Abstract
Background
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 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.
Objective
We sought to determine the binding partners of CLC/Gal-10 and elucidate its role in eosinophil biology.
Methods
Intracellular binding partners were determined by ligand blotting with CLC/Gal-10, followed by co-immunoprecipitation and co-affinity purifications. The role of CLC/Gal-10 in eosinophil function was determined by employing enzyme activity assays, confocal microscopy, and shRNA knock-out of CLC/Gal-10 expression in human CD34+ cord blood hematopoietic progenitors differentiated to eosinophils.
Results
CLC/Gal-10 interacts with both human eosinophil granule cationic ribonucleases, eosinophil-derived neurotoxin (EDN, RNS2) and eosinophil cationic protein (ECP, RNS3) , and with murine eosinophil-associated ribonucleases. The interaction is independent of glycosylation and is not inhibitory toward endoribonuclease activity. Activation of eosinophils with INF-γ induces the rapid co-localization of CLC/Gal-10 with EDN/RNS2 and CD63. ShRNA knock-down of CLC/Gal-10 in human cord blood-derived CD34+ progenitor cells impairs eosinophil granulogenesis.
Conclusions
CLC/Gal-10 functions as a carrier for the sequestration and vesicular transport of the potent eosinophil granule cationic ribonucleases during both differentiation and degranulation, enabling their intracellular packaging and extracellular functions in allergic inflammation.
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 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.
Objective
We sought to determine the binding partners of CLC/Gal-10 and elucidate its role in eosinophil biology.
Methods
Intracellular binding partners were determined by ligand blotting with CLC/Gal-10, followed by co-immunoprecipitation and co-affinity purifications. The role of CLC/Gal-10 in eosinophil function was determined by employing enzyme activity assays, confocal microscopy, and shRNA knock-out of CLC/Gal-10 expression in human CD34+ cord blood hematopoietic progenitors differentiated to eosinophils.
Results
CLC/Gal-10 interacts with both human eosinophil granule cationic ribonucleases, eosinophil-derived neurotoxin (EDN, RNS2) and eosinophil cationic protein (ECP, RNS3) , and with murine eosinophil-associated ribonucleases. The interaction is independent of glycosylation and is not inhibitory toward endoribonuclease activity. Activation of eosinophils with INF-γ induces the rapid co-localization of CLC/Gal-10 with EDN/RNS2 and CD63. ShRNA knock-down of CLC/Gal-10 in human cord blood-derived CD34+ progenitor cells impairs eosinophil granulogenesis.
Conclusions
CLC/Gal-10 functions as a carrier for the sequestration and vesicular transport of the potent eosinophil granule cationic ribonucleases during both differentiation and degranulation, enabling their intracellular packaging and extracellular functions in allergic inflammation.
| Original language | English |
|---|---|
| Pages (from-to) | 377-389.e10 |
| Number of pages | 23 |
| Journal | Journal of Allergy and Clinical Immunology |
| Volume | 146 |
| Issue number | 2 |
| Early online date | 23 Jan 2020 |
| DOIs | |
| Publication status | Published - 31 Aug 2020 |
