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Biochemical Society Transactions The galectin family of mammalian carbohydrate-binding molecules R. C. Hughes National Institute for Medical Research, Mill Hill, London NW7 IAA, U.K. I I94 T h e galectins are a family of P-galactoside-binding proteins (see [ l ] for recent review). T h e family contains nine or ten members that have distinct and dynamic patterns of expression during development, which suggests diverse and important roles in embryogenesis and tissue formation. Table 1 shows a partial list of galectin expression in adult tissues. We have focused on galectin-3, which is present on various inflammatory cells and is also widely distributed on branching epithelia, including kidney tubules, as discussed later. Structure of the galectins T h e general structure of the galectins is shown in Figure 1. T h e carbohydrate-recognition domains (CRDs) are derived from two exons and show extensive sequence homology within the family. Recent crystallographic studies show that the CRDs of galectins 1, 2, 3 and 4 are homologous 12 P-stranded structures [2]. T h e family divides into three subtypes. Galectins 1, 2 and 5 have a single CRD preceded by relatively short N-terminal domains. Under normal conditions these are dimeric molecules and hence are funcAbbreviations used: CRD, carbohydrate-recognition domain; TGB, thioglycollate broth; MDCK, MadinDerby canine kidney; HGF, human growth factor. tionally bivalent. Galectins 4, 6, 8 and 9 are constitutively bivalent: they have two CRDs joined by a linker sequence and preceded by short N-terminal domains. Galectin-3 is, so far, unique in having an extra-long N-terminal domain that is encoded within a single exon 111 and contains multiple repeats of a nine-residue sequence rich in glycine, proline and tyrosine. Structural studies, including NMR, show this domain to be largely unstructured and flexible ([3]; B. Birdsell, J. Feeney, S. Bawumia and R. C. Hughes, unpublished work). T h e function of this domain is not understood but it appears to be important for the self-assembly of galectin-3 monomers and hence a switch to multi-valency that occurs at high concentrations. Galectin-3 dimers and trimers are stabilized in solution by cross-linking of glutamine residues in the repetitive sequence to unidentified lysine residues in the globular CRD by tissue-type transglutaminase [4]. Carbohydrate binding Galectin binding to carbohydrates is calcium independent, in contrast with C-type lectins such as the selectin family. Galactose is a necessary but insufficient requirement for high-affinity binding. T h e amino acid residues in the carbohydrate-binding pocket of the CRDs responsible for galactose recognition are completely conserved for all the galectins so far sequenced [5]. Table I Partial list of galectin expression in adult tissues Galectin Tissue distribution I Skeletal/smooth muscle, motor/sensory neurons, kidney, placenta, thymus Hepatoma, GI tract (especially ileum) Activated macrophages, eosinophils, neutrophils, mast cells, epithelium of GI and respiratory tracts, some sensory neurons Intestinal and oral epithelium Erythrocytes, reticulocytes Intestinal epithelium Keratinocytes Lung, liver, kidney, heart, brain Liver, small intestine, kidney, lymphoid tissue, lung, cardiac/skeletal muscle 2 3 4 5 6 7 8 9 Volume 25 The Role of Glycosylation in Biomolecular Interactions Figure I Overall structure of the galectins The CRD encompasses the light- and dark-stippled boxes. N-terminal domains are shown as cross-hatched or unshaded boxes. The extra N-terminal domain of galedin-3 and the linker domain of other galectins are shown in black. AA, approximate numbers of amino acid residues in each domain. Exon 11 III Iv -30 -60 -50 N V VI AA Galectins 1, 2 , 7 Galectin 5 Exon II III Galectin 3 -1I5AA Repeats Y P G * * * P G A Galectins 4, 6, 8, 9 However, there is a higher level of complexity involved for carbohydrate recognition than just the central galactose. Typically the galectins bind to Type I Galb1,3 