During pregnancy, placental protein-13 (galectin-13) is highly expressed in the placenta and fetal tissue, and less so in maternal serum that is related to pre-eclampsia. To understand galectin-13 function at the molecular level, we solved its crystal structure and discovered that its dimer is stabilized by two disulfide bridges between Cys136 and Cys138 and six hydrogen bonds involving Val135, Val137, and Gln139. Native PAGE and gel filtration demonstrate that this is not a crystallization artifact because dimers also form in solution. Our biochemical studies indicate that galectin-13 ligand binding specificity is different from that of other galectins in that it does not bind β-galactosides. This is partly explained by the presence of Arg53 rather than His53 at the bottom of the carbohydrate binding site in a position that is crucial for interactions with β-galactosides. Mutating Arg53 to histidine does not re-establish normal β-galactoside binding, but rather traps cryoprotectant glycerol molecules within the ligand binding site in crystals of the R53H mutant. Moreover, unlike most other galectins, we also found that GFP-tagged galectin-13 is localized within the nucleus of HeLa and 293 T cells. Overall, galectin-13 appears to be a new type of prototype galectin with distinct properties.

Galectin-10 (Gal-10) which forms Charcot-Leyden crystals in vivo, is crucial to regulating lymph cell function. Here, we solved the crystal structures of Gal-10 and eight variants at resolutions of 1.55-2.00 Å. Structural analysis and size exclusion chromatography demonstrated that Gal-10 dimerizes with a novel global shape that is different from that of other prototype galectins (e.g., Gal-1, -2 and -7). In the Gal-10 dimer, Glu33 from one subunit modifies the carbohydrate-binding site of another, essentially inhibiting disaccharide binding. Nevertheless, glycerol (and possibly other small hydroxylated molecules) can interact with residues at the ligand binding site, with His53 being the most crucial for binding. Alanine substitution of the conserved Trp residue (Trp72) that is crucial to saccharide binding in other galectins, actually leads to enhanced erythrocyte agglutination, suggesting that Trp72 negatively regulates Gal-10 ligand binding. Overall, our crystallographic and biochemical results provide insight into Gal-10 ligand binding specificity.

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Charcot-Leyden crystal protein/Gal-10, abundantly expressed in eosinophils and basophils, is related to several immune diseases. Recently, crystallographic and biochemical studies showed that Gal-10 cannot bind lactose, because a glutamate residue (Glu33) from another monomer blocks the binding site. Moreover, Gal-10 actually forms a novel dimeric structure compared to other galectins. To investigate the role that Glu33 plays in inhibiting lactose binding, we mutated this residue to glutamine, aspartate, and alanine. The structure of E33A shows that Gal-10 can now bind lactose. In the hemagglutination assay, lactose could inhibit E33A from inducing chicken erythrocyte agglutination. Furthermore, we identified a tryptophan residue (Trp127) at the interface of homodimer that is crucial for Gal-10 dimerization. The variant W127A, which exists as a monomer, exhibited higher hemagglutination activity than wild type Gal-10. The solid phase assay also showed that W127A could bind to lactose-modified sepharose-6B, whereas wild type Gal-10 could not. This indicates that the open carbohydrate binding site of the W127A monomer can bind to lactose. In addition, the distribution of EGFP-tagged Gal-10 and its variants in HeLa cells was investigated. Because Trp72 is the highly conserved in the ligand binding sites of galectins, we used EGFP-tagged W72A to show that Gal-10 could not be transported into the nucleus, indicating that Trp72 is crucial for Gal-10 transport into that organelle.

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