Binding of Proprotein Convertase Subtilisin/Kexin Type 9 to Epidermal Growth Factor-like Repeat A of Low Density Lipoprotein Receptor Decreases Receptor Recycling and Increases Degradation

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of hepatic low density lipoprotein receptors (LDLR), the major route of clearance of circulating cholesterol. Gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, whereas loss-of-function mutations result in hypocholesterolemia and protection from heart disease. Recombinant human PCSK9 binds the LDLR on the surface of cultured hepatocytes and promotes degradation of the receptor after internalization. Here we localized the site of binding of PCSK9 within the extracellular domain of the LDLR and determined the fate of the receptor after PCSK9 binding. Recombinant human PCSK9 interacted in a sequence-specific manner with the first epidermal growth factor-like repeat (EGF-A) in the EGF homology domain of the human LDLR. Similar binding specificity was observed between PCSK9 and purified EGF-A. Binding to EGF-A was calcium-dependent and increased dramatically with reduction in pH from 7 to 5.2. The addition of PCSK9, but not heat-inactivated PCSK9, to the medium of cultured hepatocytes resulted in redistribution of the receptor from the plasma membrane to lysosomes. These data are consistent with a model in which PCSK9 binding to EGF-A interferes with an aciddependent conformational change required for receptor recycling. As a consequence, the LDLR is rerouted from the endosome to the lysosome where it is degraded.Genetic variation in proprotein convertase subtilisin/kexin type 9 (PCSK9) 4 contributes to differences in plasma levels of low density lipoprotein (LDL) cholesterol (1, 2), the primary cholesterol-carrying lipoprotein in humans. Selected missense mutations in PCSK9 cause dominant hypercholesterolemia and premature atherosclerosis (1, 3, 4), whereas loss-of-function mutations in PCSK9 reduce plasma LDL levels and protect against coronary heart disease (5-7). Studies in mice suggest that the major metabolic effect of PCSK9 is to reduce the amount of hepatic LDL receptor (LDLR), the primary conduit for the clearance of LDL from the circulation (8). Expression of recombinant PCSK9 in the livers of mice causes a reduction in hepatic LDLR protein (but not mRNA) and produces severe hypercholesterolemia (9 -11). Conversely, mice lacking PCSK9 manifest increased levels of LDLR protein in the liver and accelerated clearance of circulating LDL (12). PCSK9 is expressed predominantly in the liver, small intestine, kidney, and brain (13) and is present in human plasma (14,15). Introduction of PCSK9 into the circulation of mice through parabiosis reduces hepatic LDLR levels, which is consistent with PCSK9 interacting with the LDLR on the cell surface (14). In cultured cells, the addition of recombinant PCSK9 to the medium results in LDLR degradation, providing further evidence that PCSK9 can promote the degradation of the LDLR by acting at the cell surface (14, 16). Autocatalytic cleavage is required for PCSK9 maturation and secretion (10), but whether the catalytic activity of PCSK9 is required for LDLR d...

Versatility in ligand recognition by LDL receptor family proteins: advances and frontiers

“…A number of studies in cell culture and animal models have shown that secreted PCSK9 can reduce surface LDLR levels, and the presence of PCSK9 in the culture medium results in the redistribution of receptors from the plasma membrane to the lysosomes [26], perhaps by interfering with normal recycling of receptors after internalization (see [6] for a review).…”

“…Biochemical studies using recombinant proteins have revealed that the first EGF-like repeat of the LDLR (EGF-A) suffices for binding of recombinant, purified PCSK9, and that binding to this repeat is calcium dependent [26]. Additionally, the affinity of PCSK9 for the receptor is enhanced at endosomal pH [26,29].…”

“…Biochemical studies using recombinant proteins have revealed that the first EGF-like repeat of the LDLR (EGF-A) suffices for binding of recombinant, purified PCSK9, and that binding to this repeat is calcium dependent [26]. Additionally, the affinity of PCSK9 for the receptor is enhanced at endosomal pH [26,29]. Moreover, mutations associated with PCSK9 gain of function result in enhanced LDLR clearance from the cell surface [28,30], and at least one mutant, D374Y, exhibits ~25-fold increased affinity for the receptor at neutral pH [28,29].…”

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

“…But secreted PCSK9 might also bind to the first epidermal growth factor-like repeat (EGF-A) of the LDL receptor at the cell surface and the PCSK9/LDL receptor complex could be internalized into endosomal/lysosomal compartments [Zhang et al, 2007].…”

Hepatitis C virus and proprotein convertase subtilisin/kexin type 9: a detrimental interaction to increase viral infectivity and disrupt lipid metabolism

“…The expression of PCSK9 is high in the liver, intestine, kidney and brain, and the secreted form is present in human plasma. The function of PCSK9 is the binding of specific cell surface receptors to escort them towards intracellular acidic endosome/lysosome degradation compartments 58, 59. Specifically, PCSK9 plays a key role in the regulation of hepatic LDLR function through receptor binding and targeting to intracellular lysosomal degradation 19; this leads to reduced LDLR recycling and expression on the cell membrane, decreased plasma LDL catabolism and increased plasma LDL‐C levels.…”