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
2007; Elsevier BV; Volume: 282; Issue: 25 Linguagem: Inglês
10.1074/jbc.m702027200
ISSN1083-351X
AutoresDawei Zhang, Thomas A. Lagace, Rita Garuti, Zhenze Zhao, Meghan McDonald, Jay D. Horton, Jonathan C. Cohen, Helen H. Hobbs,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoProprotein 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 acid-dependent conformational change required for receptor recycling. As a consequence, the LDLR is rerouted from the endosome to the lysosome where it is degraded. 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 acid-dependent 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) 4The abbreviations used are: PCSK9, proprotein convertase subtilisin/kexin type 9; EGF, epidermal growth factor; LDL, low density lipoprotein; LDLR, low density lipoprotein receptor; NCLPPS, newborn calf lipoprotein-poor serum; VLDL, very low density lipoprotein; VLDLR, very low density lipoprotein receptor; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; LRP, LDL receptorrelated protein. contributes to differences in plasma levels of low density lipoprotein (LDL) cholesterol (1Abifadel M. Varret M. Rabes J.P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. Derre A. Villeger L. Farnier M. Beucler I. Bruckert E. Chambaz J. Chanu B. Lecerf J.M. Luc G. Moulin M. Weissenbach J. Prat A. Krempf M. Junien C. Seidah N.G. Boileau C. Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2320) Google Scholar, 2Horton J.D. Cohen J.C. Hobbs H.H. Trends Biochem. Sci. 2007; 32: 71-77Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar), the primary cholesterol-carrying lipoprotein in humans. Selected missense mutations in PCSK9 cause dominant hypercholesterolemia and premature atherosclerosis (1Abifadel M. Varret M. Rabes J.P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. Derre A. Villeger L. Farnier M. Beucler I. Bruckert E. Chambaz J. Chanu B. Lecerf J.M. Luc G. Moulin M. Weissenbach J. Prat A. Krempf M. Junien C. Seidah N.G. Boileau C. Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2320) Google Scholar, 3Leren T.P. Clin. Genet. 2004; 65: 419-422Crossref PubMed Scopus (216) Google Scholar, 4Timms K.M. Wagner S. Samuels M.E. Forbey K. Goldfine H. Jammulapati S. Skolnick M.H. Hopkins P.N. Hunt S.C. Shattuck D.M. Hum. Genet. 2004; 114: 349-353Crossref PubMed Scopus (279) Google Scholar), whereas loss-of-function mutations in PCSK9 reduce plasma LDL levels and protect against coronary heart disease (5Cohen J. Pertsemlidis A. Kotowski I.K. Graham R. Garcia C.K. Hobbs H.H. Nat. Genet. 2005; 37: 161-165Crossref PubMed Scopus (1120) Google Scholar, 6Cohen J.C. Boerwinkle E. Mosley T.H. Hobbs H.H. N. Engl. J. Med. 2006; 354: 34-42Crossref PubMed Scopus (2307) Google Scholar, 7Kotowski I.K. Pertsemlidis A. Luke A. Cooper R.S. Vega G.L. Cohen J.C. Hobbs H.H. Am. J. Hum. Genet. 2006; 78: 410-422Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). 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 (8Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Crossref PubMed Scopus (4551) Google Scholar). Expression of recombinant PCSK9 in the livers of mice causes a reduction in hepatic LDLR protein (but not mRNA) and produces severe hypercholesterolemia (9Maxwell K.N. Breslow J.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7100-7105Crossref PubMed Scopus (524) Google Scholar, 10Benjannet S. Rhainds D. Essalmani R. Mayne J. Wickham L. Jin W. Asselin M.C. Hamelin J. Varret M. Allard D. Trillard M. Abifadel M. Tebon A. Attie A.D. Rader D.J. Boileau C. Brissette L. Chretien M. Prat A. Seidah N.G. J. Biol. Chem. 2004; 279: 48865-48875Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar, 11Park S.W. Moon Y.A. Horton J.D. J. Biol. Chem. 2004; 279: 50630-50638Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). Conversely, mice lacking PCSK9 manifest increased levels of LDLR protein in the liver and accelerated clearance of circulating LDL (12Rashid S. Curtis D.E. Garuti R. Anderson N.N. Bashmakov Y. Ho Y.K. Hammer R.E. Moon Y.A. Horton J.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5374-5379Crossref PubMed Scopus (585) Google Scholar). PCSK9 is expressed predominantly in the liver, small intestine, kidney, and brain (13Seidah N.G. Benjannet S. Wickham L. Marcinkiewicz J. Jasmin S.B. Stifani S. Basak A. Prat A. Chretien M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 928-933Crossref PubMed Scopus (962) Google Scholar) and is present in human plasma (14Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. J. Clin. Investig. 2006; 116: 2995-3005Crossref PubMed Scopus (557) Google Scholar, 15Benjannet S. Rhainds D. Hamelin J. Nassoury N. Seidah N.G. J. Biol. Chem. 2006; 281: 30561-30572Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). 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 (14Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. J. Clin. Investig. 2006; 116: 2995-3005Crossref PubMed Scopus (557) Google Scholar). 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 (14Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. J. Clin. Investig. 2006; 116: 2995-3005Crossref PubMed Scopus (557) Google Scholar, 16Cameron J. Holla O.L. Ranheim T. Kulseth M.A. Berge K.E. Leren T.P. Hum. Mol. Genet. 2006; 15: 1551-1558Crossref PubMed Scopus (232) Google Scholar). Autocatalytic cleavage is required for PCSK9 maturation and secretion (10Benjannet S. Rhainds D. Essalmani R. Mayne J. Wickham L. Jin W. Asselin M.C. Hamelin J. Varret M. Allard D. Trillard M. Abifadel M. Tebon A. Attie A.D. Rader D.J. Boileau C. Brissette L. Chretien M. Prat A. Seidah N.G. J. Biol. Chem. 2004; 279: 48865-48875Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar), but whether the catalytic activity of PCSK9 is required for LDLR degradation has not been determined (2Horton J.D. Cohen J.C. Hobbs H.H. Trends Biochem. Sci. 2007; 32: 71-77Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar). Although the exact mecnism responsible for PCSK9-induced LDLR degradation is not known, it requires internalization of the LDLR (14Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. J. Clin. Investig. 2006; 116: 2995-3005Crossref PubMed Scopus (557) Google Scholar). Co-imnoprecipitation experiments have demonstrated that PCSK9 can interact directly with the LDLR (14Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. J. Clin. Investig. 2006; 116: 2995-3005Crossref PubMed Scopus (557) Google Scholar), but the site of binding of PCSK9 to the extracellular domain of the LDLR has not been defined. The two physiological ligands of the LDLR, apopoprotein E of VLDL and apolipoprotein B-100 of LDL, bind the ligand-binding domain of the LDLR (17Russell D.W. Brown M.S. Goldstein J.L. J. Biol. Chem. 1989; 264: 21682-21688Abstract Full Text PDF PubMed Google Scholar), which consists of seven ∼40-amino acid cysteine-rich tandem repeats. The ligand-binding domain located at the N terminus of the protein is followed by the so-called epidermal growth factor (EGF) pcursor homology domain (18Russell D.W. Schneider W.J. Yamamoto T. Luskey K.L. Brown M.S. Goldstein J.L. Cell. 1984; 37: 577-585Abstract Full Text PDF PubMed Scopus (211) Google Scholar, 19Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russell D.W. Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (1049) Google Scholar, 20Südhof T.C. Goldstein J.L. Brown M.S. Russell D.W. Science. 