Furin Is the Primary in Vivo Convertase of Angiopoietin-like 3 and Endothelial Lipase in Hepatocytes

Rachid Essalmani,Delia Susan-Resiga,Ann Chamberland,Marie-Claude Asselin,Maryssa Canuel,Daniel Constam,John W Creemers,Robert Day,Dany Gauthier,Annik Prat,Nabil G Seidah

doi:10.1074/jbc.m113.501304

Abstract

The proprotein convertases (PCs) furin, PC5/6, and PACE4 exhibit unique and/or complementary functions. Their knock-out (KO) in mice resulted in strong and specific phenotypes demonstrating that, in vivo, these PCs are unique and essential during development. However, they also exhibit redundant functions. Liver angiopoietin-like 3 (ANGPTL3) inhibits lipolysis by binding to lipoprotein lipases. It is found in the plasma as full length and truncated forms. The latter is more active and generated by cleavage at a furin-like site. Endothelial lipase (EL) binds heparin sulfate proteoglycans on cell surfaces and catalyzes the hydrolysis of HDL phospholipids. EL activity is regulated by two endogenous inhibitors, ANGPTL3 and ANGPTL4, and by PCs that inactivate EL through cleavage releasing the N-terminal catalytic and C-terminal lipid-binding domains. Herein, because furin and PC5/6 complete KOs are lethal, we used mice lacking furin or PC5/6 specifically in hepatocytes (hKO) or mice completely lacking PACE4. In primary hepatocytes, ANGPTL3 was processed into a shorter form of ANGPTL3 intracellularly by furin only, and extracellularly mainly by PACE4. In vivo, the absence of furin in hepatocytes reduced by ∼50% the circulating levels of cleaved ANGPTL3, while the lack of PACE4 had only a minor effect. Analysis of the EL processing in primary hepatocytes and in vivo revealed that it is mostly cleaved by furin. However, the lack of furin or PC5/6 in hepatocytes and complete PACE4 KO did not appreciably modify plasma HDL levels or EL activity. Thus, inhibition of furin in liver would not be expected to modify the plasma lipid profiles.

Highlights

Proprotein convertases (PCs) activate overexpressed endothelial lipase (EL) inhibitor angiopoietin-like-3 (ANGPTL3) and inactivate EL
The V5-tagged mouse ANGPTL3 was co-expressed in COS-1 cells with either furin, PC5/6, PC7, or PACE4
We analyzed the role of hepatic PCs in the cleavage-activation of ANGPTL3 [23], and the cleavage-inactivation of EL [21] in COS-1 cells and/or primary hepatocytes and plasma from KO mice

Summary

Background

Proprotein convertases (PCs) activate overexpressed endothelial lipase (EL) inhibitor angiopoietin-like-3 (ANGPTL3) and inactivate EL. Four of them: furin, PC5/6, PACE4, and PC7, are widely or ubiquitously expressed and responsible for most of the processing events occurring in the constitutive secretory pathway: trans Golgi network (TGN), cell surface, and endosomes This leads to the activation, and less frequently to the inactivation, of receptors, ligands, enzymes, viral glycoproteins, or TGF␤-like growth factors [2, 3]. ANGPTL3 is found in the plasma as a full-length protein and a truncated form generated by cleavage at a furinlike site (RAPR2242) located in the linker region between the coiled-coil and fibrinogen-like domains [23] (Fig. 1A) This truncated form exhibits enhanced inhibitory activity for EL [18], but not LPL [23], suggesting that PC-mediated cleavage of ANGPTL3 is more important for inhibiting EL versus LPL activity. Circulating HDLc levels and EL activity were not significantly affected in the latter mice, suggesting that in vivo EL cleavage by furin from hepatocytes has no major impact on HDLc

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION