Functional analysis of sites within PCSK9 responsible for hypercholesterolemia
2008; Elsevier BV; Volume: 49; Issue: 6 Linguagem: Inglês
10.1194/jlr.m800049-jlr200
ISSN1539-7262
AutoresShilpa Pandit, Doug Wisniewski, Joseph C. Santoro, Sookhee Ha, Vijayalakshmi Ramakrishnan, Rose M. Cubbon, Richard Cummings, Samuel D. Wright, Carl P. Sparrow, Ayesha Sitlani, Timothy S. Fisher,
Tópico(s)Pharmaceutical Economics and Policy
ResumoMutations within proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with dominant forms of familial hypercholesterolemia. PCSK9 binds the LDL receptor (LDLR), and addition of PCSK9 to cells promotes degradation of LDLR. PCSK9 mutant proteins associated with hypercholesterolemia (S127R and D374Y) are more potent in decreasing LDL uptake than is wild-type PCSK9. To better understand the mechanism by which mutations at the Ser127 and Asp374 residues of PCSK9 influence PCSK9 function, a limited vertical scanning mutagenesis was performed at both sites. S127R and S127K proteins were more potent in decreasing LDL uptake than was wild-type PCSK9, and each D374 mutant tested was more potent in reducing LDL uptake when the proteins were added exogenously to cells. The potencies of D374 mutants in lowering LDL uptake correlated with their ability to interact with LDLR in vitro. Combining S127R and D374Y was also found to have an additive effect in enhancing PCSK9's ability to reduce LDL uptake. Modeling of PCSK9 S127 and D374 mutations indicates that mutations that enhance PCSK9 function stabilize or destabilize the protein, respectively. In conclusion, these results suggest a model in which mutations at Ser127 and Asp374 residues modulate PCSK9's ability to regulate LDLR function through distinct mechanisms. Mutations within proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with dominant forms of familial hypercholesterolemia. PCSK9 binds the LDL receptor (LDLR), and addition of PCSK9 to cells promotes degradation of LDLR. PCSK9 mutant proteins associated with hypercholesterolemia (S127R and D374Y) are more potent in decreasing LDL uptake than is wild-type PCSK9. To better understand the mechanism by which mutations at the Ser127 and Asp374 residues of PCSK9 influence PCSK9 function, a limited vertical scanning mutagenesis was performed at both sites. S127R and S127K proteins were more potent in decreasing LDL uptake than was wild-type PCSK9, and each D374 mutant tested was more potent in reducing LDL uptake when the proteins were added exogenously to cells. The potencies of D374 mutants in lowering LDL uptake correlated with their ability to interact with LDLR in vitro. Combining S127R and D374Y was also found to have an additive effect in enhancing PCSK9's ability to reduce LDL uptake. Modeling of PCSK9 S127 and D374 mutations indicates that mutations that enhance PCSK9 function stabilize or destabilize the protein, respectively. In conclusion, these results suggest a model in which mutations at Ser127 and Asp374 residues modulate PCSK9's ability to regulate LDLR function through distinct mechanisms. Familial hypercholesterolemia (FH) is characterized by elevated levels of circulating LDL cholesterol (LDL-C), resulting in atherosclerosis and an increased risk of premature coronary heart disease. The autosomal dominant form of FH (ADH) is most often associated with loss-of-function mutations within either the LDL receptor (LDLR) or apolipoprotein B (APOB) gene, resulting in decreased hepatic clearance of LDL-C (1.Soutar A.K. Naoumova R.P. Mechanisms of disease: genetic causes of familial hypercholesterolemia.Nat. Clin. Pract. Cardiovasc. Med. 2007; 4: 214-225Crossref PubMed Scopus (462) Google Scholar). Recently, mutations within the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene were found to represent a third class of mutations associated with ADH (2.Abifadel M. Varret M. Rabes J-P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. et al.Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2221) Google Scholar). Several missense mutations (S127R, D129G, F216L, D374H, and D374Y) are associated with hypercholesterolemia and premature atherosclerosis (2.Abifadel M. Varret M. Rabes J-P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. et al.Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2221) Google Scholar, 3.Leren T. Mutations in the PCSK9 gene in Norwegian subjects with autosomal dominant hypercholesterolemia.Clin. Genet. 2004; 65: 419-422Crossref PubMed Scopus (209) Google Scholar, 4.Timms K.M. Wagner S. Samuels M. Forbey K. Goldfine H. Jammulapati S. Skolnick M. Hopkins P. Hunt S. Shattuck D. A mutation in PCSK9 causing autosomal-dominant hypercholesterolemia in a Utah pedigree.Hum. Genet. 2004; 114: 349-353Crossref PubMed Scopus (271) Google Scholar, 5.Homer V.M. Marais A.D. Charlton F. Laurie A.D. Hurndell N. Scott R. Mangili F. Sullivan D.R. Barter P.J. Rye K.A. et al.Identification and characterization of two non-secreted PCSK9 mutants associated with familial hypercholesterolemia in cohorts from New Zealand and South Africa.Atherosclerosis. 2007; 196: 659-666Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 6.Bourbon M. Alves A.C. Medeiros A.M. Silva S. Soutar A.K. Investigators of Portuguese FH StudyFamilial hypercholesterolaemia in Portugal.Atherosclerosis. 2007; 196: 633-642Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In addition, several missense (R46L, L253F, and A433T) and nonsense mutations (Y142× and C679×) are associated with low circulating LDL-C levels and a reduction in the risk of coronary heart disease (7.Cohen J. Pertsemlidis A. Kotowski I.K. Graham R. Garcia C.K. Hobbs H.H. Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9.Nat. Genet. 2005; 37: 161-165Crossref PubMed Scopus (1090) Google Scholar, 8.Berge K.E. Ose L. Leren T.P. Missense mutations in the PCSK9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1094-1100Crossref PubMed Scopus (208) Google Scholar, 9.Kotowski I. Pertsemlidis A. Luke A. Cooper R.S. Vega G.L. Cohen J.C. Hobbs H.H. A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol.Am. J. Hum. Genet. 2006; 78: 410-422Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Of note, an individual with two inactivating mutations within PCSK9 and no detectable circulating PCSK9 protein was recently identified with a significantly lower LDL-C concentration (14 mg/dl) and no observable signs of ill health due to the absence of PCSK9 (10.Zhao Z. Tuakli-Wosornu Y. Lagace T.A. Kinch L. Grishin N.V. Horton J.D. Cohen J.C. Hobbs H.H. Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote.Am. J. Hum. Genet. 2006; 79: 514-523Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar). Together, these findings demonstrate a key role for PCSK9 in regulating circulating LDL-C levels in the human population, and point to PCSK9 as an attractive target for the treatment of hypercholesterolemia (11.Rashid S. Curtis D.E. Garuti R. Anderson N.N. Bashmakov Y. Ho Y.K. Hammer R.E. Moon Y-A. Horton J.D. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9.Proc. Natl. Acad. Sci. USA. 2005; 102: 5374-5379Crossref PubMed Scopus (566) Google Scholar). PCSK9 is a secreted protein consisting of several domains found within the proprotein convertase (PC) family of serine proteases, including an N-terminal signal peptide and prodomain, followed by a catalytic domain and Cys-rich C-terminal domain (12.Seidah N.G. Chretien M. Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides.Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (692) Google Scholar, 13.Seidah N.G. Benjannet S. Wickham L. Marcinkiewicz J. Jasmin S.B. Stifani S. Basak A. Prat A. Chretien M. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver regeneration and neuronal differentiation.Proc. Natl. Acad. Sci. USA. 2003; 100: 928-933Crossref PubMed Scopus (933) Google Scholar, 14.Steiner D.F. The proprotein convertases.Curr. Opin. Chem. Biol. 1998; 2: 31-39Crossref PubMed Scopus (581) Google Scholar). Similar to other PC family members, PCSK9 undergoes autocatalytic processing within the endoplasmic reticulum (ER), resulting in cleavage of its prodomain following Q152, (FAQ↓SIP) (15.Naureckiene S. Ma L. Sreekumar K. Purandare U. Lo C.F. Huang Y. Chiang L.W. Grenier J.M. Ozenberger B.A. Jacobsen J.S. et al.Functional characterization of Narc 1, a novel proteinase related to proteinase K.Arch. Biochem. Biophys. 2003; 420: 55-67Crossref PubMed Scopus (137) Google Scholar, 16.Benjannet S. Rhainds D. Essalmani R. Mayne J. Wickham L. Jin W. Asselin M-C. Hamelin J. Varret M. Allard D. et al.NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.J. Biol. Chem. 2004; 279: 48865-48875Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar). Structural and biochemical studies of PCSK9 indicate that although other members of the PC family typically undergo a second cleavage event to release their inhibitory prodomain, the prodomain of PCSK9 remains intact following autoprocessing, with its C terminus blocking the protease catalytic site (16.Benjannet S. Rhainds D. Essalmani R. Mayne J. Wickham L. Jin W. Asselin M-C. Hamelin J. Varret M. Allard D. et al.NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.J. Biol. Chem. 2004; 279: 48865-48875Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, 17.Cunningham D. Danley D.E. Geoghegan K.F. Griffor M.C. Hawkins J.L. Subashi T.A. Varghese A.H. Ammirati M.J. Culp J.S. Hoth L.R. et al.Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.Nat. Struct. Mol. Biol. 2007; 14: 413-419Crossref PubMed Scopus (365) Google Scholar, 18.Piper D.E. Jackson S. Liu Q. Romanow W.G. Shetterly S. Thibault S.T. Shan B. Walker N.P.C. The crystal structure of PCSK9: a regulator of plasma LDL-cholesterol.Structure. 2007; 15: 545-552Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 19.Hampton E.N. Knuth M.W. Li J. Harris J.L. Lesley S.A. Spraggon G. The self-inhibited structure of full-length PCSK9 at 1.9 A reveals structural homology with resistin within the C-terminal domain.Proc. Natl. Acad. Sci. USA. 2007; 104: 14604-14609Crossref PubMed Scopus (115) Google Scholar, 20.Lagace 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. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (532) Google Scholar). PCSK9 regulates circulating LDL-C levels by reducing the number of hepatic LDLRs. Introduction of PCSK9, either into the circulation of mice via parabiosis or into cultured cells, results in LDLR degradation and hence, lowering of hepatic and cellular uptake of LDL (20.Lagace 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. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (532) Google Scholar, 21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 22.Qian Y-W. Schmidt R.J. Zhang Y. Chu S. Lin A. Wang H. Wang X. Beyer T.P. Bensch W.R. Li W. et al.Secreted proprotein convertase subtilisin/kexin-type 9 downregulates low-density lipoprotein receptor through receptor-mediated endocytosis.J. Lipid Res. 2007; 48: 1488-1498Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Interestingly, although autoprocessing of PCSK9's prodomain is required for exit from the ER and secretion of the mature protein, its serine protease activity is not required for PCSK9-mediated degradation of LDLR after PCSK9 has been secreted. Coexpression of the PCSK9 prodomain and a catalytically inactive mutant (S386A) within the catalytic domain in trans facilitated the efficient secretion of the stable heterodimer and subsequent finding that this form of PCSK9 reduced the number of LDLRs on the cell surface similarly to the wild-type protein (23.Li J. Tumanut C. Gavigan J-A. Huang W-J. Hampton E.N. Tumanut R. Suen K.F. Trauger J.W. Spraggon G. Lesley S.A. et al.Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity.Biochem. J. 2007; 406: 203-207Crossref PubMed Scopus (50) Google Scholar, 24.McNutt M.C. Lagace T.A. Horton J.D. Catalytic activity is not required for secreted PCSK9 to reduce LDL receptors in HepG2 cells.J. Biol. Chem. 2007; 282: 561-568Abstract Full Text Full Text PDF Scopus (223) Google Scholar). Although these findings do not rule out a potential role for PCSK9's catalytic activity in reducing the number of LDLRs prior to secretion, they do indicate that the protease activity of PCSK9 is not required for its effects in reducing the number of cell surface LDLRs after secretion of mature PCSK9. PCSK9 binds directly to LDLR and requires LDLR for normal cellular trafficking and endocytosis (17.Cunningham D. Danley D.E. Geoghegan K.F. Griffor M.C. Hawkins J.L. Subashi T.A. Varghese A.H. Ammirati M.J. Culp J.S. Hoth L.R. et al.Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.Nat. Struct. Mol. Biol. 2007; 14: 413-419Crossref PubMed Scopus (365) Google Scholar, 20.Lagace 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. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (532) Google Scholar, 21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 22.Qian Y-W. Schmidt R.J. Zhang Y. Chu S. Lin A. Wang H. Wang X. Beyer T.P. Bensch W.R. Li W. et al.Secreted proprotein convertase subtilisin/kexin-type 9 downregulates low-density lipoprotein receptor through receptor-mediated endocytosis.J. Lipid Res. 2007; 48: 1488-1498Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 25.Nassoury N. Blasiole D.A. Oler A.Tebon Benjannet S. Hamelin J. Poupon V. McPherson P.S. Attie A.D. Prat A. Seidah N.G. The cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on the LDLR.Traffic. 2007; 8: 718-732Crossref PubMed Scopus (205) Google Scholar). Although the region of PCSK9 responsible for binding LDLR has not been identified, the PCSK9 binding region of LDLR has been narrowed down to the epidermal growth factor-like repeat (EGF-A) within the EGF homology domain (26.Zhang D-W. Lagace T.A. Garuti R. Zhao Z. McDonald M. Horton J.D. Cohen J.C. Hobbs H.H. Binding of PCSK9 to EGF-A repeat of LDL receptor decreases receptor recycling and increases degradation.J. Biol. Chem. 2007; 282: 18602-18612Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). LDLR requires the acidic environment of endosomes to release internalized LDL; however, the binding affinity between PCSK9 and LDLR greatly increases at an acidic endosomal pH (17.Cunningham D. Danley D.E. Geoghegan K.F. Griffor M.C. Hawkins J.L. Subashi T.A. Varghese A.H. Ammirati M.J. Culp J.S. Hoth L.R. et al.Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.Nat. Struct. Mol. Biol. 2007; 14: 413-419Crossref PubMed Scopus (365) Google Scholar, 18.Piper D.E. Jackson S. Liu Q. Romanow W.G. Shetterly S. Thibault S.T. Shan B. Walker N.P.C. The crystal structure of PCSK9: a regulator of plasma LDL-cholesterol.Structure. 2007; 15: 545-552Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The gain-of-function PCSK9 mutant, D374Y, also binds LDLR with higher affinity than does wild-type protein, as measured by either Biacore or time-resolved fluorescence resonance energy transfer (TR-FRET) and is internalized into cells more efficiently than is wild-type PCSK9 (17.Cunningham D. Danley D.E. Geoghegan K.F. Griffor M.C. Hawkins J.L. Subashi T.A. Varghese A.H. Ammirati M.J. Culp J.S. Hoth L.R. et al.Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.Nat. Struct. Mol. Biol. 2007; 14: 413-419Crossref PubMed Scopus (365) Google Scholar, 20.Lagace 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. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (532) Google Scholar, 21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In addition, we and others have demonstrated that PCSK9 gain-of-function mutants S127R and D374Y are more potent in reducing the number of cell surface LDLRs, thereby lowering cellular LDL uptake (20.Lagace 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. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (532) Google Scholar, 21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Together, these findings suggest that the strength of the PCSK9-LDLR interaction is a major factor in determining the ability of PCSK9 to regulate LDLR activity and hence, serum LDL-C levels in humans. To gain further insight into the mechanistic role of sites within PCSK9 responsible for hypercholesterolemia, we undertook a limited vertical scanning mutagenesis of Ser127 and Asp374 residues and assessed the effect of such mutations on PCSK9 function. In addition, several combinations of PCSK9 gain-of-function mutants (S127R, F216L, and D374Y) were designed to assess their relative roles in PCSK9 function. Several criteria of PCSK9 function were assayed, including: processing of the PCSK9 prodomain and secretion of mature protein; the potency of purified mutant proteins in lowering LDL uptake; and the relative binding