This article is available online at http://www.jlr.org Journal of Lipid Research Volume 54, 2013 3345

Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.

of FH is the mutations in the LDL receptor (LDLR) gene ( 1 ). Most recently, mutations in the proprotein convertase subtilisin kexin-like 9 (PCSK9) genes have been found to be linked with FH ( 2–6 ). Gain-of-function mutations cause higher plasma LDL-cholesterol (LDL-C) levels and lead to accelerated atherosclerosis and premature coronary heart disease ( 2–5, 7 ). On the other hand, loss-of-function muta-tions result in low concentrations of LDL-C and protection from coronary heart disease ( 8–15 ).

PCSK9 is a 692 amino acid secreted glycoprotein that consists of a 30 amino acid signal sequence followed by a prodomain, a catalytic domain, and a C-terminal domain. The role of PCSK9 in homeostatic control of plasma LDL-C levels is dependent upon PCSK9-promoted degradation of the LDLR, thereby preventing clearance of LDL-C by the cells ( 16–21 ). Increased plasma levels of PCSK9 in mice through infusion of purifi ed PCSK9 or transgenic over-expression in the kidneys preferentially promote LDLR degradation in the liver but not in the adrenal glands ( 22, 23 ). On the other hand, knockout or knockdown expression of PCSK9 in mice leads to increased levels of LDLR protein in the liver and accelerated LDL clearance ( 24, 25 ). Stud-ies in cultured cells and parabiotic mice show that PCSK9 can promote LDLR degradation extracellularly ( 16–19 ). Overexpression of PCSK9 in cultured cells and mouse liver also induces LDLR degradation intracellularly ( 20, 21 ).

PCSK9-promoted LDLR degradation requires binding of PCSK9 to the LDLR and internalization of the receptor, but does not require the proteolytic activity of PCSK9 ( 16, 17, 26 ). Most recently, it has been shown that ubiquitina-tion of the LDLR cytoplasmic tail and the canonical endo-somal sorting complex required for traffi cking pathway

Abstract Proprotein convertase subtilisin kexin-like 9 (PCSK9) promotes the degradation of low density lipopro-tein receptor (LDLR) and plays an important role in regulat-ing plasma LDL-cholesterol levels. We have shown that the epidermal growth factor precursor homology domain A (EGF-A) of the LDLR is critical for PCSK9 binding at the cell surface (pH 7.4). Here, we further characterized the role of EGF-A in binding of PCSK9 to the LDLR. We found that PCSK9 effi ciently bound to the LDLR but not to other LDLR family members. Replacement of EGF-A in the very low density lipoprotein receptor (VLDLR) with EGF-A of the LDLR promoted the degradation of the mutant VLDLR induced by PCSK9. Furthermore, we found that PCSK9 bound to recombinant EGF-A in a pH-dependent manner with stronger binding at pH 6.0. We also identifi ed amino acid residues in EGF-A of the LDLR important for PCSK9 binding. Mutations G293H, D299V, L318D, and L318H reduced PCSK9 binding to the LDLR at neutral pH with-out effect at pH 6.0, while mutations R329P and E332G reduced PCSK9 binding at both pH values. Thus, our fi ndings reveal that EGF-A of the LDLR is critical for PCSK9 binding at the cell surface (neutral pH) and at the acidic endosomal environment (pH 6.0), but different de-terminants contribute to effi cient PCSK9 binding in differ-ent pH environments. —Gu, H-m., A. Adijiang, M. Mah, and D-w. Zhang. Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding. J. Lipid Res. 54: 3345–3357.

Supplementary key words proprotein convertase subtilisin kexin-like 9 • epidermal growth factor precursor homology domain A • ligand binding

Familial hypercholesterolemia (FH) is a common genetic disorder characterized by high cholesterol levels, specifi -cally very high low density lipoprotein (LDL), and increased risk of coronary heart disease and mortality. The main cause

This research was supported by a grant from a Grant-in-Aid for the Heart and Stroke Foundation of Canada and a research award from Pfi zer Canada. D-w.Z. is a Scholar of the Alberta Heritage Foundation for Medical Research and is supported in part by a Canadian Institutes of Health Research New Investiga-tor Award. Zhang laboratory is supported by the Canadian Foundation for Innovation.

Manuscript received 10 June 2013 and in revised form 7 October 2013.

Published, JLR Papers in Press, October 8, 2013 DOI 10.1194/jlr.M041129

Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding

Hong-mei Gu , 1 Ayinuer Adijiang , 1 Matthew Mah , and Da-wei Zhang 2

Departments of Pediatrics and Biochemistry, Group on the Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

Abbreviations: apoER, apolipoprotein E receptor; EGF-A, epider-mal growth factor precursor homology domain A; FH, familial hyperc-holesterolemia; GST, glutathione-S-transferase; HA, hemaglutinin epitope; LDL-C, LDL-cholesterol; LDLR, low density lipoprotein recep-tor; LDLR-ECD, extracellular domain of the LDL receptor; LRP, low density lipoprotein receptor-related protein; PCSK9, proprotein con-vertase subtilisin kexin-like 9; VLDLR, very low density lipoprotein receptor.

1 H-m. Gu and A. Adijiang contributed equally to this work. 2 To whom correspondence should be addressed. e-mail: dzhang@ualberta.ca

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well). After 24 h, cells were transiently transfected with expression plasmids containing cDNAs for LDLR, VLDLR, apolipoprotein E receptor (apoER)2, LRP1, LRP4, LRP6, or pcDNA 3.1( � ) vector (1.6 � g/well) using Lipofectamine 2000 or X-tremeGENE HP according to the manufacturer’s protocol. Forty-eight hours after transfection, cells were washed twice with phosphate buffered saline (PBS), incubated in 0.5 ml of DMEM medium containing 5% (v/v) newborn calf lipoprotein-poor serum, 10 � g/ml cholesterol, 1 � g/ml 25-hydroxycholesterol, and 2.0 � g/ml purifi ed wild-type PCSK9 for 2 h. The cells were then washed and lysed in 60 � l of lysis buffer (PBS, 1.5 mM MgCl 2 , 5 mM dithiothreitol, 1% (v/v) Triton X-100) containing 1× complete EDTA-free protease inhibitors. Whole cell lysate protein extracts were subjected to electrophoresis on an 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for immunoblot analysis. Immunoblotting was performed using the following antibodies: anti-LDLR monoclonal antibody HL-1 ( 34 ); monoclonal antibody (15A6) developed against full-length PCSK9 ( 16 ); anti-HA polyclonal antibody (Pierce) to detect the VLDLR, LRP6-HA; and polyclonal antibodies to detect apoER2, LRP1, and LRP4. Antibody binding was detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or donkey anti-rabbit IgG (GE Healthcare), followed by enhanced chemilumines-cence detection (ECL, Pierce). The membranes were then exposed to F-BX810 TM Blue X-Ray fi lms (Phoenix Research Products, Hayward, CA). Alternatively, antibody binding was detected us-ing IRDye-labeled goat anti-mouse or anti-rabbit IgG (LI-COR Biosciences). The signals were detected by a LI-COR Odyssey in-frared imaging system.

Degradation of receptors by PCSK9 The degradation experiment was performed as described pre-

viously ( 18 ). Briefl y, the mouse hepatoma cell line, Hepa1C1C7, was cultured in MEM � medium containing 10% (v/v) FBS at 37°C and seeded in 12-well dishes (1.5 × 10 5 cells/well). After 24 h, cells were transiently transfected with pCDNA3.1 or expres-sion plasmids containing cDNAs for wild-type or mutant LDLR or VLDLR using Lipofectamine 2000 according to the manufac-turer’s protocol. Forty-eight hours after transfection, cells were washed, incubated in 0.5 ml of MEM � medium containing 5% (v/v) newborn calf lipoprotein-poor serum, 10 � g/ml choles-terol, 1 � g/ml 25-hydroxycholesterol, and various amounts of purifi ed wild-type or mutant PCSK9 (D374Y) for the indicated time. Cells were then washed three times with ice-cold PBS and lysed in 60 � l of lysis buffer. Whole-cell lysate protein extracts were then analyzed by SDS-PAGE (8%) and immunoblotted us-ing a monoclonal anti-hLDLR antibody (HL-1).

