Publications by authors named "Alejandro J Cagnoni"

21 Publications

  • Page 1 of 1

Structural insights in galectin-1-glycan recognition: Relevance of the glycosidic linkage and the N-acetylation pattern of sugar moieties.

Bioorg Med Chem 2021 Aug 14;44:116309. Epub 2021 Jul 14.

Laboratorio de Bioquímica, Departamento de Biociencias, Facultad de Química, UdelaR, Gral. Flores, 2124, 11800 Montevideo, Uruguay. Electronic address:

Galectins, soluble lectins widely expressed intra- and extracellularly in different cell types, play major roles in deciphering the cellular glycocode. Galectin-1 (Gal-1), a prototype member of this family, presents a carbohydrate recognition domain (CRD) with specific affinity for β-galactosides such as N-acetyllactosamine (β-d-Galp-(1 → 4)-d-GlcpNAc), and mediate numerous physiological and pathological processes. In this work, Gal-1 binding affinity for β-(1 → 6) galactosides, including β-d-Galp-(1 → 6)-β-d-GlcpNAc-(1 → 4)-d-GlcpNAc was evaluated, and their performance was compared to that of β-(1 → 4) and β-(1 → 3) galactosides. To this end, the trisaccharide β-d-Galp-(1 → 6)-β-d-GlcpNAc-(1 → 4)-d-GlcpNAc was enzymatically synthesized, purified and structurally characterized. To evaluate the affinity of Gal-1 for the galactosides, competitive solid phase assays (SPA) and isothermal titration calorimetry (ITC) studies were carried out. The experimental dissociation constants and binding energies obtained were compared to those calculated by molecular docking. These analyses evidenced the critical role of the glycosidic linkage between the terminal galactopyranoside residue and the adjacent monosaccharide, as galactosides bearing β-(1 → 6) glycosidic linkages showed dissociation constants six- and seven-fold higher than those involving β-(1 → 4) and β-(1 → 3) linkages, respectively. Moreover, docking experiments revealed the presence of hydrogen bond interactions between the N-acetyl group of the glucosaminopyranose moiety of the evaluated galactosides and specific amino acid residues of Gal-1, relevant for galectin-glycan affinity. Noticeably, the binding free energies (ΔG) derived from the molecular docking were in good agreement with experimental values determined by ITC measurements (ΔG), evidencing a good correlation between theoretical and experimental approaches, which validates the in silico simulations and constitutes an important tool for the rational design of future optimized ligands.
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http://dx.doi.org/10.1016/j.bmc.2021.116309DOI Listing
August 2021

Control of intestinal inflammation by glycosylation-dependent lectin-driven immunoregulatory circuits.

Sci Adv 2021 Jun 18;7(25). Epub 2021 Jun 18.

Laboratorio de Glicómica Funcional y Molecular, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), 1428 Ciudad de Buenos Aires, Argentina.

Diverse immunoregulatory circuits operate to preserve intestinal homeostasis and prevent inflammation. Galectin-1 (Gal1), a β-galactoside-binding protein, promotes homeostasis by reprogramming innate and adaptive immunity. Here, we identify a glycosylation-dependent "on-off" circuit driven by Gal1 and its glycosylated ligands that controls intestinal immunopathology by targeting activated CD8 T cells and shaping the cytokine profile. In patients with inflammatory bowel disease (IBD), augmented Gal1 was associated with dysregulated expression of core 2 β6--acetylglucosaminyltransferase 1 () and α(2,6)-sialyltransferase 1 (), glycosyltransferases responsible for creating or masking Gal1 ligands. Mice lacking Gal1 exhibited exacerbated colitis and augmented mucosal CD8 T cell activation in response to 2,4,6-trinitrobenzenesulfonic acid; this phenotype was partially ameliorated by treatment with recombinant Gal1. While mice exhibited aggravated colitis, mice showed attenuated inflammation. These effects were associated with intrinsic T cell glycosylation. Thus, Gal1 and its glycosylated ligands act to preserve intestinal homeostasis by recalibrating T cell immunity.
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http://dx.doi.org/10.1126/sciadv.abf8630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8213219PMC
June 2021

The Tn antigen promotes lung tumor growth by fostering immunosuppression and angiogenesis via interaction with Macrophage Galactose-type lectin 2 (MGL2).

Cancer Lett 2021 Oct 16;518:72-81. Epub 2021 Jun 16.