affinities of mutant proteins to LDLR. Both energetic and molecular modeling was performed to assess the predicted effect of such mutations on PCSK9 structure. Together, our findings suggest that mutations at Asp374 of PCSK9 result in a gain-of-function phenotype predominantly due to enhancing the binding of PCSK9 to LDLR on the cell surface, whereas mutations at Ser127 may enhance PCSK9 function either at a step prior to secretion or following internalization into cells. The cloning of PCSK9 and construction of pcDNA3.1-F1-WT, pcDNA3.1-F1-S127R, and pcDNA3.1-F1-D374Y PCSK9 expression plasmids was carried out as described (21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Additional plasmids expressing mutant forms of human PCSK9 were developed by site-directed mutagenesis using the QuickChange II XL Site-Directed Mutagenesis kit (Stratagene, LaJolla, CA). The following primers were used for generating mutations from the pcDNA3.1-F1-WT starting plasmid. For plasmid pcDNA3.1-F1-S127A: F, 5′-CTTCCTGGTGAAGATGGCCGGCGACCTGCTGGAGC-3′and R, 5′-GCTCCAGCAGGTCGCCGGCCATCTTCACCAGGAAG-3′; for plasmid pcDNA3.1-F1-S127D: F, 5′-CTTCCTGGTGAAGATGGACGGCGACCTGCTGGAGC-3′ and R, 5′-GCTCCAGCAGGTCGCCGTCCATCTTCACCAGGAAG-3′; for plasmid pcDNA3.1-F1-S127K: F, 5′-CTTCCTGGTGAAGATGAAGGGCGACCTGCTGGAGC-3′ and R, 5′-GCTCCAGCAGGTCGCCCTTCATCTTCACCAGGAAG-3′; for plasmid pcDNA3.1-S127L: 5′-CTTCCTGGTGAAGATGCTGGGCGACCTGCTGGAGC-3′ and R, 5′-GCTCCAGCAGGTCGCCCAGCATCTTCACCAGGAAG-3′; for plasmid pcDNA3.1-F1-S127T: F, 5′-CTTCCTGGTGAAGATGACCGGCGACCTGCTGGAGC-3′ and R, 5′-GCTCCAGCAGGTCGCCGGTCATCTTCACCAGGAAG-3′; for plasmid pcDNA3.1-F1-D374A: F, 5′-CATTGGTGCCTCCAGCGCCTGCAGCACCTGCTTTG-3′ and R, 5′-CAAAGCAGGTGCTGCAGGCGCTGGAGGCACCAATG-3′; for plasmid pcDNA3.1-F1-D374E: F, 5′-ATTGGTGCCTCCAGCGAGTGCAGCACCTGCTTTGTG-3′ and R, 5′-CACAAAGCAGGTGCTGCACTCGCTGGAGGCACCAAT-3′; for plasmid pcDNA3.1-F1-D374F: F, 5′-CATTGGTGCCTCCAGCTTCTGCAGCACCTGCTTT-3′ and R, 5′-AAAGCAGGTGCTGCAGAAGCTGGAGGCACCAATG-3′; for plasmid pcDNA3.1-F1-D374K: F, 5′-CATTGGTGCCTCCAGCAAGTGCAGCACCTGCTTTG-3′ and R, 5′-CAAAGCAGGTGCTGCACTTGCTGGAGGCACCAATG-3′; for plasmid pcDNA3.1-F1-D374L: F, 5′-CATTGGTGCCTCCAGCCTGTGCAGCACCTGCTTTG-3′ and R, 5′-CAAAGCAGGTGCTGCACAGGCTGGAGGCACCAATG-3′. The following primers were used for generating mutations from the pcDNA3.1-F1-S127R starting plasmid. For plasmid pcDNA3.1-F1-S127R D374Y: F, 5′-CATTGGTGCCTCCAGCTACTGCAGCACCTGCTT-3′ and R, 5′-AAGCAGGTGCTGCAGTAGCTGGAGGCACCAATG-3′; for plasmid pcDNA3.1-F1-S127R F216L D374Y, use previous primers for F216L and D374Y. The following primers were used for generating mutations from the pcDNA3.1-F1-D374Y starting plasmid. For plasmid pcDNA3.1-F1-F216L D374Y: F, 5′-GAGGAGGACGGGACCCGCCTGCACAGACAGGCCAGCAAG-3′and R, 5′-CTTGCTGGCCTGTCTGTGCAGGCGGGTCCCGTCCTCCTC-3′. All PCSK9 constructs were present in a pcDNA3.1 backbone with G418 selection and carboxyl-terminal V5 and His tags (Invitrogen, Carlsbad, CA). Stable HEK293 cell lines expressing either wild-type or mutant versions of PCSK9 were created as described (21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Cells were maintained in 1× DMEM (Mediatech, Inc. Manassas, VA) containing 1 mg/ml G418 supplemented with 10% FBS. Wild-type and mutant PCSK9 proteins were purified from HEK293-generated media as described (21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). HEK293 cells (300,000/well) were plated in a 6-well plate in 1 × DMEM (Mediatech) containing 100 units of penicillin and 100 μg/ml streptomycin sulfate and supplemented with 10% FBS. The following day, cells were transfected with 1 μg of wild-type of mutant plasmid using FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. After 3 days, the medium was changed to 1 × DMEM and incubated for an additional 6 h. Media was collected, and cells were lysed in radioimmune precipitation assay buffer (Teknova, Hollister, CA) plus Complete protease inhibitor mixture (Roche Applied Science). Protein concentration was assayed using a BCA protein assay kit (Pierce, Rockford, IL). Ten micrograms of proteins from lysate or 10 μl of medium was loaded onto 10–20% Tris/glycine gels (Invitrogen). Following transfer, membranes were incubated