Purifi cation of GST:EGF-A fusion protein and ligand blotting

Wild-type EGF-A of the LDLR was expressed as recombinant glutathione-S-transferase (GST) fusion proteins using the vector pGEX-4T (GE Healthcare) in Escherichia coli BL-21-DE3 cells (EMD Biosciences, San Diego, CA) and purifi ed as described ( 17 ). Briefl y, the transformed cells were grown at 37°C, induced with 1 mM isopropylthio- � -galactoside and then harvested. The cells were lysed using a French pressure cell. The GST:EGF-A fu-sion protein was purifi ed using Glutathione Sepharose 4 Fast Flow (GE Healthcare) affi nity gel chromatography according to the manufacturer’s protocol. The protein was concentrated and further purifi ed using size-exclusion chromatography on a Tri-corn Superose 12 10/300 fast-performance liquid chromatogra-phy column (GE Healthcare). Fractions containing GST:EGF-A were concentrated using a 3 kDa MW (molecular mass) cut-off Centriplus fi lter. Protein purity was monitored by SDS-PAGE and Coomassie Brilliant Blue R-250 staining (Bio-Rad, Hercules, CA).

are not required for PCSK9-promoted LDLR degradation ( 27 ). We have shown that PCSK9 interacts with the epider-mal growth factor precursor homology domain A (EGF-A) of the LDLR at the cell surface and binds to the full-length receptor with a much higher affi nity in the acidic environ-ment of the endosome. Consequently, the receptor trans-ports from the endosome to the lysosome for degradation, rather than being recycled ( 17 ). Consistently, the crystal-lographic structures of PCSK9 and the EGF-AB of the LDLR complex reveal that the N terminus of EGF-A is as-sociated with the catalytic domain of PCSK9 ( 28–30 ). We also demonstrate that leucine at position 318 in EGF-A of the LDLR is critical for effi cient binding of PCSK9 ( 17 ). The replacement of Leu 318 in the LDLR with Asp, as it is in the very low density lipoprotein receptor (VLDLR), signifi -cantly reduces binding of PCSK9 to the LDLR. Here we further characterized the role of EGF-A of the LDLR in PCSK9 binding to the receptor. We found that Gly 293 , Asp 299 , Arg 329 , and Glu 332 in EGF-A of the LDLR contributed to PCSK9 binding at the cell surface. We also found that PCSK9 bound to recombinant EGF-A in a pH-dependent way with a stronger binding at pH 6.0.

MATERIALS AND METHODS

Materials Lipofectamine 2000 and cell culture medium were obtained

from Life Technologies. Fetal bovine serum (FBS) was purchased from Sigma. Complete EDTA-free protease inhibitors and X-tremeGENE HP were from Roche. The QuickChange site-di-rected mutagenesis kit was obtained from Agilent Technologies. All other reagents were obtained from Fisher Scientifi c unless otherwise indicated.

The recombinant wild-type human PCSK9 or mutant PCSK9 D374Y containing a FLAG tag (DYKDDDDK) at the C terminus was purifi ed from HEK-293S cells as described ( 31, 32 ). The ex-tracellular domain of the LDLR (LDLR-ECD) (amino acids 1–699) contains a six histidine residue tag at the C terminus and was purifi ed exactly as described ( 33 ). LDLR-ECD and PCSK9 were labeled with IRDye680 and IRDye800, respectively, using IRDye protein labeling kits (LI-COR Biosciences) according to the man-ufacturer’s protocol, and the proteins were visualized using an Odyssey infrared imaging system (LI-COR Biosciences).

Site-directed mutagenesis A recombinant expression vector containing the full-length

LDLR cDNA linked to pCDNA5 was used to generate the mutant forms of the LDLR using the QuikChange TM site-directed mutagen-esis kit according to the manufacturer’s instructions. The VLDLR and low density lipoprotein receptor-related protein (LRP)6 expres-sion constructs contained one copy of a hemaglutinin epitope (HA) tag (CYPYDVPDTAG) at the C terminus. The oligonucleotides con-taining the residues to be mutated were synthesized by IDT, Inc. (Coralville, IA). The presence of the desired mutation and the integrity of each construct were verifi ed by DNA sequencing.

Binding of PCSK9 to the LDLR family members The binding assay was performed as described previously ( 17 ).

Briefl y, COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM):Ham’s F12 medium (1:1 mixture) containing 10% (v/v) FBS at 37°C and seeded in 12-well dishes (1.5 × 10 5 cells/

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conserved among LDLR, VLDLR, apoER2, and LRP1. Leucine is only present in LDLR ( Fig. 1A ). Thus, we ex-pressed these LDLR family members in monkey kidney cells (COS-7 cells) and examined their ability to bind PCSK9. COS-7 cells were used to study PCSK9 binding to the LDLR because PCSK9 can bind to the LDLR but can-not induce the degradation of the receptor in this cell line ( 37 ). In addition, endogenous LDLR in COS-7 cells is low, which reduces background PCSK9 binding. The expres-sion of different LDLR family members was detected by their specifi c antibodies. As shown in Fig. 1B , when puri-fi ed PCSK9 (2 � g/ml) was added to the medium and incu-bated with the cells for 2 h at 37°C (pH 7.4), the PCSK9 association signal was detected only in cells expressing LDLR ( Fig. 1B , top, lane 2), but not in cells expressing other LDLR family members ( Fig. 1B , lanes 3–7). Next, we investigated if EGF-A of the LDLR was suffi cient to confer PCSK9-promoted receptor degradation. We have reported that VLDLR:EGF-A-LDLR, in which EGF-A of the VLDLR is replaced with EGF-A of the LDLR, binds to PCSK9 very effi ciently. However, binding of PCSK9 to LDLR:EGF-A-VLDLR, in which EGF-A of the LDLR is substituted by EGF-A from the VLDLR, is dramatically reduced ( 17 ). Therefore, the two chimeric proteins were transiently ex-pressed in mouse hepatoma cells (Hepa1C1C7) and in-cubated with PCSK9 (0.5 � g/ml) for 4 h at pH 7.4. The VLDLR that had a HA tag at its C terminus was detected by an anti-HA antibody, which recognizes both the precursor (p) and the mature (m) fully glycosylated form of the re-ceptor ( Fig. 1C ). We observed that addition of PCSK9 re-sulted in robust degradation of the mature form of hybrid VLDLR:EGF-A-LDLR ( Fig. 1C , lane 6) and the wild-type LDLR ( Fig. 1D , lane 5). Conversely, wild-type VLDLR ( Fig. 1C , lane 4) and the hybrid LDLR:EGF-A-VLDLR ( Fig. 1D , lane 3) could not be degraded by PCSK9. Thus, EGF-A of the LDLR is suffi cient to confer PCSK9-mediated degrada-tion of a receptor, when it is placed in a cell surface pro-tein that normally does not bind PCSK9 effi ciently.

Recently, it has been reported that PCSK9 promotes the degradation of both VLDLR and apoER ( 38 ). To elucidate the differences between our fi ndings and others, we tran-siently expressed the VLDLR and the LDLR in Hepa1C1C7 cells and incubated the cells with various doses of wild-type PCSK9 or mutant PCSK9 D374Y for 4 h and overnight at pH 7.4, respectively. The levels of PCSK9 in human plasma range from 0.033 to 2.988 � g/ml ( 39 ). Thus, the concen-trations of PCSK9 we tested ranged from 0.5 to 4 � g/ml. We observed that when incubated with the cells for 4 h or overnight, wild-type PCSK9 could not effi ciently induce VLDLR degradation, even at a concentration of 4 � g/ml ( Fig. 2A, B , lane 6), but effi ciently promoted LDLR degra-dation at a concentration of 0.5 � g/ml ( Fig. 2A, B , lane 12). Mutant D374Y that binds to the LDLR with a much higher affi nity could induce VLDLR degradation effi ciently at a concentration of 4 � g/ml when incubated with the cells for 4 h ( Fig. 2A , lane 10) or overnight ( Fig. 2B , lane 10). These fi ndings suggest that the VLDLR can be degraded by PCSK9, but with much less effi ciency when compared with the LDLR.

The ligand blotting assay was performed as described with mod-ifi cations ( 17 ). Briefl y, purifi ed LDL-ECD, GST, and GST:EGF-A were labeled with IRDye680 using IRDye680 protein labeling kit. The proteins were directly blotted to nitrocellulose membranes. The membranes were then cut into individual strips and blocked with PBS containing 2.5% nonfat milk. After rinsing briefl y in pH buffer [50 mM Tris maleate (pH 7.4–6.0), 75 mM NaCl, 2 mM CaCl 2 , and 2.5% nonfat milk], the strips were incubated at room temperature for 60 min with 200 ng/ml IRDye800-labeled PCSK9 in the pH buffer, followed by three 15 min washes with pH buf-fer. The signals were detected by a LI-COR Odyssey infrared imaging system.

Binding of PCSK9 to the LDLR at pH 6.0 The experiments were performed as described in ( 35 ) with

modifi cations. COS-7 cells were seeded in 12-well dishes (1.5 × 10 5 cells/well). After 24 h, cells were transiently transfected with expression plasmids containing cDNAs for wild-type or mutant LDLR and pCDNA3.1 vector using XtremeGENE HP. Forty-eight hours later, the cells were washed twice with ice-cold pH 6.0 buf-fer [50 mM Tris maleate buffer, 150 mM NaCl, 2 mM CaCl 2 , and 2.5% nonfat milk (pH 6.0)] and incubated on ice for 30 min in 0.5 ml pH 6.0 buffer. The cells were then incubated with 0.5 ml pH 6.0 buffer containing PCSK9 (0.5 � g/ml) for 1 h at 4°C, washed twice with ice-cold pH 6.0 buffer without milk, collected, and then lysed in 60 � l of lysis buffer. The whole cell lysates were subjected to SDS-PAGE (8%) and immunoblotting. The LDLR and PCSK9 were detected as described above.

Biotinylation of LDLR COS-7 cells were transiently transfected with expression plas-

mids containing cDNAs for wild-type or mutant LDLR using XtremeGENE HP. After 48 h, cell surface proteins were biotiny-lated exactly as described ( 31 ). The cells were lysed in 150 � l of lysis buffer and then subjected to centrifugation at 15,000 rpm for 5 min. A total of 50 � l of the cell lysates was saved and 100 � l of the lysates was added to 60 � l of 50% slurry of Neutravidin agarose (Pierce). The mixture was rotated overnight at 4°C. After centrifugation at 3,000 g for 5 min, the pellets were washed in ly-sis buffer three times for 10 min at 4°C. The cell surface proteins were eluted from the beads by adding 1× SDS loading buffer [31 mM Tris·HCl (pH 6.8), 1% SDS, 12.5% glycerol, and 0.0025% bromophenol] and incubated for 5 min at 85°C. Proteins were then analyzed by SDS-PAGE and immunoblotting.

Statistics All statistical analyses were carried out by GraphPad Prism ver-

sion 4.0 (GraphPad Software). Student’s t -test was used to deter-mine the signifi cant differences between groups. Signifi cance is defi ned as P < 0.05. Results are presented as mean ± SD.

RESULTS

Binding of PCSK9 to LDLR family members We have reported that PCSK9 effi ciently binds to the

LDLR but not to the VLDLR ( 17 ). The residues Asn 295 , Glu 296 , Asp 310 , Tyr 315 , and Leu 318 in EGF-A play an impor-tant role in PCSK9 binding. ( 17 ). Asn 295 , Glu 296 , Asp 310 , and Tyr 315 are the calcium binding sequences in EGF-A ( 36 ). Sequence alignment of EGF-A in LDLR family mem-bers shows that asparagine is completely conserved in all members we analyzed; glutamate and aspartate are

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(m) fully glycosylated form of the receptor. Similar amounts of mature LDLR were expressed in cells trans-fected with each construct. No PCSK9 was found associ-ated with the cells expressing the vector alone ( Fig. 3B , lane 1), mutant L318D ( Fig. 3B , lane 5), or mutant LDLR in which EGF-A in the LDLR was replaced with EGF-A from the VLDLR ( Fig. 3B , lane 3), consistent with our pre-vious fi ndings ( 17 ). Replacement of Leu 318 with either Ala ( Fig. 3B , lane 4) or Thr ( Fig. 3B , lane 6) also failed to bind PCSK9 effi ciently. Mutation of Leu 318 to either His ( Fig. 3B , lane 7) or Val ( Fig. 3B , lane 9) decreased the ability to bind PCSK9, whereas substitution of Leu 318 with a positive charged residue Arg ( Fig. 3B , lane 8) enhanced PCSK9 binding when compared with the wild-type protein. From these experiments, we concluded that the property of amino acid residue at position 318 in EGF-A of LDLR plays an important role in PCSK9 binding at the cell surface.

Effects of mutations in EGF-A on PCSK9 binding Replacement of Leu 318 with Arg enhanced PCSK9 bind-

ing, while mutation L318D reduced PCSK9 binding, suggest-ing that the charge on the amino acid side chain at position 318 in EGF-A of the LDLR affects PCSK9 binding to the

Effect of mutations of Leu 318 in EGF-A of the LDLR on PCSK9 binding

We have previously reported that replacement of Leu 318 in EGF-A of the LDLR with Asp, as it is in the VLDLR, dra-matically reduces PCSK9 binding ( 17 ). Sequence align-ment of EGF-A of the LDLR shows that the Leu residue is highly conserved among different species except for rabbit, which has a His residue at that position ( Fig. 3A ). Next, we investigated the specifi c requirement of Leu 318 in EGF-A of the LDLR for effi cient PCSK9 binding. Leu 318 was changed to fi ve other amino acids including a nega-tively charged residue Asp, as it is in the VLDLR; a Thr residue, as it is in apoER2; a positively charged residue Arg, as it is in LRP1; a neutral amino acid residue Ala, as it is in LRP4 and LRP6 ( Fig. 1A ); a His residue, as it is in the rabbit LDLR ( Fig. 3A ); and a more structurally similar resi-due Val. Each mutant or wild-type LDLR cDNA was intro-duced into COS-7 cells. Purifi ed PCSK9 was added to the medium (pH 7.4). The concentrations of PCSK9 we used in the experiments were 0.5 � g/ml because the median PCSK9 levels in normal human plasma are 0.487 � g/ml ( 39 ). As shown in Fig. 3B , the antibody used to detect the LDLR recognizes both the precursor (p) and the mature

