Galectins: a key intersection between glycobiology and immunology

Galectins are a family of evolutionarily conserved animal lectins, widely distributed from lower invertebrates to mammals. They share sequence and structure similarities in the carbohydrate recognition domain and specificity for polylactosamine-enriched glycoconjugates. In the last few years significant experimental data have been accumulated concerning their participation in different biological processes requiring carbohydrate recognition such as cell adhesion, cell growth regulation, inflammation, immunomodulation, apoptosis and metastasis. In the present review we will discuss some exciting questions and advances in galectin research, highlighting the significance of these proteins in immunological processes and their implications in biomedical research, disease diagnosis and clinical intervention. Designing novel therapeutic strategies based on carbohydrate recognition will provide answers for the treatment of autoimmune disorders, inflammatory processes, allergic reactions and tumor spreading.

GPIomics: global analysis of glycosylphosphatidylinositol-anchored molecules of Trypanosoma cruzi

Glycosylphosphatidylinositol (GPI) anchoring is a common, relevant posttranslational modification of eukaryotic surface proteins. Here, we developed a fast, simple, and highly sensitive (high attomole-low femtomole range) method that uses liquid chromatography-tandem mass spectrometry (LC-MS(n)) for the first large-scale analysis of GPI-anchored molecules (i.e., the GPIome) of a eukaryote, Trypanosoma cruzi, the etiologic agent of Chagas disease. Our genome-wise prediction analysis revealed that approximately 12% of T. cruzi genes possibly encode GPI-anchored proteins. By analyzing the GPIome of T. cruzi insect-dwelling epimastigote stage using LC-MS(n), we identified 90 GPI species, of which 79 were novel. Moreover, we determined that mucins coded by the T. cruzi small mucin-like gene (TcSMUG S) family are the major GPI-anchored proteins expressed on the epimastigote cell surface. TcSMUG S mucin mature sequences are short (56-85 amino acids) and highly O-glycosylated, and contain few proteolytic sites, therefore, less likely susceptible to proteases of the midgut of the insect vector. We propose that our approach could be used for the high throughput GPIomic analysis of other lower and higher eukaryotes. Molecular Systems Biology 7 April 2009; doi:10.1038/msb.2009.13

Differential expression of immunomodulatory galectin-1 in peripheral leukocytes and adult tissues and its cytosolic organization in striated muscle

Galectin-1 (Gal-1) is important in immune function and muscle regeneration, but its expression and localization in adult tissues and primary leukocytes remain unclear. To address this, we generated a specific monoclonal antibody against Gal-1, termed alpha hGal-1, and defined a sequential peptide epitope that it recognizes, which is preserved in human and porcine Gal-1, but not in murine Gal-1. Using alpha hGal-1, we found that Gal-1 is expressed in a wide range of porcine tissues, including striated muscle, liver, lung, brain, kidney, spleen, and intestine. In most types of cells, Gal-1 exhibits diffuse cytosolic expression, but in cells within the splenic red pulp, Gal-1 showed both cytosolic and nuclear localization. Gal-1 was also expressed in arterial walls and exhibited prominent cytosolic and nuclear staining in cultured human endothelial cells. However, human peripheral leukocytes and promyelocytic HL60 cells lack detectable Gal-1 and also showed very low levels of Gal-1 mRNA. In striking contrast, Gal-1 exhibited an organized cytosolic staining pattern within striated muscle tissue of cardiac and skeletal muscle and colocalized with sarcomeric actin on I bands. These results provide insights into previously defined roles for Gal-1 in inflammation, immune regulation and muscle biology.

