Galactoside-binding Proteins Research Articles

Galectins as pivotal components in oncogenesis and immune exclusion in human malignancies.

Galectins are galactoside-binding proteins, exerting numerous functions inside and outside the cell, particularly conferring adaptation to stress factors. For most of them, aberrant expression profiles have been reported in the context of cancer. Albeit not being oncogenic drivers, galectins can be harnessed to exacerbate the malignant phenotype. Their impact on disease establishment and progression is not limited to making cancer cells resistant to apoptosis, but is prominent in the context of the tumor microenvironment, where it fosters angiogenesis, immune escape and exclusion. This review focuses mainly on Gal-1, Gal-3 and Gal-9 for which the involvement in cancer biology is best known. It presents the types of galectin dysregulations, attempts to explain the mechanisms behind them and analyzes the different ways in which they favor tumour growth. In an era where tumour resistance to immunotherapy appears as a major challenge, we highlight the crucial immunosuppressive roles of galectins and the potential therapeutic benefits of combinatorial approaches including galectin inhibition.

Phorbol 12-myristate 13-acetate (PMA) Alters Galectin Gene Expression in Cows

Galectins (GAL) are β-galactoside binding proteins. The objective of this study was to assess the effect of PMA on the regulation of galectin gene expression in bovine blood and neutrophils. Blood was collected from 3 clinically healthy Holstein cows. Neutrophils were isolated by differential centrifugation. Blood and neutrophils were treated with 10ng/ml of PMA or maintained in phosphate-buffered saline for 30 minutes at 370 C. Pooled total RNA were transcribed to cDNA for real-time PCR. Specific primers for bovine LGALS -1, -2, -3, -4, -7, -8, -9, -11, -12, and their binding proteins (bp) LGALS3bp, T cell immunoglobulin and mucin domain 3 (TIM-3) were used. Housekeeping genes RPLP0 and UCHL5 served as internal controls. Fold changes were calculated using the Livak method. Total protein concentration was assessed using the Bicinchoninic acid assay. Secretion of GAL -1, -2, -3, -4, -8, -9 was assessed using bovine GAL specific ELISA. All tested LGALS were detected. Activation with PMA differentially modulated expression and secretion of GAL in blood and neutrophils. Expression levels of LGALS -1 and LGALS -3 was not affected. Expression of LGALS -7, -8, -9, -11, -12, and TIM-3 was differentially modulated. The secretion of GAL -3 was significantly increased in response to PMA. The most pronounced effect of PMA was observed on LGALS-9, TIM-3 expression, and the secretion of GAL -3. Thus, GAL may serve as biomarkers of cell activation with functional consequences and warrant further study.

Serum galectins as potential biomarkers of inflammatory bowel diseases.

The inflammatory bowel diseases (IBD), which include mainly Crohn's disease (CD) and ulcerative colitis (UC), are common chronic inflammatory conditions of the digestive system. The diagnosis of IBD relies on the use of a combination of factors including symptoms, endoscopy and levels of serum proteins such as C-reactive protein (CRP) or faecal calprotectin. Currently there is no single reliable biomarker to determine IBD. Galectins are a family of galactoside-binding proteins that are commonly altered in the circulation of disease conditions such as cancer and inflammation. This study investigated serum galectin levels as possible biomarkers in determining IBD and IBD disease activity. Levels of galectins-1, -2, -3, -4, -7 and -8 were analysed in 208 samples from ambulant IBD patients (97 CD, 71 UC) patients and 40 from healthy people. Disease activity was assessed using Harvey-Bradshaw Index for CD and simple clinical colitis activity index for UC. The relationship of each galectin in determining IBD and IBD disease activity were analysed and compared with current IBD biomarker CRP. It was found that serum level of galectin-1 and -3, but not galectins-2, -4, -7 and -8, were significantly higher in IBD patients than in healthy people. At cut-off of 4.1ng/ml, galectin-1 differentiated IBD from healthy controls with 71% sensitivity and 87% specificity. At cut-off of 38.5ng/ml, galectin-3 separated IBD from healthy controls with 53% sensitivity and 87% specificity. None of the galectins however were able to distinguish active disease from remission in UC or CD. Thus, levels of galectins-1 and -3 are significantly elevated in both UC and CD patients compared to healthy people. Although the increased galectin levels are not able to separate active and inactive UC and CD, they may have the potential to be developed as useful biomarkers for IBD diagnosis either alone or in combination with other biomarkers.

