The function of deciphering the biological information encoded by the glycome, which is the entire repertoire of complex sugar structures expressed by cells and tissues, is assigned in part to endogenous glycan-binding proteins or lectins. Galectins, a family of animal lectins that bind N-acetyllactosamine-containing glycans, have many roles in diverse immune cell processes, including those relevant to pathogen recognition, shaping the course of adaptive immune responses and fine-tuning the inflammatory response. How do galectins translate glycan-encoded information into tolerogenic or inflammatory cell programmes? An improved understanding of the mechanisms underlying these functions will provide further opportunities for developing new therapies based on the immunoregulatory properties of this multifaceted protein family.

Turning 'sweet' on immunity: galectin-glycan interactions in immune tolerance and inflammation.

Galectins, β-galactoside-binding animal lectins, are differentially expressed by various immune cells as well as a wide range of other cell types. Extracellularly, galectins are able to exhibit bivalent or multivalent interactions with cell-surface glycans on various immune cells and exert various effects. These include cytokine and mediator production, cell adhesion, apoptosis, and chemoattraction. In addition, they can form lattices with cell-surface glycoprotein receptors, resulting in modulation of receptor functions, including clustering and endocytosis. Intracellularly, galectins can participate in signaling pathways and modulate biologic responses. These include apoptosis, cell differentiation, and cell migration. Thus, a large body of literature indicates that galectins play important roles in the immune and inflammatory responses through regulating the homeostasis and functions of immune cells. The use of mice deficient in individual galectins has provided additional evidence for the contributions of these proteins to these responses. Current research indicates that galectins play important roles in the development of acute inflammation as well as chronic inflammation associated with allergies, autoimmune diseases, atherosclerosis, infectious processes, and cancer. Thus, recombinant proteins or specific galectin inhibitors may be used as therapeutic agents for inflammatory diseases.

Galectins: regulators of acute and chronic inflammation

Regulatory Circuits Mediated by Lectin-Glycan Interactions in Autoimmunity and Cancer

In the past decade, increasing efforts have been devoted to the study of galectins, a family of evolutionarily conserved glycan-binding proteins with multifunctional properties. Galectins function, either intracellularly or extracellularly, as key biological mediators capable of monitoring changes occurring on the cell surface during fundamental biological processes such as cellular communication, inflammation, development, and differentiation. Their highly conserved structures, exquisite carbohydrate specificity, and ability to modulate a broad spectrum of biological processes have captivated a wide range of scientists from a wide spectrum of disciplines, including biochemistry, biophysics, cell biology, and physiology. However, in spite of enormous efforts to dissect the functions and properties of these glycan-binding proteins, limited information about how structural and biochemical aspects of these proteins can influence biological functions is available. In this review, we aim to integrate structura...

When Galectins Recognize Glycans: From Biochemistry to Physiology and Back Again

Major histocompatibility complex (MHC) class I and II are glycoproteins that can present antigenic peptides at the cell surface for recognition and activation of circulating T lymphocytes. Here, the importance of the modification of protein antigens by glycans on cellular uptake, proteolytic processing, presentation by MHC and subsequent T-cell priming is reviewed. Antigen glycosylation is important for a number of diseases and vaccine design. All of the key proteins involved in antigen recognition and the orchestration of downstream effector functions are glycosylated. The influence of protein glycosylation on immune function and disease is covered.

