Prolyl Carboxypeptidase Mediates the C-Terminal Cleavage of (Pyr)-Apelin-13 in Human Umbilical Vein and Aortic Endothelial Cells

• Published in: International journal of molecular sciences Vol. 22; no. 13; p. 6698
• Main Authors: De Hert, Emilie, Bracke, An, Pintelon, Isabel, Janssens, Eline, Lambeir, Anne-Marie, Van Der Veken, Pieter, De Meester, Ingrid
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 01.07.2021; MDPI
• Subjects: (pyr)-apelin-13; ACE2; Angiotensin; Angiotensin-converting enzyme 2; Aorta; Carboxypeptidase C; Cleavage; Endothelial cells; Enzymes; human endothelial cells; IL-1β; Immunocytochemistry; Inflammation; Inhibitors; Lipopolysaccharides; Lysates; Lysosomes; Mass spectrometry; Mass spectroscopy; Metabolism; Peptides; Peptidyl-dipeptidase A; Plasma; prolyl carboxypeptidase; Tumor necrosis factor-TNF; Tumor necrosis factor-α; Umbilical vein
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms22136698

The aim of this study was to investigate the C-terminal cleavage of (pyr)-apelin-13 in human endothelial cells with respect to the role and subcellular location of prolyl carboxypeptidase (PRCP). Human umbilical vein and aortic endothelial cells, pre-treated with prolyl carboxypeptidase-inhibitor compound 8o and/or angiotensin converting enzyme 2 (ACE2)-inhibitor DX600, were incubated with (pyr)-apelin-13 for different time periods. Cleavage products of (pyr)-apelin-13 in the supernatant were identified by mass spectrometry. The subcellular location of PRCP was examined via immunocytochemistry. In addition, PRCP activity was measured in supernatants and cell lysates of LPS-, TNFα-, and IL-1β-stimulated cells. PRCP cleaved (pyr)-apelin-13 in human umbilical vein and aortic endothelial cells, while ACE2 only contributed to this cleavage in aortic endothelial cells. PRCP was found in endothelial cell lysosomes. Pro-inflammatory stimulation induced the secretion of PRCP in the extracellular environment of endothelial cells, while its intracellular level remained intact. In conclusion, PRCP, observed in endothelial lysosomes, is responsible for the C-terminal cleavage of (pyr)-apelin-13 in human umbilical vein endothelial cells, while in aortic endothelial cells ACE2 also contributes to this cleavage. These results pave the way to further elucidate the relevance of the C-terminal Phe of (pyr)-apelin-13.