Coordinated expression of galectin-3 and galectin-3-binding sites in malignant mammary tumors: implications for tumor metastasis

Galectin-3 is a glycan-binding protein that mediates cell-cell and/or cell-extracellular matrix (ECM) interactions. Although galectin-3 is implicated in the progression of various types of cancers, the mechanisms by which galectin-3 enhances metastasis remain unclear. In order to elucidate the role of galectin-3 in the complex multistage process of cancer metastasis, we examined galectin-3 and galectin-3-binding site expression in a series of 82 spontaneous canine mammary tumors (CMT) and two CMT cell lines. Benign CMT tumors exhibited strong nuclear/cytoplasmic galectin-3 immunostaining, whereas malignant CMT tumors and metastases exhibited dramatically decreased galectin-3 expression with the majority of the immunostaining confined to the cytoplasm. Interestingly, intravascular tumor cells overexpressed galectin-3 regardless of their location. CMT-U27 xenografts displayed the same pattern of galectin-3 expression found in spontaneous malignant CMT. In parallel with the downregulation of galectin-3, malignant CMT displayed an overall loss of galectin-3-binding sites in the ECM and focal expression of galectin-3-binding sites mainly detected in intravascular tumor cells and endothelium. Furthermore, loss of galectin-3-binding sites was correlated with the downregulation of GLT25D1, a beta (1-O) galactosyltransferase that modifies collagen, and upregulation of stromal galectin-1. Finally, GLT25D1 mRNA expression was strikingly downregulated in malignant CMT-U27 compared with the benign cell line, and its expression was further de-creased in a galectin-3 knockdown CMT-U27 cell line. We therefore hypothesized that the loss of galectin-3-binding sites in the ECM in conjunction with the overexpression of galectin-3 in specific tumor cell subpopulations are crucial events for the development of mammary tumor metastases.

Galectin-3 regulates peritoneal B1-cell differentiation into plasma cells

Extracellular galectin-3 participates in the control of B2 lymphocyte migration and adhesion and of their differentiation into plasma cells. Here, we analyzed the role of galectin-3 in B1-cell physiology and the balance between B1a and B1b lymphocytes in the peritoneal cavity. In galectin-3(-/-) mice, the total number of B1a lymphocytes was lower, while B1b lymphocyte number was higher as compared to wild-type mice. The differentiation of B1a cells into plasma cells was associated with their abnormal adhesion and location on the mesentery. The B220 and CD43, constitutively expressed by B1 lymphocytes, were respectively up- and downregulated in galectin-3(-/-) mice. Mononuclear cells were strongly adhered to the mesenteric membranes of both CD43(-/-) and galectin-3(-/-) mice, but in contrast to CD43(-/-) mice, the accumulation of B1 cells in peritoneal membranes in galectin-3(-/-) mice was accompanied by their functional differentiation into plasma cells. We have shown that in the absence of galectin-3, B1-cell differentiation into plasma cells is favored and the dynamic equilibrium of B1-cell populations in the peritoneum is maintained through a compensatory increase in B1b lymphocytes.

Degeneration of dystrophic or injured skeletal muscles induces high expression of Galectin-1

Muscle degenerative diseases such as Duchenne Muscular Dystrophy are incurable and treatment options are still restrained. Understanding the mechanisms and factors responsible for muscle degeneration and regeneration will facilitate the development of novel therapeutics. Several recent studies have demonstrated that Galectin-1 (Gal-1), a carbohydrate-binding protein, induces myoblast differentiation and fusion in vitro, suggesting a potential role for this mammalian lectin in muscle regenerative processes in vivo. However, the expression and localization of Gal-1 in vivo during muscle injury and repair are unclear. We report the expression and localization of Gal-1 during degenerative-regenerative processes in vivo using two models of muscular dystrophy and muscle injury. Gal-1 expression increased significantly during muscle degeneration in the murine mdx and in the canine Golden Retriever Muscular Dystrophy animal models. Compulsory exercise of mdx mouse, which intensifies degeneration, also resulted in sustained Gal-1 levels. Furthermore, muscle injury of wild-type C57BL/6 mice, induced by BaCl(2) treatment, also resulted in a marked increase in Gal-1 levels. Increased Gal-1 levels appeared to localize both inside and outside the muscle fibers with significant extracellular Gal-1 colocalized with infiltrating CD45(+) leukocytes. By contrast, regenerating muscle tissue showed a marked decrease in Gal-1 to baseline levels. These results demonstrate significant regulation of Gal-1 expression in vivo and suggest a potential role for Gal-1 in muscle homeostasis and repair.