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Adaptive immune activation: glycosylation does matter

In the present work, we followed a microarray approach to analyze the expression of glycosylation-related genes on different cell populations obtained from mouse thymus Among other genes, transcription of the two-domain type galectin-8 was detected both in thymocytes and thymic epithelial cells (TECs), which was confirmed by reverse transcriptase (RT)-PCR assays independently carried out on both cell populations Two splice variants, differing solely in the presence of a nine amino acid insertion in the linker peptide region connecting the two carbohydrate recognition domains (CRDs), were identified from purified thymocytes Expression of galectin-8 was verified at the protein level in total organ extracts by western-blots of lactosyl-Sepharose purified binders To explore the possible biological roles of locally produced galectin-8, both splice variants were recombinantly expressed in bacteria and assayed over cultured thymocytes In spite of their binding to all cell populations, addition of either isoform of galectin-8 to thymocyte cultures induced apoptosis only of the CD4 high CD8 high cells through caspases pathway activation All of these effects were prevented by the addition of thiodigalactoside (TDG) or lactose, thus indicating that the proapoptotic activity of galectin-8 was due to the specific interaction of its CRDs with defined cell surface glycans Together, our results demonstrate intrathymic expression of galectin-8 in mouse, and suggest an active role for this lectin in shaping the mature T cell repertoire

Galectin-8 Induces Apoptosis in the CD4highCD8high Thymocyte Subpopulation

Gals (galectins) are proteins with glycan affinity that are emerging as mediators of atherosclerosis. Despite the similarities in structure and sequence, different Gals exert distinct effects on their target cells. We have shown that Gal-1 triggers platelet activation, suggesting a role for Gals in thrombus formation. Since Gal-8 is expressed upon endothelial activation and also contributes to inflammation, to understand further the role of these lectins in haemostasis, we evaluated the effect of Gal-8 on human platelets. Gal-8 bound specific glycans in the platelet membrane and triggered spreading, calcium mobilization and fibrinogen binding. It also promoted aggregation, thromboxane generation, P-selectin expression and granule secretion. GP (glycoprotein) αIIb and Ib-V were identified as putative Gal-8 counter-receptors by MS. Studies performed using platelets from Glanzmann's thromboasthenia and Bernard-Soulier syndrome patients confirmed that GPIb is essential for transducing Gal-8 signalling. Accordingly, Src, PLC2γ (phospholipase C2γ), ERK (extracellular-signal-regulated kinase) and PI3K (phosphoinositide 3-kinase)/Akt downstream molecules were involved in the Gal-8 signalling pathway. Gal-8 fragments containing either the N- or C-terminal carbohydrate-recognition domains showed that activation is exerted through the N-terminus. Western blotting and cytometry showed that platelets not only contain Gal-8, but also expose Gal-8 after thrombin activation. These findings reveal Gal-8 as a potent platelet activator, supporting a role for this lectin in thrombosis and inflammation.

Human platelets express and are activated by galectin-8.

Galectin (Gal) constitute a family of carbohydrate-recognizing molecules ubiquitously expressed in mammals. In the immune system, they regulate many processes such as inflammation, adhesion, and apoptosis. Here, we report the expression in the spleen of the two same Gal-8 splice variants described previously in the thymus. Gal-8 was found to induce two separate biological activities on T lymphocytes: a robust naive CD4(+) T cell proliferation in the absence of antigen and notably, a costimulatory signal that synergized the cognate OVA peptide in DO11.10 mice transgenic for TCR(OVA). The antigen-independent proliferation induced by Gal-8 displayed increased expression of pro- and anti-inflammatory cytokines, thus suggesting the polyclonal expansion of Th1 and Th2 clones. The costimulatory effect on antigen-specific T cell activation was evidenced when the Gal and the peptide were assayed at doses suboptimal to induce T cell proliferation. By mass spectra analysis, several integrins and leukocyte surface markers, including CD45 isoforms, as well as other molecules specific to macrophages, neutrophils, and platelets, were identified as putative Gal-8 counter-receptors. Gal-8 triggered pZAP70 and pERK1/2. Moreover, pretreatment with specific inhibitors of CD45 phosphatase or ERK1/2 prevented its antigen-dependent and -independent T cell-proliferative activities. This seems to be associated with the agonistic binding to CD45, which lowers the activation threshold of the TCR signaling pathway. Taken together, our findings support a distinctive role for locally produced Gal-8 as an enhancer of otherwise borderline immune responses and also suggest that Gal-8 might fuel the reactivity at inflammatory foci.

