenvironment are primarily considered as sources of promalignant factors. The objective of our study was to define the effect of extracellular matrix (ECM) produced by normal dermal or cancer-associated fibroblasts exposed to adhesion/growth-regulatory lectin galectin-1 on human umbilical vein endothelial cells (HUVECs)., Materials and Methods: Fibroblasts were cultured for 10 days with lectin, followed by removing cellular constituents after an osmotic shock. Freshly-isolated HUVECs were placed on the ECM. In parallel, HUVECs were seeded on untreated and gelatin-coated surfaces as controls. A positive control for growth of HUVECs culture using medium supplemented with vascular endothelial growth factor completed the test panel. Cells were kept in contact to the substratum for two days and then processed for immunocytochemistry., Results: HUVECs seeded on fibroblast-generated ECM presented a comparatively high degree of proliferation. Furthermore, contact to substratum produced by tumor-associated fibroblasts led to generation of a meshwork especially rich in fibronectin., Conclusion: Galectin-1 is apparently capable to trigger ECM production favorable for growth of HUVECs, prompting further work on characterizing structural features of the ECM and in situ correlation of lectin presence, ECM constitution and neoangiogenesis., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Cancer-associated fibroblasts (CAFs) play a role in the progression of malignant tumors. They are formed by conversion of fibroblasts to smooth muscle α-actin-positive (SMA-positive) myofibroblasts. Polyamines are known to change the arrangement of the actin cytoskeleton by binding to the anionic actin. We tested the effect of the synthetic polyamine BPA-C8 on the transition of human dermal fibroblasts to myofibroblasts induced either by TGF-β1 alone or by TGF-β1 together with adhesion/growth-regulatory galectin-1. Pre-existing CAFs, myofibroblasts from pancreatitis, and rat smooth muscle cells were also exposed to BPA-C8. BPA-C8 impaired myofibroblast formation from activated fibroblasts, but it had no effect on cells already expressing SMA. BPA-C8 also reduced the occurrence of an extracellular matrix around the activated fibroblasts. The reported data thus extend current insights into polyamine activity, adding interference with tumor progression to the tumor-promoting processes warranting study., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Despite some advances, pancreatic ductal adenocarcinoma (PDAC) remains generally refractory to current treatments. Desmoplastic stroma, a consistent hallmark of PDAC, has emerged as a major source of therapeutic resistance and thus potentially promising targets for improved treatment. The glycan-binding protein galectin-1 (Gal1) is highly expressed in PDAC stroma, but its roles there have not been studied. Here we report functions and molecular pathways of Gal1 that mediate its oncogenic properties in this setting. Genetic ablation of Gal1 in a mouse model of PDAC (EIa-myc mice) dampened tumor progression by inhibiting proliferation, angiogenesis, desmoplasic reaction and by stimulating a tumor-associated immune response, yielding a 20% increase in relative lifesplan. Cellular analyses in vitro and in vivo suggested these effects were mediated through the tumor microenvironment. Importantly, acinar-to-ductal metaplasia, a crucial step for initiation of PDAC, was found to be regulated by Gal1. Mechanistic investigations revealed that Gal1 promoted Hedgehog pathway signaling in PDAC cells and stromal fibroblasts as well as in Ela-myc tumors. Taken together, our findings establish a function for Gal1 in tumor-stroma crosstalk in PDAC and provide a preclinical rationale for Gal1 targeting as a microenvironment-based therapeutic strategy., (©2014 American Association for Cancer Research.)

The human lectin galectin-1 (hGal-1) translates sugar signals, that is, β-galactosides, into effects on the level of cells, for example, growth regulation, and has become a model for studying binding of biopharmaceutically relevant derivatives. Bound-state conformations of Galβ-C-(1→3)-Glcβ-OMe (1) and its βGal-(1→3)-βGlc-OMe disaccharide parent compound were studied by using NMR spectroscopy (transferred (TR)-NOESY data), assisted by docking experiments and molecular dynamics (MD) simulations. The molecular recognition process involves a conformational selection event. Although free C-glycoside access four distinct conformers in solution, hGal-1 recognizes shape of a local minimum of compound 1, the syn-Φ/syn-Ψ conformer, not the structure at global minimum. MD simulations were run to explain, in structural terms, the observed geometry of the complex., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: One route of translating the information encoded in the glycan chains of cellular glycoconjugates into physiological effects is via receptor (lectin) binding. A family of endogenous lectins, sharing folding, a distinct sequence signature and affinity for β-galactosides (thus termed galectins), does so effectively in a context-dependent manner., Areas Covered: An overview is given on the multifunctional nature of galectins, with emphasis on galectin-1. The broad range of functions includes vital processes such as adhesion via glycan bridging, glycoconjugate transport or triggering signaling relevant, for example, for growth regulation. Besides distinct glycoconjugates, this lectin can also interact with certain proteins so that it can target counterreceptors at all sites of location, that is, in the cytoplasm and/or nucleus, at both sides of the membrane or extracellularly. Approaches to strategically exploit galectin activities with therapeutic intentions are outlined., Expert Opinion: The wide versatility of sugar coding and the multifunctionality of galectin-1 explain why considering to turn the protein into a therapeutic target is an ambitious aim. Natural pathways shaped by physiologic master regulators (e.g., the tumor suppressor p16(INK4a)) are suggested to teach inspiring lessons as to how the lectin might be recruited to clinical service.

