Catalytic Activity Is Not Required for Secreted PCSK9 to Reduce Low Density Lipoprotein Receptors in HepG2 Cells

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9), a member of the proteinase K subfamily of subtilases, promotes internalization and degradation of low density lipoprotein receptors (LDLRs) after binding the receptor on the surface of hepatocytes. PCSK9 has autocatalytic activity that releases the prodomain at the N terminus of the protein. The prodomain remains tightly associated with the catalytic domain as the complex transits the secretory pathway. It is not known whether enzymatic activity is required for the LDLR-reducing effects of PCSK9. Here we expressed the prodomain together with a catalytically inactive protease domain in cells and purified the protein from the medium. The ability of the catalytically inactive PCSK9 to bind and degrade LDLRs when added to culture medium of human hepatoma HepG2 cells at physiological concentrations was similar to that seen using wild-type protein. Similarly, a catalytic-dead version of a gain-of-function mutant, PCSK9(D374Y), showed no loss of activity compared with a catalytically active counterpart; both proteins displayed ϳ10-fold increased activity in degradation of cell surface LDLRs compared with wild-type PCSK9. We conclude that the ability of PCSK9 to degrade LDLRs is independent of catalytic activity and suggest that PCSK9 functions as a chaperone to prevent LDLR recycling and/or to target LDLRs for lysosomal degradation.

Novel Domain Interaction Regulates Secretion of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Protein

“…Fig. 2A shows that deletion of the entire PD protein abolished both the secretion and LDLR-reducing activity of PCSK9, consistent with previous reports (27,28). Separate deletions of PD residues 31-40, 41-50, and 51-60 did not significantly affect PCSK9 processing, secretion, or function, except for a modest effect of ⌬51-60 on autoprocessing.…”

“…The structures of several other serine proteases have been solved in the mature form, where the prodomain is not part of the catalytically active protein (23)(24)(25)(26). Finally, biochemical data show that the catalytic activity of CA is not required for the LDLR-reducing activity in cells (27,28). This also makes PCSK9 different from other serine proteases (23)(24)(25)(26), and thus, its mode of action cannot be deduced from infamily comparisons.…”

Mutations and polymorphisms in the proprotein convertase subtilisin kexin 9 (PCSK9) gene in cholesterol metabolism and disease

“…Thus, PCSK9 functions as a chaperone to prevent LDL receptor recycling and/or target LDL receptors for lysosomal degradation. Furthermore, the ability of PCSK9 to degrade the LDL receptor is independent of its catalytic activity [McNutt et al, 2007;Li et al, 2007]. The role of PCSK9 serine protease activity seems limited to catalyzing the selfcleavage of pro-PCSK9, which is essential for PCSK9 secretion from liver cells and for the reduction of the number of LDL receptors [Zhang et al, 2007].…”

“…The 74-kDa precursor form of PCSK9 undergoes intramolecular autocatalytic cleavage in the endoplasmic reticulum (ER), which produces a 60-kDa catalytic fragment. Autocatalytic cleavage of the zymogen in the ER is essential for transport from this compartment and for secretion [Benjannet et al, 2004;McNutt et al, 2007]. The cleaved prodomain of 14 kDa remains associated with the catalytic domain, permitting the mature protein to move from the ER into the secretory pathway and assisting in the proper folding and the regulation of catalytic activity of the enzyme [McNutt et al, 2007].…”

“…Autocatalytic cleavage of the zymogen in the ER is essential for transport from this compartment and for secretion [Benjannet et al, 2004;McNutt et al, 2007]. The cleaved prodomain of 14 kDa remains associated with the catalytic domain, permitting the mature protein to move from the ER into the secretory pathway and assisting in the proper folding and the regulation of catalytic activity of the enzyme [McNutt et al, 2007]. Also, the 60-kDa mature and secreted form is cleaved at the motif RFHRk218 in an 53-kDa inactivated fragment by other proprotein convertases, particularly furin and/or PC5/6A [Benjannet et al, 2006].…”