Good evidence suggest roles of galectins in cancer, immunity and inflammation, and development, but a unifying picture of their biological function is lacking. Instead galectins appear to have a particularly diverse, bewildering but intriguing array of activities both inside and outside cells--"clear truths and mysteries are inextricably twined". Fortunately this has not discouraged but rather enthused a large number of good galectin researchers, some of which have contributed to this special issue of Glycoconjugate Journal to provide a personal, critical status of the field. Here we will give a brief introduction to the galectins as a protein family with some comments on nomenclature.

Galectin-8 has two different carbohydrate recognition domains (CRDs), the N-terminal Gal-8N and the C-terminal Gal-8C linked by a peptide, and has various effects on cell adhesion and signaling. To understand the mechanism for these effects further, we compared the binding activities of galectin-8 in solution with its binding and activation of cells. We used glycan array analysis to broaden the specificity profile of the two galectin-8 CRDs, as well as intact galectin-8s (short and long linker), confirming the unique preference for sulfated and sialylated glycans of Gal-8N. Using a fluorescence anisotropy assay, we examined the solution affinities for a subset of these glycans, the highest being 50 nM for NeuAcalpha2,3Lac by Gal-8N. Thus, carbohydrate-protein interactions can be of high affinity without requiring multivalency. More importantly, using fluorescence polarization, we also gained information on how the affinity is built by multiple weak interactions between different fragments of the glycan and its carrier molecule and the galectin CRD subsites (A-E). In intact galectin-8 proteins, the two domains act independently of each other in solution, whereas at a surface they act together. Ligands with moderate or weak affinity for the isolated CRDs on the array are bound strongly by intact galectin-8s. Also galectin-8 binding and signaling at cell surfaces can be explained by combined binding of the two CRDs to low or medium affinity ligands, and their highest affinity ligands, such as sialylated galactosides, are not required.

No abstract

The galectins are a family of [small beta]-galactoside-binding proteins that have been implicated in cancer and inflammation processes. Herein, we report the synthesis of a library of 28 compounds that was tested for binding to galectins-1, -3, -7, -8N and -9N. An aromatic nucleophilic substitution reaction between 1,5-difluoro-2,4-dinitrobenzene and a galacto thiol gave 5-fluoro-2,4-dinitrophenyl 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-galactopyranoside. This versatile intermediate was then modified in a two dimensional manner: either by further substitution of the second fluoride by amines or thiols, or by reduction of the nitro groups and acylation of the resulting amines, or both. Deacetylation then gave a library of aromatic beta-galactosides that showed variable inhibitory activity against the different galectins, as shown by screening with a fluorescence-polarisation assay. Particularly efficient inhibitors were found against galectin-7, while less impressive enhancements of inhibitor affinity over methyl beta-D-galactopyranoside were found for galectin-1, -3, -8N and -9N. The best inhibitors against galectin-7 showed significantly higher affinity (K(d) as low as 140 microM) than both beta-methyl galactoside (K(d) 4.8 mM) and the unsubstituted beta-phenyl thiogalactoside (non-inhibitory). The best inhibitors against galectin-7 were poor against the other galectins and thus have potential as structurally simple and selective tools for dissecting biological functions of galectin-7.

Galectin-8 has two carbohydrate recognition domains (CRDs), both of which bind beta-galactosides, but have different fine specificity for larger saccharides. Previously we found that both CRDs were needed for efficient cell surface binding and signaling by soluble galectin-8, but unexpectedly binding of the N-CRD to its best ligands, alpha2-3-sialylated galactosides, was not needed. In search for another role for this fine specificity, we now compared endocytosis of galectin-8 in Chinese hamster ovary (CHO) cells and in a mutant (Lec2) lacking sialylated glycans, by fluorescence microscopy. Galectin-8 was endocytosed in both cells by a non-clathrin and non-cholesterol dependent pathway, but surprisingly, the pathway after endocytosis differed dramatically. In wild type (wt) cells, galectin-8 was found along the plasma membrane, near the nucleus, and in small vesicles. In the Lec2 cells, galectin-8 was found in larger vesicles evenly spread in the cell, but not along the plasma membrane or near the nucleus. A galectin-8 mutant with an N-CRD having reduced affinity to sialylated glycans and increased affinity for other glycans, gave a Lec2 like pattern in the wt CHO cells, but a wt pattern in the Lec2 cells. Moreover, the pattern of galectin-3 after endocytosis differed from that of both the wt and mutant galectin-8. These data clearly demonstrate a role of galectin fine specificity for intracellular targeting.