Tumor stroma is an active part influencing the biological properties of malignancies via molecular cross-talk. Cancer-associated fibroblasts play a significant role in this interaction. These cells frequently express smooth muscle actin and can be classified as myofibroblasts. The adhesion/growth-regulatory lectin galectin-1 is an effector for their generation. In our study, we set the presence of smooth muscle actin-positive cancer-associated fibroblasts in relation to this endogenous lectin and an in vivo competitor (galectin-3). In squamous cell carcinomas of head and neck, upregulation of galectin-1 presence was highly significantly correlated to presence of smooth muscle actin-positive cancer-associated fibroblasts in the tumor (p = 4 × 10(-8)). To pinpoint further correlations on the molecular level, we applied microarray analyses to the transcription profiles of the corresponding tumors. Significant correlations of several transcripts were detected with the protein level of galectin-1 in the cancer-associated fibroblasts. These activated genes (MAP3K2, TRIM23, PTPLAD1, FUSIP1, SLC25A40 and SPIN1) are related to known squamous-cell-carcinoma poor-prognosis factors, NF-κB upregulation and splicing downregulation. These results provide new insights into the significance of presence of myofibroblasts in squamous cell carcinoma., (Copyright © 2012 UICC.)

Spectroscopic monitoring is applied to detect structural alterations for homodimeric adhesion/growth-regulatory galectins. Mammalian galectin-1 and the avian ortholog CG-1B, due to their distinct patterns of cysteine positioning, can undergo oxidation. When monitoring tryptophan fluorescence anisotropy comparatively, an indicator of structural changes affecting rotational diffusion, segmental motion and/or fluorescence life time, reductions are seen in both cases upon oxidation. The decrease was especially marked for the human protein, more than 2-fold compared to the avian lectin. Using this approach to analyze binding of lactose, equilibrium and kinetic binding constants of both proteins were similar. This result is corroborated by fluorescence correlation spectroscopy with labeled proteins. Of note, the diffusion constant of CG-1B increased by 5.6% in the presence of lactose, as has been seen for the human protein. When processing the other two homodimeric avian galectins (CG-1A, CG-2) accordingly it was revealed that sequence homology does not translate into identical behavior. The diffusion constant of CG-1A was not affected, a slight decrease (-3.8%) was observed for CG-2. Obviously, alterations induced by oxidation and responses to ligand binding are different between these closely related proteins. Methodologically, the two spectroscopic techniques are proven to be sensitive and robust sensors for detecting intergalectin differences., (Copyright © 2012. Published by Elsevier Masson SAS.)

Tumour suppressor p16(INK4a) is known to exert cell-cycle control via cyclin-dependent kinases. An emerging aspect of its functionality is the orchestrated modulation of N/O-glycosylation and galectin expression to induce anoikis in human Capan-1 pancreatic carcinoma cells. Using chemoselective N/O-glycan enrichment technology (glycoblotting) and product characterization, we first verified a substantial decrease in sialylation. Tests combining genetic (i.e. transfection with α2,6-sialyltransferase-specific cDNA) or metabolic (i.e. medium supplementation with N-acetylmannosamine to track down a bottleneck in sialic acid biosynthesis) engineering with cytofluorometric analysis of lectin binding indicated a role of limited substrate availability, especially for α2,6-sialylation, which switches off reactivity for anoikis-triggering homodimeric galectin-1. Quantitative MS analysis of protein level changes confirmed an enhanced galectin-1 presence along with an influence on glycosyltransferases (β1,4-galactosyltransferase-IV, α2,3-sialyltransferase-I) and detected p16(INK4a) -dependent down-regulation of two enzymes in the biosynthesis pathway for sialic acid [i.e. the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) and N-acetylneuraminic acid 9-phosphate synthase] (P < 0.001). By contrast, quantitative assessment for the presence of nuclear CMP-N-acetylneuraminic acid synthase (which is responsible for providing the donor for enzymatic sialylation that also acts as feedback inhibitor of the epimerase activity of GNE) revealed a trend for an increase. Partial restoration of sialylation in GNE-transfected cells supports the implied role of sialic acid availability for the glycophenotype. Fittingly, the extent of anoikis was reduced in double-transfected (p16(INK4a) /GNE) cells. Thus, a second means of modulating cell reactivity to the growth effector galectin-1 is established in addition to the common route of altering α2,6-sialyltransferase expression: regulating enzymes of the pathway for sialic acid biosynthesis., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence, robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r(0) was about 0.2, altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited, no labeling is needed. Owing to tryptophan's presence in carbohydrate-binding sites, also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases, glycosidases), this assay procedure can have relevance beyond galectins.

