Cleavage of endothelial lipase by the proprotein convertases yields N-terminal 40 kDa-fragment devoid of enzymatic activity and bridging function

Martin Gauster, Andelko Hrzenjak, Katja Schick, and Sasa Frank

Institut of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University, Graz 8010

Corresponding Author: sasa.frank{at}meduni-graz.at

Mature endothelial lipase (EL) is a 68 kDa-glycoprotein. In HepG2 cells infected with adenovirus encoding human EL, the mature EL was detectable in the cell lysates and heparin releasable fractions. In contrast, cell media of these cells contained two EL-fragments: a N-terminal 40 kDa- and a C-terminal 28 kDa-fragment. N-terminal protein sequencing of the His-tagged 28 kDa-fragment revealed that EL is cleaved on the C-terminus of the sequence R-N-K-R³³⁰Ø, the consensus cleavage sequence for mammalian proprotein convertases (pPCs). Replacement of Arg³³⁰, with Ser by site directed mutagenesis totally abolished EL-processing. EL-processing could efficiently be attenuated by specific inhibitors of pPCs, $alpha$ 1-Antitrypsin Portland ( $alpha$ 1-PDX) and $alpha$ 1-Antitrypsin variant AVRR. Co-expression of PCs furin, PC6A and PACE4 with EL, resulted in a complete conversion of the full-length EL to truncated 40 kDa-fragment. Exogenously added EL was also processed by cells and the processing could be attenuated by $alpha$ 1-PDX. The expressed N-terminal 40 kDa-fragment of EL (EL-40) harboring the catalytic site, failed to hydrolyze ¹⁴C-NEFA from ¹⁴C -dipalmitoyl-phosphatidycholine (PC)- labeled HDL. EL-40 was incapable of bridging ¹²⁵I-labeled HDL to the cells and had no impact on the plasma lipid concentration when overexpressed in mice. Thus, our results demonstrate that pPCs are involved in the inactivation process of EL.

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