Trypanosoma cruzi cleaves galectin-3 N-terminal domain to suppress its innate microbicidal activity.

Galectin-3 is the best-characterized member of galectins, an evolutionary conserved family of galactoside-binding proteins that play central roles in infection and immunity, regulating inflammation, cell migration and cell apoptosis. Differentially expressed by cells and tissues with immune privilege, they bind not only to host ligands, but also to glycans expressed by pathogens. In this regard, we have previously shown that human galectin-3 recognizes several genetic lineages of the protozoan parasite Trypanosoma cruzi, the causal agent of Chagas' disease or American trypanosomiasis. Herein we describe a molecular mechanism developed by T. cruzi to proteolytically process galectin-3 that generates a truncated form of the protein lacking its N-terminal domain-required for protein oligomerization-but still conserves a functional carbohydrate recognition domain (CRD). Such processing relies on specific T. cruzi proteases, including Zn-metalloproteases and collagenases, and ultimately conveys profound changes in galectin-3-dependent effects, as chemical inhibition of parasite proteases allows galectin-3 to induce parasite death in vitro. Thus, T. cruzi might have established distinct mechanisms to counteract galectin-3-mediated immunity and microbicide properties. Interestingly, non-pathogenic T. rangeli lacked the ability to cleave galectin-3, suggesting that during evolution two genetically similar organisms have developed different molecular mechanisms that, in the case of T. cruzi, favoured its pathogenicity, highlighting the importance of T. cruzi proteases to avoid immune mechanisms triggered by galectin-3 upon infection. This study provides the first evidence of a novel strategy developed by T. cruzi to abrogate signalling mechanisms associated with galectin-3-dependent innate immunity.

Galectin Secretion and Modulation in Sheep Blood

Galectins (GAL) are β-galactoside binding proteins that can modulate both pregnancy and the response to pathogens. Increased susceptibility to infection by pathogens is linked to periparturient immune suppression and a periparturient rise (PPR) in parasite eggs on pasture. The possible role of GAL in periparturient immune relaxation and PRR needs definition.  The objective of this study was to evaluate galectin secretion and its relationship to measures of the immune relaxation and PRR in periparturient sheep. Samples were collected from pregnant St. Croix sheep (n=6) weekly at days -21 to + 21 relative to lambing. Fecal samples were collected and evaluated for strongyle and coccidia parasite eggs. The concentration of IgA and IgE coproantibodies, total microbial DNA, Bifidobacteria and Lactobacillus levels in fecal samples were used as indicators of gut health.  Blood samples were collected by jugular venipuncture and assessed for Packed Cell Volume (PCV), total and differential white blood cell counts. Total protein concentrations and protein profile were evaluated in serum. Secretion of GAL 1, 3, 9 and 14 were evaluated using ELISAs. Data were analyzed by one-way ANOVA and statistical significance was declared at P <0.05. Galectins tested were secreted in sheep blood. Differential modulation of GAL secretion and correlation with periparturient immune suppression and parasite infection was observed. Galectin secretion was modulated by the periparturient period, type and status of parasite infection. This first insight into a possible role of secreted galectins in periparturient immune relaxation and PRR improves the understanding of the immune response, informs development of management programs and therapeutics and presents Galectin profiles as biomarkers with diagnostic potential.

Expression profiling and microbial ligand binding analysis of galectin-4 in turbot (Scophthalmus maximus L.)