By definition, adhesion/growth-regulatory galectins are known for their ability to bind β-galactosides such as Galβ(1 → 4)Glc (lactose). Indications for affinity of human galectin-1 to α-linked digalactosides pose questions on the interaction profile with such bound ligands and selection of the galactose moiety for CH-π stacking. These issues are resolved by a combination of (15)N-(1)H heteronuclear single quantum coherence (HSQC) chemical shift and saturation transfer difference nuclear magnetic resonance (STD NMR) epitope mappings with docking analysis, using the α(1 → 3/4)-linked digalactosides and also Galα(1 → 6)Glc (melibiose) as test compounds. The experimental part revealed interaction with the canonical lectin site, and this preferentially via the non-reducing-end galactose moiety. Low-energy conformers appear to be selected without notable distortion, as shown by molecular dynamics simulations. With the α(1 → 4) disaccharide, however, the typical CH-π interaction is significantly diminished, yet binding appears to be partially compensated for by hydrogen bonding. Overall, these findings reveal that the type of α-linkage in digalactosides has an impact on maintaining CH-π interactions and the pattern of hydrogen bonding, explaining preference for the α(1 → 3) linkage. Thus, this lectin is able to accommodate both α- and β-linked galactosides at the same site, with major contacts to the non-reducing-end sugar unit., (© The Author 2011. Published by Oxford University Press. All rights reserved.)

Aim: To define specific staining patterns for the adhesion/growth-regulatory lectin tandem-repeat-type galectin-8 in hypopharyngeal and laryngeal tumor progression and relate these parameters to galectins 1, 3 and 7 in the quest to explore the galectin network., Materials and Methods: The level of expression of galectin-8 was determined immunohistochemically in a series of 18 and 16 cases of tumor-free epithelium, 24 and 10 cases of low-grade dysplasia, 22 and 15 cases of high-grade dysplasia located in peri-tumoral area of 74 and 37 hypopharyngeal and laryngeal carcinomas., Results: Marked upregulation in galectin-8 staining intensity and immunopositive area in malignancy versus dysplasia was seen in hypopharyngeal cancer (p<10(-6)), in laryngeal cancer for labeling index and high-grade dysplasia/carcinoma (p<10(-6)). No correlation to recurrence was delineated., Conclusion: The presented data revealed a divergence within the galectin-1, -3, -7 and -8 network during tumor progression.

Purpose: Adhesion and migration of dislocated retinal pigment epithelial (RPE) cells are initial steps in the pathogenesis of proliferative vitreoretinopathy (PVR). The role of the endogenous lectin, galectin-1, in attachment, spreading, and migration of human RPE cells was investigated from a therapeutic perspective., Methods: Human RPE cells were treated with galectin-1 concentrations that ranged 0-250 microg/ml. Cell viability was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay. Galectin-1 binding to the RPE cells was investigated by immunocytochemistry. Attachment of RPE cells was assessed on 96-well plates coated with laminin, or fibronectin, or galectin-1, or the glycoprotein-lectin combinations and subsequent MTT-testing. RPE migration in the absence or presence of galectin-1 on the respective substrates was tested using a modified Boyden chamber assay with platelet derived growth factor (PDGF)-BB as the chemoattractant. Cellular spreading was characterized by cytoplasmic halo formation of RPE cells after three hours in contact with the surface coating., Results: Galectin-1 bound to the cell surface. Binding could be inhibited by a beta-galactoside. MTT assays revealed no toxicity within control limits for the concentration range tested. When added to the medium, galectin-1 dose-dependently inhibited RPE cell attachment, spreading, and migration by more than 70%, irrespective of the substratum tested. When coated onto the plastic surface, galectin-1 alone impaired spreading and migration of RPE cells, and reduced attachment to and migration on fibronectin by up to 80%., Conclusions: Galectin-1 inhibits RPE cell attachment, migration, and spreading in vitro with no apparent cytotoxicity. This activity of the endogenous effector deserves consideration as a potential therapeutic agent for the prevention of PVR.

