Crystallization of Galectin-8 Linker Reveals Intricate Relationship between the N-terminal Tail and the Linker

Abstract

Galectin-8 (Gal-8) plays a significant role in normal immunological function as well as in cancer. This lectin contains two carbohydrate recognition domains (CRD) connected by a peptide linker. The N-terminal CRD determines ligand binding specificity, whereas the linker has been proposed to regulate overall Gal-8 function, including multimerization and biological activity. Here, we crystallized the Gal-8 N-terminal CRD with the peptide linker using a crystallization condition that contains Ni2+. The Ni2+ ion was found to be complexed between two CRDs via crystal packing contacts. The coordination between Ni2+ and Asp25 plays an indirect role in determining the structure of β-strand F0 and in influencing the linker conformation which could not be defined due to its dynamic nature. The linker was also shortened in situ and crystallized under a different condition, leading to a higher resolution structure refined to 1.08 Å. This crystal structure allowed definition of a short portion of the linker interacting with the Gal-8 N-terminal tail via ionic interactions and hydrogen bonds. Observation of two Gal-8 N-terminal CRD structures implies that the N-terminal tail and the linker may influence each other’s conformation. In addition, under specific crystallization conditions, glycerol could replace lactose and was observed at the carbohydrate binding site. However, glycerol did not show inhibition activity in hemagglutination assay.

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

“…The alignment showed that several key amino acid residues (Arg53, Arg55, and His57) in the ligand binding site of Gal-13 were not conserved as are residues in Gal-3 and Gal-8N carbohydrate-binding domain (CRD) that bind lactose. Here, we structurally superimposed Gal-13 and Gal-3 [32] and Gal-8N CRD [33] (Figure 1). Gal-13 has Arg53, Arg55 and His57, whereas Gal-3 has His158, Asn160, and Arg162, and Gal-8N has His65, Asn67, and Arg69 in those positions.…”

“…His158 and His65 (located at the same positions as Arg53 in Gal-13) at the bottom of the ligand binding site of Gal-3 and Gal-8 N CRD make important hydrogen bonds with galactose O4 to stabilize the binding [34,35]. Arg162 in Gal-3 and Arg69 in Gal-8N (located at the same position as His57 in Gal-13) also could stabilize the pyran ring of galactose [32,33]. Gal-13 has such a dramatically different ligand binding site compared with Gal-3 and Gal-8N that this protein cannot bind lactose.…”

Galectins and Ovarian Cancer

“…Galectin-8 is one of the tandem repeat galectins that contain two CRDs connected by a linker chain [ 140 , 141 ]. Galectin-8 is expressed in several tissues, including the lung, liver, kidney, brain, and myocardium [ 142 ].…”

Galectin Targeted Therapy in Oncology: Current Knowledge and Perspectives

“…Gal-8 is a type of tandem-repeat galectin with two CRDs one at C- and another at N-terminal region joined by a polypeptide linker. The terminal domains are responsible for recognizing and binding ligands whereas the linking peptide regulates biological functions and it has a multimerization function [ 135 ]. Alternative splicing of the linker region results in the formation of a peptide of different length, which determines the formation of the various Gal-8 isoforms: Gal-8S (short liner region), Gal-8M (medium linker region) and Gal-8L (long linker region) [ 136 , 137 ].…”

“…Studies performed in hepatocellular carcinoma (HCC) cell models indicated that silencing of Gal-9, by small interfering RNAs (siRNA), resulted in increased proliferation and migration [ 162 ]. Additionally, patients with positive Gal-9 expression had longer survival times than those with negative lesions [ 135 ].…”