GlcNAc or Type I1 GalP1,4 GlcNAc chains, and with higher-affinity polylactosamine chains. However, extension at the nonreducing end of the disaccharide units with NeuNAca2,3 or with GalNAccr1,3 and Fuccr1,Z substituents enhances affinity to galectin-3 but has a smaller effect or reduces galectin-1 binding [6,7]. Hence, human blood group type A epitopes are good ligands for galectin-3 but not galectin-1. Recent homology modelling and oligosaccharide docking studies together with site-directed mutagenesis have identified likely contact residues in the extended binding pocket of galectin-3 CRD responsible for recognition of these more complex carbohydrates (K. Henrick, S. Bawumia, E. A. M. Barboni, B. Mehul and R. C. Hughes, unpublished work). These residues include, in the hamster sequence: Arg- 13 and Glu- 160 for Fuccc1,Z; Arg-139 and Ile-141 for GalNAca1,3; and Arg-139, Glu-230 and Ser-232 for NeuNAca2,3 substituents on the primary galactose. Secretion The galectins are found in the cell cytoplasm. They are made on free ribosomes [8], lack trans- membrane sequences or signal sequences for co-translational transfer into the endoplasmic reticulum and are not glycosylated although several have consensus sites for N-glycosylation. Despite these features galectins are secreted from cells by a novel, incompletely understood mechanism independent of the classical secretory pathway through the endoplasmic reticulumGolgi complex [9- 111. Immunocytochemical studies have indicated that galectin-1 and galectin-3, before secretion, accumulate at sites at the cytoplasmic side of plasma membranes [9,10,12]. This step is rate limiting in galectin-3 secretion from macrophages as well as from Cos cells transfected with galectin-3 constructs, and is strongly up-regulated by heat shock and calcium ionophores [10,13]. The next step in galectin secretion appears to be evagination of plasma membranes specifically at those sites [9,10,12,13], a process that, at least for galectin3, critically requires N-terminal domains. Thus, Cos cells transfected with galectin-3 CRD cDNA can be induced to deliver the expressed protein to plasma membrane sites, but evagination and subsequent secretion are blocked [ 131. Furthermore, fusion proteins of a cytoplasmic indicator protein such as chloramphenicol acetyltransferase with the N-terminal half of galectin-3 are very efficiently exported from transfected Cos cells [ 131. Pulse-chase and cytochemical data I997 I195 Biochemical Society Transactions I196 indicate that the final steps in galectin secretion are the pinching off of evaginating plasma membrane domains and the release, either spontaneous or assisted, of galectin from the externalized vesicles. Thus a light vesicular fraction isolated on sucrose gradients from conditioned medium of transfected Cos cells secreting hamster galectin-3, or mouse macrophages secreting endogenous galectin-3, contains lectin in a protease-resistant form unless detergent is added to the incubation mixture [13]. Under physiological conditions galectin-3 is rather quickly released (half-life 60-90 min) from the vesicles into a soluble form that is fully sensitive to protease. T h e results suggest that vesicular release plays a significant role in secretion of galectin-3, although the possibility that direct transfer through the plasma membrane at the evaginating sites or elsewhere also takes place cannot be excluded. In yeast, a novel transmembrane protein was recently identified by expression cloning that appears to be critical for galectin-1 secretion from transfected cells [ 141. Extracellular functions Once exported from the cell, galectins are free to combine with appropriately glycosylated proteins at cell surfaces or in the extracellular matrix. Recent evidence indicates that galectins play diverse and potentially important roles subsequent to their ligation of biologically active receptors. In particular, galectin-3 appears to be a multi-purpose signalling molecule. It modulates growth and apoptosis of human leukaemia cells [15]. When