Background & Aims: Tissue plasminogen activator (tPA) exerts many different functions in addition to its role in fibrinolysis. In pancreatic ductal adenocarcinoma (PDA), tPA is overexpressed and plays an important role in proliferation, invasion, and angiogenesis. tPA interaction with cell membrane receptors has been related to increased proteolytic activity and to signal transduction through nonenzymatic mechanisms. The aim was to analyze the role of galectin-1 (Gal-1), an endogenous lectin that also is overexpressed in PDA, as a new functional receptor for tPA., Methods: Gal-1/tPA interaction was analyzed using surface plasmon resonance and pull-down assays. Pancreatic cells and tumors were used to study Gal-1 expression and localization by Western blot and immunostaining. Down-regulation of Gal-1 by small interference RNA was used to analyze the involvement of Gal-1/tPA interaction in extracellular signal-regulated kinase 1/2 activation, cell proliferation, and invasion in pancreatic and fibroblastic cells., Results: Gal-1/tPA interaction is direct, specific, and of high affinity. Gal-1 moderately increases the catalytic activity of tPA. High Gal-1 levels were detected in PDA cells in culture, where it concentrates at the migration front, and in tissues, where it is expressed in epithelial cells and in the stroma. Down-regulation of Gal-1 abolished the effects of tPA on extracellular signal-regulated kinase 1/2 activation, cell proliferation, and invasion, both in pancreatic and in tumor-derived fibroblasts., Conclusions: These findings support a new molecular mechanism by which Gal-1 interaction with tPA contributes to PDA progression involving both transformed epithelial cells and tumor fibroblasts.

The enzymatic activity of matrix metalloproteinase-9 (MMP-9) suggests that its presence in hypopharyngeal and laryngeal squamous cell carcinomas (HSCCs, LSCCs) could have prognostic value. We tested this hypothesis by quantitative morphometric analysis of immunohistochemical staining in histological sections of 73 stage IV HSCCs and 45 LSCCs (30 cases of stage I/II, 15 cases of stage IV). As compared to tumour-free epithelium an increase for the labelling index in LSCCs reached statistical significance (p=0.04). Specimens of Reinke's edema were strongly higher in this parameter compared to tumour-free tissue area (p=0.000001), underscoring an association between the level of MMP-9 expression and inflammation. Focusing on patients' recurrence status we identified thresholds for the labelling index of 10% for HSCCs and 18% for LSCCs, both indicating rapid recurrence and dismal prognosis unless surpassed. When relating data for MMP-9 to those for three adhesion/growth-regulatory galectins, a positive correlation with galectin-7 expression was detected in LSCCs. This finding suggests a possible potential role of this endogenous lectin as inducer of MMP-9 gene expression in situ. Of note, galectin-1 expression was negatively correlated with MMP-9 and that of galectin-3, a substrate of MMP-9, not related. In conclusion our study delineated a prognostic role of MMP-9 immunodetection in high-stage HSCCs and in LSCCs when separating patients by a distinct threshold for the labelling index. Moreover, it indicated associations between MMP-9 and multifunctional galectins-1 and -7 in situ.

Background: Galectin-1 has been found to modulate lymphocyte invasion in inflammation and to be involved in angiogenesis in models, thus prompting examination of its clinical relevance in laryngeal cancer., Patients and Methods: Immunohistochemical processing of tissue sections (n=53) from patients with stage I/II (n=35) and stage IV (n=18) laryngeal squamous cell carcinoma (LSCC) with a specific anti-galectin-1 antibody and monitoring of CD45/CD31 positivity was combined with quantitative morphometric analysis., Results: Lectin presence in the tumor and endothelial cells was positively correlated, while a negative relationship to the number of CD45-positive lymphocytes was demonstrated. No association was seen with the extent of neovascularization. The mean optical density (MOD) of lectin-dependent staining in the tumor stroma was significantly increased compared to normal stroma., Conclusion: Galectin-1 was not associated with angiogenesis in the studied cohort while galectin-1 in endothelial cells may negatively influence lymphocyte invasion and the mean optical density for the stromal galectin-1 signal is up-regulated in tumors.