Laboratorio de Inmunomodulación y Desarrollo de Vacunas, Departamento de Inmunobiología, Facultad de Medicina, Universidad de La República, Montevideo, Uruguay. Electronic address:

Tn is a tumor-associated carbohydrate antigen that constitutes both a diagnostic tool and an immunotherapeutic target. It originates from interruption of the mucin O-glycosylation pathway through defects involving, at least in part, alterations in core-1 synthase activity, which is highly dependent on Cosmc, a folding chaperone. Tn antigen is recognized by the Macrophage Galactose-type Lectin (MGL), a C-type lectin receptor present on dendritic cells and macrophages. Specific interactions between Tn and MGL shape anti-tumoral immune responses by regulating several innate and adaptive immune cell programs. In this work, we generated and characterized a variant of the lung cancer murine cell line LL/2 that expresses Tn by mutation of the Cosmc chaperone gene (Tn LL/2). We confirmed Tn expression by lectin glycophenotyping and specific anti-Tn antibodies, verified abrogation of T-synthase activity in these cells, and confirmed its recognition by the murine MGL2 receptor. Interestingly, Tn LL/2 cells were more aggressive in vivo, resulting in larger and highly vascularized tumors than those generated from wild type Tn LL/2 cells. In addition, Tn tumors exhibited an increase in CD11c F4/80 cells with high expression of MGL2, together with an augmented expression of IL-10 in infiltrating CD4 and CD8 T cells. Importantly, this immunosuppressive microenvironment was dependent on the presence of MGL2 cells, since depletion of these cells abrogated tumor growth, vascularization and recruitment of IL-10 T cells. Altogether, our results suggest that expression of Tn in tumor cells and its interaction with MGL2-expressing CD11cF4/80 cells promote immunosuppression and angiogenesis, thus favoring tumor progression.
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http://dx.doi.org/10.1016/j.canlet.2021.06.012DOI Listing
October 2021

Galectin-1 fosters an immunosuppressive microenvironment in colorectal cancer by reprogramming CD8 regulatory T cells.

Proc Natl Acad Sci U S A 2021 May;118(21)

Laboratorio de Inmunopatología, Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, C1428ADN Buenos Aires, Argentina;

Colorectal cancer (CRC) represents the third most common malignancy and the second leading cause of cancer-related deaths worldwide. Although immunotherapy has taken center stage in mainstream oncology, it has shown limited clinical efficacy in CRC, generating an urgent need for discovery of new biomarkers and potential therapeutic targets. Galectin-1 (Gal-1), an endogenous glycan-binding protein, induces tolerogenic programs and contributes to tumor cell evasion of immune responses. Here, we investigated the relevance of Gal-1 in CRC and explored its modulatory activity within the CD8 regulatory T cell (Treg) compartment. Mice lacking Gal-1 ( ) developed a lower number of tumors and showed a decreased frequency of a particular population of CD8CD122PD-1 Tregs in the azoxymethane-dextran sodium sulfate model of colitis-associated CRC. Moreover, silencing of tumor-derived Gal-1 in the syngeneic CT26 CRC model resulted in reduced number and attenuated immunosuppressive capacity of CD8CD122PD-1 Tregs, leading to slower tumor growth. Moreover, stromal Gal-1 also influenced the fitness of CD8 Tregs, highlighting the contribution of both tumor and stromal-derived Gal-1 to this immunoregulatory effect. Finally, bioinformatic analysis of a colorectal adenocarcinoma from The Cancer Genome Atlas dataset revealed a particular signature characterized by high CD8 Treg score and elevated Gal-1 expression, which delineates poor prognosis in human CRC. Our findings identify CD8CD122PD-1 Tregs as a target of the immunoregulatory activity of Gal-1, suggesting a potential immunotherapeutic strategy for the treatment of CRC.
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http://dx.doi.org/10.1073/pnas.2102950118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8166155PMC
May 2021

Crystal structures of peanut lectin in the presence of synthetic β-N- and β-S-galactosides disclose evidence for the recognition of different glycomimetic ligands.

Acta Crystallogr D Struct Biol 2020 Nov 13;76(Pt 11):1080-1091. Epub 2020 Oct 13.

Fundación Instituto Leloir, IIBBA-CONICET, Avenida Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina.

Carbohydrate-lectin interactions are involved in important cellular recognition processes, including viral and bacterial infections, inflammation and tumor metastasis. Hence, structural studies of lectin-synthetic glycan complexes are essential for understanding lectin-recognition processes and for the further design of promising chemotherapeutics that interfere with sugar-lectin interactions. Plant lectins are excellent models for the study of the molecular-recognition process. Among them, peanut lectin (PNA) is highly relevant in the field of glycobiology because of its specificity for β-galactosides, showing high affinity towards the Thomsen-Friedenreich antigen, a well known tumor-associated carbohydrate antigen. Given this specificity, PNA is one of the most frequently used molecular probes for the recognition of tumor cell-surface O-glycans. Thus, it has been extensively used in glycobiology for inhibition studies with a variety of β-galactoside and β-lactoside ligands. Here, crystal structures of PNA are reported in complex with six novel synthetic hydrolytically stable β-N- and β-S-galactosides. These complexes disclosed key molecular-binding interactions of the different sugars with PNA at the atomic level, revealing the roles of specific water molecules in protein-ligand recognition. Furthermore, binding-affinity studies by isothermal titration calorimetry showed dissociation-constant values in the micromolar range, as well as a positive multivalency effect in terms of affinity in the case of the divalent compounds. Taken together, this work provides a qualitative structural rationale for the upcoming synthesis of optimized glycoclusters designed for the study of lectin-mediated biological processes. The understanding of the recognition of β-N- and β-S-galactosides by PNA represents a benchmark in protein-carbohydrate interactions since they are novel synthetic ligands that do not belong to the family of O-linked glycosides.
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http://dx.doi.org/10.1107/S2059798320012371DOI Listing
November 2020

