Galectins are a class of proteins that bind specifically to β-galactoside sugars, such as N-acetyllactosamine (Galβ1-3GlcNAc or Galβ1-4GlcNAc), which can be bound to proteins by either N-linked or O-linked glycosylation. They are also termed S-type lectins due to their dependency on disulphide bonds for stability and carbohydrate binding. There have been about 15 galectins discovered in mammals, encoded by the LGALS genes, which are numbered in a consecutive manner. Only galectin-1, -2, -3, -4, -7, -7B, -8, -9, -9B, 9C, -10, -12, -13, -14, and -16 have been identified in humans. Galectin-5 and -6 are found in rodents, whereas galectin-11 and -15 are uniquely found in sheep and goats. Members of the galectin family have also been discovered in other mammals, birds, amphibians, fish, nematodes, sponges, and some fungi. Unlike the majority of lectins they are not membrane bound, but soluble proteins with both intra- and extracellular functions. They have distinct but overlapping distributions but found primarily in the cytosol, nucleus, extracellular matrix or in circulation. Although many galectins must be secreted, they do not have a typical signal peptide required for classical secretion. The mechanism and reason for this non-classical secretion pathway is unknown.
Structure
There are three different forms of galectin structure: dimeric, tandem or chimera. Dimeric galectins, also called prototypical galectins, are homodimers, consisting of two identical galectin subunits that have associated with one another. The galectins that fall under this category are galectin-1, -2, -5, -7, -10, -11, -14 and -15. Tandem galectins contain at least two distinct carbohydrate recognition domains (CRD) within one polypeptide, thus are considered intrinsically divalent. The CRDs are linked with a small peptide domain. Tandem galectins include galectin-4, -6, -8, -9 and -12. The final galectin is galectin-3 which is the only galectin found in the chimera category in vertebrates. Galectin-3 has one CRD and a long non-lectin domain. Galectin-3 can exist in monomeric form or can associate via the non-lectin domain into multivalent complexes up to a pentameric form. This allows galectin-3 to bridge effectively between different ligands and form adhesive networks. The formation of multimers is concentration dependent. When Galectin-3 is at a low concentration it is monomeric and likely to inhibit adhesion. It binds to adhesion proteins such as integrins and blocks further binding to other cells or the extracellular matrix. When concentrations of galectin-3 are high it forms large complexes that assist in adhesion by bridging between cells or cells and the extracellular matrix. Many isoforms of galectins have been found due to different splicing variants. For example, Galectin-8 has seven different mRNAs encoding for both tandem and dimeric forms. The type of galectin-8 that is expressed is dependent on the tissue. Galectin-9 has three different isoforms which differ in the length of the linker region.
The galectin carbohydrate recognition domain (CRD) is constructed from beta-sheet of about 135 amino acids. The two sheets are slightly bent with 6 strands forming the concave side and 5 strands forming the convex side. The concave side forms a groove in which the carbohydrate ligand can bind, and which is long enough to hold about a linear tetrasaccharide.
Ligand binding Galectins essentially bind to glycans featuring galactose and its derivatives. However, physiologically, they are likely to require lactose or N-acetyllactosamine for significantly strong binding. Generally, the longer the sugar the stronger the interactions. For example, galectin-9 binds to polylactosamine chains with stronger affinity than to an N-acetyllactosamine monomer. This is because more Van der Waals interactions can occur between sugar and binding pocket. Carbohydrate binding is calcium independent, unlike C-type lectins. The strength of ligand binding is determined by a number of factors: The multivalency of both of ligand and the galectin, the length of the carbohydrate and the mode of presentation of ligand to carbohydrate recognition domain. Different galectins have distinct binding specificities for binding oligosaccharides depending on the tissue in which they are expressed and the function that they possess. However, in each case, galactose is essential for binding. Crystallisation experiments of galectins in complex with N-acetyllactosamine show that binding arises due to hydrogen bonding interactions from the carbon-4 and carbon-6 hydroxyl groups of galactose and carbon-3 of N-acetylglucosamine (GlcNAc) to the side chains of amino acids in the protein. They cannot bind to other sugars such as mannose because this sugar will not fit inside the carbohydrate recognition domain without steric hindrance. Due to the nature of the binding pocket, galectins can bind terminal sugars or internal sugars within a glycan. This allows bridging between two ligands on the same cell or between two ligands on different cells.
Function Galectins are a large family with relatively broad specificity. Thus, they have a broad variety of functions including mediation of cell–cell interactions, cell–matrix adhesion and transmembrane signalling. Their expression and secretion is well regulated, suggesting they may be expressed at different times during development. There are no serious defects when individual galectin genes are deleted in knock-out mouse models. This is because there is substantial overlap for the essential functions. The list of functions for galectins is extensive and it is unlikely they have all been discovered. A handful of the main functions are described below.