Galectin-3 is a multifunctional protein with modular design. A distinct expression profile was determined in various murine organs when set into relation to homodimeric galectins-1 and -7. Fittingly, the signature of putative transcription-factor-binding sites in the promoter region of the galectin-3 gene affords a toolbox for a complex combinatorial regulation, distinct from the respective sequence stretches in galectins-1 and -7. A striking example for cell-type specificity was the ovary, where these two lectins were confined to the surface epithelium. Immunohistochemically, galectin-3 was found in macrophages of the cortical interstitium between developing follicles and medullary interstitium, matching the distribution of the F4/80 antigen. With respect to atresia and luteolysis strong signals in granulosa cells of atretic preantral but not antral follicles and increasing positivity in corpora lutea upon regression coincided with DNA fragmentation. Labeled galectin-3 revealed lactose-inhibitable binding to granulosa cells. Also, slender processes of vital granulosa cells which extended into the zona pellucida were positive. This study demonstrates cell-type specificity and cycle-associated regulation for galectin-3 with increased presence in atretic preantral follicles and in late stages of luteolysis.

Combinatorial (glyco)peptide libraries offer the possibility to define effective inhibitors of protein (lectin)-glycan interactions. If a (glyco)peptide surpasses the inhibitory potency of the free sugar, then the new peptide-lectin contacts underlying the affinity enhancement may guide further rational drug design. Focusing on the adhesion/growth regulatory human galectins 1 and 3, a screening of three combinatorial solid-phase (glyco)peptide libraries, containing Gal(beta1-O)Thr, Gal(beta1-S)Cys/Gal(beta1-N)Asn, and Lac(beta1-O)Thr, with the fluorescently labeled lectins had led to a series of lead compounds. To define the inhibitory potency of a selection of resynthesized (glyco)peptides systematically, a surface plasmon resonance-based inhibition assay with immobilized asialofetuin was set up. (Glyco)Peptides with up to 66-fold potency relative to free lactose as inhibitor were characterized. The presence of lactose in the most effective glycopeptides indicated the presence of affinity-enhancing peptide-lectin contacts. In addition to drug design, they may be helpful for fine-structural analysis of the binding sites.

Background: Since the histological expression of galectins is increased in thyroid carcinoma, determination of their serum levels may provide useful preoperative information. The goal of this study was to determine if a difference in galectin serum levels could be detected between benign and malignant nodular thyroid diseases., Design: Using validated ELISAs, the concentrations of several galectins were prospectively measured in serum samples from 30 healthy individuals and preoperatively in 90 patients with thyroid disease. Seventy-one patients had multiple thyroid nodules (MTN), 13 patients had a single thyroid nodule (STN), and 6 patients had Graves' disease. Nine of 71 patients with MTN had fine-needle aspiration biopsy (FNAB) of their nodules and in 7 patients a "benign" diagnosis was made, in 0 patient a "malignant" diagnosis was made, and in 2 patients a "suspicious" diagnosis was made. Six of 13 patients with STN had FNAB of their nodules and in 2 patients a "benign" diagnosis was made, in 3 patients a "malignant" diagnosis was made, and in 1 patient a "suspicious" diagnosis was made., Results: Thyroid disease was associated with higher levels of galectins-1 and -3 compared to normal subjects. Using a threshold value of 3.2 ng/mL as a cut-off point, the measurement of serum galectin-3 separated micro- and macropapillary thyroid carcinoma (PAP&#95;CA) from patients with nonmalignant thyroid disease with 74% specificity, 73% sensitivity, 57% positive predictive value, and 85% negative predictive value. Elevated serum galectin-3 concentrations (>3.2 ng/mL) detected 87% of macropapillary thyroid carcinomas and 67% of micropapillary thyroid carcinomas., Conclusions: Serum levels of galectins-1 and -3 are relatively high in patients with thyroid malignancy but there is considerable overlap in serum galectin-3 concentrations between those with benign and malignant nodular thyroid disease and, to a lesser extent, between those with and without nodular thyroid disease.

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.

The conformational behavior of the C-glycoside analogue of N-acetyl-lactosamine, beta-C-Gal-(1-->4)-beta-GlcNAc-OMe, 1, has been studied using a combination of molecular mechanics calculations and NMR spectroscopy (J and NOE data). It is shown that the C-disaccharide populates three distinctive conformational families in solution, the major one being the anti-psi conformation. Of note, this conformation is only marginally populated for the O-disaccharide. Due to its conspicuous role in the regulation of adhesion, growth and tissue invasion of tumors and its avid binding to N-acetyl-lactosamine human, galectin-1 was tested as a receptor. This endogenous lectin recognizes a local minimum of 1, the syn-PhiPsi conformer, and thus a conformational selection process is correlated with the molecular recognition event.

Expression of the tumor suppressor p16(INK4a) after stable transfection can restore the susceptibility of epithelial tumor cells to anoikis. This property is linked to increases in the expression and cell-surface presence of the fibronectin receptor. Considering its glycan chains as pivotal signals, we assumed an effect of p16(INK4a) on glycosylation. To test this hypothesis for human Capan-1 pancreatic carcinoma cells, we combined microarray for selected glycosyltransferase genes with 2D chromatographic glycan profiling and plant lectin binding. Major differences between p16-positive and control cells were detected. They concerned expression of beta1,4-galactosyltransferases (down-regulation of beta1,4-galactosyltransferases-I/V and up-regulation of beta1,4-galactosyltransferase-IV) as well as decreased alpha2,3-sialylation of O-glycans and alpha2,6-sialylation of N-glycans. The changes are compatible with increased beta(1)-integrin maturation, subunit assembly and binding activity of the alpha(5)beta(1)-integrin. Of further functional relevance in line with our hypothesis, we revealed differential reactivity towards endogenous lectins, especially galectin-1. As a result of reduced sialylation, the cells' capacity to bind galectin-1 was enhanced. In parallel, the level of transcription of the galectin-1 gene increased conspicuously in p16(INK4a)-positive cells, and even figured prominently in a microarray on 1996 tumor-associated genes and in proteomic analysis. The cells therefore gain optimal responsiveness. The correlation between genetically modulated galectin-1 levels and anoikis rates in engineered transfectants inferred functional significance. To connect these findings to the fibronectin receptor, galectin-1 was shown to be co-immunoprecipitated. We conclude that p16(INK4a) orchestrates distinct aspects of glycosylation that are relevant for integrin maturation and reactivity to an endogenous effector as well as the effector's expression. This mechanism establishes a new aspect of p16(INK4a) functionality.

Monitoring of gene-expression profiles is assumed to refine tumor characterization of laryngeal squamous cell carcinomas (LSCCs) with a therapeutic perspective. This is especially expected for adhesion/growth-regulatory effectors such as galectins, a class of endogenous lectins. Using computer-assisted microscopy, we investigated the prognostic value contributed by the quantitative determination of the immunohistochemical levels of expression of galectin-1, -3 and -7 in a series of 62 LSCCs including 42 low- and 20 high-stage LSCCs. As galectin-1 may have a key role leading to a tumor escape from immune surveillance, we also investigated whether or not the level of galectin-1 expression correlated with lymphocyte infiltration in LSCCs. The immunohistochemical determination of expression of galectin-1 is of prognostic value in human squamous laryngeal cancers. LSCCs that display high levels of galectin-1 have worse prognoses than laryngeal cancers with low levels of galectin-1 expression. Elevation of galectin-1 levels in laryngeal cancers can contribute to the process of tumor immune escape by killing the activated T-cells and other protumoral activities such as promoting motility or activity of oncogenic H-Ras proteins. The quantitative determination of galectin-1 in LSCCs is an independent prognostic marker when opposed to TNM staging. It has the potential to identify patients unlikely to benefit from T-cell-mediated immunotherapy, although the definitive effector function from its pro- and antitumoral activity profile has not been delineated.

The branched pentasaccharide chain of ganglioside GM1 is a prominent cell surface ligand, for example, for cholera toxin or tumor growth-regulatory homodimeric galectins. This activity profile via protein recognition prompted us to examine the binding properties of peptides with this specificity. Our study provides insights into the mechanism of molecular interaction of this thus far unexplored size limit of the protein part. We used three pentadecapeptides in a combined approach of mass spectrometry, NMR spectroscopy and molecular modelling to analyze the ligand binding in solution. Availability of charged and hydrophobic functionalities affected the intramolecular flexibility of the peptides differently. Backfolding led to restrictions in two cases; the flexibility was not reduced significantly by association of the ligand in its energetically privileged conformations. Major contributions to the interaction energy arise from the sialic acid moiety contacting Arg/Lys residues and the N-terminal charge. Considerable involvement of stacking between the monovalent ligand and aromatic rings could not be detected. This carbohydrate binding strategy is similar to how an adenoviral fiber knob targets sialylated glycans. Rational manipulation for an affinity enhancement can now be directed to reduce the flexibility, exploit the potential for stacking and acquire the cross-linking capacity of the natural lectins by peptide attachment to a suitable scaffold.