Synthesis of N-acetylglucosamine and N-acetylallosamine resorcinarene-based multivalent β-thio-glycoclusters: unexpected affinity of N-acetylallosamine ligands towards Wheat Germ Agglutinin.

Org Biomol Chem 2020 Sep 28;18(35):6853-6865. Epub 2020 Aug 28.

Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Química Orgánica, Intendente Güiraldes 2160 (C1428EHA), Buenos Aires, Argentina.

Herein, we report the synthesis of calix[4]resorcinarene-based multivalent ligands bearing β-S-GlcNAc and β-S-AllNAc recognition elements. A clickable β-S-AllNAc derivative was successfully prepared from a β-thioalkynyl GlcNAc precursor, making use of a 2,3-oxazoline intermediate, easily formed by intramolecular displacement of a triflate group located at the 3-position by the 2-N-acetate group. By reaction of these alkynyl-functionalized derivatives with an octaazido-calix[4]resorcinarene macrocycle having undecyl chains, two octavalent glycoclusters exposing the epimeric N-acetylhexosamines were obtained. In addition, a related calix[4]resorcinarene-based glycocluster having methyl groups instead of undecyl chains and β-S-GlcNAc residues was also synthesized. After an initial evaluation of the interaction of the undecyl-functionalized β-S-GlcNAc octavalent derivative with Wheat Germ Agglutinin (WGA) by a turbidimetry experiment, the interaction of the three synthesized glycoclusters towards WGA was studied by Isothermal Titration Calorimetry. The results showed a favorable effect due to the presence of the undecyl chains in terms of affinity. Surprisingly, the β-S-AllNAc octavalent compound showed the highest affinity among the evaluated glycoclusters, showing for the first time that WGA interacts with β-AllNAc-bearing ligands. Molecular docking studies of β-AllNAc with WGA in comparison with β-GlcNAc contributed to the understanding of the atomic interactions responsible for this unexpected affinity.
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http://dx.doi.org/10.1039/d0ob01498bDOI Listing
September 2020

Full-length galectin-8 and separate carbohydrate recognition domains: the whole is greater than the sum of its parts?

Biochem Soc Trans 2020 06;48(3):1255-1268

Instituto de Química y Fisicoquímica Biológicas Prof. Dr. Alejandro Paladini (UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.

Galectin-8 (Gal-8) is a tandem-repeat type galectin with affinity for β-galactosides, bearing two carbohydrate recognition domains (CRD) connected by a linker peptide. The N- and C-terminal domains (Gal-8N and Gal-8C) share 35% homology, and their glycan ligand specificity is notably dissimilar: while Gal-8N shows strong affinity for α(2-3)-sialylated oligosaccharides, Gal-8C has higher affinity for non-sialylated oligosaccharides, including poly-N-acetyllactosamine and/ or A and B blood group structures. Particularly relevant for understanding the biological role of this lectin, full-length Gal-8 can bind cell surface glycoconjugates with broader affinity than the isolated Gal-8N and Gal-8C domains, a trait also described for other tandem-repeat galectins. Herein, we aim to discuss the potential use of separate CRDs in modelling tandem-repeat galectin-8 and its biological functions. For this purpose, we will cover several aspects of the structure-function relationship of this protein including crystallographic structures, glycan specificity, cell function and biological roles, with the ultimate goal of understanding the potential role of each CRD in predicting full-length Gal-8 involvement in relevant biological processes.
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http://dx.doi.org/10.1042/BST20200311DOI Listing
June 2020

An adipose tissue galectin controls endothelial cell function via preferential recognition of 3-fucosylated glycans.

FASEB J 2020 01 26;34(1):735-753. Epub 2019 Nov 26.

Laboratorio de Glicómica Funcional y Molecular, Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (IBYME-CONICET), Buenos Aires, Argentina.