Apoptosis Galectins are distinct in that they can regulate cell death both intracellularly and extracellularly. Extracellularly, they cross link glycans on the outside of cells and transduce signals across the membrane to directly cause cell death or activate downstream signaling that triggers apoptosis. Intracellularly, they can directly regulate proteins that control cell fate. Many galectins have roles in apoptosis: One essential way galectins regulate apoptosis is to control positive and negative selection of T cells in the thymus. This process prevents the circulation of T cells that are self-reactive and recognise self antigen. Both galectin-1 and galectin-9 are secreted by epithelial cells in the thymus and mediate T cell apoptosis. T cell death is also necessary to kill activated and infected T cells after an immune response. This is also mediated by galectin-1 and galectin-9. Galectin-1 binds many proteins on the T cell surface, but specifically CD7, CD43 and CD45 are involved in apoptosis. Galectin-7 is expressed under the p53 promoter and may have a key role in regulating apoptosis of keratinocytes after DNA damage, such as that caused by UV radiation. Galectin-12 expression induces apoptosis of adipocytes. Galectin-3 has been shown to be the only galectin with anti-apoptotic activity, proven by knock-out in mice increasing rates of apoptosis. Intracellularly, galectin-3 can associate with Bcl-2 proteins, an antiapoptotic family of proteins, and thus may enhance Bcl-2 binding to the target cell. On the other hand, galectin-3 can also be pro-apoptotic and mediate T cell and neutrophil death.
Embryonic development The interaction of galectin-1 and galectin-8, produced by avian limb bud mesenchymal cells, plays a key role in mediating the development of the vertebrate limb skeleton. These proteins interact with the cells and with one another through a reaction-diffusion-adhesion mechanism, which promotes the patterning and structural organization of the developing limb. Phylogenomic analysis suggests that evolutionary modifications of each of these proteins and the acquisition of new regulatory sequences were responsible for morphological transformations, including the fin-limb transition.
Suppression of T-cell receptor activation Galectin-3 has an essential role in negatively regulating T cell receptor (TCR) activation. Crosslinking of T cell receptors and other glycoproteins by galectin-3 on the membrane of T cells prevents clustering of TCRs and ultimately suppresses activation. This prevents auto-activation. Experiments in transgenic mice with deficient N-acetylglucosamine transferase V (GnTV) have increased susceptibility to autoimmune diseases. GnTV is the enzyme required to synthesise polylactosamine chains, which are the ligand for galectin-3 on T cell receptors. This knock-out means galectin-3 cannot prevent auto-activation of TCR so T cells are hypersensitive. Also within the immune system, galectins have been proven to act as chemoattractants to immune cells and activate secretion of inflammatory cytokines.
Adhesion Galectins can both promote and inhibit integrin-mediated adhesion. To enhance integrin-mediated adhesion, they cross link between two glycans on different cells. This brings the cells closer together so integrin binding occurs. They can also hinder adhesion by binding to two glycans on the same cell, which blocks the integrin binding site. Galectin-8 is specific for the glycans bound to integrin and has a direct role in adhesion as well as activating integrin-specific signaling cascades.
Nuclear pre-mRNA splicing Galectin-1 and galectin-3 have been found, surprisingly, to associate with nuclear ribonucleoprotein complexes including the spliceosome. Studies revealed that galectin-1 and -3 are required splicing factors, since removal of the galectins by affinity chromatography with lactose resulted in loss of splicing activity. It appears that the splicing capability of galectins is independent of their sugar-binding specificities. Site-directed mutagenesis studies to the carbohydrate recognition domain removes glycan binding but does not prevent association with the spliceosome.
Galectins in control of ESCRT, mTOR, AMPK, and autophagy Cytoplasmic galectin-8 and galectin-9 have been shown to control mTOR (mTORC1) and PRKAA (AMPK) in response to lysosomal membrane damage. Lysosomal perforation and other endomembrane damage can be inflicted by various agents such as some chemicals yielding osmotically active products, crystalline silica, possibly amyloid aggregates and cytoplasmic organic or inorganic crystals, as well as intracellular microbial pathogens such as Mycobacterium tuberculosis; such injury can be modeled using membrane-permeant dipeptide precursors that polymerize in lysosomes,. Under resting, homeostatic conditions galectin-8 interacts with mTOR, which in its active state resides on the cytosolic (cytofacial) side of lysosomal membranes. However, under lysosome damaging conditions leading to exposure of the exofacially, i.e., lumenaly, oriented glycans (glycoproteins and glycolipids), galectin-8 recognizes the cytofacial glycans exposed due to membrane damage and releases mTOR. Galectin-8 instead now binds to the mTOR-regulatory complex located on the lysosomal membrane including SLC38A9, LAMTOR1, and RRAGA/RRAGB (RagA/B). This complex loses affinity for mTOR causing mTOR's inactivation and translocation from the lysosome to the cytosol. The damage-responsive complex containing galectin-8, SLC38A9, LAMTOR1, and RRAGA/RRAGB is known as GALTOR. Galectin-3 and galectin-8 also interact with autophagy receptor-regulator TRIM16 that assembles autophagy initiation machinery on damaged lysosomes, whereas galectin-8 also interacts with the autophagy receptor CALCOCO2 (NDP52) recognizing Salmonella-damaged vacuole.
The functional roles of galectins in cellular response to membrane damage are expanding, e.g. Galectin-3 recruits ESCRTs to damaged lysosomes so that lysosomes can be repaired. This occurs before autophagy is induced to repair endosomes and lysosomes lest they are removed by autophagy.
Galectins and disease