Characterisation of endothelin converting enzyme-1 shedding from endothelial cells

Abstract 

The aim of this study was to determine if endothelin converting enzyme-1 (ECE-1) like other members of this metalloprotease family undergoes ectodomain shedding. The release/shedding of catalytically active ECE-1 was measured by monitoring the fluorescence resulting from the cleavage of a specific quenched fluorescent substrate. Catalytically active ECE-1 was detected in the media of human umbilical vein endothelial cells, and was confirmed by mass spectrometry based assays. Specificity of cleavage was confirmed by using both narrow and broad specificity inhibitors. In conclusion we demonstrate and characterize for the first time, ECE-1 shedding from the surface of endothelial cells.

Abbreviations: ET-1, endothelin-1, ECE-1, endothelin converting enzyme-1, ACE-2, angiotensin converting enzyme-2, MMP, matrix metalloproteinase, ADAM, a distintegrin and metalloproteinases, PKC, protein kinase C, HUVEC, human umbilical vein endothelial cells, TIMP, tissue inhibitors of metalloproteinase, NEP, neutral endopeptidase, QFS, quenched fluorescent substrate, PMA, phorbol 12-myristate 13-acetate

Keywords: Endothelin, ECE-1, Fluorescence, Ectodomain shedding, Mass spectrometry