Galectin-8 binding to alpha-1 antitrypsin is a physiological mechanism in healthy individuals but exacerbates the symptoms of alpha-1 antitrypsin deficiency.

Abstract

Alpha-1 Antitrypsin (AAT) is a serine protease inhibitor that protects lung tissue by neutralizing neutrophil elastase. Galectin-8 (Gal-8) is a tandem-repeat galectin with N- and C-terminal carbohydrate recognition domains (CRDs) that bind β-galactoside-containing N-glycans. Both proteins co-localize in pulmonary and circulatory systems, suggesting a physiological interaction. Here, we demonstrate a glycan-dependent binding between Gal-8 and AAT using pull-down assays, mass spectrometry, isothermal titration calorimetry, and size-exclusion chromatography. Importantly, binding of Gal-8 impairs AAT's protease inhibitory activity, with the N-terminal CRD of Gal-8 sufficient to disrupt AAT function. Kinetic analyses show that Gal-8 inhibits AAT and enhances trypsin activity, as evidenced by a decrease in K_m and an increase in catalytic efficiency (k_cat/K_m). In cell assays, Gal-8 restored trypsin-mediated proteolysis and induced cell detachment in HeLa and CHO cells despite AAT presence, confirming biological relevance. Leveraging this interaction, we developed a lactose-mediated elution method to purify AAT from human and bovine serum using Gal-8 immobilized on Ni-NTA beads. Moreover, a stable CHO cell line expressing recombinant AAT (∼2 g/L) exhibited glycosylation comparable to serum AAT and retained Gal-8 binding. Our findings reveal Gal-8 as a novel modulator of AAT activity and present a glycan-specific strategy for AAT purification, with implications for biotherapeutic production and quality control.