Fig. 1. Binding of PCSK9 to the LDLR family members. A: Sequence alignment of EGF-A of LDLR family members. The sequence alignment was performed by ClustalW2. Amino acid residues that play an impor-tant role in PCSK9 binding to the LDLR ( 17 ) are shown in bold. B: Binding of PCSK9 to the LDLR family members. The experiment was performed as described in Materials and Methods. Briefl y, COS-7 cells tran-siently expressing the LDLR family members were incubated with PCSK9 (2.0 � g/ml) at pH 7.4. The whole lysates were analyzed by SDS-PAGE (8%) and immunoblotted using a monoclonal anti-hLDLR (HL-1), a polyclonal anti-HA (LRP6 and VLDLR), a polyclonal anti-apoER2, LRP1, LRP4, or a monoclonal anti-PCSK9 antibody (15A6). V, cells were transfected with the empty vector pCDNA3.1; LR, LDLR; VLR, VLDLR; AER, apoER2; LP1, LRP1; LP4, LRP4; LP6, LRP6. C, D: PCSK9-induced degradation of receptors. The experiment was performed as described in Materials and Methods. Briefl y, Hepa1C1C7 cells transiently expressing wild-type (WT) or mutant VLDLR or LDLR were incubated with PCSK9 (0.5 � g/ml) for 2 h at 37°C and pH 7.4. The whole lysates were then analyzed by SDS-PAGE (8%) and immunoblotted. VLDLR was detected by a polyclonal anti-HA antibody. m, mature fully glycosylated form of the VLDLR; p, precursor of the VLDLR. LDLR was detected by a monoclonal anti-hLDLR (HL-1). Calnexin was detected by a polyclonal antibody and used as a loading control. Similar results were obtained from at least one more independent experiment .

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on the amino acid side chains. However, Pro 320 and Arg 329 are mutated to Arg and Pro, respectively, in FH patients. Thus, we also examined the effect of these two FH mutations on PCSK9 binding. PCSK9 (0.5 � g/ml) was added to COS-7 cells transiently expressing wild-type or mutant LDLR and incubated with the cells for 2 h at 37°C (pH 7.4). Overex-pression of wild-type LDLR signifi cantly enhanced PCSK9 binding ( Fig. 4B , lane 1; Fig. 4C , lane 2). Binding of PCSK9 to the cells expressing mutant LDLRs including D299V ( Fig. 4B , lane 3), R329P ( Fig. 4B , lane 7), G293H ( Fig. 4C , lane 3), and E332G ( Fig. 4C , lane 8) was signifi cantly reduced, while binding of PCSK9 to N309K was increased

receptor at the cell surface. Thus, we investigated the poten-tial effects of changes in the charge on amino acid side chains at other positions in EGF-A of the LDLR on PCSK9 binding. We focused on amino acid residues in EGF-A that are differ-ent between the LDLR and the VLDLR. As shown in Fig. 4A , 15 amino acid residues are different between the LDLR and the VLDLR, among which 11 residues including Leu 318 change the charge on amino acid side chains. We replaced these amino acid residues in the LDLR with their corresponding residues in the VLDLR ( Fig. 4A ). The corre-sponding residues of Pro 320 and Arg 329 in the VLDLR are Ala and Lys, respectively, which do not change the charge

Fig. 2. PCSK9-induced receptor degradation. A, B: PCSK9-induced receptor degradation for 4 h and over-night. The experiment was performed as described in Materials and Methods. Briefl y, Hepa1C1C7 cells tran-siently expressing VLDLR or LDLR were incubated with various concentrations of PCSK9 for 4 h (A) or overnight (B) at 37°C. VLDLR was detected by a polyclonal anti-HA antibody. LDLR was detected by a mono-clonal anti-hLDLR (HL-1). m, mature fully glycosylated form of the receptors; p, precursor of the receptors. V, cells were transfected with the empty vector pCDNA3.1. Binding of antibody was detected by IRDye800-labeled goat anti-mouse IgG and IRDye680-labeled goat anti-rabbit IgG and imaged on a LI-COR Odyssey sys-tem. Calnexin was detected by a polyclonal antibody and used as a loading control. Similar results were obtained from at least two more independent experiments. The bottom fi gures in (A) and (B) are representative ones of protein levels. The top fi gures in (A) and (B) are percentage of densitometry of the receptors. The densi-tometry of VLDLR and LDLR signals was determined by a LI-COR Odyssey system. Relative densitometry was the ratio of densitometry of VLDLR and LDLR in the presence of a different amount of PCSK9 to that of VLDR and LDLR in the absence of PCSK9 (0). The percentage of densitometry of VLDR and LDLR in the absence of PCSK9 (0) was defi ned as 100%. Values are mean ± SD of three or more independent experiments.

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the lysosome for degradation ( 17 ). Most recently, it has been shown that the C-terminal domain of PCSK9 interacts with the ligand binding repeats of the LDLR under acidic conditions ( 35, 40 ), which may contribute to the stronger binding between PCSK9 and the LDLR in the endosome. Here we investigated if PCSK9 bound to recombinant EGF-A in a pH-dependent manner. EGF-A was purifi ed as GST fusion proteins and shown as a single band ( Fig. 5A ). Same amounts of IRDye680-labeled purifi ed LDLR-ECD that contains the extracellular domain of the LDLR (amino acids 1–699), GST, and GST:EGF-A fusion protein were di-rectly blotted to nitrocellulose membranes ( Fig. 5B ). The membranes were cut into individual strips and then incu-bated with IRDye800-labeled PCSK9 at different pH values. As shown in Fig. 5C , PCSK9 bound to the LDLR-ECD in a pH-dependent manner. The binding was stronger at pH 6.0 ( Fig. 5C , top), consistent with previous reports ( 16, 17 ). There was no detectable PCSK9 binding to GST at all pH values tested ( Fig. 5C , middle). The binding pattern of PCSK9 to GST:EGF-A was similar to that of LDLR-ECD; the strongest binding was at pH 6.0 ( Fig. 5C , bottom). To con-fi rm these fi ndings, we examined binding of labeled PCSK9 (200 ng/ml) to six different concentrations of GST:EGF-A (10, 25, 50, 100, 250, and 500 ng) at pH 7.4 and pH 6.0. The densitometry of PCSK9 binding was determined. Apparent K m and V max were obtained from the curve fi t of the data to a nonlinear regression (one site binding equation). The ap-parent V max values of PCSK9 binding to GST:EGF-A at pH 7.4 and at pH 6.0 were comparable (22 and 27 arbitrary units/h for pH 7.4 and pH 6.0, respectively) ( Fig. 5D ). The apparent K m values of PCSK9 binding to GST:EGF-A at pH 7.4 and at pH 6.0 were 747 and 307 ng, respectively ( Fig. 5D ). We also analyzed the binding data with the Scatchard plot (inserts in Fig. 5D ). Similar results were obtained. The apparent K m and V max values of PCSK9 binding to GST:EGF-A were 966 ng and 28 arbitrary units/h for pH 7.4 and 298 ng and 30 arbitrary units/h for pH 6.0, respectively. Thus, PCSK9 has increased affi nity toward the purifi ed recombi-nant GST:EGF-A at pH 6.0.

We next examined the effect of mutations in EGF-A of the LDLR on PCSK9 binding to the receptor in an acidic environment. The binding assay was performed at 4°C and under pH 6.0 to minimize the internalization of the LDLR and to mimic PCSK9 binding in the endosomal environ-ment. This assay has been widely used to study PCSK9-LDLR binding and LDL-LDLR binding ( 35, 41, 42 ). COS-7 cells transiently expressing wild-type or mutant LDLR were incubated with ice-cold pH buffer (pH 6.0) containing PCSK9 (0.5 � g/ml) for 1 h at 4°C. PCSK9 and the LDLR in the whole cell lysates were then determined by im-munoblotting. We found that mutations R329P ( Fig. 6A , lane 7) and E332G ( Fig. 6B , lane 8) led to a signifi cant reduction in PCSK9 binding at pH 6.0. Other mutations including D299V ( Fig. 6A , lane 3), G293H ( Fig. 6B , lane 3), and N309K ( Fig. 6B , lane 4) that affected PCSK9 bind-ing at pH 7.4 ( Fig. 4 ) had no signifi cant effect on PCSK9 binding at pH 6.0. Mutation of Leu 318 to His, Asp, or Arg also had no effect on PCSK9 binding at pH 6.0 ( Fig. 6C ), even though these mutations signifi cantly affected PCSK9

( Fig. 4C , lane 4). Other mutations had no signifi cant effect on PCSK9 binding. Thus, Gly 293 , Asp 299 , Asn 309 , Arg 329 , and Glu 332 , like Leu 318 , also involve in PCSK9 binding to the LDLR.