Upon overnutrition, adipocytes activate a homeostatic program to adjust anabolic pressure. An inflammatory response enables adipose tissue (AT) expansion with concomitant enlargement of its capillary network, and reduces energy storage by increasing insulin resistance. Galectin-12 (Gal-12), an endogenous lectin preferentially expressed in AT, plays a key role in adipocyte differentiation, lipolysis, and glucose homeostasis. Here, we reveal biochemical and biophysical determinants of Gal-12 structure, including its preferential recognition of 3-fucosylated structures, a unique feature among members of the galectin family. Furthermore, we identify a previously unanticipated role for this lectin in the regulation of angiogenesis within AT. Gal-12 showed preferential localization within the inner side of lipid droplets, and its expression was upregulated under hypoxic conditions. Through glycosylation-dependent binding to endothelial cells, Gal-12 promoted in vitro angiogenesis. Moreover, analysis of in vivo AT vasculature showed reduced vascular networks in Gal-12-deficient (Lgals12) compared to wild-type mice, supporting a role for this lectin in AT angiogenesis. In conclusion, this study unveils biochemical, topological, and functional features of a hypoxia-regulated galectin in AT, which modulates endothelial cell function through recognition of 3-fucosylated glycans. Thus, glycosylation-dependent programs may control AT homeostasis by modulating endothelial cell biology with critical implications in metabolic disorders and inflammation.
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http://dx.doi.org/10.1096/fj.201901817RDOI Listing
January 2020

Dual knockdown of Galectin-8 and its glycosylated ligand, the activated leukocyte cell adhesion molecule (ALCAM/CD166), synergistically delays in vivo breast cancer growth.

Biochim Biophys Acta Mol Cell Res 2019 08 21;1866(8):1338-1352. Epub 2019 Mar 21.

Instituto de Química y Fisicoquímica Biológicas Prof. Dr. Alejandro Paladini (CONICET-UBA), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina. Electronic address:

Galectin-8 (Gal-8), a 'tandem-repeat'-type galectin, has been described as a modulator of cellular functions including adhesion, spreading, growth arrest, apoptosis, pathogen recognition, autophagy, and immunomodulation. We have previously shown that activated leukocyte cell adhesion molecule (ALCAM), also known as CD166, serves as a receptor for endogenous Gal-8. ALCAM is a member of the immunoglobulin superfamily involved in cell-cell adhesion through homophilic (ALCAM-ALCAM) and heterophilic (i.e. ALCAM-CD6) interactions in different tissues. Here we investigated the physiologic relevance of ALCAM-Gal-8 association and glycosylation-dependent mechanisms governing these interactions. We found that silencing of ALCAM in MDA-MB-231 triple negative breast cancer cells decreases cell adhesion and migration onto Gal-8-coated surfaces in a glycan-dependent fashion. Remarkably, either Gal-8 or ALCAM silencing also disrupted cell-cell adhesion, and led to reduced tumor growth in a murine model of triple negative breast cancer. Moreover, structural characterization of endogenous ALCAM N-glycosylation showed abundant permissive structures for Gal-8 binding. Importantly, we also found that cell sialylation controls Gal-8-mediated cell adhesion. Altogether, these findings demonstrate a central role of either ALCAM or Gal-8 (or both) in controlling triple negative breast cancer.
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http://dx.doi.org/10.1016/j.bbamcr.2019.03.010DOI Listing
August 2019

Enzymatic synthesis of non-natural trisaccharides and galactosides; Insights of their interaction with galectins as a function of their structure.

Carbohydr Res 2019 Jan 3;472:1-15. Epub 2018 Nov 3.

Laboratorio de Bioquímica, Departamento de Biociencias, Facultad de Química, UdelaR, Gral. Flores, 2124, Montevideo, Uruguay. Electronic address:

Galectins are a family of carbohydrate-recognizing proteins that by interacting with specific glycoepitopes can mediate important biological processes, including immune cell homeostasis and activation of tolerogenic circuits. Among the different members of this family, Galectin 1 and 3 have shown pro-tumorigenic effects, being overexpressed in numerous neoplasic diseases, proving to be relevant in tumor immune escape, tumor progression and resistance to drug-induced apoptosis. Thus, generation of specific glycosides that could inhibit their pro-tumorigenic ability by blocking their carbohydrate recognition domain is one of the current major challenges in the field. Considering that galectin-ligand binding strength is closely related to the ligand structure, analysis of this relationship provides valuable information for rational design of high-affinity ligands that could work as effective galectin inhibitors. Taking profit of the ability of glycosidases to catalyze transglycosylation reactions we achieved the enzymatic synthesis of β-d-Galp-(1 → 6)-β-d-Galp-(1 → 4)-d-Glcp(2), a mixture of β-d-Galp-(1 → 6)-β-d-Glcp-(1 → 4)-d-Glcp(5) and β-d-Galp-(1 → 3)-β-d-Glcp-(1 → 4)-d-Glcp(6), and finally benzyl β-d-galactopyranoside (9), with reaction yields between 16 and 27%. All the galactosides were purified, and characterized using H and C nuclear magnetic resonance spectroscopy. Docking results performed between the synthesized compounds and human Galectin 1 (hGal-1) and human Galectin 3 (hGal-3) showed that the replacement of a glucose moiety linked to the terminal galactose with a galactose moiety, decreases the affinity for these galectins. Moreover, regarding the interglycosidic bond the most favorable β-Gal linkage seems to be β(1 → 4) followed by β(1 → 3) and β(1 → 6) for hGal-1, and β(1 → 4) followed by β(1 → 6) and β(1 → 3) for hGal-3. These results were in accordance with the IC50 values obtained with in vitro solid phase inhibition assays. Therefore, docking results obtained in this work proved to be a very good approximation for predicting binding affinity of novel galactosides.
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http://dx.doi.org/10.1016/j.carres.2018.10.011DOI Listing
January 2019

