Removal of acidic residues of the prodomain of PCSK9 increases its activity towards the LDL receptor

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and mediates intracellular degradation of the LDLR. The amino-terminus of mature PCSK9, residues 31–53 of the prodomain, has an inhibitory effect on this function of PCSK9, but the underlying mechanism is not fully understood. In this study, we have identified two highly conserved negatively charged segments (residues 32–40 and 48–50, respectively) within this part of the prodomain and performed deletions and substitutions to study their importance for degradation of the LDLRs.Deletion of the acidic residues of the longest negatively charged segment increased PCSK9’s ability to degrade the LDLR by 31%, whereas a modest 8% increase was observed when these residues were mutated to uncharged amino acids. Thus, both the length and the charge of this part of the prodomain were important for its inhibitory effect. Deletion of the residues of the shorter second negatively charged segment only increased PCSK9’s activity by 8%. Substitution of the amino acids of both charged segments to uncharged residues increased PCSK9’s activity by 36%. These findings indicate that the inhibitory effect of residues 31–53 of the prodomain is due to the negative charge of this segment. The underlying mechanism could involve the binding of this peptide segment to positively charged structures which are important for PCSK9’s activity. One possible candidate could be the histidine-rich C-terminal domain of PCSK9.     

Investigations on the evolutionary conservation of PCSK9 reveal a functionally important protrusion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interferes with the recycling of low-density lipoprotein (LDL) receptor (LDLR). This leads to LDLR degradation and reduced cellular uptake of plasma LDL. Naturally occurring human PCSK9 loss-of-function mutations are associated with low levels of plasma LDL cholesterol and a reduced risk of coronary heart disease. PCSK9 gain-of-function mutations result in lower LDL clearance and increased risk of atherosclerosis. The exact mechanism by which PCSK9 disrupts the normal recycling of LDLR remains to be determined. In this study, we have assembled homologs of human PCSK9 from 20 vertebrates, a cephalochordate and mollusks in order to search for conserved regions of PCSK9 that may be important for the PCSK9-mediated degradation of LDLR. We found a large, conserved protrusion on the surface of the PCSK9 catalytic domain and have performed site-directed mutagenesis experiments for 13 residues on this protrusion. A cluster of residues that is important for the degradation of LDLR by PCSK9 was identified. Another cluster of residues, at the opposite end of the conserved protrusion, appears to be involved in the physical interaction with a putative inhibitor of PCSK9. This study identifies the residues, sequence segments and surface patches of PCSK9 that are under strong purifying selection and provides important information for future studies of PCSK9 mutants and for investigations on the function of this regulator of cholesterol homeostasis.     

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.     

The cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on the LDLR

Mutations in the proprotein convertase PCSK9 gene are associated with autosomal dominant familial hyper- or hypocholesterolemia. These phenotypes are caused by a gain or loss of function of proprotein convertase subtilisin kexin 9 (PCSK9) to elicit the degradation of the low-density lipoprotein receptor (LDLR) protein. Herein, we asked whether the subcellular localization of wild-type PCSK9 or mutants of PCSK9 and the LDLR would provide insight into the mechanism of PCSK9-dependent LDLR degradation. We show that the LDLR is the dominant partner in regulating the cellular trafficking of PCSK9. In cells lacking the LDLR, PCSK9 localized in the endoplasmic reticulum (ER). In cells expressing the LDLR, PCSK9 sorted to post-ER compartments (i.e. endosomes in cell lines and Golgi apparatus in primary hepatocytes), where it colocalized with the LDLR. In cell lines, PCSK9 also colocalized with the LDLR at the cell surface, requiring the presence of the C-terminal Cys/His-rich domain of PCSK9. We provide evidence that PCSK9 promotes the degradation of the LDLR by an endocytic mechanism, as small interfering RNA-mediated knockdown of the clathrin heavy chain reduced the functional activity of PCSK9. We also compared the subcellular localization of natural mutants of PCSK9 with that of the wild-type enzyme in human hepatic (HuH7) cells. Whereas the mutants associated with hypercholesterolemia (S127R, F216L and R218S) localized to endosomes/lysosomes, those associated with hypocholesterolemia did not reach this compartment. We conclude that the sorting of PCSK9 to the cell surface and endosomes is required for PCSK9 to fully promote LDLR degradation and that retention in the ER prevents this activity. Mutations that affect this transport can lead to hyper- or hypocholesterolemia.     

Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH

The protein PCSK9 (proprotein convertase subtilisin/kexin type 9) is a key regulator of low-density lipoprotein receptor (LDLR) levels and cardiovascular health. We have determined the crystal structure of LDLR bound to PCSK9 at neutral pH. The structure shows LDLR in a new extended conformation. The PCSK9 C-terminal domain is solvent exposed, enabling cofactor binding, whereas the catalytic domain and prodomain interact with LDLR epidermal growth factor(A) and β-propeller domains, respectively. Thus, PCSK9 seems to hold LDLR in an extended conformation and to interfere with conformational rearrangements required for LDLR recycling.     

A two-step binding model of PCSK9 interaction with the low density lipoprotein receptor

PCSK9 (proprotein convertase subtilisin-like/kexin type 9) is an emerging target for pharmaceutical intervention. This multidomain protein interacts with the LDL receptor (LDLR), promoting receptor degradation. Insofar as PCSK9 inhibition induces a decrease in plasma cholesterol levels, understanding the nature of the binding interaction between PCSK9 and the LDLR is of critical importance. In this study, the ability of PCSK9 to compete with apoE3 N-terminal domain-containing reconstituted HDL for receptor binding was examined. Whereas full-length PCSK9 was an effective competitor, the N-terminal domain (composed of the prodomain and catalytic domain) was not. Surprisingly, the C-terminal domain (CT domain) of PCSK9 was able to compete. Using a direct binding interaction assay, we show that the PCSK9 CT domain bound to the LDLR in a calcium-dependent manner and that co-incubation with the prodomain and catalytic domain had no effect on this binding. To further characterize this interaction, two LDLR fragments, the classical ligand-binding domain (LBD) and the EGF precursor homology domain, were expressed in stably transfected HEK 293 cells and isolated. Binding assays showed that the PCSK9 CT domain bound to the LBD at pH 5.4. Thus, CT domain interaction with the LBD of the LDLR at endosomal pH constitutes a second step in the PCSK9-mediated LDLR binding that leads to receptor degradation.     

Novel domain interaction regulates secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein

PCSK9 (proprotein convertase subtilisin/kexin type 9) has emerged as a novel therapeutic target for hypercholesterolemia due to its LDL receptor (LDLR)-reducing activity. Although its structure has been solved, the lack of a detailed understanding of the structure-function relation hinders efforts to develop small molecule inhibitors. In this study, we used mutagenesis and transfection approaches to investigate the roles of the prodomain (PD) and the C-terminal domain (CD) and its modules (CM1-3) in the secretion and function of PCSK9. Deletion of PD residues 31-40, 41-50, or 51-60 did not affect the self-cleavage, secretion, or LDLR-degrading activity of PCSK9, whereas deletion of residues 61-70 abolished all of these functions. Deletion of the entire CD protein did not impair PCSK9 self-cleavage or secretion but completely abolished LDLR-degrading activity. Deletion of any one or two of the CD modules did not affect self-cleavage but influenced secretion and LDLR-reducing activity. Furthermore, in cotransfection experiments, a secretion-defective PD deletion mutant (ΔPD) was efficiently secreted in the presence of CD deletion mutants. This was due to the transfer of PD from the cotransfected CD mutants to the ΔPD mutant. Finally, we found that a discrete CD protein fragment competed with full-length PCSK9 for binding to LDLR in vitro and attenuated PCSK9-mediated hypercholesterolemia in mice. These results show a previously unrecognized domain interaction as a critical determinant in PCSK9 secretion and function. This knowledge should fuel efforts to develop novel approaches to PCSK9 inhibition.     

Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in plasma cholesterol regulation through modulation of low density lipoprotein receptor (LDLR) levels. Naturally occurring mutations can lead to hyper- or hypocholesterolemia in human. Recently, we reported that PCSK9 was also able to modulate CD81 in Huh7 cells. In the present study, several gain-of-function and loss-of-function mutants as well as engineered mutants of PCSK9 were compared for their ability to modulate the cell surface expression of LDLR and CD81. Although PCSK9 gain-of-function D374Y enhanced the degradation both receptors, D374H and D129N seemed to only reduce LDLR levels. In contrast, mutations in the C-terminal hinge-cysteine-histidine-rich domain segment primarily affected the PCSK9-induced CD81 degradation. Furthermore, when C-terminally fused to an ACE2 transmembrane anchor, the secretory N-terminal catalytic or hinge-cysteine-histidine-rich domain domains of PCSK9 were able to reduce CD81 and LDLR levels. These data confirm that PCSK9 reduces CD81 levels via an intracellular pathway as reported for LDLR. Using immunocytochemistry, a proximity ligation assay, and co-immunoprecipitation, we found that the cell surface level of PCSK9 was enhanced upon overexpression of CD81 and that both PCSK9 and LDLR interact with this tetraspanin protein. Interestingly, using CHO-A7 cells lacking LDLR expression, we revealed that LDLR was not required for the degradation of CD81 by PCSK9, but its presence strengthened the PCSK9 effect.     

Effects of the prosegment and pH on the activity of PCSK9: evidence for additional processing events

PCSK9, a target for the treatment of dyslipidemia, enhances the degradation of the LDL receptor (LDLR) in endosomes/lysosomes, up-regulating LDL-cholesterol levels. Whereas the targeting and degradation of the PCSK9-LDLR complex are under scrutiny, the roles of the N- and C-terminal domains of PCSK9 are unknown. Although autocatalytic zymogen processing of PCSK9 occurs at Gln(152)↓, here we show that human PCSK9 can be further cleaved in its N-terminal prosegment at Arg(46)↓ by an endogenous enzyme of insect High Five cells and by a cellular mammalian protease, yielding an ～4-fold enhanced activity. Removal of the prosegment acidic stretch resulted in ～3-fold higher binding to LDLR in vitro, in ≥4-fold increased activity on cellular LDLR, and faster cellular internalization in endosome/lysosome-like compartments. Finally, swapping the acidic stretch of PCSK9 with a similar one found in the glycosylphosphatidylinositol-anchored heparin-binding protein 1 does not impair PCSK9 autoprocessing, secretion, or activity and confirmed that the acidic stretch acts as an inhibitor of PCSK9 function. We also show that upon short exposure to pH values 6.5 to 5.5, an ～2.5-fold increase in PCSK9 activity on total and cell surface LDLR occurs, and PCSK9 undergoes a second cleavage at Arg(248), generating a two-chain PCSK9-ΔN(248). At pH values below 5.5, PCSK9 dissociates from its prosegment and loses its activity. This pH-dependent activation of PCSK9 represents a novel pathway to further activate PCSK9 in acidic endosomes. These data enhance our understanding of the functional role of the acidic prosegment and on the effect of pH in the regulation of PCSK9 activity.     

Role of ubiquitination in PCSK9-mediated low-density lipoprotein receptor degradation

The proprotein convertases subtilisin kexin 9 (PCSK9) binds to the epidermal growth factor domain A (EGF-A) of low-density lipoprotein receptor (LDLR) and leads to its destruction. However, the intracellular processes leading to LDLR degradation have not been fully delineated. In this report, we show that PCSK9 treatment can lead to ubiquitination of LDLR, which was enhanced in the presence of proteasome inhibitor MG132. Furthermore, LDLR protein carrying mutations in the C-terminal ubiquitination sites was resistant to PCSK9-mediated degradation. Our data suggest that the ubiquitination system is involved in PCSK9-induced LDLR degradation.     

Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels

The proprotein convertase subtilisin/kexin-type 9 (PCSK9), which promotes degradation of the hepatic low density lipoprotein receptor (LDLR), is now recognized as a major player in plasma cholesterol metabolism. Several gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, and thus, inhibition of PCSK9-induced degradation of the LDLR may be used to treat this deadly disease. Herein, we discovered an endogenous PCSK9 binding partner by Far Western blotting, co-immunoprecipitation, and pull-down assays. Following two-dimensional gel electrophoresis and mass spectrometry analysis, we demonstrated that PCSK9 binds to a approximately 33-kDa protein identified as annexin A2 (AnxA2) but not to the closely related annexin A1. Furthermore, our functional LDLR assays and small hairpin RNA studies show that AnxA2 and the AnxA2.p11 complex could prevent PCSK9-directed LDLR degradation in HuH7, HepG2, and Chinese hamster ovary cells. Immunocytochemistry revealed that PCSK9 and AnxA2 co-localize at the cell surface, indicating a possible competition with the LDLR. Structure-function analyses demonstrated that the C-terminal cysteine-histidine-rich domain of PCSK9 interacts specifically with the N-terminal repeat R1 of AnxA2. Mutational analysis of this 70-amino acid-long repeat indicated that the RRTKK81 sequence of AnxA2 is implicated in this binding because its mutation to AATAA81 prevents its interaction with PCSK9. To our knowledge, this work constitutes the first to show that PCSK9 activity on LDLR can be regulated by an endogenous inhibitor. The identification of the minimal inhibitory sequence of AnxA2 should pave the way toward the development of PCSK9 inhibitory lead molecules for the treatment of hypercholesterolemia.     

The M2 Module of the Cys-His-rich Domain (CHRD) of PCSK9 Protein Is Needed for the Extracellular Low-density Lipoprotein Receptor (LDLR) Degradation Pathway

PCSK9 enhances the cellular degradation of the LDL receptor (LDLR), leading to increased plasma LDL cholesterol. This multidomain protein contains a prosegment, a catalytic domain, a hinge region, and a cysteine-histidine rich domain (CHRD) composed of three tightly packed modules named M1, M2, and M3. The CHRD is required for the activity of PCSK9, but the mechanism behind this remains obscure. To define the contribution of each module to the function of PCSK9, we dissected the CHRD structure. Six PCSK9 deletants were generated by mutagenesis, corresponding to the deletion of only one (ΔM1, ΔM2, ΔM3) or two (ΔM12, ΔM13, ΔM23) modules. Transfection of HEK293 cells showed that all deletants were well processed and expressed compared with the parent PCSK9 but that only those lacking the M2 module were secreted. HepG2 cells lacking endogenous PCSK9 (HepG2/shPCSK9) were used for the functional analysis of the extracellular or intracellular activity of PCSK9 and its deletants. To analyze the ability of the deletants to enhance the LDLR degradation by the intracellular pathway, cellular expressions revealed that only the ΔM2 deletant retains a comparable total LDLR-degrading activity to full-length PCSK9. To probe the extracellular pathway, HepG2/shPCSK9 cells were incubated with conditioned media from transfected HEK293 or HepG2/shPCSK9 cells, and cell surface LDLR levels were analyzed by FACS. The results showed no activity of any secreted deletant compared with PCSK9. Thus, although M2 is dispensable for secretion, its presence is required for the extracellular activity of PCSK9 on cell surface LDLR.     

PCSK9 Prosegment Chimera as Novel Inhibitors of LDLR Degradation

The proprotein convertase PCSK9, a target for the treatment of hypercholesterolemia, is a negative regulator of the LDL receptor (LDLR) leading to its degradation in endosomes/lysosomes and up-regulation of plasma LDL-cholesterol levels. The proprotein convertases, a family of nine secretory serine proteases, are first synthesized as inactive zymogens. Except for PCSK9, all other convertases are activated following the autocatalytic excision of their inhibitory N-terminal prosegment. PCSK9 is unique since the mature enzyme exhibits a cleaved prosegment complexed with the catalytic subunit and has no protease activity towards other substrates. Similar to other convertases, we hypothesized that the in trans presence of the PCSK9 prosegment would interfere with PCSK9''s activity on the LDLR. Since the prosegment cannot be secreted alone, we engineered a chimeric protein using the Fc-region of human IgG1 fused to the PCSK9 prosegment. The expression of such Fcpro-fusion protein in HEK293 and HepG2 cells resulted in a secreted protein that binds PCSK9 and markedly inhibits its activity on the LDLR. This was observed by either intracellular co-expression of PCSK9 and Fcpro or by an extracellular in vitro co-incubation of Fcpro with PCSK9. Structure-function studies revealed that the inhibitory function of Fcpro does not require the acidic N-terminal stretch (residues 31–58) nor the C-terminal Gln₁₅₂ of the prosegment. Fcpro likely interacts with the prosegment and/or catalytic subunit of the prosegment≡PCSK9 complex thereby allosterically modulating its function. Our data suggest a novel strategic approach for the design and isolation of PCSK9 inhibitors.     

The crystal structure of PCSK9: a regulator of plasma LDL-cholesterol

Proprotein convertase subtilisin kexin type 9 (PCSK9) has been shown to be involved in the regulation of extracellular levels of the low-density lipoprotien receptor (LDLR). Although PCSK9 is a subtilase, it has not been shown to degrade the LDLR, and its LDLR-lowering mechanism remains uncertain. Here we report the crystal structure of human PCSK9 at 2.3 A resolution. PCSK9 has subtilisin-like pro- and catalytic domains, and the stable interaction between these domains prevents access to PCSK9's catalytic site. The C-terminal domain of PCSK9 has a novel protein fold and may mediate protein-protein interactions. The structure of PCSK9 provides insight into its biochemical characteristics and biological function.     

PCSK9结构与功能

前蛋白转化酶枯草溶菌素9（PCSK9）基因属于前蛋白转化酶（PC）家族,由信号肽、前结构域、催化结构域和羧基末端结构域组成.大量研究发现,PCSK9能介导低密度脂蛋白受体（LDLR）降解,调节血浆LDL胆固醇（LDL-C）水平;而PCSK9的两类主要突变,功能获得型、功能缺失型可分别导致高胆固醇血症和低胆固醇血症. 因而研究PCSK9对相关心血管疾病的防治有重要意义. PCSK9结构特性与其生化功能密切相关,突变致使其调节胆固醇代谢的机制更为复杂.本文旨在总结PCSK9结构与功能的分子生物学特性,并指出目前研究中存在的问题,以利对PCSK9的进一步探索.     