Galectins are a family of galactoside-binding proteins with an affinity for β-galactosides, involved in mediating fundamental processes including development, inflammation, cell migration and apoptosis. Galectin-4 is a member of tendem-repeat galectins, plays vital roles in intestinal epithelial barrier. Here, one galectin-4 gene was captured in turbot (SmLgals4) contains a 1197 bp open reading frame (ORF). In comparison to other species, SmLgals4 showed the highest similarity and identity both to large yellow croaker. The genomic structure analysis showed that SmLgals4 had conserved exons in the CRD domains compared to other vertebrate species. The syntenic analysis revealed that galectin-4 had the same neighboring genes across all the selected species, which suggested the synteny encompassing galectin-4 region during vertebrate evolution. Subsequently, SmLgals4 was widely expressed in all the examined tissues, with the highest expression level in intestine and the lowest expression level in skin. In addition, SmLgals4 was significantly down-regulated in intestine following both Gram-negative bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmLgals4 showed strong binding ability to all the examined microbial ligands. Taken together, our results suggested SmLgals4 plays vital roles in fish intestinal immune responses against infection, but the detailed roles of galectin-4 in teleost are still lacking, further studies are needed to be carried out to characterize whether galectin-4 plays similar roles in teleost intestinal immunity.

Bactericidal activity of fish galectin 4 derived membrane-binding peptide tagged with oligotryptophan

Galectins belong to the family of galactoside-binding proteins which act as pathogen recognition receptors by recognizing and binding to the carbohydrate present in the bacterial membranes. In this study, a Galectin-4 sequence was identified from the constructed cDNA library of Channa striatus and its structural features were reported. Gene expression analysis revealed that CsGal4 was highly expressed in liver and strongly induced by Epizootic Ulcerative Syndrome (EUS) causing pathogens such as Aphanomyces invadans, Aeromonas hydrophila and a viral analogue, poly I:C. To understand the antimicrobial role of putative dimerization site of CsGal4, the region was chemically synthesized and its bactericidal effect was determined. G4 peptide exhibited a weak bactericidal activity against Vibrio harveyi, an important aquaculture pathogen. We have also determined the bactericidal activity of the dimerization site by tagging pentamer oligotryptophan (W5) at the C-terminal of G4 peptide. Flow cytometry analysis revealed that G4W induced drastic reduction in cell counts than G4. Electron microscopic images showed membrane blebbings in V. harveyi which indicated the membrane disrupting activity of G4W. Interestingly, both the peptides did not exhibit any hemolytic activity and cytotoxicity towards peripheral blood cells of Channa striatus and the activity was specific only towards the bacterial membrane. Our results suggested that addition of W5 at the C-terminal of membrane-binding peptide remarkably improved its membrane disrupting activity.

Promoter SNPs rs116896264 and rs73933062 form a distinct haplotype and are associated with galectin-4 overexpression in colorectal cancer.

Galectin-4 is a member of the galectin family which consists of 15 galactoside-binding proteins. Previously, galectin-4 has been shown to have a role in cancer progression and metastasis and it is found upregulated in many solid tumours, including colorectal cancer (CRC). Recently, the role in the metastatic process was suggested to be via promoting cancer cells to adhere to blood vascular endothelium. In the present study, the regulatory region of LGALS4 (galectin-4) in seven colon cell lines was investigated with respect to genetic variation that could be linked to expression levels and therefore a tumourigenic effect. Interestingly, qRT-PCR and sequencing results revealed that galectin-4 upregulation is associated with SNPs rs116896264 and rs73933062. By use of luciferase reporter- and pull-down assays, we confirmed the association between the gene upregulation and the two SNPs. Also, using pull-down assay followed by mass spectrometry, we found that the presence rs116896264 and rs73933062 is changing transcription factors binding sites. In order to assess the frequencies of the two SNPs among colon cancer patients and healthy individuals, we genotyped 75 colon cancer patients, 12 patients with adenomatous polyposis and 17 patients with ulcerative colitis and we performed data mining in the 1000 genomes databank. We found the two SNPs co-occuring in 21% of 75 CRC patients, 0 out of 12 patients of adenomatous polyposis, and 6 out of 17 patients (35%) with ulcerative colitis. Both in the patient samples and in the 1000 genomes project, the two SNPs were found to co-occur whenever present (D' = 1).

