Tumor galectinology: Insights into the complex network of a family of endogenous lectins

β-Galactosides of cell surface glycoconjugates are docking sites for endogenous lectins of the galectin family. In cancer cells, primarily galectins-1 and -3 have been studied to date. With the emergence of insights into their role in growth control, resistance to or induction of apoptosis and invasive behavior the notion is supported that they can be considered as functional tumor markers. In principle, the same might hold true for the other members of the galectin family. But their expression in tumors has hitherto been a subject of attention only to a very limited extent. Pursuing our concept to define the complexity of the galectin network in cancer cells and the degree of functional overlap/divergence with diagnostic/therapeutic implications, we have introduced comprehensive RT-PCR monitoring to map their galectin gene expression. The data on so far less appreciated galectins in this context such as galectins-4 and -8 vindicate this approach. They, too, attach value to extend the immunohistochemical panel accordingly. Our initial histopathological and cell biological studies, for example on colon cancer progression, prove the merit of this procedure. Aside from the detection of gene expression profiles by RT-PCR, the detailed molecular biological monitoring yielded further important information. We describe different levels of regulation of galectin production in colon cancer cells in the cases of the tandem-repeat-type galectins-8 and -9. Isoforms for them are present with insertions into the peptide linker sequence attributed to alternative splicing. Furthermore, variants with distinct amino acid substitutions (galectin-8, Po66-CBP, PCTA-1, CocaI/II and galectin-9/ecalectin) and generation of multiple mRNA species, notably those coding for truncated galectin-8 and -9 versions with only one lectin site, justify to portray these two family members not as distinct individuals but as groups. In aggregate, the ongoing work to thoroughly chart the galectin network and to disentangle the individual functional contributions is expected to make its mark on our understanding of the malignant phenotype in certain tumor types. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of a novel Drosophila melanogaster galectin. Expression in developing immune, neural, and muscle tissues.

We have cloned and characterized the first galectin to be identified in Drosophila melanogaster. The amino acid sequence of Drosophila galectin showed striking sequence similarity to invertebrate and vertebrate galectins and contained amino acids that are crucial for binding beta-galactoside sugars. Confirming its identity as a galectin family member, the Drosophila galectin bound beta-galactoside sugars. Structurally, the Drosophila galectin was a tandem repeat galectin containing two carbohydrate recognition domains connected by a unique peptide link. This divalent structure suggests that like mammalian galectins, Drosophila galectin may mediate cell-cell communication or facilitate cross-linking of receptors to trigger signal transduction events. The Drosophila galectin was very abundant in embryonic, larval, and adult Drosophila. During embryogenesis, Drosophila galectin had a unique and specific tissue distribution. Drosophila galectin expression was concentrated in somatic and visceral musculature and in the central nervous system. Similar to other insect lectins, Drosophila galectin may function in both embryogenesis and in host defense. Drosophila galectin was expressed by hemocytes, circulating phagocytic cells, suggesting a role for Drosophila galectin in the innate immune system.     

Tumor galectinology: insights into the complex network of a family of endogenous lectins

Beta-Galactosides of cell surface glycoconjugates are docking sites for endogenous lectins of the galectin family. In cancer cells, primarily galectins-1 and -3 have been studied to date. With the emergence of insights into their role in growth control, resistance to or induction of apoptosis and invasive behavior the notion is supported that they can be considered as functional tumor markers. In principle, the same might hold true for the other members of the galectin family. But their expression in tumors has hitherto been a subject of attention only to a very limited extent. Pursuing our concept to define the complexity of the galectin network in cancer cells and the degree of functional overlap/divergence with diagnostic/therapeutic implications, we have introduced comprehensive RT-PCR monitoring to map their galectin gene expression. The data on so far less appreciated galectins in this context such as galectins-4 and -8 vindicate this approach. They, too, attach value to extend the immunohistochemical panel accordingly. Our initial histopathological and cell biological studies, for example on colon cancer progression, prove the merit of this procedure. Aside from the detection of gene expression profiles by RT-PCR, the detailed molecular biological monitoring yielded further important information. We describe different levels of regulation of galectin production in colon cancer cells in the cases of the tandem-repeat-type galectins-8 and -9. Isoforms for them are present with insertions into the peptide linker sequence attributed to alternative splicing. Furthermore, variants with distinct amino acid substitutions (galectin-8, Po66-CBP, PCTA-1, CocaI/II and galectin-9/ecalectin) and generation of multiple mRNA species, notably those coding for truncated galectin-8 and -9 versions with only one lectin site, justify to portray these two family members not as distinct individuals but as groups. In aggregate, the ongoing work to thoroughly chart the galectin network and to disentangle the individual functional contributions is expected to make its mark on our understanding of the malignant phenotype in certain tumor types.     