Glycosylation-dependent galectin-receptor interactions promote infection.

Proc Natl Acad Sci U S A 2018 06 11;115(26):E6000-E6009. Epub 2018 Jun 11.

Laboratorio de Inmunopatología, Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, C1428ADN Buenos Aires, Argentina;

() constitutes the most prevalent sexually transmitted bacterium worldwide. Chlamydial infections can lead to severe clinical sequelae including pelvic inflammatory disease, ectopic pregnancy, and tubal infertility. As an obligate intracellular pathogen, has evolved multiple strategies to promote adhesion and invasion of host cells, including those involving both bacterial and host glycans. Here, we show that galectin-1 (Gal1), an endogenous lectin widely expressed in female and male genital tracts, promotes infection. Through glycosylation-dependent mechanisms involving recognition of bacterial glycoproteins and -glycosylated host cell receptors, Gal1 enhanced attachment to cervical epithelial cells. Exposure to Gal1, mainly in its dimeric form, facilitated bacterial entry and increased the number of infected cells by favoring - and -host cell interactions. These effects were substantiated in vivo in mice lacking Gal1 or complex β1-6-branched -glycans. Thus, disrupting Gal1--glycan interactions may limit the severity of chlamydial infection by inhibiting bacterial invasion of host cells.
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http://dx.doi.org/10.1073/pnas.1802188115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042088PMC
June 2018

Multivalent sialylation of β-thio-glycoclusters by Trypanosoma cruzi trans sialidase and analysis by high performance anion exchange chromatography.

Glycoconj J 2016 10 15;33(5):809-18. Epub 2016 Jun 15.

CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, 1428, Buenos Aires, Argentina.

The synthesis of multivalent sialylated glycoclusters is herein addressed by a chemoenzymatic approach using the trans-sialidase of Trypanosoma cruzi (TcTS). Multivalent β-thio-galactopyranosides and β-thio-lactosides were used as acceptor substrates and 3'-sialyllactose as the sialic acid donor. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was shown to be an excellent technique for the analysis of the reaction products. Different eluting conditions were optimized to allow the simultaneous resolution of the sialylated species, as well as their neutral precursors. The TcTS efficiently transferred sialyl residues to di, tri, tetra and octa β-thiogalactosides. In the case of an octavalent thiolactoside, up to six polysialylated compounds could be resolved. Preparative sialylation reactions were performed using the tetravalent and octavalent acceptor substrates. The main sialylated derivatives could be unequivocally assigned by MALDI mass spectrometry. Inhibition of the transfer to the natural substrate, N-acetyllactosamine, was also studied. The octalactoside caused 82 % inhibition of sialic acid transfer when we used equimolar concentrations of donor, acceptor and inhibitor.
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http://dx.doi.org/10.1007/s10719-016-9676-0DOI Listing
October 2016

Turning-Off Signaling by Siglecs, Selectins, and Galectins: Chemical Inhibition of Glycan-Dependent Interactions in Cancer.

Front Oncol 2016 13;6:109. Epub 2016 May 13.

Laboratorio de Glicómica Funcional y Molecular, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) , Buenos Aires , Argentina.

Aberrant glycosylation, a common feature associated with malignancy, has been implicated in important events during cancer progression. Our understanding of the role of glycans in cancer has grown exponentially in the last few years, concurrent with important advances in glycomics and glycoproteomic technologies, paving the way for the validation of a number of glycan structures as potential glycobiomarkers. However, the molecular bases underlying cancer-associated glycan modifications are still far from understood. Glycans exhibit a natural heterogeneity, crucial for their diverse functional roles as specific carriers of biologically relevant information. This information is decoded by families of proteins named lectins, including sialic acid-binding immunoglobulin (Ig)-like lectins (siglecs), C-type lectin receptors (CLRs), and galectins. Siglecs are primarily expressed on the surface of immune cells and differentially control innate and adaptive immune responses. Among CLRs, selectins are a family of cell adhesion molecules that mediate interactions between cancer cells and platelets, leukocytes, and endothelial cells, thus facilitating tumor cell invasion and metastasis. Galectins, a family of soluble proteins that bind β-galactoside-containing glycans, have been implicated in diverse events associated with cancer biology such as apoptosis, homotypic cell aggregation, angiogenesis, cell migration, and tumor-immune escape. Consequently, individual members of these lectin families have become promising targets for the design of novel anticancer therapies. During the past decade, a number of inhibitors of lectin-glycan interactions have been developed including small-molecule inhibitors, multivalent saccharide ligands, and more recently peptides and peptidomimetics have offered alternatives for tackling tumor progression. In this article, we review the current status of the discovery and development of chemical lectin inhibitors and discuss novel strategies to limit cancer progression by targeting lectin-glycan interactions.
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http://dx.doi.org/10.3389/fonc.2016.00109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4865499PMC
May 2016

Glycosylation-dependent binding of galectin-8 to activated leukocyte cell adhesion molecule (ALCAM/CD166) promotes its surface segregation on breast cancer cells.

Biochim Biophys Acta 2016 10 26;1860(10):2255-68. Epub 2016 Apr 26.

Institute of Biochemistry and Biophysics (IQUIFIB), UBA-CONICET, Biological Chemistry Department, School of Pharmacy and Biochemistry, UBA, Buenos Aires, Argentina. Electronic address:

Background: We previously demonstrated that the activated leukocyte cell adhesion molecule (ALCAM/CD166) can interact with galectin-8 (Gal-8) in endothelial cells. ALCAM is a member of the immunoglobulin superfamily that promotes homophilic and heterophilic cell-cell interactions. Gal-8 is a "tandem-repeat"-type galectin, known as a matricellular protein involved in cell adhesion. Here, we analyzed the physical interaction between both molecules in breast cancer cells and the functional relevance of this phenomenon.

Methods: We performed binding assays by surface plasmon resonance to study the interaction between Gal-8 and the recombinant glycosylated ALCAM ectodomain or endogenous ALCAM from MDA-MB-231 breast cancer cells. We also analyzed the binding of ALCAM-silenced or control breast cancer cells to immobilized Gal-8 by SPR. In internalization assays, we evaluated the influence of Gal-8 on ALCAM surface localization.

Results: We showed that recombinant glycosylated ALCAM and endogenous ALCAM from breast carcinoma cells physically interacted with Gal-8 in a glycosylation-dependent fashion displaying a differential behavior compared to non-glycosylated ALCAM. Moreover, ALCAM-silenced breast cancer cells exhibited reduced binding to Gal-8 relative to control cells. Importantly, exogenously added Gal-8 provoked ALCAM segregation, probably trapping this adhesion molecule at the surface of breast cancer cells.

Conclusions: Our data indicate that Gal-8 interacts with ALCAM at the surface of breast cancer cells through glycosylation-dependent mechanisms.

General Significance: A novel heterophilic interaction between ALCAM and Gal-8 is demonstrated here, suggesting its physiologic relevance in the biology of breast cancer cells.
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http://dx.doi.org/10.1016/j.bbagen.2016.04.019DOI Listing
October 2016

Re-wiring regulatory cell networks in immunity by galectin-glycan interactions.

FEBS Lett 2015 Nov 6;589(22):3407-18. Epub 2015 Sep 6.

Laboratory of Immunopathology and Functional Glycomics, Institute of Biology and Experimental Medicine (IBYME), CONICET, C1428 Buenos Aires, Argentina; School of Exact and Natural Sciences, University of Buenos Aires, C1428 Buenos Aires, Argentina. Electronic address:

Programs that control immune cell homeostasis are orchestrated through the coordinated action of a number of regulatory cell populations, including regulatory T cells, regulatory B cells, myeloid-derived suppressor cells, alternatively-activated macrophages and tolerogenic dendritic cells. These regulatory cell populations can prevent harmful inflammation following completion of protective responses and thwart the development of autoimmune pathology. However, they also have a detrimental role in cancer by favoring escape from immune surveillance. One of the hallmarks of regulatory cells is their remarkable plasticity as they can be positively or negatively modulated by a plethora of cytokines, growth factors and co-stimulatory signals that tailor their differentiation, stability and survival. Here we focus on the emerging roles of galectins, a family of highly conserved glycan-binding proteins in regulating the fate and function of regulatory immune cell populations, both of lymphoid and myeloid origins. Given the broad distribution of circulating and tissue-specific galectins, understanding the relevance of lectin-glycan interactions in shaping regulatory cell compartments will contribute to the design of novel therapeutic strategies aimed at modulating their function in a broad range of immunological disorders.
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http://dx.doi.org/10.1016/j.febslet.2015.08.037DOI Listing
November 2015

Synthesis of divalent ligands of β-thio- and β-N-galactopyranosides and related lactosides and their evaluation as substrates and inhibitors of Trypanosoma cruzi trans-sialidase.

