Endothelial lipase is inactivated upon cleavage by the members of the proprotein convertase family.
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: an N-terminal 40 kDa fragment 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 RNKR330, the consensus cleavage sequence for mammalian proprotein convertases (pPCs). Replacement of Arg-330 with Ser by site-directed mutagenesis totally abolished EL processing. EL processing could efficiently be attenuated by specific inhibitors of pPCs, alpha1-antitrypsin Portland (alpha1-PDX) and alpha1-antitrypsin variant AVRR. Coexpression of the pPCs furin, PC6A, and PACE4 with EL resulted in a complete conversion of the full-length EL to a truncated 40 kDa fragment. Exogenously added EL was also processed by cells, and the processing could be attenuated by alpha1-PDX. The expressed N-terminal 40 kDa fragment of EL (EL-40) harboring the catalytic site failed to hydrolyze [14C]NEFA from [14C]dipalmitoyl-PC-labeled HDL. EL-40 was incapable of bridging 125I-labeled HDL to the cells and had no impact on plasma lipid concentration when overexpressed in mice. Thus, our results demonstrate that pPCs are involved in the inactivation process of EL.
Gauster
Martin
M
Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University, Graz A-8010, Austria.
Hrzenjak
Andelko
A
Schick
Katja
K
Frank
Sasa
S
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002855:Chromatography, Thin Layer; D017931:DNA Primers; D006801:Humans; D006868:Hydrolysis; D008049:Lipase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008055:Lipids / Q000097:blood; D008075:Lipoproteins, HDL / Q000097:blood; D051379:Mice; D016297:Mutagenesis, Site-Directed; D043484:Proprotein Convertases / Q000378:metabolism*
D017931:DNA Primers; D008055:Lipids; D008075:Lipoproteins, HDL; C410382:LIPG protein, human; D008049:Lipase; C498741:Lipg protein, mouse; D043484:Proprotein Convertases