Galectinomics: finding themes in complexity

With the rapid explosion of genomic sequence databases, there has been an equivalent boom in genomics, the use of sequence information to define and compare gene families and their organization across diverse species. Such expansion of the galectin family by "galectinomics" to include many new members is reviewed here. The galectin gene family is evolutionarily ancient with representatives in vertebrates, invertebrates, and even in protists. Although the identification of many novel galectin relatives in widely divergent organisms (including Arabidopsis, Drosophila, Caenorhabditis, Danio, Xenopus, and human) has added significantly to the size and complexity of this intriguing protein family, several common themes arise, which suggest promising new research targets.     

Biochemical and molecular characterization of galectins from zebrafish (Danio rerio): notochord-specific expression of a prototype galectin during early embryogenesis

Galectins are a family of beta-galactoside-binding lectins that on synthesis are either translocated into the nucleus or released to the extracellular space. Their developmentally regulated expression, extracellular location, and affinity for extracellular components (such as laminin and fibronectin) suggest a role in embryonic development, but so far this has not been unequivocally established. Zebrafish constitute an ideal model for developmental studies because of their external fertilization, transparent embryos, rapid growth, and availability of a large collection of mutants. As a first step in addressing the biological roles in zebrafish embryogenesis, we identified and characterized members of the three galectin types: three protogalectins (Drgal1-L1, Drgal1-L2, Drgal1-L3), one chimera galectin (Drgal3), and one tandem-repeat galectin (Drgal9-L1). Like mammalian prototype galectin-1, Drgal1-L2 preferentially binds to N-acetyllactosamine. Genomic structure of Drgal1-L2 revealed four exons, with the exon-intron boundaries conserved with the mammalian galectin-1. Interestingly, this gene also encodes an alternatively spliced form of Drgal1-L2 that lacks eight amino acids near the carbohydrate-binding domain. Zebrafish galectins exhibited distinct patterns of temporal expression during embryo development. Drgal1-L2 is expressed postbud stage, and its expression is strikingly specific to the notochord. In contrast, Drgal1-L1 is expressed maternally in the oocytes. Drgal1-L3, Drgal3, and Drgal9-L1 are expressed both maternally and zygotically, ubiquitously in the adult tissues. The distinct temporal and spatial patterns of expression of members of the zebrafish galectin repertoire suggest that each may play distinct biological roles during early embryogenesis.     

Satellite Glial Cells and Astrocytes,a Comparative Review

Astroglia are neural cells, heterogeneous in form and function, which act as supportive elements of the central nervous system; astrocytes contribute to all aspects of neural functions in health and disease. Through their highly ramified processes, astrocytes form close physical contacts with synapses and blood vessels, and are integrated into functional syncytia by gap junctions. Astrocytes interact among themselves and with other cells types (e.g., neurons, microglia, blood vessel cells) by an elaborate repertoire of chemical messengers and receptors; astrocytes also influence neural plasticity and synaptic transmission through maintaining homeostasis of neurotransmitters, K⁺ buffering, synaptic isolation and control over synaptogenesis and synaptic elimination. Satellite glial cells (SGCs) are the most abundant glial cells in sensory ganglia, and are believed to play major roles in sensory functions, but so far research into SGCs attracted relatively little attention. In this review we compare SGCs to astrocytes with the purpose of using the vast knowledge on astrocytes to explore new aspects of SGCs. We survey the main properties of these two cells types and highlight similarities and differences between them. We conclude that despite the much greater diversity in morphology and signaling mechanisms of astrocytes, there are some parallels between them and SGCs. Both types serve as boundary cells, separating different compartments in the nervous system, but much more needs to be learned on this aspect of SGCs. Astrocytes and SGCs employ chemical messengers and calcium waves for intercellular signaling, but their significance is still poorly understood for both cell types. Both types undergo major changes under pathological conditions, which have a protective function, but an also contribute to disease, and chronic pain in particular. The knowledge obtained on astrocytes is likely to benefit future research on SGCs.