Characterization of binding of PCSK9 to purifi ed EGF-A We have shown that PCSK9 interacts with EGF-A of the

LDLR at the cell surface and binds to the full-length recep-tor with a much higher affi nity in the acidic environment of the endosome. Consequently, the receptor is redirected to

Fig. 3. Binding of PCSK9 to wild-type and mutant LDLR. A: Se-quence alignment of EGF-A of LDLR among different species. The sequence alignment was performed by ClustalW2. Leu 318 in the LDLR is in bold. B: Binding of PCSK9 to the LDLR. The experi-ment was performed as described in the legend to Fig. 1B except that COS-7 cells transiently expressing wild-type (WT) or mutant LDLR were incubated with PCSK9 (0.5 � g/ml) for 2 h at 37°C and pH 7.4. LDLR and PCSK9 were detected by HL-1 and 15A6, respec-tively. Antibody binding was detected using HRP-conjugated goat anti-mouse IgG, followed by ECL. m, mature fully glycosylated form of the LDLR; p, precursor of the LDLR. Calnexin was detected by a polyclonal antibody. Binding of antibody was detected by IRDye800-labeled goat anti-rabbit IgG and imaged on a LI-COR Odyssey system. The bottom fi gure in (B) is representative one of protein levels . V, cells were transfected with the empty vector pCD-NA3.1. Similar results were obtained from at least two more in-dependent experiments. The top fi gure in (B) is percentage of relative densitometry of PCSK9 binding signal. The densitometry of PCSK9 and LDLR signals was determined by Image J Analysis Software. The relative densitometry of PCSK9 binding was the ratio of PCSK9 densitometry to LDLR densitometry. Percentage of the relative densitometry of PCSK9 binding was the percentage of the relative densitometry of PCSK9 binding to mutant LDLR to that of PCSK9 binding to wild-type LDLR. The percentage of the rela-tive densitometry of PCSK9 binding to wild-type LDLR was de-fi ned as 100%. Values are mean ± SD of three or more independent experiments.

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The effect of conserved histidine and lysine residues in YWTD on PCSK9-promoted LDLR degradation

Our data showed that mutations D299V, G293H, N309K, and L318R in EGF-A affected PCSK9 binding at pH 7.4 but not at pH 6.0, suggesting that these residues may not contribute signifi cantly to PCSK9 binding in the acidic endosomal environment. Given that PSCK9 binds to the LDLR with a much higher affi nity in the endosome ( 17 ), other parts of the LDLR may contribute to PCSK9 binding at the low pH environment. YWTD has been shown to in-teract with the prodomain of PCSK9 ( 30 ). We have previ-ously reported that the LDLR lacking YWTD binds PCSK9,

binding at pH 7.4 ( Fig. 3 ). Given that mutations R329P and E332G dramatically reduced PCSK9 binding at both pH values, we examined whether the two mutations af-fected the cell surface expression of the LDLR via labeling the cells with biotin. The cell surface proteins were then precipitated using NeutrAvidin beads and then immunob-lotted for the LDLR. As shown in Fig. 6D , the cell surface levels of wild-type and mutant LDLR (pellets) were pro-portional to the levels of mature forms of the receptors in the whole cell lysates, indicating that the two mutations had no effect on the traffi cking of the LDLR to the plasma membrane.

Fig. 4. Effect of mutations in EGF-A of the LDLR on PCSK9 binding. A: Sequence alignment of EGF-A of the LDLR and the VLDLR. The sequence align-ment was performed by ClustalW2. Mutations shown in (B) are underlined in (A ). Mutations shown in (C) are bold italic in (A). B, C: Binding of PCSK9 to wild-type (WT) and mutant LDLR was performed as described in Materials and Methods. COS-7 cells tran-siently expressing wild-type or mutant LDLR were incubated with PCSK9 (0.5 � g/ml) for 2 h at 37°C and pH 7.4. LDLR and PCSK9 were detected by HL-1 and 15A6, respectively. Calnexin was detected by a polyclonal antibody. The binding of antibody was detected by IRDye800-labeled goat anti-mouse IgG and IRDye680-labeled goat anti-rabbit IgG and im-aged on a LI-COR Odyssey system. The bottom fi g-ures in (B) and (C) are representative ones of protein levels. m, mature fully glycosylated form of the LDLR; p, precursor of the LDLR; V, cells were transfected with the empty vector pCDNA3.1. The top fi gures in panels (B) and (C) are percentage of relative densi-tometry of PCSK9 binding signal. The relative densi-tometry of PCSK9 binding was the ratio of PCSK9 densitometry to LDLR densitometry. Percentage of the relative densitometry of PCSK9 binding was the percentage of the relative densitometry of PCSK9 binding to mutant LDLR to that of PCSK9 binding to wild-type LDLR. The percentage of the relative densitometry of PCSK9 binding to wild-type LDLR was defi ned as 100%. Values are mean ± SD of three independent experiments.

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investigated whether Lys 560 and Lys 582 participated into PCSK9’s action on the LDLR. Lys 560 and Lys 582 were re-placed by Met individually (K560M and K582M) and the mutants were transiently expressed in Hepa1C1C7 cells. As shown in Fig. 7B , these mutations had little detectable effect on PCSK9-promoted degradation of the LDLR. Thus, the three conserved histidine residues and the two lysine residues are not required for PCSK9-promoted deg-radation of the LDLR.

DISCUSSION

The data reported here provide direct evidence for the critical role of EGF-A of the LDLR in PCSK9-mediated degradation of the receptor. First, PCSK9 only effi ciently

but cannot be subjected to PCSK9-induced degradation ( 18 ). The crystallographic structure of the LDLR at the low pH suggests that His 190 , His 562 , and His 586 serve as pH sensors to promote closure of the receptor under acidic conditions ( 33 ). Thus, we changed His 190 to Asp, His 562 to Arg, and His 586 to Lys simultaneously to examine the role of these residues in PCSK9’s action on the LDLR. The mutant receptor (H190DH562RH586K) was expressed in Hepa1C1C7 cells at a similar level as the wild-type receptor ( Fig. 7A , lanes 3 and 5). Addition of PCSK9 to the medium resulted in a robust reduction in mature wild-type and mu-tant LDLR ( Fig. 7A , lanes 4 and 6). In addition, it has been reported that two highly conserved lysine residues, Lys 560 and Lys 582 , present in YWTD play an essential role in the release of ligand from the LDLR ( 42 ). Therefore, we also

