Isolation and characterization of the circulating truncated form of PCSK9[S]

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 purified 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 definitive characterization of the composition and activity of the truncated form of PCSK9 found in human serum.