MABS2160-100UG Sigma-AldrichAnti-NARC-1 (PCSK9) Antibody, clone 13D3

PCSK9 is a secreted protein that influences plasma levels of low-density lipoprotein cholesterol (LDL-C) and susceptibility to coronary heart disease. PCSK9 is present in human plasma, but the factors that contribute to differences in plasma concentrations of PCSK9 and how they impact on the levels of lipoproteins have not been well-characterized.The aim of the study was to measure PCSK9 levels in a large, ethnically diverse population (n = 3138) utilizing a sensitive and specific sandwich ELISA.We conducted an observational study in the Dallas Heart Study, a multiethnic, probability-based sample of Dallas County.Plasma levels of PCSK9 varied over approximately 100-fold range (33-2988 ng/ml; median, 487 ng/ml). Levels were significantly higher in women (517 ng/ml) than in men (450 ng/ml), and in postmenopausal women compared to premenopausal women (P < 0.0001), irrespective of estrogen status. Plasma levels of PCSK9 correlated with plasma levels of LDL-C (r = 0.24) but explained less than 8% of the variation in LDL-C levels (r(2) = 0.073). Other factors that correlated with PCSK9 levels included plasma levels of triglycerides, insulin, and glucose. Individuals with loss-of-function mutations in PCSK9 and reduced plasma levels of LDL-C also had significantly lower plasma levels of PCSK9 after adjusting for age, gender, and LDL-C levels (P < 0.0001).Multiple metabolic and genetic factors contribute to variation in plasma levels of PCSK9 in the general population. Although levels of PCSK9 correlate with plasma levels of LDL-C, they account for only a small proportion of the variation in the levels of this lipoprotein.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that controls plasma LDL cholesterol levels by posttranslational regulation of the LDL receptor (LDLR). Previously, we showed that PCSK9 binds specifically to an EGF-like repeat (EGF-A) in LDLR and reroutes the receptor from endosomes to lysosomes rather than to the cell surface. Here, we defined the regions in LDLR and PCSK9 that are required for receptor degradation and examined the relationship between PCSK9 binding and LDLR conformation. Addition of PCSK9 to cultured hepatocytes promoted degradation of WT LDLR and of receptors lacking up to four ligand binding domains, EGF-B or the clustered O-linked sugar region. In contrast, LDLRs lacking the entire ligand binding domain or the beta-propeller domain failed to be degraded, although they bound and internalized PCSK9. Using gel filtration chromatography, we assessed the effects of PCSK9 binding on an acid-dependent conformational change that happens in the extracellular domain of the LDLR. Although PCSK9 prevented the reduction in hydrodynamic radius of the receptor that occurs at a reduced pH, the effect was not sufficient for LDLR degradation. A truncated version of PCSK9 containing the prodomain and the catalytic domain, but not the C-terminal domain, bound the receptor but did not stimulate LDLR degradation. Thus, domains in both the LDLR and PCSK9 that are not required for binding (or internalization) are essential for PCSK9-mediated degradation of the LDLR.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of subtilases that reduces the number of LDL receptors (LDLRs) in liver through an undefined posttranscriptional mechanism. We show that purified PCSK9 added to the medium of HepG2 cells reduces the number of cell-surface LDLRs in a dose- and time-dependent manner. This activity was approximately 10-fold greater for a gain-of-function mutant, PCSK9(D374Y), that causes hypercholesterolemia. Binding and uptake of PCSK9 were largely dependent on the presence of LDLRs. Coimmunoprecipitation and ligand blotting studies indicated that PCSK9 and LDLR directly associate; both proteins colocalized to late endocytic compartments. Purified PCSK9 had no effect on cell-surface LDLRs in hepatocytes lacking autosomal recessive hypercholesterolemia (ARH), an adaptor protein required for endocytosis of the receptor. Transgenic mice overexpressing human PCSK9 in liver secreted large amounts of the protein into plasma, which increased plasma LDL cholesterol concentrations to levels similar to those of LDLR-knockout mice. To determine whether PCSK9 was active in plasma, transgenic PCSK9 mice were parabiosed with wild-type littermates. After parabiosis, secreted PCSK9 was transferred to the circulation of wild-type mice and reduced the number of hepatic LDLRs to nearly undetectable levels. We conclude that secreted PCSK9 associates with the LDLR and reduces hepatic LDLR protein levels.