Identification of key amino acid residues determining ligand binding specificity, homodimerization and cellular distribution of human Galectin-10

Abstract

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.

Charcot-Leyden crystal protein/galectin-10 interacts with cationic ribonucleases and is required for eosinophil granulogenesis

“…64 Subsequent studies demonstrated that CLC/Gal-10 can weakly (nonspecifically) bind to carbohydrate under several conditions, as in its monomeric state. 65,66 In addition, recent findings indicate that Gal-10 dimerizes with a novel global shape that is different from that of other prototype galectins (eg, Gal-1, Gal-2, and Gal-7) and may essentially inhibit disaccharide binding. 65,67 The lack of binding to ß-galactosides FIG 6.…”

“…65,66 In addition, recent findings indicate that Gal-10 dimerizes with a novel global shape that is different from that of other prototype galectins (eg, Gal-1, Gal-2, and Gal-7) and may essentially inhibit disaccharide binding. 65,67 The lack of binding to ß-galactosides FIG 6. CLC/Gal-10-deficient (knockdown) eosinophils have fewer secondary granules, a nonproliferative phenotype, decreased MBP-1 expression, and increased secretion of EDN in response to PAF stimulation.…”

A Brief History of Charcot-Leyden Crystal Protein/Galectin-10 Research

“…However, three contradictory results showed that the weak carbohydrate binding activity may be attributed to wild type Gal-10 binding to the cross-linked agarose (or Sepharose) matrix and not to lactose [ 19 , 25 , 27 ], and the crystal structure of Gal-10 showed that it could bind mannose [ 23 ]. Recently, we generated a variant of Gal-10 (W127A) that exists as a monomer and can weakly bind to lactose-modified Sepharose [ 28 ].…”

“…Gel filtration validated the formation of Gal-10 dimers in solution [ 25 ]. In addition, a key residue (Trp127) helps mediate Gal-10 dimerization [ 28 ]. In fact, mutation of Trp127 to alanine abolishes Gal-10 dimer formation.…”

“…In addition, the crystal structure showed that Gal-10 can co-crystallize with mannose, but not lactose [ 23 ]. However, the variant (W127A), which exists as a monomer, can bind to lactose-modified agarose beads [ 28 ]. By investigating the homodimeric structure of Gal-10, we found that a glutamate residue (Glu33) from the S-face of one monomer directly occupies the carbohydrate binding site of another monomer.…”