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

• Published in: Journal of allergy and clinical immunology 2020-08, Vol.146 (2), p.377-389.e10
• Main Authors: Grozdanovic, Milica M., Doyle, Christine B., Liu, Li, Maybruck, Brian T., Kwatia, Mark A., Thiyagarajan, Nethaji, Acharya, K. Ravi, Ackerman, Steven J.
• Format: Article
• Language: English
• Subjects: Animals; Antigens, CD34 - metabolism; Cells, Cultured; Charcot-Leyden; Cytoplasmic Granules - metabolism; ECP; EDN; Eosinophil Cationic Protein - metabolism; Eosinophil-Derived Neurotoxin - metabolism; Eosinophils; Eosinophils - immunology; galectins; Galectins - metabolism; Glycoproteins - metabolism; granulogenesis; Granuloma - metabolism; Hematopoietic Stem Cells - physiology; Humans; Hypersensitivity - metabolism; Lysophospholipase - metabolism; Mice; Protein Binding; ribonucleases; RNase 2; RNase 3
• ISSN: 0091-6749; 1097-6825
• DOI: 10.1016/j.jaci.2020.01.013

The human eosinophil Charcot-Leyden crystal (CLC) protein is a member of the Galectin superfamily and is also known as galectin-10 (Gal-10). CLC/Gal-10 forms the distinctive hexagonal bipyramidal crystals that are considered hallmarks of eosinophil participation in allergic responses and related inflammatory reactions; however, the glycan-containing ligands of CLC/Gal-10, its cellular function(s), and its role(s) in allergic diseases are unknown. We sought to determine the binding partners of CLC/Gal-10 and elucidate its role in eosinophil biology. Intracellular binding partners were determined by ligand blotting with CLC/Gal-10, followed by coimmunoprecipitation and coaffinity purifications. The role of CLC/Gal-10 in eosinophil function was determined by using enzyme activity assays, confocal microscopy, and short hairpin RNA knockout of CLC/Gal-10 expression in human CD34+ cord blood hematopoietic progenitors differentiated to eosinophils. CLC/Gal-10 interacts with both human eosinophil granule cationic ribonucleases (RNases), namely, eosinophil-derived neurotoxin (RNS2) and eosinophil cationic protein (RNS3), and with murine eosinophil-associated RNases. The interaction is independent of glycosylation and is not inhibitory toward endoRNase activity. Activation of eosinophils with INF-γ induces the rapid colocalization of CLC/Gal-10 with eosinophil-derived neurotoxin/RNS2 and CD63. Short hairpin RNA knockdown of CLC/Gal-10 in human cord blood–derived CD34+ progenitor cells impairs eosinophil granulogenesis. CLC/Gal-10 functions as a carrier for the sequestration and vesicular transport of the potent eosinophil granule cationic RNases during both differentiation and degranulation, enabling their intracellular packaging and extracellular functions in allergic inflammation. [Display omitted]