added to human monocytes exogenously at low, probably monomeric concentrations, galectin-3 triggers superoxide production and potentiates the lipopolysaccharide-induced release of interleukin-1 up to 2-fold [16,17]. At higher concentrations it binds to and cross-links IgE receptors on basophilic cells and induces cytotoxicity towards intracellular parasites, degranulation and serotonin release [ 18,191. Galectin3 binds to the NCA 160 surface antigen of human neutrophils, a member of the carcinoembryonic antigen family of cell-adhesion molecules, and triggers an oxidative burst [ZO]. On human T-cells (Jurkat cells) the lectin binds to the heavy chain of the CD98 T-cell activation antigen and induces an uptake of extracellular calcium [21]. Of course, a direct link between galectin-3 ligation of these and other surface antigens and the biological effect is not implied, except the effect on mast cells which is most Volume 25 likely mediated by IgE cross-linking as are other degranulation-effectors. However, the results amply illustrate the diversity of surface receptors recognized by galectin-3 and the range of downstream effects reported. Galectins also bind to certain glycoforms of extracellular matrix components, such as laminin and fibronectin [7] or tenascin [22] as well as cell-surface adhesion molecules. A major receptor on mouse macrophages is the a-subunit of the C D l l b / l 8 integrin, the Mac 1 antigen [23]. Interestingly, surface expression of galectin-3 is a developmental marker on primary peritoneal mouse macrophages [ 11,24,25]. Although it is made and secreted by cells at various stages of activation elicited with different inflammatory agents, surface expression is rather tightly restricted to peritoneal macrophages elicited by thioglycollate broth (TGB). This implies that glycosylation of galectin-3 receptors, including CD1 lb, is modulated developmentally and that provision of high-affinity lectin-binding glycans on these receptors may be specifically associated with elicitation by TGB. A correlation of these findings with the reported up-regulation of a specific a 1,3-gaIactosyl transferase gene in TGBelicited mouse macrophages [26] is rather striking, although so far no direct linkage between the two observations has been described. However, the transferase would be capable of extending P-galactose terminals on N-glycans of surface receptors such as C D l l b with a1,3 galactose substituents, thereby increasing their affinity for galectin-3 and boosting surface retention; this applies especially under high-stringency conditions such as those which are likely to occur in tissues. In humans, where the a 1,3 galactosyl transferase is absent, other high-affinity glycans may, by analogy, appear transiently on activated macrophages. In pulmonary fibrosis a subset of alveolar macrophages transiently expressing glycan structures recognized by the plant lectin DBA and possibly related to blood-group A epitopes, have been identified [27]. TGB-elicited macrophages may represent a population of cells that have been recently recruited into the tissue space by extravasation through an endothelial monolayer and penetration of the underlying basement membrane. T h e C D l l b / l 8 integrin is a key player in these events and modulation of its activity by ligation with galectin-3 is an intriguing possibility. Several studies have shown that galectins, when added exogenously to various cell-adhesion or motility The Role of Glycosylation in Biomolecular Interactions assays, can either antagonize or promote adhesions in a concentration-dependent manner [ 11,281. For example, galectin-1 and galectin-3 can weaken cell interactions with laminin substrata in simple in vitro assays. In the former case a direct inhibitory effect on the ct7/fil integrin, the major laminin receptor in the myoblasts used for this study, was shown. Similarly the migration of human metastatic breast tumour cell lines through a Matrigel barrier in a Transwell assay is increased by nanomolar concentrations of galectin-3 