Surface binding of galectin family members has the potential to link distinct glycan structures to growth regulation. Therefore, we addressed the antiproliferative potential of galectin-1 (Gal-1) in a panel of carcinoma cell lines. We discovered growth inhibition by Gal-1 in epithelial tumor cell lines from different origins and provide evidence that this effect requires functional interaction with the alpha5beta1 integrin. Antiproliferative effects result from inhibition of the Ras-MEK-ERK pathway and consecutive transcriptional induction of p27. We have further identified two Sp1-binding sites in the p27 promoter as crucial for Gal-1 responsiveness. Inhibition of the Ras-MEK-ERK cascade by Gal-1 increased Sp1 transactivation and DNA binding due to reduced threonine phosphorylation of Sp1. Furthermore, Gal-1 induced p21 transcription and selectively increased p27 protein stability. Gal-1-mediated accumulation of p27 and p21 inhibited cyclin-dependent kinase 2 activity and ultimately resulted in G(1) cell cycle arrest and growth inhibition. These data define a novel mechanism whereby Gal-1 regulates epithelial tumor cell homeostasis via carbohydrate-dependent interaction with the alpha5beta1 integrin.

The current study focused on galectins (-1, -3, -4, -7, and -8) and deliberately performed immunohistochemical fingerprinting to explore their complexity in a context of experimental renal carcinogenesis. The diethylstilbestrol (DES)-induced renal tumors in male Syrian hamster kidney (SHKT) represent a unique animal model for the study of estrogen-dependent renal malignancies. Kidney sections of DES-treated hamsters (3 days to 11 months of DES exposure) were analyzed by immunohistochemistry using a panel of non-crossreactive antibodies raised against galectins-1, -3, -4, -7, and -8. Levels of expression were quantitatively determined by using computer-assisted microscopy on immunostained tissue sections. Except for galectin-4, all above mentioned galectins were expressed in kidney tumors. Small clusters of galectin-1-positive, most likely preneoplastic cells at the corticomedullary junction were already evident 1 week after DES administration. Galectin-1 and -3 expression was apparently associated with the first steps of the neoplastic transformation, because small tumorous buds were found to be positive after 1 month of treatment. In contrast, galectins-7 and -8 were detected in large tumors and medium-sized tumors, respectively, thereby indicating an involvement in later stages of DES-induced SHKT. Galectins-1, -3, -7, and -8 were also detected by immunofluorescence staining in the HKT-1097 cell line established from SHKT, thus illustrating the stability of galectin expression in tumor cells. Our data document the presence and differential regulation of galectins in the course of renal tumorigenesis in the model of DES-induced SHKT.

Human galectin-1 is a potent multifunctional effector that participates in specific protein-carbohydrate and protein-protein (lipid) interactions. By determining its X-ray structure, we provide the basis to define the structure of its ligand-binding pocket and to perform rational drug design. We have also analysed whether single-site mutations introduced at some distance from the carbohydrate recognition domain can affect the lectin fold and influence sugar binding. Both the substitutions introduced in the C2S and R111H mutants altered the presentation of the loop, harbouring Asp123 in the common "jelly-roll" fold. The orientation of the side-chain was inverted 180 degrees and the positions of two key residues in the sugar-binding site of the R111H mutant were notably shifted, i.e. His52 and Trp68. Titration calorimetry was used to define the decrease in ligand affinity in both mutants and a significant increase in the entropic penalty was found to outweigh a slight enhancement of the enthalpic contribution. The position of the SH-groups in the galectin appeared to considerably restrict the potential to form intramolecular disulphide bridges and was assumed to be the reason for the unstable lectin activity in the absence of reducing agent. However, this offers no obvious explanation for the improved stability of the C2S mutant under oxidative conditions. The noted long-range effects in single-site mutants are relevant for the functional divergence of closely related galectins and in more general terms, the functionality definition of distinct amino acids.

Ras biological activity necessitates membrane anchorage that depends on the Ras farnesyl moiety and is strengthened by Ras/galectin-1 interactions. We identified a hydrophobic pocket in galectin-1, analogous to the Cdc42 geranylgeranyl-binding cavity in RhoGDI, possessing homologous isoprenoid-binding residues, including the critical L11, whose RhoGDI L77 homologue changes dramatically on Cdc42 binding. By substituting L11A, we obtained a dominant interfering galectin-1 that possessed normal carbohydrate-binding capacity but inhibited H-Ras GTP-loading and extracellular signal-regulated kinase activation, dislodged H-Ras(G12V) from the cell membrane, and attenuated H-Ras(G12V) fibroblast transformation and PC12-cell neurite outgrowth. Thus, independently of carbohydrate binding, galectin-1 cooperates with Ras, whereas galectin-1(L11A) inhibits it.

