Resetting the ligand binding site of placental protein 13/galectin-13 recovers its ability to bind lactose

Abstract

Placental protein 13/galectin-13 (Gal-13) is highly expressed in placenta, where its lower expression is related to pre-eclampsia. Recently, the crystal structures of wild-type Gal-13 and its variant R53H at high resolution were solved. The crystallographic and biochemical results showed that Gal-13 and R53H could not bind lactose. Here, we used site-directed mutagenesis to re-engineer the ligand binding site of wild-type Gal-13, so that it could bind lactose. Of six newly engineered mutants, we were able to solve the crystal structures of four of them. Three variants (R53HH57R, R53HH57RD33G and R53HR55NH57RD33G had the same two mutations (R53 to H, and H57 to R) and were able to bind lactose in the crystal, indicating that these mutations were sufficient for recovering the ability of Gal-13 to bind lactose. Moreover, the structures of R53H and R53HR55N show that these variants could co-crystallize with a molecule of Tris. Surprisingly, although these variants, as well as wild-type Gal-13, could all induce hemagglutination, high concentrations of lactose could not inhibit agglutination, nor could they bind to lactose-modified Sepharose 6b beads. Overall, our results indicate that Gal-3 is not a normal galectin, which could not bind to β-galactosides. Lastly, the distribution of EGFP-tagged wild-type Gal-13 and its variants in HeLa cells showed that they are concentrated in the nucleus and could be co-localized within filamentary materials, possibly actin.

Structure–function studies of galectin‐14, an important effector molecule in embryology

“…At position 55 in Gal‐13 and Gal‐14, there is an arginine residue, whereas in Gal‐1, Gal‐3, and Gal‐8, there is an asparagine at the same position. Previously, we mutated Arg55 in Gal‐13 to an asparagine and found that this mutation did not influence lactose binding [17]. This indicates that the residues at this position might regulate the ligand‐binding specificity of galectins.…”

“…In Gal‐13, there is also a histidine residue (His57) in the lactose‐binding site. However, previous Gal‐13 studies showed that only the variant having an arginine at this position could bind lactose [17]. In fact, when both Arg53 and His57 in Gal‐13 were mutated to His53 and Arg57, Gal‐13 regained its ability to bind lactose.…”

“…Our biochemical and crystallographic studies demonstrated that the replacement of His with Arg explains the lack of lactose binding. Upon mutating this arginine to histidine, we observed that Gal‐13 regained its ability to bind lactose [17].…”

“…Gal-4, Gal-8, Gal-9, and Gal-12 are tandem-repeat galectins with two CRDs linked by a short linker peptide [10]. Prototype galectins Gal-1, Gal-2, Gal-7, and Gal-10 form dimers via noncovalent interactions [12][13][14][15], whereas Gal-13 dimers are stabilized by two intermolecular disulfide bonds [16,17]. Amino acid sequence alignments of Gal-1, Gal-2, Gal-7, Gal-10, and show that they are highly conserved, thus explaining their highly conserved monomer structures as well [18].…”

The Structural Biology of Galectin-Ligand Recognition: Current Advances in Modeling Tools, Protein Engineering, and Inhibitor Design

“…Figure 3C shows that these could be due to the fact it lacks 4 out of the 6 most conserved residues, the previously described His, Asn, Arg, and Trp of subsite C. A second special case is Gal-13 (also known as Placenta Protein 13), which has proven to be unable to bind lactose by both crystallographic and biochemical analysis. However, the Gal-13 R53H-H57R double mutant (PDB id: 6a62) was proven to recover the lactose-binding capabilities (Su et al, 2018a), thus suggesting that it possibly changed its binding capacity due to a few recent evolutionary steps. The structural explanation behind this clever work of re-engineering lays in the fact that in Gal-13 position 53 corresponds to the key His and position 57 to the Arg of subsite C, which as mentioned above are necessary for hydrogen bonding the axial C4-OH.…”