consistent with a weakening of cell-substratum adhesions, but is inhibited at higher concentrations [29]. T h e simplest interpretation of these findings is that galectins may sterically block integrin-matrix interactions upon binding to one or more of the interacting partners. At higher concentrations bivalent galectins may stimulate adhesions by cross-linking specifically glycosylated cell surface and matrix proteins. Galectin-3 and epithelial polarity Galectin-3 is expressed widely in epithelia, usually on apical plasma membranes domains of polarized tissues [30]. In the kidney its expression is regulated developmentally. Using reverse transcription-PCR the first transcripts in mouse embryonic kidney are detected at Ell-12, expression is maximal at E15-16 and decreases in newborns (P. Winyard, A. s. Wolf, Q. Bao and R. C. Hughes, unpublished work). Immunocytochemistry of sections of newborn kidney shows that galectin-3 expression is highest in ureteric bud derivatives i.e., collecting ducts and connecting segments of distal tubules. These results suggest that galectin-3 may play a role in establishing or maintaining the epithelial architecture of collecting ducts during kidney differentiation. Recent work, using the collecting ductderived Madin-Darby canine kidney (MDCK) cell line as a model, is consistent with this idea [31]. When cells are seeded at low density within three-dimensional collagen gels, the cells grow clonally and form small aggregates that, in time, become organized into polarized cysts with a central lumen and a basal surface in contact with the surrounding matrix, as well as a laminin-rich basement membrane deposited by the cells. Treatment of the cyst cultures with human growth factor (HGF) scatter factor induces the cysts to form sprouts that by further cell division and migration elongate into tubule-like structures that eventually fuse and form a large syncy- tium. Immunocytochemistry shows that galectin-3 is excluded from the lumenal/apical surface of the cysts whereas the lateral and basal surfaces are heavily stained. In the HGF/scatter factor-treated cultures the baso-lateral staining in the cyst bodies is retained but there is a marked down-regulation of galectin-3 expression at the sprout sites and in the forming tubules. Thus high galectin-3 expression is associated with sites of tight adhesions in the developing epithelium, leading to the possibility that the lectin may serve to synergize with or activate other adhesive interactions. These interactions include cell-cell adhesions at lateral surfaces mediated by cadherin family members and cellmatrix adhesions at basal surfaces involving integrins and matrix components. At sites of cell movement and re-organization occurring during sprouting and tubule formation, where strength of adhesion would need to be lessened, lectin expression is reduced at the interactive plasma membrane domains. In support of this idea treatment of cyst cultures in MDCK cells with galectin-3-blocking antibodies does indeed speed up the growth of the cysts as compared with the control culture. Conversely addition of high concentrations of recombinant galectin-3 retards cyst growth. Similarly, a ricin-resistant MDCK cell line that produces and secretes wild-type levels of galectin-3, but fails to galactosylate glycoproteins, forms cysts in collagen cultures that grow very quickly and adopt very irregular shapes (Q. Bao and R. C. Hughes, unpublished work). These results show that loss of galectin-3 binding receptors at the cell surface or within the extracellular matrix is correlated with abnormal cyst formation in this experimental system. Some recent findings relate these results to the potential roles of galectin-3 in formation of epithelia in the embryonic kidney (P. Winyard, A. S. Wolf, Q. Bao and R. C. Hughes, unpublished work). During human kidney ontogeny galectin-3 is first expressed in the epithelia of the mesonephric duct and is absent in the intermediate mesoderm. In human metanephric kidney galectin-3 is found predominantly at