https://jlb.onlinelibrary.wiley.com/doi/pdf/10.1189/jlb.0908529

Galectin-8 provides costimulatory and proliferative signals to T lymphocytes

Galectins, a family of mammalian lectins, have emerged as key regulators of the immune response. We previously demonstrated that galectin (Gal)-8, from the tandem-repeat subgroup, exerts two well-defined effects on mouse naive peripheral CD4 T cells: Ag-specific costimulation and Ag-independent proliferation. These stimulatory signals on naive T cells have not been described for any other Gal. Therefore, we investigated whether Gal-1 and Gal-3, two prominent members of the Gal family, share the stimulatory effects exerted by Gal-8 on naive T cells. We found that Gal-1 costimulated Ag-specific T cell responses similarly to Gal-8, as evaluated in the DO11.10 TCR OVA -transgenic mouse model, by acting simultaneously on APCs and target CD4 T cells. In contrast, Gal-3 failed to costimulate Ag-specific T cell responses; moreover, it antagonized both Gal-1 and Gal-8 signals. We observed that both Gal-1 and Gal-3 were unable to induce Ag-independent proliferation; however, when two Gal-1 molecules were covalently fused, the resulting chimeric protein efficiently promoted proliferation. This finding indicates that Gal-1 might eventually induce proliferation and, moreover, stresses the requirement of a tandem-repeat structure. Remarkably, a single dose of recombinant Gal-1 or Gal-8 administered together with a suboptimal Ag dose to DO11.10 mice strengthened weak responses in vivo. Taken together, these findings argue for the participation of Gals in the initiation of the immune response and allow the postulation of these lectins as enhancers of borderline Ag responses, thus representing potential adjuvants for vaccine formulations.

Redundant and Antagonistic Functions of Galectin-1, -3, and -8 in the Elicitation of T Cell Responses

Gal (galectin)-8 is a tandem-repeat Gal containing N-CRDs (Nterminal carbohydrate-recognition domains) and C-CRDs (C-terminal carbohydrate-recognition domains) with differential glycan-binding specificity fused by a linker peptide. Gal-8 has two distinct effects on CD4 T-cells: at high concentrations it induces antigen-independent proliferation, whereas at low concentrations it co-stimulates antigen-specific responses. Associated Gal-8 structural requirements were dissected in the present study. Recombinant homodimers N–N (two N-terminal CRD chimaera) and C–C (two C-terminal CRD chimaera), but not single C-CRDs or N-CRDs, induced proliferation; however, single domains induced co-stimulation. These results indicate that the tandem-repeat structure was essential only for the proliferative effect, suggesting the involvement of lattice formation, whereas co-stimulation could be mediated by agonistic interactions. In both cases, C–C chimaeras displayed higher activity than Gal-8, indicating that the C-CRD was mainly involved, as was further supported by the strong inhibition of proliferation and co-stimulation in the presence of blood group B antigen, specifically recognized by this domain. Classic Gal inhibitors (lactose and thiodigalactoside) prevented proliferation but not co-stimulatory activity, which was inhibited by 3-O-β-D-galactopyranosyl-D-arabinose. Interestingly, Gal-8 induced proliferation of naive human CD4 T-cells, varying from non- to high-responder individuals, whereas it promoted cell death of phytohaemagglutinin or CD3/CD28 pre-activated cells. The findings of the present study delineate the differential molecular requirements for Gal-8 activities on T-cells, and suggest a dual activity relying on activation state.

Valentina Cattaneo

Papers

Galectin-8 Induces Apoptosis in the CD4highCD8high Thymocyte Subpopulation

Human platelets express and are activated by galectin-8.

Galectin-8 provides costimulatory and proliferative signals to T lymphocytes

Redundant and Antagonistic Functions of Galectin-1, -3, and -8 in the Elicitation of T Cell Responses

Galectin-8 tandem-repeat structure is essential for T-cell proliferation but not for co-stimulation.