Identification of cathepsin G in the generation of elastase-resistant fragment of vascular endocan: involvement in the regulation of LFA-1-dependent cascade
© BioMed Central Ltd 2009
Published: 11 November 2009
The migration of polymorphonuclear neutrophils (PMN) into inflamed tissue requires fine interactions with the endothelial cell surface. The PMN serine proteases cathepsin G (CG), neutrophil elastase (NE) and proteinase 3 (PR3) were originally thought to play a role by the cleavage of endothelial cell proteins that control the PMN firm adhesion and the transendothelial cell migration. However, how these proteases participate in leukocyte adhesion and transmigration remains controversial. Vascular endocan, also called esm1, is a restricted endothelial cell-secreted proteoglycan constituted by a protein core of 20 kDa and by a unique glycosaminoglycan chain of dermatan sulphate (DS). Endocan is preferentially expressed in lung and kidney tissues. Endocan binds to its leukocytic receptor, the LFA-1 integrin, with an affinity of 18 nM, and inhibits the LFA-1-ICAM-1 interactions. Its expression is upregulated by the proinflammatory mediators TNF, IL-1, and lipopolysaccharide (LPS). In human sepsis, the serum endocan increases from fivefold to 30-fold the normal value and correlates with bad prognosis. Here, we examined the role of PMN-derived proteases in the degradation of endocan.
Human endocan is produced by overexpressing HEK 293 cell lines and purified by anion exchange and affinity chromatographies. Proteolysis is performed by addition of endocan with PMA-activated PMN supernatants, purified CG, NE, or PR3. Endocan degradation is evaluated by ELISA, western blot and MALDI-TOF mass spectrometry. Binding assay on Jurkat cells is performed as described previously. Endocan-derived p14 fragments in human serum were detected by immunoprecipitation.
We demonstrate that CG, NE but not PR3 degrade endocan. We show that the degradation profiles of endocan by CG or NE are different. We interestingly identify a novel peptide fragment of endocan of 14 kDa, named p14, which represents the main endocan degradation product. This p14 results from the specific cleavage of the full-length endocan by CG. The generation of this particular fragment strictly required the presence of the DS chain on full-length endocan. Furthermore, this p14 fragment becomes resistant to NE. We also demonstrate that p14 inhibits the binding of endocan to Jurkat cells. Finally, we present evidence that p14 could be detected in patient serum suffering from an acute PMN-mediated disease like sepsis.
The results suggest that the expression of CG by PMN shortly after their activation, by modifying the micro-environment in degrading vascular endocan, may participate in the complex network that controls tissue infiltration of leukocytes during sepsis.