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.

Endogenous lectins induce effects on cell growth by binding to antennae of natural glycoconjugates. These complex carbohydrates often present more than one potential lectin-binding site in a single chain. Using the growth-regulatory interaction of the pentasaccharide of ganglioside GM(1) with homodimeric galectin-1 on neuroblastoma cell surfaces as a model, we present a suitable strategy for addressing this issue. The approach combines NMR spectroscopic and computational methods and does not require isotope-labeled glycans. It involves conformational analysis of the two building blocks of the GM(1) glycan, i.e., the disaccharide Galbeta1-3GalNAc and the trisaccharide Neu5Acalpha2-3Galbeta1-4Glc. Their bound-state conformations were determined by transferred nuclear Overhauser enhancement spectroscopy. Next, measurements on the lectin-pentasaccharide complex revealed differential conformer selection regarding the sialylgalactose linkage in the tri- versus pentasaccharide (Phi and Psi value of -70 degrees and 15 degrees vs 70 degrees and 15 degrees, respectively). To proceed in the structural analysis, the characteristic experimentally detected spatial vicinity of a galactose unit and Trp68 in the galectin's binding site offered a means, exploiting saturation transfer from protein to carbohydrate protons. Indeed, we detected two signals unambiguously assigned to the terminal Gal and the GalNAc residues. Computational docking and interaction energy analyses of the entire set of ligands supported and added to experimental results. The finding that the ganglioside's carbohydrate chain is subject to differential conformer selection at the sialylgalactose linkage by galectin-1 and GM(1)-binding cholera toxin (Phi and Psi values of -172 degrees and -26 degrees, respectively) is relevant for toxin-directed drug design. In principle, our methodology can be applied in studies aimed at blocking galectin functionality in malignancy and beyond glycosciences.

The bioactivity of galectin-1 in cell growth regulation and adhesion prompted us to answer the questions whether ligand presence and a shift to an aprotic solvent typical for bioaffinity chromatography might alter the shape of the homodimeric human lectin in solution. We used small angle neutron and synchrotron x-ray scattering studies for this purpose. Upon ligand accommodation, the radius of gyration of human galectin-1 decreased from 19.1 +/- 0.1 A in the absence of ligand to 18.2 +/- 0.1 A. In the aprotic solvent dimethyl sulfoxide, which did not impair binding capacity, galectin-1 formed dimers of a dimer, yielding tetramers with a cylindrical shape. Intriguingly, no dissociation into subunits occurred. In parallel, NMR monitoring was performed. The spectral resolution was in accord with these data. In contrast to the properties of the human protein, a nonhomologous agglutinin from mistletoe sharing galactose specificity was subject to a reduction in the radius of gyration from approximately 62 A in water to 48.7 A in dimethyl sulfoxide. Evidently, the solvent caused opposite responses in the two tested galactoside-binding lectins with different folding patterns. We have hereby proven that ligand presence and an aprotic solvent significantly affect the shape of galectin-1 in solution.

A thorough characterization of the properties of squamous epithelial cells is necessary in order to improve our understanding of the functional aspects of normal development and malignant aberrations. Up to now, studies have focused almost exclusively on monitoring distinct protein markers. With our growing awareness of the coding function of glycan chains of cellular glycoconjugates and their interaction with receptors (lectins) in situ, defining the glycophenotype of these cells has become an important issue. Whereas the commonly applied plant lectins are tools used to map the presence and localization of biochemically defined saccharide epitopes, the introduction of endogenous (mammalian) lectins to this analysis enables us to take the step from monitoring the presence of glycan to understanding the functional implications by revealing ligand properties of the detected epitope for tissue lectin. Thus, in this study we investigated a distinct aspect of glycosylation using plant and mammalian lectins, i.e. the linkage type of sialylation. We first mapped the expression profile of the type of sialylation (alpha2,3- or alpha2,6-linked) by plant lectins. Based on the hypothesis that this factor regulates accessibility of ligands for endogenous lectins we introduced two labeled galectins to this study. Galectin-3 (but not galectin-1) binding was related to cell differentiation in normal adult and developing epithelia, cultured epidermal cells, and carcinomas derived from these epithelia. The presented data suggest that alpha2,6-linked N-acetyl-D-neuraminic acid moieties could serve to mask galectin-3-reactive glycoepitopes. As a consequence, monitoring of the linkage type of sialic acid in glycans by plant lectins therefore has implications for the extent of glycan reactivity with endogenous lectins, pointing to a potential function of changes in sialylation type beyond these cell and lectin systems.