Molecular characterization of Galectin-8 from Nile tilapia (Oreochromis niloticus Linn.) and its response to bacterial infection.

Galectins belong to the family of galactoside-binding proteins and play a major role in the immune and inflammatory responses of vertebrates and invertebrates. The galectin family is divided into three subtypes based on molecular structure; prototypes, chimera types, and tandem-repeated types. We isolated and characterized the cDNA of galectin-8 (OnGal-8) in Nile tilapia (Oreochromis niloticus). OnGal-8 consisted of a 966 bp open reading frame (ORF) that encoded a 321 amino acid protein (43.47 kDa). Homology and phylogenetic tree analysis suggested the protein was clustered with galectin-8s from other animal species and shared at least 56.8% identity with salmon galectin-8. Structurally, the amino acid sequence included two distinct N- and C- terminus carbohydrate recognition domains (CRDs) of 135 and 133 amino acids, respectively, that were connected by a 39 amino acid polypeptide linker. The N- and C-CRDs contained two conserved WG-E-I and WG-E-T motifs, suggesting they have an important role in mediating the specific interactions between OnGal-8 and saccharide moieties such as β-galactoside. The structure of OnGal-8 was characterized by a two-fold symmetric pattern of 10-and 12-stranded antiparallel ß-sheets of both N- and C-CRDs, and the peptide linker presumably formed a random coil similar to the characteristic tandem-repeat type galectin. The expression of OnGal-8 in healthy fish was highest in the skin, intestine, and brain. Experimental challenge of Nile tilapia with S. agalactiae resulted in significant up-regulation of OnGal-8in the spleen after 5 d. Our results suggest that OnGal-8 is involved in the immune response to bacterial infection.

Abstract 4136: Development of a new and sensitive detection technology for galectin cancer proteins.

Abstract The galectin protein family consists of fifteen galactoside-binding proteins that are present in all classes of life where they function as cell-cell and cell-matrix interaction mediators. Galectins are however also highly overexpressed in various processes related to cancer, including apoptosis, angiogenesis and metastasis. Galectins are therefore excellent targets for tumor diagnostics and medical prognostic purposes. Current proteomic tools, e.g. Polymerase Chain Reaction (PCR) and Enzyme-linked Immunosorbent Assay (ELISA), are based on protein abundance rather than protein activity. In contrast, we have developed a galectin detection assay that visualizes galactoside-binding, i.e. functional, galectins using PamChip® microarray technology. Firstly, antibodies directed against different galectins are efficiently immobilized on the microarray surface by the Fc-binding protein A/G. Then galectin-1 was captured from a spiked cell lysate that is added to the microarray and the unbound proteins are washed away. The galectin presence is subsequently quantified with a general fluorescent ligand that is only bound by active galectins. Several ligands based on lactose and lactosamine were designed and tested. With the best ligands it was possible to detect galectin quantities in the low nanogram range using only 1 μg or less of precious anti-galectin antibodies per measurement. These results are truly promising for the use of the newly developed galectin detection technology in a clinical setting. Citation Format: Hilde van Hattum, Rob Ruijtenbeek, Riet Hilhorst, Roland Pieters. Development of a new and sensitive detection technology for galectin cancer proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4136. doi:10.1158/1538-7445.AM2013-4136

Role of Galectin-3 in Thyroid Cancer

Copyright: © 2012 Htwe TT. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Thyroid cancer (TC) is the most common endocrine malignancy with a rising incidence. It has a wide spectrum of clinical behavior and varied therapeutic responsiveness. Among the various forms of TC, papillary thyroid cancer (PTC) accounts for 85% of all TCs. It is the most common form compared to follicular carcinoma (FCA) and other rare forms such as medullary carcinoma (MCA), anaplastic carcinoma (ANA), poorly differentiated carcinoma (PDTC), undifferentiated thyroid carcinoma (UDTC), Hurthle cell carcinoma (HCA) and Insular carcinoma (Ins). In the past, PTC and FCA were generally identified by the difference in their clinical nature and the characteristic morphological features by using the conventional way of microscopy, following the guidelines from the World Health Organization (WHO). PTC is identified by its peculiar nuclear features and the papillary pattern whereas in FCA it is from its follicular pattern. The main criterion for the diagnosis of PTC is the occurrence of enlarged, round to oval shaped overlapping ground-glass nuclei, well known as OrphanAnnie eyes. The delicate papillary pattern of growth and the presences of laminated calcifications (psammoma bodies) are also diagnostic. For the FCA it is based on its closely packed follicular pattern and its tendency in invasiveness. However, there is a difficulty in identifying one sub type of TC known as papillary thyroid carcinoma follicular variant (PTCFC). Henceforth, with the development in immunohistochemical (IHC) techniques, experiments have been performed to find out novel molecular markers for TC, where Galectin-3 (Gal-3) has been identified as the most useful tumor marker among other galactosidebinding proteins such as Gal-1 and Gal-7.

Combining Glycomimetic and Multivalent Strategies toward Designing Potent Bacterial Lectin Inhibitors

As part of ongoing activities toward the design of potent and selective ligands against galactoside-binding proteins from animal, bacterial, and plant lectins, a systematic investigation involving the synthesis and binding evaluations of a series of original β-C-galactopyranoside mimetics is described. The multivalent presentation of partly optimized candidates on various dendritic scaffolds through Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAc) has also been achieved. Biophysical investigations based on isothermal titration calorimetry (ITC) have indicated a dissociation constant in the low micromolar range for the best optimized monovalent conjugate (K(d)=37 μM). The results thus confirmed that stable C-galactosides could represent efficient synthetic glycomimetics of natural α-linked oligosaccharidic inhibitors of PA-IL lectin (Lec A) from the pathogenic Pseudomonas aeruginosa. Striking enhancements in the avidity of the glycoconjugates were also observed for tri-, hexa-, and nonavalent derivatives, among which the most potent exhibited dissociation constants below 500 nM, corresponding to a 400-fold increase in affinity compared with the β-D-Gal-O-Me used as reference. To deepen our understanding of the binding mode of the best glycomimetics involved in the recognition process, molecular modeling studies, docking calculations, and NMR diffusion measurements have been performed. Although favorable complementary interactions induced by the addition of the hydrophobic aglycon might explain the affinity enhancement, experimental determination of the size and the topology of the multivalent conjugates further supported the formation of aggregative complexes as a major multivalent binding mode. This work represents a systematic and comprehensive study towards a thorough understanding of the protein-carbohydrate interactions involved in Pseudomonas aeruginosa infection, and as such should prove useful for the development of stable and optimized anti-adhesive agents.

The role of galectins in colorectal cancer progression

Galectins constitute a family of 15 mammalian galactoside-binding proteins that share a consensus amino acid sequence in their carbohydrate binding sites. They are multi-functional molecules and are expressed widely in human tissues. Four galectins: galectin -1, -3, -4 and -8 are expressed in the human colon and rectum and their expressions show significant changes during colorectal cancer development and metastasis. These changes in galectin expression correlate with alterations in cancer cell growth, apoptosis, cell-cell and cell-matrix interactions and angiogenesis. This review summaries current knowledge of the expression and roles of these galectins in the progression of human colorectal cancer and discusses the relevance of galectins and their ligands as potential therapeutic targets for cancer treatment.