Genome-wide association study identifies loci influencing concentrations of liver enzymes in plasma.

Concentrations of liver enzymes in plasma are widely used as indicators of liver disease. We carried out a genome-wide association study in 61,089 individuals, identifying 42 loci associated with concentrations of liver enzymes in plasma, of which 32 are new associations (P = 10(-8) to P = 10(-190)). We used functional genomic approaches including metabonomic profiling and gene expression analyses to identify probable candidate genes at these regions. We identified 69 candidate genes, including genes involved in biliary transport (ATP8B1 and ABCB11), glucose, carbohydrate and lipid metabolism (FADS1, FADS2, GCKR, JMJD1C, HNF1A, MLXIPL, PNPLA3, PPP1R3B, SLC2A2 and TRIB1), glycoprotein biosynthesis and cell surface glycobiology (ABO, ASGR1, FUT2, GPLD1 and ST3GAL4), inflammation and immunity (CD276, CDH6, GCKR, HNF1A, HPR, ITGA1, RORA and STAT4) and glutathione metabolism (GSTT1, GSTT2 and GGT), as well as several genes of uncertain or unknown function (including ABHD12, EFHD1, EFNA1, EPHA2, MICAL3 and ZNF827). Our results provide new insight into genetic mechanisms and pathways influencing markers of liver function.

Episodes in carbohydrate chemistry : from β-linked mannosides to glycoconjugates

Carbohydrates are one of the most abundant classes of biomolecules on earth. In the initial stages of research on carbohydrates much effort was focused on investigation and determination of the structural aspects and complex nature of individual monosaccharides. Later on, development of protective group strategies and methods for oligosaccharide synthesis became the main topics of research. Today, the methodologies developed early on are being utilized in the production of carbohydrates for biological screening events. This multidisciplinary approach has generated the new discipline of glycobiology which focuses on research related to the appearance and biological significance of carbohydrates. In more detail, studies in glycobiology have revealed the essential roles of carbohydrates in cell-cell interactions, biological recognition events, protein folding, cell growth and tumor cell metastasis. As a result of these studies, carbohydrate derived diagnostic and therapeutic agents are likely to be of growing interest in the future. In this doctoral thesis, a journey through the fundamentals of carbohydrate synthesis is presented. The research conducted on this journey was neither limited to the study of any particular phenomena nor to the addressing of a single synthetic challenge. Instead, the focus was deliberately shifted from time to time in order to broaden the scope of the thesis, to continue the learning process and to explore new areas of carbohydrate research. Throughout the work, several previously reported synthetic protocols, especially procedures related to glycosylation reactions and protective group manipulations, were evaluated, modified and utilized or rejected. The synthetic molecules targeted within this thesis were either required for biological evaluations or utilized to study phenomena occuring in larger molecules. In addition, much effort was invested in the complete structural characterization of the synthesized compounds by a combination of NMR spectroscopic techniques and spectral simulations with the PERCH-software. This thesis provides the basics of working with carbohydrate chemistry. In more detail, synthetic strategies and experimental procedures for many different reactions and guidelines for the NMR-spectroscopic characterization of oligosaccharides and glycoconjugates are provided. Therefore, the thesis should prove valuable to researchers starting their own journeys in the ever expanding field of carbohydrate chemistry.

Galectin-1, an alternative signal for T cell death, is increased in activated macrophages

Galectin-1 belongs to an evolutionarily conserved family of animal ß-galactoside-binding proteins, which exert their functions by crosslinking the oligosaccharides of specific glycoconjugate ligands. During the past decade, attempts to identify the functional role of galectin-1 suggested participation in the regulation of the immune response. Only in the last few years has the molecular mechanism involved in these properties been clearly elucidated, revealing a critical role for galectin-1 as an alternative signal in the generation of T cell death. In the present study we will discuss the latest advances in galectin research in the context of the regulation of the immune response, not only at the central level but also at the periphery. Moreover, we will review the purification, biochemical properties and functional significance of a novel galectin-1-like protein from activated rat macrophages, whose expression is differentially regulated according to the activation state of the cells. The novel role of a carbohydrate-binding protein in the regulation of apoptosis is providing a breakthrough in galectin research and extending the interface between immunology, glycobiology and clinical medicine.

Jacalin interaction with human immunoglobulin A1 and bovine immunoglobulin G1: Affinity constant determined by piezoelectric biosensoring

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aspergillus niger beta-Glucosidase Has a Cellulase-like Tadpole Molecular Shape INSIGHTS INTO GLYCOSIDE HYDROLASE FAMILY 3 (GH3) beta-GLUCOSIDASE STRUCTURE AND FUNCTION

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Jacalin interaction with human immunoglobulin A1 and bovine immunoglobulin G1: Affinity constant determined by piezoelectric biosensoring

The affinity of the d-galactose-binding lectin from Artocarpus heterophyllus lectin, known as jacalin, with immonuglobulins (Igs) was determined by biofunctionalization of a piezoelectric transducer. This piezoelectric biofunctionalized transducer was used as a mass-sensitive analytical tool, allowing the real-time binding analysis of jacalin-human immunoglobulin A1 (IgA(1)) and jacalin-bovine IgG(1) interactions from which the apparent affinity constant was calculated. The strategy was centered in immobilizing jacalin on the gold electrode's surface of the piezoelectric crystal resonator using appropriate procedures based on self-assembling of 11-mercaptoundecanoic acid and 2-mercaptoethanol thiol's mixture, a particular immobilization strategy by which it was possible to avoid cross-interaction between the proteins over electrode's surface. The apparent affinity constants obtained between jacalin-human IgA(1) and jacalin-bovine IgG(1) differed by 1 order of magnitude [(8.0 +/- 0.9) x 10(5) vs (8.3 +/- 0.1) x 10(6) L mol(-1)]. On the other hand, the difference found between human IgA(1) and human IgA(2) interaction with jacalin, eight times higher for IgA(1), was attributed to the presence of O-linked glycans in the IgA(1) hinge region, which is absent in IgA(2). Specific interaction of jacalin with O-glycans, proved to be present in the human IgA(1) and hypothetically present in bovine IgG(1) structures, is discussed as responsible for the obtained affinity values.

Glycomic analysis of human mast cells, eosinophils and basophils

In allergic diseases such as asthma, eosinophils, basophils and mast cells, through release of preformed and newly generated mediators, granule proteins and cytokines, are recognized as key effector cells. While their surface protein phenotypes, mediator release profiles, ontogeny, cell trafficking and genomes have been generally explored and compared, there has yet to be any thorough analysis and comparison of their glycomes. Such studies are critical to understand the contribution of carbohydrates to the induction and regulation of allergic inflammatory responses and are now possible using improved technologies for detecting and characterizing cell-derived glycans. We thus report here the application of high-sensitivity mass spectrometric-based glycomics methodologies to the analysis of N-linked glycans derived from isolated populations of human mast cells, eosinophils and basophils. The samples were subjected to matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) screening analyses and MALDI-TOF/TOF sequencing studies. Results reveal substantive quantities of terminal N-acetylglucosamine containing structures in both the eosinophil and the basophil samples, whereas mast cells display greater relative quantities of sialylated terminal epitopes. For the first time, we characterize the cell surface glycan structures of principal allergic effector cells, which by interaction with glycan-binding proteins (e.g. lectins) have the possibility to dictate cellular functions, and might thus have important implications for the pathogenesis of inflammatory and allergic diseases.