     

Galectin‐3, a marker for vacuole lysis by invasive pathogens

Shigella bacteria invade macrophages and epithelial cells and following internalization lyse the phagosome and escape to the cytoplasm. Galectin‐3, an abundant protein in macrophages and epithelial cells, belongs to a family of beta‐galactoside‐binding proteins, the galectins, with many proposed functions in immune response, development, differentiation, cancer and infection. Galectins are synthesized as cytosolic proteins and following non‐classical secretion bind extracellular beta‐galactosides. Here we analysed the localization of galectin‐3 following entry of Shigella into the cytosol and detected a striking phenomenon. Very shortly after bacterial invasion, intracellular galectin‐3 accumulated in structures in vicinity to internalized bacteria. By using immuno‐electron microscopy analysis we identified galectin‐3 in membranes localized in the phagosome and in tubules and vesicles that derive from the endocytic pathway. We also demonstrated that the binding of galectin‐3 to host N‐acetyllactosamine‐containing glycans, was required for forming the structures. Accumulation of the structures was a type three secretion system‐dependent process. More specifically, existence of structures was strictly dependent upon lysis of the phagocytic vacuole and could be shown also by Gram‐positive Listeria and Salmonella sifA mutant. We suggest that galectin‐3‐containing structures may serve as a potential novel tool to spot vacuole lysis.     

Morphological manifestations of local functional activation of astrocytes induced by transient global cerebral ischemia

The goal of the work was to study changes of structural and cytochemical organization of activated hippocampal astrocytes in the rat exposed to transient global ischemia of the brain. Intermediate filament proteins immunocytochemistry revealed functional activation of astrocytes of dorsal hippocampus 7 days following the ischemia, which was manifested as changes of size and shape of the cells and processes and accumulation of intermediate filament proteins GFAP and nestin. This is accompanied by formation of two populations of activated astrocytes: GFAP-positive astrocytes, which are more abundant and nestin-positive astrocytes distributed predominantly in the area of massive loss of neural cells. The obtained data suggest that astrocytes activated post-ischemically obtain properties typical for immature cells of nervous tissue, but lack of morphological signs of dedifferentiation do not support their contribution to reparative neurogenesis in the hippocampus.     

Control of Angiogenesis by Galectins Involves the Release of Platelet-Derived Proangiogenic Factors

Platelets contribute to vessel formation through the release of angiogenesis-modulating factors stored in their α-granules. Galectins, a family of lectins that bind β-galactoside residues, are up-regulated in inflammatory and cancerous tissues, trigger platelet activation and mediate vascularization processes. Here we aimed to elucidate whether the release of platelet-derived proangiogenic molecules could represent an alternative mechanism through which galectins promote neovascularization. We show that different members of the galectin family can selectively regulate the release of angiogenic molecules by human platelets. Whereas Galectin (Gal)-1, -3, and -8 triggered vascular endothelial growth factor (VEGF) release, only Gal-8 induced endostatin secretion. Release of VEGF induced by Gal-8 was partially prevented by COX-1, PKC, p38 and Src kinases inhibitors, whereas Gal-1-induced VEGF secretion was inhibited by PKC and ERK blockade, and Gal-3 triggered VEGF release selectively through a PKC-dependent pathway. Regarding endostatin, Gal-8 failed to stimulate its release in the presence of PKC, Src and ERK inhibitors, whereas aspirin or p38 inhibitor had no effect on endostatin release. Despite VEGF or endostatin secretion, platelet releasates generated by stimulation with each galectin stimulated angiogenic responses in vitro including endothelial cell proliferation and tubulogenesis. The platelet angiogenic activity was independent of VEGF and was attributed to the concerted action of other proangiogenic molecules distinctly released by each galectin. Thus, secretion of platelet-derived angiogenic molecules may represent an alternative mechanism by which galectins promote angiogenic responses and its selective blockade may lead to the development of therapeutic strategies for angiogenesis-related diseases.     