Molecular characterization of proprotein convertase subtilisin/kexin type 9-mediated degradation of the LDLR

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that promotes degradation of cell surface LDL receptors (LDLRs) in selected cell types. Here we used genetic and pharmacological inhibitors to define the pathways involved in PCSK9-mediated LDLR degradation. Inactivating mutations in autosomal recessive hypercholesterolemia (ARH), an endocytic adaptor, blocked PCSK9-mediated LDLR degradation in lymphocytes but not in fibroblasts. Thus, ARH is not specifically required for PCSK9-mediated LDLR degradation. Knockdown of clathrin heavy chain with siRNAs prevented LDLR degradation. In contrast, prevention of ubiquitination of the LDLR cytoplasmic tail, inhibition of proteasomal activity, or disruption of proteins required for lysosomal targeting via macroautophagy (autophagy related 5 and 7) or the endosomal sorting complex required for trafficking (ESCRT) pathway (hepatocyte growth factor-regulated Tyr-kinase substrate and tumor suppressor gene 101) failed to block PCSK9-mediated LDLR degradation. These findings are consistent with a model in which the LDLR-PCSK9 complex is internalized via clathrin-mediated endocytosis and then routed to lysosomes via a mechanism that does not require ubiquitination and is distinct from the autophagy and proteosomal degradation pathways. Finally, the PCSK9-LDLR complex appears not to be transported by the canonical ESCRT pathway.     

A novel splicing variant of proprotein convertase subtilisin/kexin type 9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is the most recently identified member of the proprotein convertase family. Genetic and cell biology studies have suggested a critical role of PCSK9 in regulating low-density lipoprotein receptor (LDLR) protein levels and thus modulating plasma LDL cholesterol. Recent data on the molecular basis for PCSK9 action support the model in which PCSK9 is self-cleaved, secreted, and tightly bound to the EGF-A repeat of LDLR extracellular domain. PCSK9 binding to LDLR is essential for the ensuing receptor-mediated endocytosis and is speculated to lock LDLR in a specific conformation that favors degradation in lysosomal compartment instead of recycling back to plasma membrane. We report here a novel human PCSK9 splicing variant, which we named PCSK9sv. PCSK9sv had an in-frame deletion of the eighth exon of 58 amino acids and was expressed in multiple tissues, including liver, small intestine, prostate, uterus, brain, and adipose tissue. Unlike wild-type PCSK9, which is secreted, PCSK9sv expressed in human embryonic kidney HEK293 cells failed to process the prosegment intracellularly and thus was not secreted into the medium. Examination of potential functions revealed that PCSK9sv did not change the LDLR protein levels. Two mutations that have been reported in humans with the associated changes in plasma LDL cholesterol were within exon 8, and thus the expression and function of the two mutants were studied. Both N425S and A443T mutants were processed normally, secreted, and reduced LDLR levels. However, the physiological function of this novel splicing variant of PCSK9 has yet to be determined.     

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.     

Low Density Lipoprotein Binds to Proprotein Convertase Subtilisin/Kexin Type-9 (PCSK9) in Human Plasma and Inhibits PCSK9-mediated Low Density Lipoprotein Receptor Degradation

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a secreted protein that binds to the epidermal growth factor-like-A domain of the low density lipoprotein receptor (LDLR) and mediates LDLR degradation in liver. Gain-of-function mutations in PCSK9 are associated with autosomal dominant hypercholesterolemia in humans. Size-exclusion chromatography of human plasma has shown PCSK9 to be partly associated with undefined high molecular weight complexes within the LDL size range. We used density gradient centrifugation to isolate LDL in plasma pooled from 5 normolipidemic subjects and report that >40% of total PCSK9 was associated with LDL. Binding of fluorophore-labeled recombinant PCSK9 to isolated LDL in vitro was saturable with a K_D ∼ 325 nm. This interaction was competed >95% by excess unlabeled PCSK9, and competition binding curves were consistent with a one-site binding model. An N-terminal region of the PCSK9 prodomain (amino acids 31–52) was required for binding to LDL in vitro. LDL dose-dependently inhibited binding and degradation of cell surface LDLRs by exogenous PCSK9 in HuH7 cells. LDL also inhibited PCSK9 binding to mutant LDLRs defective at binding LDL. These data suggest that association of PCSK9 with LDL particles in plasma lowers the ability of PCSK9 to bind to cell surface LDLRs, thereby blunting PCSK9-mediated LDLR degradation.