1985; 228: 815-822Crossref PubMed Scopus (626) Google Scholar). The EGF precursor domain contains two cysteine-rich EGF-like domains (EGF-A and EGF-B) separated from a third EGF repeat (EGF-C) by a β-propeller domain (21Springer T. J. Mol. Biol. 1998; 283: 837-862Crossref PubMed Scopus (170) Google Scholar, 22Jeon H. Meng W. Takagi J. Eck M.J. Springer T.A. Blacklow S.C. Nat. Struct. Biol. 2001; 8: 499-504Crossref PubMed Scopus (186) Google Scholar). The EGF precursor homology domain is required for the LDLR to dissociate from lipoproteins in the endosome after the complex is internalized (23Davis C.G. Goldstein J.L. Südhof T.C. Anderson R.G.W. Russell D.W. Brown M.S. Nature. 1987; 326: 760-765Crossref PubMed Scopus (328) Google Scholar). Immediately downstream of the EGF precursor homology domain is a threonine- and serine-rich region, to which multiple O-linked sugars are attached, which is followed by the transmembrane domain and a relatively short cytoplasmic tail that contains all the sequences required for receptor clustering in clathrincoated pits and for internalization of the receptor (24Davis C.G. Lehrman M.A. Russell D.W. Anderson R.G.W. Brown M.S. Goldstein J.L. Cell. 1986; 45: 15-24Abstract Full Text PDF PubMed Scopus (272) Google Scholar, 25Davis C.G. van Driel I.R. Russell D.W. Brown M.S. Goldstein J.L. J. Biol. Chem. 1987; 262: 4075-4082Abstract Full Text PDF PubMed Google Scholar). In 2002, Rudenko et al. (26Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Science. 2002; 298: 2353-2358Crossref PubMed Scopus (399) Google Scholar) crystallized the extracellular domain of the receptor at an acidic pH, providing new insights into the mechanism by which the LDLR delivers cholesterol to cells without being degraded in the process. At neutral pH, the ligand-binding repeats are predicted to be extended away from the EGF precursor homology domain and accessible to lipoproteins. When the pH falls, as occurs in the endosome, the ligand-binding domain forms a physical association with the EGF precursor homology domain. The acid-dependent conformational change in the LDLR releases the lipoprotein from the ligandbinding domain and signals the receptor to return to the cell surface. As a first step in determining how PCSK9 binding to the extracellular domain of the LDLR results in receptor degradation, we mapped the site in the LDLR that interacts with PCSK9. We provide evidence that a direct interaction between PCSK9 and the EGF-A repeat in the LDLR interferes with the recycling of the receptor protein from the endosome to the cell surface, resulting in the receptors being rerouted to the lysosome for degradation. Materials—The culture medium and fetal bovine serum were obtained from Meditech, Inc. (Herndon, VA). Complete EDTA-free protease inhibitors were purchased from Roche Applied Science; Nonidet P-40 was purchased from Calbiochem (La Jolla, CA). All other chemicals and reagents were obtained from Sigma-Aldrich unless otherwise indicated. Site-directed Mutagenesis—A recombinant expression vector containing the full-length LDLR cDNA linked to the SV40 early promoter (pLDLR2) (19Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russell D.W. Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (1049) Google Scholar) or the coding region of the receptor cDNA driven by the human cytomegalovirus immediate early region promoter (pLDLR17) was used to generate the mutant forms of the LDLR, including deletions in the ligandbinding domain (27Esser V. Limbrid L.E. Brown M.S. Goldstein J.L. Russell D.W. J. Biol. Chem. 1988; 263: 13282-13290Abstract Full Text PDF PubMed Google Scholar), the EGF precursor homology domain (23Davis C.G. Goldstein J.L. Südhof T.C. Anderson R.G.W. Russell D.W. Brown M.S. Nature. 1987; 326: 760-765Crossref PubMed Scopus (328) Google Scholar), and the clustered O-linked sugar domain (28Davis C.G. Elhammer A. Russell D.W. Schneider W.J. Kornfeld S. Brown M.S. Goldstein J.L. J. Biol. Chem. 1986; 261: 2828-2838Abstract Full Text PDF PubMed Google Scholar). The VLDL receptor (VLDLR) expression construct (pVLDLR) contained one copy of a hemagglutinin epitope tag (CYPYDVPDTAG) (29Tokiwa G. Tyers M. Volpe T. Futcher B. Nature. 1994; 371: 342-345Crossref PubMed Scopus (167) Google Scholar) at the C terminus. Mutations in EGF-A were generated using pLDLR17 or pVLDLR and the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The sequences of the oligonucleotides containing the residues to be mutated (underlined) were synthesized by IDT, Inc. (Coralville, IA) and are provided in supplemental Table S1. The presence of the desired mutation and the integrity of each construct were verified by DNA sequencing. Binding of PCSK9 to the LDLR or VLDLR—Simian COS-M cells were seeded in 60-mm dishes (1.5 × 106 cells/dish) in 4 ml of DMEM (glucose, 1 g/liter) containing 10% (v/v) fetal bovine serum. After 24 h, the cells were transfected with expression plasmids for wild-type or mutant LDLR or VLDLR (8 μg/dish) using Lipofectamine 2000 (20 μl of Lipofectamine/dish) according to the manufacturer's protocol. After 48 h, the cells were washed twice with Dulbecco's phosphate-buffered saline (PBS; Meditech, Inc.) and then incubated in 2 ml of DMEM (glucose, 1 g/liter) containing 5% (v/v) newborn calf lipoprotein-poor serum (NCLPPS), 10 μg/ml cholesterol, 1 μg/ml 25-hydroxycholesterol, and 0.5 μg/ml purified PCSK9. The PCSK9 used in these experiments contained a FLAG tag at the C terminus and was purified using anti-FLAG M2 affinity gel chromatography (Sigma), followed by size exclusion chromatography on a Tricorn Superose 6 10/300 fast performance liquid chromatography column (GE Healthcare, Piscataway, NJ), as previously described (14Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. J. Clin. Investig. 2006; 116: 2995-3005Crossref PubMed Scopus (557) Google Scholar). The cells were incubated with PCSK9 for 2 h, washed three times with ice-cold PBS, and lysed in 200 μl of lysis buffer (PBS, 1.5 mm MgCl2, 5 mm dithiothreitol, 1% (v/v) Triton X-100). The cell lysates were subjected to SDS-PAGE (8%) and transferred to nitrocellulose membranes (GE Healthcare) by electroblotting. Immunoblotting was performed using the following antibodies: an anti-LDLR polyclonal anti-serum (3143) directed against the 14 amino acids at the C terminus of the LDLR (30Russell D.W. Schneider W.J. Yamamamoto T. Luskey K.L. Brown M.S. Goldstein J.L. Cell. 1984; 37: 577-585Abstract Full Text PDF PubMed Scopus (241) Google Scholar), a monoclonal antibody (15A6) developed against full-length PCSK9 (14Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. J. Clin. Investig. 2006; 116: 2995-3005Crossref PubMed Scopus (557) Google Scholar), and an anti-HA monoclonal antibody to detect the VLDLR (12CA5; Roche Applied Science). Antibody binding was detected using horseradish peroxidase-conjugated goat anti-mouse IgG or donkey anti-rabbit IgG (GE Healthcare), followed by enhanced chemiluminescence detection (Pierce). The membranes were then exposed to F-BX810™ Blue X-Ray films (Phoenix Research Products, Hayward, CA). Biotinylation of Wild-type and Mutant LDLR—COS-M cells were transiently transfected with expression plasmids containing cDNAs for wild-type or mutant LDLR (1 μg/35-mm dish) using FuGENE 6 (Roche Applied Science; 6 μl of FuGENE/35-mm dish) according to the manufacturer's protocol. After 24 h, the cell surface proteins were biotinylated exactly as previously described (14Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. J. Clin. Investig. 2006; 116: 2995-3005Crossref PubMed Scopus (557) Google Scholar). The cells were lysed in 150 μl of lysis buffer and then subjected to centrifugation at 15,000 rpm for 5 min in a microcentrifuge to remove insoluble debris. A total of 60 μl of the cell lysate was saved. Ninety μl of lysate was added to 60 μl of a 50% slurry of neutravidin-agarose and 410 μl of lysis buffer. The mixture was rotated overnight at 4 °C. After centrifugation at 3,000 × g for 5 min, the pellets were washed in lysis 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, 0.0025% bromphenol) and incubated for 5 min at 90 °C. The proteins were then analyzed by SDS-PAGE (8%) and immunoblotted using a monoclonal anti-hLDLR antibody (HL-1) directed against the linker sequence between the forth and fifth LDLR ligand-binding repeat (31van Driel I.R. Davis C.G. Goldstein J.L. Brown M.S. J. Biol. Chem. 1987; 262: 16127-16134Abstract Full Text PDF PubMed Google Scholar). Recombinant Adenoviral Vector Expression of LDLR in WIF-B Cells—Recombinant adenoviral vectors containing cDNAs for wild-type and mutant forms of the LDLR were generated by in vitro cre-lox recombination as described previously (32Zhang D.W. Graf G.A. Gerard R.D. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2006; 281: 4507-4516Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). WIF-B cells that were kindly provided by Dr. Ann Hubbard (Johns Hopkins University) were cultured in 6-well plates as previously described (33Graf G.A. Li W.-P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. J. Clin. Investig. 2002; 110: 659-669Crossref PubMed Scopus (300) Google Scholar). After 8 days, the cells were infected with viruses expressing β-galactosidase, the wild-type LDLR or mutant LDLR (1.4 × 109 particles/35-mm dish). After 48 h, the cells were washed twice with PBS and incubated in 1 ml of culture medium containing 5% (v/v) NCLPPS, 10 μg/ml cholesterol, 1 μg/ml 25-hydroxycholesterol, and 1 μg/ml purified recombinant PCSK9 for 4 h. The cells were washed three times with ice-cold PBS and lysed in 150 μl of lysis buffer. Whole cell protein extracts were then subjected to SDS-PAGE (8%) and immunoblot analysis to detect the LDLR (HL-1) and PCSK9 (15A6). Transferrin receptor was detected using a monoclonal antibody (Invitrogen). Purification of GST:EGF-A Fusion Proteins—Wild-type and mutant (D310E) EGF-A of the LDLR were expressed as recombinant GST fusion proteins using the vector pGEX-4T (GE Healthcare) in Escherichia coli BL-21-DE3 cells (EMD Biosciences, San Diego, CA). The transformed cells were grown at 37 °C in LB medium containing ampicillin (50 μg/ml) and induced with 1 mm isopropyl β-d-thiogalactopyranoside for 5 h when the A600 reached 0.8. The cells were harvested by centrifugation at 5,000 × g for 20 min at 4 °C. The cell pellets were resuspended and incubated on ice for 30 min in 25 ml of PBS containing 0.1 mm phenylmethylsulfonyl fluoride and 1 mg/ml lysozyme. The cells were lysed using a French pressure cell operated at 1500 p.s.i. (1 p.s.i. = 6.9 kPa). The lysate was centrifuged at 16,500 × g for 30 min. The supernatants were filtered through a 0.22-μm filter, and GST:EGF-A fusion protein was purified using Glutathione-Sepharose 4 Fast Flow (GE Healthcare) affinity gel chromatography according to the manufacturer's protocol. The fusion protein was eluted with 25 ml of 50 mm Tris-HCl, pH 8.8, for 2 h at room temperature, and concentrated using a Centriplus filter (3-kDa molecular mass cut-off; Millipore, Billerica, MA). The fusion protein was further purified using size exclusion chromatography on a Tricorn Superose 12 10/300 fast performance liquid chromatography column (GE Healthcare). Fractions containing the fusion protein were concentrated using a 3-kDa molecular mass cut-off Centriplus filter. Protein purity was monitored by SDS-PAGE and Coomassie Brilliant Blue R-250 staining (Bio-Rad). Co-immunoprecipitation of Purified GST:EGF-A Fusion Protein and PCSK9—Purified wild-type or mutant GST:EGF-A (1 μg) and PCSK9 (1 μg) were incubated in 500 μl of immunoprecipitation buffer (PBS, 1% Tween 20 and protease inhibitors) overnight at 4 °C on a rotator in the presence of an anti-GST polyclonal antibody (Novus, Littleton, CO) or an anti-PCSK9 polyclonal antibody (295A) (14Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. J. Clin. Investig. 2006; 116: 2995-3005Crossref PubMed Scopus (557) Google Scholar) and protein A-agarose (Replicon, Waltham, MA). After centrifugation at 3,000 × g for 5 min, the supernatants were removed and retained. The pellets were washed in immunoprecipitation buffer three times for 10 min at 4 °C, and the proteins were eluted using 1× SDS loading buffer containing 5% β-mercaptoethanol (pellets). The proteins were then analyzed by SDS-PAGE (12%), and immunoblotting. PCSK9 was detected using a monoclonal antibody, 15A6. GST:EGF-A was detected using an anti-GST monoclonal antibody (Invitrogen). Ligand Blotting and pH Dependence—Purified extracellular domain of the LDLR (amino acids 1-699) was kindly provided by J. Deisenhofer (University of Texas Southwestern Medical Center). The LDLR protein (200 ng) was size-fractionated under nonreducing conditions by 8% SDS-PAGE, transferred to nitrocellulose, and cut into individual strips. Blocking buffer (PBS plus 2.5% nonfat milk) was added to each strip. After 30 min, the strips were rinsed briefly in pH buffer (50 mm Trismaleate, pH 7.0 to 5.2, or 50 mm sodium citrate, pH 5.0, 75 mm NaCl, 2 mm CaCl2, and 2.5% nonfat milk) and then incubated at room temperature for 60 min with 1.0 μg/ml 125I-PCSK9 (0.5 μCi/μg) in the pH buffer in the absence or presence of 50 μg/ml nonradiolabeled PCSK9. Following three 15-min washes with pH buffer, the strips were dried and exposed to a PhosphorImager plate. The resulting signals were quantified using a Molecular Dynamics Storm 820 system (GE Healthcare). Specific binding represents total binding (absence of excess nonradiolabeled PCSK9) minus nonspecific binding (presence of excess nonradiolabeled PCSK9). Immunofluorescence of WIF-B Cells—WIF-B cells were grown as previously described (33Graf G.A. Li W.-P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. J. Clin. Investig. 2002; 110: 659-669Crossref PubMed Scopus (300) Google Scholar) on glass coverslips for 6-8 days. The medium was changed to F12 Coon's modification medium (Sigma-Aldrich) plus 5% human lipoprotein-poor serum, and the cells were cultured for an additional 48 h. On the day of the experiment the cells were incubated with 5 μg/ml of either native or heat-inactivated (for 10 min at 95 °C) recombinant PCSK9. The coverslips were rinsed with cold PBS and were fixed using 4% (w/v) paraformaldehyde diluted in PBS at 4 °C for 5 min. The cells were permeabilized in methanol at 4 °C for 10 min and then incubated in PBS plus 1% (w/v) bovine serum albumin (Buffer A) for 30 min followed by an overnight incubation at 4 °C in Buffer A plus rabbit anti-bovine LDLR anti-serum (0.5 μg/ml) (4548) (34Beisiegel U. Kita T. Anderson R.G. Schneider W.J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1981; 256: 4071-4078Abstract Full Text PDF PubMed Google Scholar) and monoclonal antibodies to early endosome antigen 1 (1:100) (BD Transduction Laboratories, San Jose, CA), bovine mannose-6-phosphate receptor (1:100) (Affinity Bioreagents, Golden, CO), human lysosomal protein Cathepsin D (1:50) (Santa Cruz Biotechnology, Santa Cruz, CA), and human transferrin receptor (1:200) (Invitrogen). The