Beilstein J Org Chem 2014 19;10:3073-3086. Epub 2014 Dec 19.

CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, 1428 Buenos Aires, Argentina.

In this work we describe the synthesis of mono- and divalent β-N- and β-S-galactopyranosides and related lactosides built on sugar scaffolds and their evaluation as substrates and inhibitors of the Trypanosoma cruzi trans-sialidase (TcTS). This enzyme catalyzes the transfer of sialic acid from an oligosaccharidic donor in the host, to parasite βGalp terminal units and it has been demonstrated that it plays an important role in the infection. Herein, the enzyme was also tested as a tool for the chemoenzymatic synthesis of sialic acid containing glycoclusters. The transfer reaction of sialic acid was performed using a recombinant TcTS and 3'-sialyllactose as sialic acid donor, in the presence of the acceptor having βGalp non reducing ends. The products were analyzed by high performance anion exchange chromatography with pulse amperometric detection (HPAEC-PAD). The ability of the different S-linked and N-linked glycosides to inhibit the sialic acid transfer reaction from 3'-sialyllactose to the natural substrate N-acetyllactosamine, was also studied. Most of the substrates behaved as good acceptors and moderate competitive inhibitors. A di-N-lactoside showed to be the strongest competitive inhibitor among the compounds tested (70% inhibition at equimolar concentration). The usefulness of the enzymatic trans-sialylation for the preparation of sialylated ligands was assessed by performing a preparative sialylation of a divalent substrate, which afforded the monosialylated compound as main product, together with the disialylated glycocluster.
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http://dx.doi.org/10.3762/bjoc.10.324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311708PMC
February 2015

Design and synthesis of hydrolytically stable multivalent ligands bearing thiodigalactoside analogues for peanut lectin and human galectin-3 binding.

J Org Chem 2014 Jul 25;79(14):6456-67. Epub 2014 Jun 25.

CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires , Pabellón 2, Ciudad Universitaria 1428 Buenos Aires, Argentina.

Herein, we describe the design and synthesis of a novel family of hydrolytically stable glycoclusters bearing thiodigalactoside (TDG) analogues as recognition elements of β-galactoside binding lectins. The TDG analogue was synthesized by thioglycosylation of a 6-S-acetyl-α-D-glucosyl bromide with the isothiouronium salt of 2,3,4,6-tetra-O-acetyl-β-D-galactose. Further propargylation of the TDG analogue allowed the coupling to azido-functionalized oligosaccharide scaffolds through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) under microwave activation. The final mono-, di-, and tetravalent ligands were resistant to enzymatic hydrolisis by Escherichia coli β-galactosidase. Binding affinities to peanut agglutinin and human galectin-3 were measured by isothermal titration calorimetry which showed K(a) constants in the micromolar range as well as a multivalent effect. Monovalent ligand exhibited a binding affinity higher than that of thiodigalactoside. Docking studies performed with a model ligand on both β-galactoside binding lectins showed additional interactions between the triazole ring and lectin amino acid residues, suggesting a positive effect of this aromatic residue on the biological activity.
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http://dx.doi.org/10.1021/jo500883vDOI Listing
July 2014

Synthesis and biological activity of divalent ligands based on 3-deoxy-4-thiolactose, an isosteric analogue of lactose.

Org Biomol Chem 2013 Sep;11(33):5500-11

CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II, Ciudad Universitaria, 1428, Buenos Aires, Argentina.

We report here the synthesis of divalent ligands containing 3-deoxy-4-thiolactose. This thiodisaccharide has been synthesized using the Michael addition of β-1-thiogalactose to the α,β-unsaturated system of sugar-derived dihydropyranones, followed by the reduction of the remaining carbonyl group. We were able to control the configuration (S) of the stereocenter linked to sulfur (C-4) of the reducing end by conducting the thioglycosylation at high temperature or by isomerization during the reduction of the 2-ulose thiodisaccharide with NaBH4/THF. The energy profile for this reaction on a model compound was calculated. The anomeric position of the 3-deoxy-4-thiolactose was functionalized with a terminal alkyne, which was coupled to azide-containing sugar scaffolds through CuAAC reaction to afford mono- and divalent ligands. The final products were competitive inhibitors of E. coli β-galactosidase in the micromolar range. Their binding affinities to peanut agglutinin (PNA) were determined by isothermal calorimetry, which showed a clear decrease in the Ka values for monovalent derivatives compared to lactose. This report contributes to establishing the role of a particular hydroxyl group of lactose in sugar-protein recognition processes.
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http://dx.doi.org/10.1039/c3ob41074aDOI Listing
September 2013

Escherichia coli β-galactosidase inhibitors through modifications at the aglyconic moiety: experimental evidence of conformational distortion in the molecular recognition process.

Chemistry 2013 Mar 19;19(13):4262-70. Epub 2013 Feb 19.

Chemical and Physical Biology, Centro de Investigationes Biològicas, CSIC, Ramiro de Maetzu 9, 28040 Madrid, Spain.

Herein, we describe the use of thioglycosides as glycosidase inhibitors by employing novel modifications at the reducing end of these glycomimetics. The inhibitors display a basic galactopyranosyl unit (1→4)-bonded to a 3-deoxy-4-thiopentopyranose moiety. The molecular basis of the observed inhibition has been studied by using a combination of NMR spectroscopy and molecular modeling techniques. It is demonstrated that these molecules are not recognized by Escherichia coli β-galactosidase in their ground-state conformation, with a conformational selection process taking place. In fact, the observed conformational distortion depends on the chemical nature of the compounds and results from the rotation around the glycosidic linkage (variation of Φ or Ψ) or from the deformation of the six-membered ring of the pentopyranose. The bound conformations of the ligand are adapted in the enzymatic pocket with a variety of hydrogen-bond, van der Waals, and stacking interactions.
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http://dx.doi.org/10.1002/chem.201203673DOI Listing
March 2013

Synthesis of multivalent glycoclusters from 1-thio-β-D-galactose and their inhibitory activity against the β-galactosidase from E. coli.

J Org Chem 2011 May 8;76(9):3064-77. Epub 2011 Apr 8.

CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.

The synthesis of multivalent glycoclusters, designed to be compatible with biological systems, is reported. A variety of 1-thio-β-D-galactosides linked to a terminal triple bond through oligoethyleneglycol chains of variable lengths has been synthesized. Also, azide-containing oligosaccharide scaffolds were prepared from trehalose, maltose, and maltotriose by direct azidation with NaN(3)/PPh(3)/CBr(4). Click reaction between the thiogalactoside residues and the azide scaffolds under microwave irradiation afforded a family of glycoclusters containing 1 to 4 residues of 1-thio-β-D-galactose. The yields went from moderate to excellent, depending on the valency of the desired product. Deacetylation with Et(3)N/MeOH/H(2)O led to the final products. Complete characterization of the products was performed by NMR spectroscopy and HR-MS techniques. Their activities as inhibitors of β-galactosidase from E. coli were determined by using the Lineweaver-Burk method. The use of hydrophilic carbohydrate scaffolds for the synthesis of multivalent galactosides represents an interesting approach to improve their pharmacokinetics and bioavailability. In addition, the presence of the thioglycosidic bond will improve their stability in biological fluids.
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http://dx.doi.org/10.1021/jo102421eDOI Listing
May 2011

Synthesis of pentopyranosyl-containing thiodisaccharides. Inhibitory activity against beta-glycosidases.

Bioorg Med Chem 2009 Sep 26;17(17):6203-12. Epub 2009 Jul 26.

CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, 1428-Buenos Aires, Argentina.

Beta-(1-->4)-thiodisaccharides formed by a pentopyranose unit as reducing or non reducing end have been synthesized using a sugar enone derived from a hexose or pentose as Michael acceptor of a 1-thiopentopyranose or 1-thiohexopyranose derivatives. Thus, 2-propyl per-O-acetyl-3-deoxy-4-S-(beta-D-Xylp)-4-thiohexopyranosid-2-ulose (3) and benzyl per-O-acetyl-3-deoxy-4-S-(beta-D-Galp)-4-thiopentopyranosid-2-ulose (11) were obtained in almost quantitative yields. The carbonyl function of these uloses was reduced with NaBH(4) or K-Selectride, and the stereochemical course of the reduction was highly dependent on the reaction temperature, reducing agent and solvent. Unexpectedly, reduction of 3 with NaBH(4)-THF at 0 degrees C gave a 3-deoxy-4-S-(beta-D-Xylp)-4-thio-alpha-D-ribo-hexopyranoside derivative (6) as major product (74% yield), with isomerization of the sulfur-substituted C-4 stereocenter of the pyranone. Reduction of 11 gave always as major product the benzyl 3-deoxy-4-S-(Galp)-4-thio-beta-D-threo-pentopyranoside derivative 14, which was the only product isolated (80% yield) in the reduction with K-Selectride in THF at -78 degrees C. Deprotection of 14 and its epimer at C-2 (13) afforded, respectively the free thiodisaccharides 19 and 18. They displayed strong inhibitory activity against the beta-galactosidase from Escherichia coli. Thus, compound 18 proved to be a non-competitive inhibitor of the enzyme (K(i)=0.80 mM), whereas 19 was a mixed-type inhibitor (K(i)=32 microM).
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http://dx.doi.org/10.1016/j.bmc.2009.07.055DOI Listing
September 2009
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