Natural killer (NK) cells are critical components of the innate immune system. Their primary role is to induce apoptosis in target cells, such as cancerous or virally infected cells. These targets are recognized through interactions between activating or inhibitory receptors on the NK cell surface. Among the activating receptors is the natural cytotoxicity receptor NKp46. Several ligands for this receptor have been identified, including the epithelial adhesin Epa1 from the yeast Candida glabrata. Invasive candidiasis caused by this yeast is a significant complication for patients with hematological diseases. The interaction between NKp46 and Epa1 is thought to depend specifically on an O-glycan at threonine 225 of NKp46. To elucidate the molecular details of this interaction, we optimized the recombinant production of soluble NKp46 and Epa1, generated glycosylation variants of multiple NKp46 mutants, and evaluated the role of NKp46 glycosylation in Epa1 binding using microscale thermophoresis and isothermal titration calorimetry. Additionally, for the first time, we provide a comprehensive glycosylation profile of NKp46, determined through mass spectrometry of intact glycopeptides obtained by O-glycoprotease and trypsin proteolysis. Our findings demonstrate that the NKp46 stalk is glycosylated at multiple sites, involving both an N-glycan and more than one O-glycan. These glycans are critical for the interaction with Epa1, providing NK cells with enhanced sensitivity to Candida glabrata epitopes.

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 12840 Prague Czech Republic

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