Ligand Reduces Galectin-1 Sensitivity to Oxidative Inactivation by Enhancing Dimer Formation

Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell surface exposure of phosphatidylserine (PS), a ligand that targets cells for phagocytic removal, in the absence of apoptosis. Gal-1 monomer-dimer equilibrium appears to modulate Gal-1-induced PS exposure, although the mechanism underlying this regulation remains unclear. Here we show that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired dimerization and fails to induce cell surface PS exposure while retaining the ability to recognize carbohydrates and signal Ca²⁺ flux in leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1 dimerization. Continual incubation of leukocytes with Gal-1 resulted in gradual oxidative inactivation with concomitant loss of cell surface PS, whereas rapid oxidation prevented mGal-1 from inducing PS exposure. Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to signal sustained PS exposure in leukocytes and mGal-1 to signal both Ca²⁺ flux and PS exposure. Taken together, these results demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidative inactivation and provides a mechanism whereby ligand partially protects Gal-1 from oxidation.Immunological homeostasis relies on efficient contraction of activated leukocytes following an inflammatory episode. Several factors, including members of the galectin and tumor necrosis factor families (1, 2), regulate leukocyte turnover by inducing apoptotic cell death. In contrast, several galectin family members, in particular galectin-1 (Gal-1),² uniquely regulate neutrophil turnover by inducing phosphatidylserine (PS) exposure, which normally sensitizes apoptotic cells to phagocytic removal (3, 4), independent of apoptosis, a process recently termed preaparesis (5).Previous studies suggested that dimerization may be required for Gal-1-induced PS exposure, as a mutant form of Gal-1 (mGal-1) containing two point mutations within the dimer interface, C2S and V5D (C2S,V5D), displays impaired Gal-1 dimerization and fails to induce PS exposure (6). However, the manner in which monomer-dimer equilibrium regulates Gal-1 signaling remains unclear. Previous studies suggest that dimerization may be required for efficient cross-linking of functional receptors or the formation of signaling lattices (7–9). Consistent with this, monomeric mutants of several other galectins fail to induce PS exposure or signal leukocytes (4, 8). Gal-1 signaling of PS exposure requires initial signaling events, such as mobilization of intracellular Ca²⁺ followed by sustained receptor engagement (10). Although mGal-1 fails to induce PS exposure (6), whether mGal-1 can induce these initial signaling events remains unknown (10).In addition to directly regulating signaling, monomer-dimer equilibrium may also regulate other aspects of Gal-1 function. Unlike many other proteins involved in the regulation of immunity, Gal-1 displays unique sensitivity to oxidative inactivation (11–15). Although engagement of ligand partially protects Gal-1 from oxidation (15), the impact of Gal-1 oxidation on signaling remains enigmatic. During oxidation, Gal-1 forms three distinct intramolecular disulfide bridges that facilitate profound conformational changes that preclude ligand binding and Gal-1 dimerization (12–14), suggesting that monomerdimer equilibrium may also regulate Gal-1 sensitivity to oxidative inactivation.Previous studies utilized dithiothreitol (DTT) in treatment conditions to protect Gal-1 from oxidative inactivation (16, 17). Indeed, failure to include DTT precluded Gal-1-induced death in T cells (3, 18), suggesting that Gal-1 undergoes rapid oxidation in vivo in the absence of reducing conditions. However, DTT itself can induce apoptosis in leukocytes (19), leaving questions regarding the impact of Gal-1 oxidation on these signaling events. In contrast, recent studies utilizing iodoacetamide-alkylated Gal-1 (iGal-1), previously shown to protect Gal-1 from oxidative inactivation (20–29), demonstrated that DTT actually primes cells to become sensitive to Gal-1-induced apoptosis regardless of Gal-1 sensitivity to oxidation (5).As the engagement of leukocyte ligands requires glycan recognition and oxidation precludes this binding (11, 15), understanding the impact of oxidation on Gal-1 signals will facilitate a greater appreciation of the factors that govern Gal-1 oxidation and therefore function. Our results demonstrate that Gal-1 monomer-dimer equilibrium provides a key regulatory point controlling both Gal-1 sensitivity to oxidation and its ability to signal PS exposure in leukocytes. These results provide novel insights into Gal-1 function and explain at a biochemical level the mechanisms regulating Gal-1 oxidative inactivation and signaling.