Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells.
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 approximately 10-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.
McNutt
Markey C
MC
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Lagace
Thomas A
TA
Horton
Jay D
JD
D002384:Catalysis; D002460:Cell Line; D045744:Cell Line, Tumor; D000939:Epitopes / Q000737:chemistry; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*
D000939:Epitopes; D018832:Molecular Chaperones; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases