Báo cáo khoa học: Investigations on the evolutionary conservation of PCSK9 reveal a functionally important protrusion pot

Investigations on the evolutionary conservation of PCSK9 reveal a functionally important protrusion Jamie Cameron 1 , Øystein L. Holla 1 , Knut Erik Berge 1, *, Mari Ann Kulseth 1 , Trine Ranheim 1 , Trond P. Leren 1 and Jon K. Laerdahl 2 1 Medical Genetics Laboratory, Department of Medical Genetics, Rikshospitalet University Hospital, Oslo, Norway 2 Centre for Molecular Biology and Neuroscience (CMBN), Institute of Medical Microbiology, Rikshospitalet University Hospital, Oslo, Norway An elevated level of plasma low-density lipoprotein (LDL) cholesterol is a major risk factor for coronary heart disease. The key factor regulating the level of LDL cholesterol is the cell surface LDL receptor (LDLR) [1]. The number of LDLRs is regulated at the transcriptional level [1] but is also post-transcription- ally regulated by proprotein convertase subtilisin⁄ kexin type 9 (PCSK9) [2], also known as NARC-1 [3]. Over- expression of PCSK9 in mice leads to reduced levels of LDLR and increased levels of LDL cholesterol [2,4,5], whereas mice with no functional PCSK9 have increased levels of LDLR and reduced levels of LDL cholesterol [6]. Some aspects of the mechanism by which PCSK9 regulates the number of LDLRs have recently been identified. Secreted PCSK9 binds to the epidermal growth factor-like repeat A (EGF-A) of the extra- cellular domain of the LDLR [7]. PCSK9 bound to the Keywords evolutionary conservation; LDL cholesterol; LDL receptor; PCSK9; structural bioinformatics Correspondence J. K. Laerdahl, Centre for Molecular Biology and Neuroscience (CMBN), Institute of Medical Microbiology, Rikshospitalet University Hospital, NO-0027 Oslo, Norway Fax: +47 22 84 47 82 Tel: +47 22 84 47 84 E-mail: j.k.lardahl@medisin.uio.no (Received 3 April 2008, revised 9 May 2008, accepted 16 June 2008) doi:10.1111/j.1742-4658.2008.06553.x Proprotein convertase subtilisin ⁄ kexin type 9 (PCSK9) interferes with the recycling of low-density lipoprotein (LDL) receptor (LDLR). This leads to LDLR degradation and reduced cellular uptake of plasma LDL. Naturally occurring human PCSK9 loss-of-function mutations are associated with low levels of plasma LDL cholesterol and a reduced risk of coronary heart disease. PCSK9 gain-of-function mutations result in lower LDL clearance and increased risk of atherosclerosis. The exact mechanism by which PCSK9 disrupts the normal recycling of LDLR remains to be determined. In this study, we have assembled homologs of human PCSK9 from 20 ver- tebrates, a cephalochordate and mollusks in order to search for conserved regions of PCSK9 that may be important for the PCSK9-mediated degra- dation of LDLR. We found a large, conserved protrusion on the surface of the PCSK9 catalytic domain and have performed site-directed mutagenesis experiments for 13 residues on this protrusion. A cluster of residues that is important for the degradation of LDLR by PCSK9 was identified. Another cluster of residues, at the opposite end of the conserved protrusion, appears to be involved in the physical interaction with a putative inhibitor of PCSK9. This study identifies the residues, sequence segments and surface patches of PCSK9 that are under strong purifying selection and provides important information for future studies of PCSK9 mutants and for inves- tigations on the function of this regulator of cholesterol homeostasis. Abbreviations CRD, cysteine-rich domain of PCSK9, i.e. the C-terminal domain; EGF-A, epidermal growth factor-like repeat A of LDLR; EST, expressed sequence tag; LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor; PC, proprotein convertase; PCSK9, proprotein convertase subtilisin ⁄ kexin type 9; WT, wild-type. *[Correction added on 16 July 2008, after first online publication: the author name has been amended] FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS 4121 LDLR is internalized by endocytosis [7,8], and bound PCSK9 somehow disrupts the recycling of the LDLR. As a consequence, the LDLR is transferred to the lysosomes for degradation [7]. PCSK9 belongs to a superfamily of subtilisin-like