PCSK9 binds to the LDLR and directs it into a lysosomal degradation pathway rather than the recycling pathway

Journal of Lipid Research Volume 55, 2014 1505 Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc. modulating the levels of LDL receptor (LDLR) ( 11–14 ). PCSK9 binds to the LDLR and directs it into a lysosomal degradation pathway rather than the recycling pathway ( 15–18 ). This activity of PCSK9 on the LDLR is independent of its catalytic activity ( 18 ). The synthesis of PCSK9 and LDLR is induced by HMG-CoA reductase inhibitors, such as statins, under the control of the sterol-regulated transcription factor, SREBP2 ( 19–20 ). Treatment with statins increases both hepatic LDLR content and circulating levels of PCSK9 ( 21–22 ). Therefore, increased PCSK9 appears to limit both statin-induced increases in LDLR content and the resulting reduction of plasma LDL-C in humans. PCSK9 is initially synthesized as a 74 kDa proprotein, from which proteolytic cleavage of the 14 kDa N-terminal prodomain results in the 60 kDa mature form consisting of the N-terminal catalytic domain and the C-terminal domain ( 13 ). PCSK9 is expressed in multiple tissues, including liver, intestine, kidney, and cerebellum, of which the liver appears to be the major source of the circulating protein ( 23–25 ). The liver is also the major site of regulation of plasma LDL-C by PCSK9 ( 24, 26–28 ). Clearance of PCSK9 from the circulation is thought to be rapid, based on the 5 min half-life of the recombinant human protein injected into mice ( 26, 28 ). The clearance is presumed to be mediated primarily by the LDLR, as the half-life of recombinant PCSK9 is prolonged when injected into LDLR-defi cient mice ( 26 ). Decreased half-life of a mutant form of PCSK9 (D374Y) that has higher LDLR degradation activity also supports this notion ( 26 ). The proteolytic cleavage of PCSK9 may represent a second mechanism of its clearance. A truncated form of PCSK9 with a molecular mass of 52–55 kDa has been observed and represents up to 40% of the total circulating PCSK9 in human serum ( 29, 30 ). Studies in mice with Abstract Proprotein convertase subtilisin-kexin type 9 (PCSK9) is a secreted protein which regulates serum LDL cholesterol. It circulates in human and rodent serum in an intact form and a major truncated form. Previous in vitro studies involving the expression of human PCSK9 genetic variants and in vivo studies of furin knockout mice suggest that the truncated form is a furin cleavage product. However, the circulating truncated form of PCSK9 has not been isolated and characterized. Utilizing antibodies which bind to either the catalytic domain or the C-terminal domain of PCSK9, the truncated PCSK9 was isolated from serum. MS was used to determine that this form of PCSK9 is a product of in vivo cleavage at Arg218 resulting in pyroglutamic acid formation of the nascent N terminus corresponding to Gln219 of intact PCSK9. We also determined that the truncated PCSK9 in serum lacked the N-terminal segment which contains amino acids critical for LDL receptor binding. A truncated PCSK9, expressed and purifi ed from HEK293 cells with identical composition as the circulating truncated protein, was not active in inhibition of LDL uptake by HepG2 cells. These studies provide a defi nitive characterization of the composition and activity of the truncated form of PCSK9 found in human serum. —Han, B., P. I. Eacho, M. D. Knierman, J. S. Troutt, R. J. Konrad, X. Yu, and K. M. Schroeder. Isolation and characterization of the circulating truncated form of PCSK9. J. Lipid Res. 2014. 55: 1505 – 1514 .