Poster Presentation 9th General Meeting of the International Proteolysis Society 2015

Prolyl carboxypeptidase activity, expression and localization in primary murine and human cells  (#140)

Kaat Kehoe 1 , Wendy Theelen 2 , Heidi Noels 2 , Gwendolyn Vliegen 1 , Yannick Waumans 1 , Jan Gielis 3 , Anne-Marie Lambeir 1 , Ingrid De Meester 1
  1. Department of Pharmaceutical Sciences, Laboratory of Medical Biochemistry, University of Antwerp, Antwerp, Belgium
  2. Institute for Molecular Cardiovascular Research, RWTH Aachen University, Aachen, Germany
  3. Department of Thoracic and Vascular Surgery; Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp, Antwerp, Belgium

Background: Prolylcarboxypeptidase (PRCP) is a serine carboxypeptidase that cleaves off C-terminal amino acids adjacent to proline or alanine. PRCP is mostly found in the lysosomes of the cells, or as a soluble enzyme in plasma, or bound to the plasma membrane of endothelial cells. As such PRCP is capable of changing the biological activity of its physiological substrates being angiotensin 2 and 3, des-Arg9-bradykinin and a-melanocyte-stimulating hormone 1-13. Here, we aim to evaluate the activity, expression and localization of PRCP in primary murine and human cells.

Methods: Primary monocytes and macrophages were derived from the bone marrow of wildtype Swiss mice. Primary human monocytes, macrophages, lymphocytes were isolated from buffy coats using ficoll-paque density gradient centrifugation followed by red blood cell lysis and dextran sedimentation to isolate the granulocytes. Resting macrophages were further differentiated into activated pro-inflammatory M1 macrophages upon LPS/IFN-γ stimulation for 24 h. Granulocytes were activated with PMA for 30 min. Primary human aortic endothelial and smooth muscle cells were bought from a commercial supplier. PRCP activity was measured using a validated RP-HPLC assay1. PRCP expression was assessed via western blotting. Immunofluorescence was performed to identify the cellular localization of PRCP.

Results: Murine and human PRCP activity and expression is significantly upregulated upon monocyte to macrophage differentiation, implying a role for the enzyme in the immune respons. PRCP activity is high in human macrophages, smoothe muscle cells and granulocytes compared with monocytes, lymphocytes and endothelial cells. PMA stimulation of granulocytes decreases PRCP activity, indicating the secretion of PRCP. Immunofluorescene revealed that PRCP is located in the lysosomes of monocytes and macrophages and is not anchored to the membrane of these cells.

Conclusion: The knowledge of PRCPs activity, expression and localization in blood cells will improve our insight in its role in the turnover of circulating peptides.

  1. K. Kehoe et al. Validation of a specific prolylcarboxypeptidase activity assay and its suitability for plasma and serum measurements, Analytical Biochemistry 443(2), 232-239 (2013).