serine proteases and is the ninth mammalian member identified in the proprotein convertase (PC) family [3]. The PC zymogens have an N-terminal signal sequence, a prodomain, a catalytic subtilisin-like domain, and a C-terminal domain [9]. They undergo autocatalytic cleavage in the endoplasmic reticulum, but the prodo- main remains noncovalently bound to the catalytic domain. In PCSK9, the backbone is cut between Gln152 and Ser153 [4], and autocatalysis, as well as correct folding of the protein, is necessary for secretion of PCSK9 [3]. Unlike other convertases, PCSK9 does not appear to undergo a second autocatalytic event resulting in the release of an active protease [4,10]. Instead, the prodomain remains tightly bound to the mature, cleaved PCSK9 after secretion. It has been shown that the enzymatic activity of PCSK9 is not necessary for its regulation of the LDLR [11,12]. The finding that individuals without any detectable plasma PCSK9 are healthy and develop normally [13,14] sug- gests that drugs targeting PCSK9 might represent a promising new class of LDL cholesterol-lowering drugs. The crystal structure of free PCSK9 has recently been determined, and showed the catalytic domain to have high structural similarity to other subtilisin-like serine proteases [10,15,16]. The prodomain (resi- dues 31–152) is tightly bound to the catalytic domain (residues 153–449), hindering access to the active site catalytic triad, Asp186, His226, and Ser386. The N-ter- minal part of the prodomain (residues 31–60) is struc- turally disordered. The C-terminal cysteine-rich domain (CRD) is built from three modules arranged with quasi-three-fold rotational symmetry, where each module forms a two-sheet b-sandwich comprising six b-strands. Each b-sandwich of this pseudo-propeller fold is structurally homologous to the C-terminal region of resistin [15] and is held together by three structurally conserved disulfide bonds. In humans, various mutations in the PCSK9 gene have been found to cause autosomal dominant hypo- cholesterolemia or hypercholesterolemia [4,13,17–24]. For mutations that do not affect PCSK9 folding or secretion, these effects appear to be largely mediated by different affinities of the mutant PCSK9s for the LDLR [11,25]. However, another level of complexity has been added with the recent finding that PCSK9 itself is cleaved by the PC furin, and, to a lesser extent, by PC5 ⁄ 6A [26]. PCSK9 is cleaved between resi- dues 218 and 219 in what has been shown to be a structurally disordered loop on the surface of the PCSK9 catalytic domain [10,15,16]. Furin-cleaved PCSK9 is inactive in degrading LDLR, and naturally occurring gain-of-function mutations such as R215H [24], F216L and R218S [17,27] are likely to be gain-of- function mutations due to reduced furin cleavage. The exact mechanism by which PCSK9 binds to the LDLR and disrupts the normal recycling of the LDLR remains to be determined. One strategy to elucidate the underlying mechanism is to study how mutations in the PCSK9 gene affect the PCSK9-mediated degra- dation of the LDLR. Candidate residues for being of functional importance for macromolecular interactions involving PCSK9 are those that are highly conserved between different species, especially conserved residues that are solvent-exposed in unbound PCSK9 and that do not appear to be important for protein folding. Specific and functionally important protein–protein interactions between PCSK9 and other macromole- cules are likely to be mediated through a contact area with complementary shape, hydrophobicity and charges for the two protein surfaces. Mutations that change the properties of the interacting PCSK9 surface will result in altered, usually weakened and less specific interaction with LDLR or another binding partner. Consequently, residues involved in protein–protein interactions will be more conserved during evolution than other surface-exposed residues. We therefore extracted the sequences of homologs of human PCSK9 from public sequence databases in order to search for functional regions by mapping phylogenetic informa- tion onto the known protein structure. PCSK9 is present in the proteome of most verte- brates