Caenorhabditis elegans Galectins LEC-6 and LEC-10 Interact with Similar Glycoconjugates in the Intestine

Galectins are a family of metazoan proteins that show binding to various β-galactoside-containing glycans. Because of a lack of proper tools, the interaction of galectins with their specific glycan ligands in the cells and tissues are largely unknown. We have investigated the localization of galectin ligands in Caenorhabditis elegans using a novel technology that relies on the high binding specificity between galectins and their endogenous ligands. Fluorescently labeled recombinant galectin fusions are found to bind to ligands located in diverse tissues including the intestine, pharynx, and the rectal valve. Consistent with their role as galactoside-binding proteins, the interaction with their ligands is inhibited by galactose or lactose. Two of the galectins, LEC-6 and LEC-10, recognize ligands that co-localize along the intestinal lumen. The ligands for LEC-6 and LEC-10 are absent in three glycosylation mutants bre-1, fut-8, and galt-1, which have been shown to be required to synthesize the Gal-β1,4-Fuc modifications of the core N-glycans unique to C. elegans and several other invertebrates. Both galectins pull down the same set of glycoproteins in a manner dependent on the presence of these carbohydrate modifications. Endogenous LEC-6 and LEC-10 are expressed in the intestinal cells, but they are localized to different subcellular compartments that do not appear to overlap with each other or with the location of their glycan targets. An altered subcellular distribution of these ligands is found in mutants lacking both galectins. These results suggest a model where LEC-6 and LEC-10 interact with glycoproteins through specific glycans to regulate their cellular fate.

Expression of galectin-3 in non-small cell lung cancer (NSCLC): Prognostic significance in adenocarcinoma

9671 Background: Expression of galectin-3, which belongs to a family of galactoside-binding proteins, has been associated with malignant transformation, cancer progression, and protection against apoptosis in several malignancies such as breast cancer. Methods: We investigated the expression of galectin-3 and its association with clinicopatholgic characteristics including disease-free survival (DFS) and overall survival (OS) in 104 patients (pts) with locally advanced NSCLC who underwent surgical resection (stage II: 47, IIIA: 40, IIIB: 10, IV: 7) using immunohistochemistry. Eighty-two pts (79%) received adjuvant treatment (chemotherapy: 9, radiotherapy: 17, both: 56). Results: High expression of galectin-3 was observed in 46 pts (44%), which was significantly associated with adenocarcinoma (p=0.001) and large primary tumor size (>4 cm) (p=0.033). With a median follow-up of 45 months (20–114 months) in survivors, 4-year DFS and OS of total pts were 31% and 43%, respectively, excluding 6 deaths from postoperative complications. High expression of galectin-3 was not associated with poor DFS (p=0.233) or OS (p=0.391). However, in pts with adenocarcinoma (36 pts) or adenosquamous carcinoma (7 pts), high expression of galectin-3 was significant independent predictor of poor DFS (p=0.021) in addition to male gender (p=0.026) and advanced stage (p=0.013). Conclusions: High expression of galectin-3 was significantly associated with poor DFS in locally advanced NSCLC pts with adenocarcioma. No significant financial relationships to disclose.

Synthesis of a phenyl thio-β-d-galactopyranoside library from 1,5-difluoro-2,4-dinitrobenzene: discovery of efficient and selective monosaccharide inhibitors of galectin-7

The galectins are a family of [small beta]-galactoside-binding proteins that have been implicated in cancer and inflammation processes. Herein, we report the synthesis of a library of 28 compounds that was tested for binding to galectins-1, -3, -7, -8N and -9N. An aromatic nucleophilic substitution reaction between 1,5-difluoro-2,4-dinitrobenzene and a galacto thiol gave 5-fluoro-2,4-dinitrophenyl 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-galactopyranoside. This versatile intermediate was then modified in a two dimensional manner: either by further substitution of the second fluoride by amines or thiols, or by reduction of the nitro groups and acylation of the resulting amines, or both. Deacetylation then gave a library of aromatic beta-galactosides that showed variable inhibitory activity against the different galectins, as shown by screening with a fluorescence-polarisation assay. Particularly efficient inhibitors were found against galectin-7, while less impressive enhancements of inhibitor affinity over methyl beta-D-galactopyranoside were found for galectin-1, -3, -8N and -9N. The best inhibitors against galectin-7 showed significantly higher affinity (K(d) as low as 140 microM) than both beta-methyl galactoside (K(d) 4.8 mM) and the unsubstituted beta-phenyl thiogalactoside (non-inhibitory). The best inhibitors against galectin-7 were poor against the other galectins and thus have potential as structurally simple and selective tools for dissecting biological functions of galectin-7.