lumenal/apical domains of ureteric bud branches in the nephrogenic cortex. Interestingly, this subcellular localization shifts to a more baso-lateral pattern as the medullary collecting duct lineage matures. However, in human multicystic dysplastic kidney disease the malformed dysplastic tubules and cysts retain an immature apical expression of I997 I197 Biochemical Society Transactions I198 galectin-3. It is tempting to see parallels in these expression patterns occurring during normal and diseased kidney differentiation and the MDCK model. Galectin-3 on basal domains of maturing collecting duct epithelia would be in a position to influence cell interactions with basement membranes, particularly laminin, that is firmly implicated in epithelial maturation based on in vitro studies [32]. Conversely, apically expressed galectin-3 would be functionless as a negative growth regulator of cyst expansion. Further studies are directed at defining the signals involved in targeted secretion of galectin-3 from apical or baso-lateral domains of polarizing epithelia and the effects of recombinant lectin and blocking antibodies on ureteric b u d development in explants of kidneys of normal and dysplastic mice as well as mice having null mutations in the galectin-3 gene [331. 1 Barondes, S. H., Cooper, D. N. W., Gitt, M. A. and Leffler, H. (1994) J. Biol. Chem. 269, 20807-20810 2 Lobasonov, Y. D. and Rini, J. M. (1997) Trends Glycosci. Glycotech. 9, 145-154 3 Mehul, B., Bawumia, S., Martin, S. R. and Hughes, R. C. (1994) J. Biol. Chem. 269, 18250-18258 4 Mehul, B., Bawumia, S. and Hughes, R. C. (1995) FEBS Lett. 300, 160-164 5 Ahmed, H. and Vesta, G. R. (1994) Glycobiology 4, 545-549 6 Sparrow, C. P., Leffler, H. and Barondes, S. H. (1987) J. Biol. Chem. 262, 7383-7390 7 Sato, S. and Hughes, R. C. (1992) J. Biol. Chem. 267,6938-6990 8 Wilson, T. J. G., Firth, M. N., Powell, J. T. and Harrison, F. L. (1989) Biochem. J. 261,847-852 9 Cooper, D. N. W. and Barondes, S. H. (1990) J. Cell Biol. 110, 1681-1691 10 Sato, S., Burdett, I. and Hughes, R. C. (1993) Exp. Cell Res. 207, 8-18 11 Sato, S. and Hughes, R. C. (1994) J. Biol. Chem. 269, 4424-4430 12 Harrison, F. L. and Wilson, T. J. (1992) J. Cell Sci. 101,635-646 13 Mehul, B. and Hughes, R. C. (1997) J. Cell Sci. 110, 1169-1 178 Volume 25 14 Cleves, A. E., Cooper, D. N. W., Barondes, S. €1. and Kelly, R. B. (1996) J. Cell Biol. 133, 1017-1026 15 Yang, R.-Y., Hsu, D. K. and Li, F.-T. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 6737-6742 16 Jeng, K.-C. G., Frigieri, L. G. and Liu, F.-U. (1994) Immunol. Lett. 42, 113-1 16 17 Liu, F.-T., Hsu, D. K., Zuberi, R. I., Kuwabara, I., Chi, E. Y. and Henderson, W. R. (1995) Am. J. Pathol. 147, 1016-1028 18 Zuberi, R. I., Frigeri, L. G. and Liu, F.-T. (1994) Cell. Immunol. 156, 1-12 19 Truong, M.-J., Gruart, V., Liu, F.-T., Prin, L., Capron, A. and Capron, M. (1993) Eur. J. Immunol. 23, 3230-3235 20 Yamaoka, A., Kuwabara, I., Frigeri, L. G. and Liu, F.-T. (1995) J. Immunol. 154, 3479-3487 21 Dong, S. and Hughes, R. C. (1997) FEBS Lett. 395, 165-169 22 Probstmeier, K., Montag, D. and Schachner, M. (1995) J. Neurochem. 64,2465-2472 23 Dong, S. and Hughes, R. C. (1997) Glycoconjugate J. 14, 267-274 24 Ho, M.-K. and Springer, T. A. (1982) J. Immunol. 128, 1221- 1228 25 Hughes, R. C. (1994) Glycobiology 4,5-12 26 Joziasse, D. H., Shaper, N. L., Kim, D., van den Eijnden, D. H. and Shaper, J. H. (1992) J. Biol. Chem. 267, 5534-5541 27 Kasper, M. and Hughes, R. C. (1996) J. Pathol. 179, 309-316 28 Cooper, D. N. W. (1997) Trends Glycosci. Glycotech. 9, 57-68 29 Le Marer, N. and Hughes, R. C. (1996) J. Cell. Physiol. 168, 51-58 30 Fowlis, D., Colnot, C., Ripoche, M. A. and Poirier, F. (1995) Devel. Dynamics 203, 241-251 31 Bao, Q. and Hughes, R. C. (1995) J. Cell Sci. 108, 279 1 -2800 32 Klein, G., Langegger, M., Timpl, R. and Ekblom, P. (1988) Cell 55, 331-341 33 Colnot, C., Ripoche, D., Cannon, V., Scaervu, F., Cooper, D. N. W. and Poirier, F. (1997) Trends Glycosci. Glycotech. 9, 31-40 _ _ _ _ _ _ _ ~ Received 26 June 1997