as well as in the invertebrate Branchiostoma flor- idae. Whereas most residues exposed on the PCSK9 surface appear to be under limited selective pressure in vertebrates, a large protrusion on the catalytic domain contains a number of absolutely conserved residues. This protrusion could play an important role in specific macromolecular interactions, e.g. for the inter- action with and degradation of the LDLRs. We have therefore performed site-directed mutagenesis of 13 residues within this protrusion in order to study how the mutant PCSK9s affect uptake of LDL. We found that the conserved residues cluster in two groups: one group causes PCSK9 gain-of-function mutations, whereas the remaining residues are located in a small patch giving rise to mutations of the loss-of-function type. Our data suggest that the conserved protrusion is involved in two separate specific macromolecular inter- actions of importance for the PCSK9-mediated degra- dation of the LDLRs. Conserved protrusion on PCSK9 J. Cameron et al. 4122 FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS Results PCSK9 has homologs in chordates and mollusks Homologs of human PCSK9 were extracted from a number of public databases, including the NCBI non- redundant protein and expressed sequence tag (EST) databases [28], uniprot [29], the ensembl resources [30], and some sequencing project databases. Protein sequences homologous to full-length human PCSK9 from many vertebrates were found, including primates, rat, mouse, squirrel (Spermophilus tridecemlineatus), and a number of other placental mammals, opossum (Monodelphis domestica), chicken, the Carolina anole lizard (Anolis carolinensis), frogs (Xenopus tropicalis ⁄ Xenopus laevis), and the fish species Oryzias latipes, Danio rerio, Tetraodon nigroviridis, and Takifugu rubri- pes (see supplementary Doc. S1). No vertebrate with more than a single PCSK9 homolog was found. Data from the Florida lancelet (B. floridae) sequenc- ing project, containing both genomic and EST sequences, indicate at least two potential homologs of full-length PCSK9 in this organism. B. floridae is a representative of the cephalochordates, one of the three chordate subphyla, the other two being verte- brates and urochordates. No homologs of full-length PCSK9 were detected in any urochordate, e.g. in the fairly well-studied Ciona intestinalis, or in any other invertebrates. The C-terminal domain of PCSK9, the CRD, was not found in any vertebrate protein apart from PCSK9 itself. However, homologs of the CRD appear to be present in proteins in the marine Califor- nia sea slug (Aplysia californica) and in the freshwater snail Biomphalaria glabrata. These CRD homologs were extracted from ESTs from the Aplysia EST project and the Biomphalaria sequencing project (see supplementary Doc. S1). The sequence data for the PCSK9 homologs are given in the supplementary Table S1. Multiple sequence alignments for all PCSK9 homologs were generated (Fig. 1 and supplementary Figs S1 and S2). Bovines might be lacking a functional PCSK9 Sequence searching with human PCSK9 in the NCBI EST databases gave highly significant hits in human, mouse, rat, dog, chicken, frog, fish and lancelet ESTs. However, there was not a single detected PCSK9 homolog in 1.3 million bovine EST sequences. In a recent Bos taurus genome assembly (Btau_3.1) from the Baylor College of Medicine Human Genome Sequencing Center, we detected a genomic sequence on Bos taurus chromosome 3 with high similarity to human PCSK9 exons 8–12. These putative PCSK9 exons have insertions ⁄ deletions and nonsynonymous mutations in regions that are absolutely conserved in all other vertebrates, including fish, and there appears to be a premature stop codon in putative bovine exon 10. Traces sequenced in both directions on the genome are available in the NCBI Trace Archive that supports the stop codon in exon 10. The Btau_3.1 ver- sion of the bovine genome is a preliminary assembly based on approximately 26 million reads and  7· sequence coverage. Close to 95% of bovine ESTs were contained in the assembled contigs, indicating that less than one in 20 Bos taurus protein-coding genes are missing in this assembly. The