Ligand binding and complex formation of galectin-3 is modulated by pH variations.

Galectin-3-dependent clusters or lattices are formed at the surface as well as in distinct organelles of eukaryotic cells. Incorporation into membrane proximal networks can fix glycoproteins within subcellular domains or sort them into distinct transport pathways. In the present paper we analysed the effect of acidification on the sugar binding and self-oligomerization of galectin-3. Using a fluorescence anisotropy assay we measured decreasing galectin-3 affinities to the blood group antigen GalNAcα1-3(Fucα1-2)Galß1-4Glc under low pH conditions. Binding to the strong interaction partner N-acetyl-D-lactosamine was also lost at pH 5.0, whereas the less efficient ligand lactose was still able to bind. This indicates that variations in the binding specificity to distinct glycans can be observed by altering the pH. The formation of galectin-3-based complexes by interaction with the multivalent glycoproteins asialofetuin or transferrin was also obliterated at acidic pH and the ligand-binding affinity itself was modulated by oligomerization of the lectin. When galectin-3 was added to giant plasma membrane vesicles from the apical surface of epithelial cells, pH modulation could generate or eliminate the formation of membrane domains enriched with p75(NTR) (neurotrophin receptor p75). In conclusion, the results of the present study suggest that the formation and composition of galectin-3 networks can be fine-tuned by changes in the environmental pH.

pH-dependent recycling of galectin-3 at the apical membrane of epithelial cells.

The ß-galactoside binding protein galectin-3 is highly expressed in a variety of epithelial cell lines. Polarized MDCK cells secrete this lectin predominantly into the apical medium by non-classical secretion. Once within the apical extracellular milieu, galectin-3 can re-enter the cell followed by passage through endosomal organelles and modulate apical protein sorting. Here, we could show that galectin-3 is internalized by non-clathrin mediated endocytosis. Within endosomal organelles this pool associates with newly synthesized neurotrophin receptor in the biosynthetic pathway and assists in its membrane targeting. This recycling process is accompanied by transient interaction of galectin-3 with detergent insoluble membrane microdomains in a lactose- and pH-dependent manner. Moreover, in the presence of lactose, apical sorting of the neurotrophin receptor is affected following endosomal deacidification. Taken together, our results suggest that internalized galectin-3 directs the subcellular targeting of apical glycoproteins by membrane recycling.

Changes in the expression and subcellular distribution of galectin-3 in clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma, a solid growing tumor, is the most common tumor in human kidney. Evaluating the usefulness of ß-galactoside binding galectin-3 as a diagnostic marker for this type of cancer could open avenues for preventive and therapeutic strategies by employing specific inhibitors of the lectin. To study a putative correlation between the extent of galectin-3 and the development of clear cell renal cell carcinoma, we monitored the quantity and distribution of this lectin in tissue samples from 39 patients. METHODS: Galectin-3 concentrations in normal, intermediate and tumor tissues were examined by immunofluorescence microscopy and on immunoblots with antibodies directed against galectin-3 and renal control proteins. The cell nuclei were isolated to determine quantities of galectin-3 that were transferred into this compartment in normal or tumor samples. RESULTS: Immunofluorescence data revealed a mosaic pattern of galectin-3 expression in collecting ducts and distal tubules of normal kidney. Galectin-3 expression was significantly increased in 79% of tumor samples as compared to normal tissues. Furthermore, we observed an increase in nuclear translocation of the lectin in tumor tissues. CONCLUSIONS: Our data indicate that changes in the cellular level of galectin-3 correlate with the development of clear cell renal cell carcinoma, which is in line with previously published data on this specific type of tumor. In most of these studies the lectin tends to be highly expressed in tumor tissues. Furthermore, this study suggests that the increase in the proportion of galectin-3 affects the balance from a cytosolic distribution towards translocation into the nucleus.

Trafficking of galectin-3 through endosomal organelles of polarized and non-polarized cells.

In epithelial cells, the ß-galactoside-binding lectin galectin-3 mediates the non-raft-dependent glycoprotein targeting to the apical membrane domain. In this study, we aimed to identify intracellular compartments involved in the trafficking of galectin-3. By studying fluorescent fusion proteins in living cells, we could show that galectin-3 accumulates intracellularly in acidified endosomes. Total internal reflection fluorescence microscopy studies of the apical surface of polarized MDCK cells revealed that galectin-3 is enriched in tubular and vesicular Rab11-positive recycling endosomes in the vicinity of the apical cell surface. These endosomal organelles are candidate compartments for the association between galectin-3 and exocytic apical cargo.