Fig. 5. Binding of PCSK9 to recombinant EGF-A. A: Purifi cation of GST:EGF-A fusion protein. GST:EGF-A fusion protein was purifi ed using Glutathione Sepharose 4 Fast Flow affi nity gel chromatography, followed by size-exclusion chromatography on a Tricorn Superose 12 10/300 column as described in Materials and Methods. Purifi ed proteins were then subjected to SDS-PAGE (12%) and detected with Coomassie Brilliant Blue R-250 staining. The size of GST:EGF-A is about 28 kDa. (GST tag is about 23 kDa, EGF-A is about 4.4 kDa). B, C: Binding of PCSK9 to purifi ed LDLR-ECD, GST, and GST:EGF-A. The same amount of IRDye680-labeled LDLR-ECD, GST, or GST-EGF-A (200 ng) was applied directly on a nitrocellulose membrane (B). The membrane was cut into individual strips. The strips were blocked and rinsed briefl y in the pH buffer indicated, and then incubated for 1 h at room temperature with 200 ng/ml PCSK9-IRDye800 in the pH buf-fer. Following washes, the strips were imaged on a LI-COR Odyssey infrared imaging system. PCSK9 binding is shown in (C). Similar results were obtained from one more independent experiment. D: Binding of PCSK9 to purifi ed GST-EGF-A. The experiment was performed as described in the legend to Fig. 5C except that various amounts of GST-EGF-A (0, 10, 25, 50, 100, 250, and 500 ng) were applied directly on a nitrocel-lulose membrane. The membrane was cut into individual strips. The strips were blocked and rinsed briefl y in pH 7.4 or 6.0 buffer, and then incubated for 1 h at room temperature with 200 ng/ml PCSK9-IRDye800 in the pH buffer. Following washes, the strips were imaged and the densitometry of PCSK9 signals was deter-mined by a LI-COR Odyssey system. Mean values of densitometry were plotted using GraphPad Prism version 4.0 with nonlinear regression (curve fi t, equation; one site binding) and with the Scatchard plots (inserts) PCSK9 binding at pH 6.0 ( � ). PCSK9 binding at pH 7.4 ( � ). Values are mean ± SD of three independent experiments.

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demonstrated that highly conserved histidine residues (His 190 in LR5, His 562 and His 586 in YWTD) and lysine resi-dues (Lys 560 and Lys 582 in YWTD), which play an important role in the release of ligand LDL from the receptor, were not required for PCSK9’s action on the LDLR ( Fig. 7 ).

Previously, we have reported that the association of PCSK9 with COS-M cells is detectable only in cells ex-pressing the LDLR, but not in cells expressing the VLDLR after addition of PCSK9 at a physiological concentration (0.5 � g/ml) ( 17 ). Most recently, Shan et al. ( 43 ) and Poirier et al. ( 38 ) reported that PCSK9 could bind to the VLDLR and the apoER. It is possible that binding of PCSK9 to the VLDLR might be too weak to be detected in our pre-vious experiment when we added a physiological con-centration of PCSK9 to COS-M cells. Thus, in the current study, we incubated COS-7 cells expressing various LDLR family members with the medium containing 2 � g/ml of

bound to the LDLR among the LDLR family members we tested ( Fig. 1B ). Second, EGF-A from the LDLR was suffi -cient to confer VLDLR degradation after addition of PCSK9 at a normal physiological concentration (0.5 � g/ml) ( Fig. 1C ). Third, wild-type PCSK9 could not induce VLDLR degradation even at a concentration of 4 � g/ml with overnight incubation ( Fig. 2 ). In addition, we dem-onstrated that PCSK9 bound to recombinant EGF-A in a pH-dependent manner with greater binding effi ciency at pH 6.0 ( Fig. 5 ). Replacement of amino acid residues Gly 293 , Asp 299 , and Leu 318 in EGF-A of the LDLR with their corre-sponding residues in the VLDLR signifi cantly reduced PCSK9 binding at pH 7.4 without effects on PCSK9 bind-ing at pH 6.0 ( Figs. 4, 6 ). On the other hand, substitution of Glu 332 , with its corresponding amino acid residues in the VLDLR (E332G) and FH mutation R329P, reduced PCSK9 binding at pH 7.4 and 6.0 ( Figs. 4, 6 ). Finally, we

Fig. 6. Effect of mutations in EGF-A of the LDLR on PCSK9 binding at pH 6.0. A–C: Binding of PCSK9 to wild-type and mutant LDLR was performed as described in Materials and Methods. COS-7 cells transiently expressing wild-type or mutant LDLR were incubated with pH 6.0 buffer containing PCSK9 (0.5 � g/ml) for 1 h at 4°C. LDLR and PCSK9 were detected by HL-1 and 15A6, respectively. Calnexin was detected by a polyclonal antibody. The binding of antibody was detected by IRDye800-labeled goat anti-mouse IgG and IRDye680-labeled goat anti-rabbit IgG and imaged on a LI-COR Odyssey system. V, cells were transfected with the empty vector pCDNA3.1. Similar results were obtained in at least two more independent experiments. The bottom fi gures in (A–C) are representative ones of protein levels. m, mature fully glycosylated form of the LDLR; p, precursor of the LDLR. The top fi gures in (A–C) are percentage of relative densitometry of PCSK9 binding signal that was determined as described in the legend to Fig. 3 . Percentage of the relative densitometry of PCSK9 binding was the percentage of the relative densitometry of PCSK9 binding to mutant LDLR to that of PCSK9 binding to wild-type LDLR that was set at 100%. Values are mean ± SD of three independent experiments. D: Biotinylation of the LDLR. COS-7 cells transiently expressing wild-type (WT) or mutant LDLR were biotinylated exactly as described. Biotinylated proteins from the cell surface (pellets) and proteins from the whole cell lysate were analyzed by SDS-PAGE (8%) and immunoblotting. LDLR was detected using HL-1 and calnexin was detected with a polyclonal antibody. V, cells were transfected with the empty vector pCDNA3.1. Similar results were obtained in two independent experiments.

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LDLR ( 30 ). EGF-A of the VLDLR is identical in humans and mice. Most recently, Surdo et al. ( 30 ) reported that the prodomain of PCSK9 contacts with the YWTD domain of the LDLR via van der Waals interactions. There is 98% amino acid identity in YWTD between human and mouse VLDLR. More studies are needed to determine if these dif-ferent amino acid residues affect PCSK9 binding to the VLDLR. Taken together, these fi ndings indicate that the physiological role of PCSK9 to either VLDLR or apoER is still uncertain. The plasma levels of PCSK9 in people with-out statin treatment range from 33 to 2988 ng/ml ( 39 ). It has been shown that plasma levels of PCSK9 are increased in patients treated with atorvastatin ( 46, 47 ). Thus, it is possible that PCSK9 may promote VLDLR degradation in individuals with high plasma levels of PCSK9 or gain-of-function PCSK9 mutants, especially while under statin treatment.

Previously, we reported that replacement of Leu 318 in the LDLR with Asp, as it is in the VLDLR, signifi cantly re-duces PCSK9 binding, and mutation of the corresponding Asp in the VLDLR to Leu increases PCSK9 binding ( 17 ). Here, we observed that replacement of Asn 309 in LDLR with Lys, as it is in the VLDLR, increased PCSK9 binding. However, mutation of the Lys residue in the VLDLR to its corresponding residue in the LDLR, Asn, has no effect on PCSK9 binding ( 17 ). Thus it appears that Leu at position 318 of EGF-A of the LDLR plays a critical role in binding of PCSK9 to the receptor. Indeed, we found that substitu-tion of Leu 318 in EGF-A of the LDLR with other residues including Asp, Thr, and Ala, as they are in VLDLR, apoER2, and LRP4/6, respectively, reduced PCSK9 binding. How-ever, replacement of Leu 318 with Arg, as it is in LRP1, en-hanced PCSK9 binding ( Fig. 3B ). Sequence alignment of EGF-A of LDLR family membranes reveals that EGF-A in LRP1 contains amino acid residues that are required for binding of PCSK9 (Asn 295 , Glu 296 , and Asp 310 ; Fig. 1A , bold) ( 17 ). However, no detectable binding was observed in COS-7 cells overexpressing LRP1 ( Fig. 1B ), suggesting