Purification and cDNA cloning of Xenopus liver galectins and their expression

We have characterized galectin family proteins in adult tissues of Xenopus laevis and purified 14-kDa and 36-kDa proteins from the liver. The liver galectins showed comparable hemagglutination activities to those of mammalian galectins. Furthermore, we isolated five galectin cDNAs from a Xenopus liver library. These cDNAs revealed that X. laevis galectins (xgalectins) form a family consisting of at least proto and tandem repeat types based on their domain structures, like the mammalian galectin family. Two proto-type xgalectins, -Ia and -Ib, exhibited a high sequence identity (91%) with each other at the amino acid level and were most similar (49-50% identity) to human galectin-1. From their sequence similarity and ubiquitous tissue distributions, xgalectins-Ia and -Ib both seemed to be Xenopus homologues of mammalian galectin-1. Three tandem repeat-type xgalectins were newly identified. Two of them, xgalectins-IIa and -IIIa, seemed to be homologous to human galectins-4 and -9, respectively, judging from their high sequence similarities (42-50% identity). However, xgalectin-IVa seemed to be a novel type. Distributions of mRNAs of xgalectins were analyzed by northern hybridization. In addition to adult tissues, either of three tandem repeat-type xgalectins were expressed in whole embryos. Moreover, amino acid sequence analysis of liver proteins indicated that xgalectins-Ia, -IIa, and -IIIa are produced as abundant galectins in the adult liver.     

Shedding light on the immunomodulatory properties of galectins: Novel regulators of innate and adaptive immune responses

Galectins are a large family of structurally related β-galactoside-binding proteins that play a pivotal role in the control of cell differentiation, proliferation, activation and apoptosis of many different cell types including immune cells. By crosslinking specific glycoconjugates, different members of the galectin family behave as pro-inflammatory or anti-inflammatory “cytokine-like” mediators, acting at different levels of innate and adaptive immune responses. Here we will review recent advances on the role of galectins in key events of the immune and inflammatory response, such as tolerance induction, cell cycle progression, cell adhesion, chemotaxis, antigen presentation and apoptosis. In particular we will examine the influence of individual members of the galectin family in the physiology of different immune cell types involved in innate and adaptive immune responses. Moreover, we will discuss the importance of these sugar-binding proteins as therapeutic targets in Th1- and Th2-mediated immune disorders, an exciting area for future research. Published in 2004.     

Simulating cellular galectin networks by mixing galectins in vitro reveals synergistic activity

BackgroundEven though members of the family of adhesion/growth-regulatory galectins are increasingly detected to be co-expressed, they are still being routinely tested separately. The recent discovery of heterodimer formation among galectins-1, -3, and -7 in mixtures prompts further study of their functional activities in mixtures.MethodsCell agglutination, galectin binding to cells, as well as effects on cell proliferation, onset of apoptosis and migration were determined in assays using various cell types and mixtures of galectins-1, -3, and -7.ResultsEvidence for a more than additive increases of experimental parameters was consistently obtained.ConclusionTesting galectins in mixtures simulates the situation of co-expression in situ and reveals unsuspected over-additive activities. This new insight is relevant for analyzing galectin functionality in (patho)physiological conditions.     

Small leucine rich proteoglycan family regulates multiple signalling pathways in neural development and maintenance

The small leucine-rich repeat proteoglycan (SLRPs) family of proteins currently consists of five classes, based on their structural composition and chromosomal location. As biologically active components of the extracellular matrix (ECM), SLRPs were known to bind to various collagens, having a role in regulating fibril assembly, organization and degradation. More recently, as a function of their diverse proteins cores and glycosaminoglycan side chains, SLRPs have been shown to be able to bind various cell surface receptors, growth factors, cytokines and other ECM components resulting in the ability to influence various cellular functions. Their involvement in several signaling pathways such as Wnt, transforming growth factor-β and epidermal growth factor receptor also highlights their role as matricellular proteins. SLRP family members are expressed during neural development and in adult neural tissues, including ocular tissues. This review focuses on describing SLRP family members involvement in neural development with a brief summary of their role in non-neural ocular tissues and in response to neural injury.     

Shedding light on the immunomodulatory properties of galectins: novel regulators of innate and adaptive immune responses

Galectins are a large family of structurally related beta-galactoside-binding proteins that play a pivotal role in the control of cell differentiation, proliferation, activation and apoptosis of many different cell types including immune cells. By crosslinking specific glycoconjugates, different members of the galectin family behave as pro-inflammatory or anti-inflammatory "cytokine-like" mediators, acting at different levels of innate and adaptive immune responses. Here we will review recent advances on the role of galectins in key events of the immune and inflammatory response, such as tolerance induction, cell cycle progression, cell adhesion, chemotaxis, antigen presentation and apoptosis. In particular we will examine the influence of individual members of the galectin family in the physiology of different immune cell types involved in innate and adaptive immune responses. Moreover, we will discuss the importance of these sugar-binding proteins as therapeutic targets in Th1- and Th2-mediated immune disorders, an exciting area for future research.