with anti-V5 primary antibody (1:5000; Invitrogen) and alkaline phosphatase-conjugated anti-mouse IgG (H + l) (1:3000; Promega, Madison, WI). Bands were detected using a one-step nitro blue tetazolium/5-bromo-4-chloro-3-indolyl phosphate kit (Pierce) according to the manufacturer's instructions. LDL was isolated from healthy human volunteers as previously described (21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). LDL was labeled as described previously with either a fluorescent 3,3′-dioctadecylindocarbocyanine (DiI) particle (Molecular Probes, Carlsbad, CA) (21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) or Alexa Fluor 546 as follows. Prior to labeling, LDL (0.5–2 mg/ml) was dialyzed against 200 mM sodium carbonate, pH 8.5, 100 mM NaCl, and 1 mM EDTA. Alexa Fluor 546-labeled carboxylic acid, succinimidyl ester (Invitrogen) was dissolved in Me2SO at 10 mg/ml. LDL was then combined with a 25× molar excess of Alexa Fluor 546-labeled NHS and set at room temperature for 5 h protected from the light. The reaction was stopped with 15 μl of 1 M Tris, pH 8.0. Labeled alexa fluor-LDL was dialyzed against PBS and 1 mM EDTA. Anti-V5 antibody was labeled Alexa Fluor 647 as described previously (21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). LDLR was also labeled as described previously (21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), except that 5 equivalents of Eu(8044)-DTA (Perkin-Elmer, Waltham, MA) were used in place of 20 equivalents of Eu(W1024)-ITC in the original labeling. LDL uptake was measured in HEK293 cells stably expressing the pcDNA3.1 vector alone as previously described (21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The only difference in this report is that both DiI- and AF546-labeled LDL was used to measure cellular LDL uptake. The amount of LDL uptake measured by the HEK293 cell line and the regulation of LDL uptake by added PCSK9 protein was identical with either LDL preparation (data not shown). PCSK9-LDLR interactions were measured by TR-FRET (27.Mathis G. Rare earth cryptates and homogeneous fluoroimmunoassays with human sera.Clin. Chem. 1993; 39: 1953-1959Crossref PubMed Scopus (363) Google Scholar, 28.Mathis G. Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer.Clin. Chem. 1995; 41: 1391-1397Crossref PubMed Scopus (319) Google Scholar) as described (21.Fisher T.S. Surdo P.Lo Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.PCSK9-dependent LDL receptor regulation: effects of pH and LDL.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Briefly, reactions took place in 50 μl total volume in 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.1 mM CaCl2, and 0.05% (w/v) BSA. A preformed complex of Alexa Fluor 647-labeled anti-V5 antibody (40 nM) and Eu+3-labeled recombinant LDLR ectodomain (R and D Systems, Minneapolis, MN) (3–5 nM, or 20,000 B counts) was combined with increasing amounts of purified wild-type, Ser127, or Asp374 mutant PCSK9 protein. The data were fit to a sigmoidal dose-response curve by nonlinear regression after normalization of the relative ratios to 100% of maximum fluorescence transfer for each protein. Molecular modeling was performed in order to compare the energetics of wild-type and mutant proteins using a recently solved structure of processed PCSK9 (17.Cunningham D. Danley D.E. Geoghegan K.F. Griffor M.C. Hawkins J.L. Subashi T.A. Varghese A.H. Ammirati M.J. Culp J.S. Hoth L.R. et al.Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.Nat. Struct. Mol. Biol. 2007; 14: 413-419Crossref PubMed Scopus (365) Google Scholar). Single point mutations of Ser127 of the prodomain and Asp374 of the catalytic domain were made in silico using the Protein Design module in the Quanta program (29.Oldfield T.J. Waterpaugh P.E.B.K. D. Crystallographic Computing 7. Oxford University Press, Oxford, UK1996Google Scholar). After each mutation at the Ser127 and Asp374 positions, a conformational search of the mutated side chain was made to find the optimal position of the mutated side chain residue. Using the JG protocol in the MIX modeling environment at Merck and Co.
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