PCSK9 for 2 h. However, we still observed PCSK9 binding only in cells expressing the LDLR but not in cells express-ing other LDLR family members tested, including the VLDLR and the apoER ( Fig. 1B ). The different results may be simply accounted for by the different protocols used in each study. We incubated COS-7 cells for 2 h with the medium containing purifi ed PSCK9 so that we could control the amount of proteins used. Poirier et al. ( 38 ) incubated CHO-7 cells overnight in the conditional me-dium isolated from HEK293 cells expressing PCSK9. Shan et al. ( 43 ) used an in vitro assay by mixing purifi ed recep-tors and purifi ed PCSK9 together. To elucidate these dif-ferent fi ndings, we incubated the cells expressing the VLDLR with various doses of wild-type and mutant PCSK9 D374Y for 4 h or overnight. We observed that only mutant D374Y that has a much higher affi nity for the LDLR, but not wild-type PCSK9, induced VLDLR degradation at a concentration of 4 � g/ml ( Fig. 2 ). Similarly, wild-type PCSK9 (0.5 � g/ml) could not induce the degradation of mutant LDLR in which EGF-A was replaced by EGF-A of VLDLR ( Fig. 1D ), but promoted the degradation of mu-tant VLDLR, in which EGF-A was substituted with EGF-A of LDLR ( Fig. 1C ). These results indicate that PCSK9 may promote VLDLR degradation, but with much less effi -ciency when compared with the LDLR. The VLDLR and apoER are essential during mouse cerebellar development ( 44 ). Most recently, Roubtsova et al. ( 45 ) reported that PCSK9 induces VLDLR degradation in mouse adipose tis-sue. Pcsk9 � / � mice show higher cell surface expression of VLDLR and accumulate more visceral adipose tissue ( 45 ). However, absence of PCSK9 in humans is not associated with any obvious phenotypes except for hypocholester-olemia ( 14, 24 ). The underlying mechanism for the differ-ent phenotypes observed in mice and in humans is unclear. The overall sequence homology between mouse and hu-man VLDLR is high, with 97% amino acid identity. The interaction between PCSK9 and LDLR mainly happens be-tween the catalytic domain of PCSK9 and EGF-A of the

Fig. 7. The effects of mutations of His residues (A) and Lys residues (B) in the LDLR on PCSK9-promoted LDLR degradation. The experiments were carried out as described in the legend to Fig. 2 except that Hep-a1C1C7 cells transiently expressing wild-type (WT) or mutant LDLR were incubated with PCSK9 (2 � g/ml) for 4 h at 37°C (pH 7.4). LDLR was detected by a monoclonal anti-hLDLR (HL-1). Calnexin was detected by a polyclonal antibody. V, cells were transfected with the empty vector pCDNA3.1. Antibody binding was de-tected using HRP-conjugated goat anti-mouse IgG or donkey anti-rabbit IgG, followed by ECL and exposure to fi lms. m, mature fully glycosylated form of the LDLR; p, precursor of the LDLR. Similar results were obtained from at least two more independent experiments .

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conditions ( 33 ), and play an important role in the release of bound LDL ( 42 ). YWTD of the LDLR also interacts with the prodomain of PCSK9 ( 30 ), and is required for PCSK9-induced LDLR degradation ( 18 ). However, unlike the sit-uation of LDL binding and releasing, substitution of His 190 to Asp, His 562 to Arg, and His 586 to Lys simultaneously or mutation of the two highly conserved lysine residues, Lys 560 and Lys 582 , in YWTD to Met had no effect on PCSK9-pro-moted LDLR degradation, indicating that these residues have no essential roles in PCSK9’s action on the receptor.

FH mutation H306Y increases PCSK9 binding ( 48 ). Here, we found that FH mutation P320R had no effect on PCSK9 binding while R329P reduced PCSK9 binding at pH 7.4 and 6.0. The nuclear magnetic resonance structure of EGF-AB reveals that mutation R329P may disrupt the geometry of the region of the calcium binding site in EGF-A ( 36 ). The integrity of the calcium-binding site in EGF-A is important for PCSK9 binding ( 17 ). Thus, R329P may impair PCSK9 binding through disruption of the cal-cium-binding site in EGF-A of the LDLR. The crystallo-graphic structures of PCSK9-EGF-AB complex reveal that PCSK9 interacts with the N-terminal EGF-A ( 28–30 ). Asp 299 forms a salt bridge to the N-terminal amine of Ser 153 in PCSK9. We found that mutation D299V reduced PCSK9 binding. Zhang et al. ( 49 ) reported that replacement of Asp 299 with Ser (D299S) in recombinant EGF-A has no sig-nifi cant effect on PCSK9 binding. Thus, it is possible that D299V, but not D299S, disrupts the salt bridge to Ser 153 in PCSK9, leading to a reduction in PCSK9 binding. Asn 309 contributes to PCSK9 binding through forming a hydro-gen bond to Thr 377 in PCSK9. Replacement of Asn 309 with Lys (N309K) increases PCSK9 binding at pH 7.4, consis-tent with the previous fi nding that replacement of Asn 299 with a positively charged residue Arg or Lys in recombi-nant EGF-A improves binding affi nity for PCSK9 ( 49 ). Mu-tation N309K introduces a positive charge in the side chain that may stabilize the negative charge on the side chain of Asp 374 in PCSK9; meanwhile mutation N309K retains the hydrogen bond to Thr 377 in PCSK9. Thus, mutation N309K enhances PCSK9 binding. The side chain of Leu 318 reaches out and forms a van der Waals interaction with Cys 378 in PCSK9 ( 28 ). Like mutation N309K, mutation of Leu 318 to Arg also introduces a positive charge in the side chain, which may stabilize the negatively charged side chain of Asp 374 in PCSK9, thereby enhancing PCSK9 binding. The side chain of Asp 374 in PCSK9 is stabilized by His 306 in EGF-A via a salt bridge at low pH ( 29 ). Thus, mutations N309K and L318R did not increase PCSK9 binding at pH 6.0 ( Fig. 6 ). Glu 332 is the last amino acid residue in the C terminus of EGF-A. PCSK9 primarily interacts with the N-terminal EGF-A and does not contact with the C terminus of EGF-A. Gly 293 is the fi rst amino acid residue in EGF-A. Gly 293 also does not contact with PCSK9. Thus, it is unlikely that Gly 293 and Glu 332 contribute signifi cantly to binding of PCSK9 to the LDLR via direct interactions. G293H had no effect on PCSK9 binding at pH 6.0 and E332G had no ef-fect on the traffi cking of the LDLR to the cell surface, sug-gesting that the two mutations did not result in a major perturbation of the structure of the protein. However, we

that there may be some other determinants in EGF-A of the LDLR that contribute to effi cient PCSK9 binding. We did observe that in addition to Leu 318 , replacement of Gly 293 , Asp 299 , and Glu 332 in EGF-A of the LDLR with their corresponding amino acid residues in the VLDLR signifi -cantly reduced PCSK9 binding at pH 7.4 ( Fig. 4 ). LRP1 has His, Ser, and Cys at the corresponding positions ( Fig. 1A ), which may cause low-affi nity binding of PCSK9.