Unlocking the secrets of galectins: a challenge at the frontier of glyco-immunology

Over the last decade, we have witnessed an explosion of information regarding the function of glycoconjugates, carbohydrate-binding proteins, and the elucidation of the sugar code. This progress has yielded not only important insights into fundamental areas of glycobiology but has also influenced other fields such as immunology and molecular medicine. A family of galactoside-binding proteins, called galectins, has emerged recently as a novel kind of bioactive molecules with powerful, immunoregulatory functions. Different members of this family have been shown to modulate positively or negatively multiple steps of the inflammatory response, such as cell-matrix interactions, cell trafficking, cell survival, cell-growth regulation, chemotaxis, and proinflammatory cytokine secretion. To introduce a comprehensive overview of these new advances, here we will explore the molecular mechanisms and biochemical pathways involved in these functions. We will also examine the role of these proteins in the modulation of different pathological processes, such as chronic inflammation, autoimmunity, infection, allergic reactions, and tumor spreading. Understanding the intimate mechanisms involved in galectin functions will help to delineate selective and novel strategies for disease intervention and diagnosis.

Lectin-mediated drug targeting: selection of valency, sugar type (Gal/Lac), and spacer length for cluster glycosides as parameters to distinguish ligand binding to C-type asialoglycoprotein receptors and galectins.

Common oligosaccharides of cellular glycoconjugates are ligands for more than one type of endogenous lectin. Overlapping specificities to beta-galactosides of C-type lectins and galectins can reduce target selectivity of carbohydrate-ligand-dependent drug targeting. The purpose of this study is to explore distinct features of ligand presentation and structure for design of cluster glycosides to distinguish between asialoglycoprotein-specific (C-type) lectins and galectins. Extent of binding of labeled sugar receptors to two types of matrix-immobilized (neo)glycoproteins and to cells was evaluated in the absence and presence of competitive inhibitors. This panel comprised synthetic mono-, bi-, and trivalent glycosides with two spacer lengths and galactose or lactose as ligand part. In contrast to C-type lectins of hepatocytes and macrophages, bi- and trivalent glycosides do not yield a notable glycoside cluster effect for galectins-1 and -3. Also, these Ca2+-independent galactoside-binding proteins prefer to home in on lactose-bearing glycosides relative to galactose as ligand, while spacer length requirements were rather similar. Trivalent cluster glycosides with Gal/GalNAc as ligand markedly distinguish between C-type lectins and galectins. Undesired side reactivities to galectins for C-type lectin drug delivery will thus be minimal.

Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties.

Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-beta-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)(2)-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential of dendrimers for applications as lectin-targeting device, as attested by these observations.

Galectin-1 and galectin-3 expression in human bladder transitional-cell carcinomas.

Galectin-1 and galectin-3 are galactoside-binding proteins involved in different steps of tumor progression and potential targets for therapy. We have investigated the expression of these galectins in 38 human bladder transitional-cell carcinomas of different histological grade and clinical stage and in 5 normal urothelium samples. Galectin-1 mRNA levels were highly increased in most high-grade tumors compared with normal bladder or low-grade tumors. Western blot and immuno-histochemical analysis of normal and neoplastic tissues revealed a higher content of galectin-1 in tumors. Galectin-3 mRNA levels were also increased in most tumors compared with normal urothelium, but levels were comparable among tumors of different histological grade.