coverslips were washed three times for 5 min with 0.1% bovine serum albumin in PBS (Buffer B) and then incubated for 1 h in buffer A with Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 568-conjugated goat anti-mouse IgG (1 μg/ml) (Invitrogen). The coverslips were rinsed in Buffer B prior to mounting with a 4′,6′-diamino-2-phenylindole containing mounting medium (Vectashield, Vector Laboratories, Burlingame, CA) and imaging the sections using a 100× 1.3 objective on a confocal microscope (Zeiss LSM510 Meta 2P). PCSK9 Binding to the Extracellular Domain of the LDLR—Previously, we showed that PCSK9 binds the LDLR on the surface of cells (14Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. J. Clin. Investig. 2006; 116: 2995-3005Crossref PubMed Scopus (557) Google Scholar). To map the site of PCSK9 binding to the LDLR, we used cultured monkey kidney cells (COS-M cells) to express either wild-type LDLR or LDLRs lacking each (or all) of the ligand-binding repeats. Purified human PCSK9 (0.5 μg/ml) was added to the medium, and after 2 h the cells were lysed and subjected to duplicate immunoblotting to detect the LDLR and PCSK9 (Fig. 1A). The antibody used to detect the LDLR recognizes both the precursor (∼120 kDa) and the mature, fully glycosylated form (∼160 kDa) of the receptor (35Tolleshaug H. Hobgood K.K. Brown M.S. Goldstein J.L. Cell. 1983; 32: 941-951Abstract Full Text PDF PubMed Scopus (142) Google Scholar), as well as partially glycosylated forms of the receptor that are often seen when the receptor is expressed in COS-M cells and HEK-293 cells (27Esser V. Limbrid L.E. Brown M.S. Goldstein J.L. Russell D.W. J. Biol. Chem. 1988; 263: 13282-13290Abstract Full Text PDF PubMed Google Scholar). Similar amounts of mature LDLR were expressed in cells transfected with each construct. No PCSK9 was found associated with the cells expressing the vector alone (lane 1), whereas PCSK9 bound to cells expressing the wild-type LDLR (lane 2) and in cells expressing LDLRs lacking one or all of the ligand-binding repeats (lanes 3-10). From these experiments we concluded that the ligand-binding repeats were not required for binding of PCSK9 to the LDLR. Next, we examined the effect of deleting portions of the EGF precursor homology domain of the LDLR on PCSK9 binding. No PCSK9 was detected in cells expressing a LDLR missing the first EGF-like repeat, EGF-A (Fig. 1B, lane 3) or the entire EGF precursor homology domain (lane 7), whereas PCSK9 bound to cells with receptors lacking EGF-B (lane 4), the β-propeller domain (lane 5) or the clustered O-linked sugar region (lane 10). Deletion of the third EGF-like repeat (EGF-C) disrupted the intracellular trafficking of the LDLR so that no mature, fully glycosylated form of the receptor was seen in these cells (see below), and no PCSK9 binding was detected (lane 6). These results are consistent with a requirement for EGF-A for PCSK9 binding to the LDLR. EGF-A Is Required for PCSK9 Binding to the LDLR—Protein structures determined by solution nuclear magnetic resonance and x-ray crystallography (36Kurniawan N.D. Aliabadizadeh K. Brereton I.M. Kroon P.A. Smith R. J. Mol. Biol. 2001; 311: 341-356Crossref PubMed Scopus (28) Google Scholar, 37Saha S. Boyd J. Werner J.M. Knott V. Handford P.A. Campbell I.D. Downing A.K. Structure. 2001; 9: 451-456Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 38Malby S. Pickering R. Saha S. Smallridge R. Linse S. Downing A.K. Biochemistry. 2001; 40: 2555-2563Crossref PubMed Scopus (29) Google Scholar) have indicated that the N-terminal region of the EGF-A repeat contains a calcium ion-binding site (Fig. 2). The residues that coordinate calcium binding include As
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