PCSK9 binds to the LDLR in a pH-dependent manner with a greater affi nity at low pH. Recently, several studies reported that the ligand binding repeats in the LDLR might interact with the C terminus of PCSK9 at low pH and subsequently contribute a higher affi nity to PCSK9 in the acidic endosomal environment. In the present study, we observed that PCSK9 bound to purifi ed EGF-A more strongly at pH 6.0 than at pH 7.4 ( Fig. 5 ). Our binding experiments indicate that binding of PCSK9 to recombi-nant GST:EGF-A was increased more than 2-fold at pH 6.0, consistent with previous fi ndings that the pH 6.0 binding environment leads to a 3.8-fold increase in the binding affi nity of the recombinant EGF-AB fragment to PCSK9, when compared with pH 7.4 ( 28 ). Taken together, these fi ndings suggest that EGF-A also contributes to the higher affi nity between PCSK9 and the LDLR at the acidic envi-ronment of the endosome. Interestingly, mutations G293H and D299V reduced PCSK9 binding at pH 7.4, but had no detectable effect on PCSK9 binding at pH 6.0, and muta-tion N309K enhanced PCSK9 binding only at pH 7.4 even though EGF-A bound to PCSK9 more strongly at pH 6.0 ( Figs. 4 and 6 ), suggesting that determinants in EGF-A of the LDLR required for effi cient PCSK9 binding are differ-ent at different pH environments. The overall structures of the PCSK9-EGF-AB complex at neutral and low pH are highly similar ( 28, 29 ). However, conformation rearrange-ments happen for EGF-A ( 29 ). For example, His 306 in EGF-A of the LDLR forms an intramolecular hydrogen bond with Ser 305 at neutral pH ( 29 ), but forms an intermo-lecular salt bridge with Asp 374 in PCSK9 at pH 4.8 ( 28 ). Thus, it is possible that the delivery of the PCSK9-LDLR complex from physiological neutral pH to acidic pH in the endosome leads to conformational changes in EGF-A, which may result in different amino acid residues in EGF-A involved in PCSK9 binding at an acidic pH. Alternatively, these residues may still contribute to PCSK9 binding at pH 6.0, but the other parts of the LDLR, such as the ligand binding repeats, also interact with PCSK9 at the acidic en-dosomal environment ( 35, 40 ), which may compensate for the loss of contributions from these residues in EGF-A at pH 6.0. Yamamoto, Lu, and Ryan ( 40 ) proposed a two-step binding model for interaction between PCSK9 and the LDLR. The catalytic domain of PCSK9 interacts with EGF-A of the LDLR at the cell surface. The conformation of the LDLR is changed when the receptor is exposed to the low pH endosomal environment. The ligand binding repeats of the LDLR then interact with the positively charged C terminus of PCSK9, enhancing PCSK9 binding at the acidic endosomal environment. It has been shown that His 190 in LR5 and His 562 and His 586 in YWTD serve as pH sensors to promote closure of the receptor under acidic

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15 . Abifadel , M. , J. P. Rabes , S. Jambart , G. Halaby , M. H. Gannage-Yared , A. Sarkis , G. Beaino , M. Varret , N. Salem , S. Corbani , et al . 2009 . The molecular basis of familial hypercholesterolemia in Lebanon: spectrum of LDLR mutations and role of PCSK9 as a modifi er gene. Hum. Mutat. 30 : E682 – E691 .

16 . Lagace , T. A. , D. E. Curtis , R. Garuti , M. C. McNutt , S. W. Park , H. B. Prather , N. N. Anderson , Y. K. Ho , R. E. Hammer , and J. D. Horton . 2006 . Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice. J. Clin. Invest. 116 : 2995 – 3005 .

17 . Zhang , D. W. , T. A. Lagace , R. Garuti , Z. Zhao , M. McDonald , J. D. Horton , J. C. Cohen , and H. H. Hobbs . 2007 . Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recy-cling and increases degradation. J. Biol. Chem. 282 : 18602 – 18612 .

18 . Zhang , D. W. , R. Garuti , W. J. Tang , J. C. Cohen , and H. H. Hobbs . 2008 . Structural requirements for PCSK9-mediated degradation of the low-density lipoprotein receptor. Proc. Natl. Acad. Sci. USA . 105 : 13045 – 13050 .

19 . Cameron , J. , O. L. Holla , T. Ranheim , M. A. Kulseth , K. E. Berge , and T. P. Leren . 2006 . Effect of mutations in the PCSK9 gene on the cell surface LDL receptors. Hum. Mol. Genet. 15 : 1551 – 1558 .

20 . Maxwell , K. N. , E. A. Fisher , and J. L. Breslow . 2005 . Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment. Proc. Natl. Acad. Sci. USA . 102 : 2069 – 2074 .

21 . Park , S. W. , Y. A. Moon , and J. D. Horton . 2004 . Post-transcriptional regulation of low density lipoprotein receptor protein by propro-tein convertase subtilisin/kexin type 9a in mouse liver. J. Biol. Chem. 279 : 50630 – 50638 .

22 . Grefhorst , A. , M. C. McNutt , T. A. Lagace , and J. D. Horton . 2008 . Plasma PCSK9 preferentially reduces liver LDL receptors in mice. J. Lipid Res. 49 : 1303 – 1311 .

23 . Luo , Y. , L. Warren , D. Xia , H. Jensen , T. Sand , S. Petras , W. Qin , K. S. Miller , and J. Hawkins . 2009 . Function and distribution of circulating human PCSK9 expressed extrahepatically in transgenic mice. J. Lipid Res. 50 : 1581 – 1588 .

24 . Rashid , S. , D. E. Curtis , R. Garuti , N. N. Anderson , Y. Bashmakov , Y. K. Ho , R. E. Hammer , Y. A. Moon , and J. D. Horton . 2005 . Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc. Natl. Acad. Sci. USA . 102 : 5374 – 5379 .

25 . Frank-Kamenetsky , M. , A. Grefhorst , N. N. Anderson , T. S. Racie , B. Bramlage , A. Akinc , D. Butler , K. Charisse , R. Dorkin , Y. Fan , et al . 2008 . Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. Proc. Natl. Acad. Sci. USA . 105 : 11915 – 11920 .

26 . McNutt , M. C. , T. A. Lagace , and J. D. Horton . 2007 . Catalytic activ-ity is not required for secreted PCSK9 to reduce low density lipo-protein receptors in HepG2 cells. J. Biol. Chem. 282 : 20799 – 20803 .

27 . Wang , Y. , Y. Huang , H. H. Hobbs , and J. C. Cohen . 2012 . Molecular characterization of proprotein convertase subtilisin/kexin type 9-mediated degradation of the LDLR. J. Lipid Res. 53 : 1932 – 1943 .

28 . Kwon , H. J. , T. A. Lagace , M. C. McNutt , J. D. Horton , and J. Deisenhofer . 2008 . Molecular basis for LDL receptor recognition by PCSK9. Proc. Natl. Acad. Sci. USA . 105 : 1820 – 1825 .

29 . Bottomley , M. J. , A. Cirillo , L. Orsatti , L. Ruggeri , T. S. Fisher , J. C. Santoro , R. T. Cummings , R. M. Cubbon , P. Lo Surdo , A. Calzetta , et al . 2009 . Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial hypercholes-terolemia mutants. J. Biol. Chem. 284 : 1313 – 1323 .

cannot exclude a possibility that the two mutations may cause subtle conformational changes in EGF-A, reducing PCSK9 binding indirectly.

In summary, we characterized the role of EGF-A of the LDLR in PCSK9 binding and identifi ed several amino acid residues in EGF-A that contribute to PCSK9 binding. Among them, we found that mutations L318R and N309K increased PCSK9 binding. Biochemistry and crystallogra-phy studies reveal that EGF-A directly interacts with PCSK9 ( 17, 28, 29 ). Purifi ed EGF-AB of the LDLR and synthetic EGF-A peptide can inhibit PCSK9-promoted LDLR degra-dation in HepG2 cells ( 29, 43, 48 ). Studies in cultured cells and parabiotic mice demonstrate that PCSK9 can promote LDLR degradation extracellularly ( 16–19 ). Therefore, the EGF-A domain that contains only 40 amino acid residues is a very good target for inhibiting PCSK9-mediated LDLR degradation. However, EGF-A binds to PCSK9 with a rela-tively low affi nity, which makes it less attractive. Thus, the identifi cation of mutations in EGF-A that can enhance PCSK9 binding will help develop a peptide homologous to EGF-A that can bind to PCSK9 with a high affi nity.

The authors are deeply grateful to Drs. Jonathan Cohen and Helen Hobbs (University of Texas Southwestern Medical Cen-ter at Dallas) for their great training, support, and helpful discussion. The authors also thank Christina Zhao for technical assistance and Marni Devlin-Moses for help in manuscript editing.

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