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

Abstract

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.

The Role of Apelin–APJ System in Diabetes and Obesity

“…It can prevent apelin-13 from degradation by exopeptidase and exert long-term biological effects ( 4 ). Therefore, [pyr 1 ]-apelin-13 is considered a physiological ligand for APJ due to its higher anti-degradation properties ( 18 ). Apelin-17 was found to be the most potent inducer of APJ internalization, and the removal of a single amino acid at the C-terminus can abolish this process ( 19 ).…”

Prolyl Carboxypeptidase Activity Is Present in Human Adipose Tissue and Is Elevated in Serum of Obese Men with Type 2 Diabetes

“…Prolyl carboxypeptidase (PRCP, EC 3.4.16.2), a lysosomal carboxypeptidase, is of interest in metabolic disorders due to its role in the C-terminal cleavage of α-melanocyte stimulating hormone (MSH) 1–13 and (pyr)-apelin-13 [ 1 , 2 , 3 ]. α-MSH 1–13 is an anorexigenic peptide, derived from the polypeptide precursor proopiomelanocortin (POMC), that suppresses appetite and stimulates metabolism by acting on postsynaptic melanocortin receptors MC3R and MC4R in the hypothalamus [ 4 ].…”

“…For example, the apelin peptide increases glucose uptake and insulin sensitivity [ 7 ]. PRCP cleaves (pyr)-apelin-13 to form (pyr)-apelin-13 (1–12) [ 2 , 3 ]. Although many efforts were made in the past to elucidate the exact role of the C-terminal Phe of (pyr)-apelin-13, it is still not clear whether (pyr)-apelin-13 is a more active isoform than (pyr)-apelin-13 (1–12) [ 3 , 9 ].…”

“…PRCP cleaves (pyr)-apelin-13 to form (pyr)-apelin-13 (1–12) [ 2 , 3 ]. Although many efforts were made in the past to elucidate the exact role of the C-terminal Phe of (pyr)-apelin-13, it is still not clear whether (pyr)-apelin-13 is a more active isoform than (pyr)-apelin-13 (1–12) [ 3 , 9 ]. PRCP gene trap mice have higher hypothalamic α-MSH 1–13 levels and a leaner phenotype compared to controls.…”

“…Furthermore, an upregulation of PRCP mRNA expression was seen in human lipopolysaccharide-stimulated endothelial cells [ 20 ]. Pro-inflammatory stimulation of human endothelial cells also induced PRCP secretion, while its intracellular activity level remained constant [ 3 ]. Moreover, PRCP activity is already described in different immune cells like macrophages, T-cells and B-cells [ 15 ].…”