above findings suggest that the region on bovine chromosome 3 with homology to PCSK9 is a remnant of a PCSK9 pseudogene, and that extant Bos taurus might be lacking functional PCSK9. Site-directed mutagenesis of residues in a conserved protrusion on PCSK9 On the basis of a multiple sequence alignment of 18 vertebrate PCSK9 homologs, residue conservation was mapped onto a PCSK9 structural model with the consurf tool [31,32] (Fig. 2). Residue conservation on the solvent-exposed PCSK9 surface is limited. The exception is a large protrusion on the catalytic domain with a surface area of roughly 1500 A ˚ 2 (Fig. 2B). Approximately half of this protrusion, the part closest to the prodomain, is built from the struc- turally disordered loop Gly213–Arg218 (Fig. 2A) and residues partially covered by this loop (Fig. 2C). Evo- lutionarily conserved regions on protein surfaces are likely to be of functional importance, such as being involved in specific interactions with other macro- molecules. We therefore performed site-directed muta- genesis of the 13 most conserved residues in this region (i.e. residues on yellow, blue, pink and green background in Fig. 2C) and investigated how these mutations affected PCSK9 secretion and the internali- zation of LDL. To study whether the mutant PCSK9s were autocat- alytically cleaved and secreted in a normal fashion, HepG2 cells were transiently transfected with mutant PCSK9 plasmids harboring each of the 13 different mutations. The amounts of pro-PCSK9 and mature PCSK9 in cell lysates were determined by western blot analysis using an antibody to PCSK9. In cells express- ing wild-type (WT) PCSK9, two bands of 73 kDa and 64 kDa were observed, which correspond to pro- PCSK9 and the mature form of PCSK9, respectively (Fig. 3). Unlike the catalytically inactive mutant J. Cameron et al. Conserved protrusion on PCSK9 FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS 4123 S386A-PCSK9, the 13 new PCSK9 mutants appeared to be autocatalytically cleaved in a fashion similar to that of WT-PCSK9. Moreover, all 13 mutant PCSK9s, except for C375A-PCSK9 and C378A-PCSK9, were secreted in a normal fashion (Fig. 3). The amount of C378A-PCSK9 in the culture media was markedly reduced, whereas no C375A-PCSK9 was observed in the media. It is likely that C375A-PCSK9 and C378A- PCSK9 are completely or partially, respectively, retained in the endoplasmic reticulum due to abnormal protein folding caused by disruption of the disulfide bond bridging the residues Cys375 and Cys378. Effect of PCSK9 mutants on the internalization of LDL and on PCSK9 cleavage by furin To study the effects of the 13 PCSK9 mutants on the PCSK9-mediated degradation of the LDLR, we used transiently transfected HepG2 cells and studied the amount of LDL internalization by flow cytometry. HepG2 cells transfected with WT-PCSK9, empty plas- mid, the catalytically inactive S386A-PCSK9 plasmid [3,23] or one of the two gain-of-function plasmids, S127R-PCSK9 and D374Y-PCSK9 [23], were used as controls (Fig. 4). Internalization of LDL by cells A B C Fig. 1. Multiple sequence alignments of human PCSK9 homologs from vertebrates, a cephalochordate (B. floridae) and the mollusks Aplysia and Biomphalaria, showing the signal sequence and N-terminus of the prodomain (A), two segments of the catalytic domain (B), and the full CRD, the C-terminal domain (C). Conserved residues are indicated by numbering referring to human PCSK9. The catalytic triad residues are marked with an asterisk. The full alignment and sequence data are given in supplementary Figs S1, S2 and supplementary Table S1. Conserved protrusion on PCSK9 J. Cameron et al. 4124 FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS expressing these control plasmids was comparable to previous findings [23,24]. Cells expressing the two mutants C375A-PCSK9 and C378A-PCSK9 internalized 19% and 14% more LDL, respectively, than cells expressing WT-PCSK9. Thus, as expected for PCSK9 mutants that are secreted at markedly reduced levels, the two mutants present as loss-of-function type. Cells expressing R194A-PCSK9, D238A-PCSK9, T377A-PCSK9 or F379A-PCSK9 also internalized more LDL than cells expressing WT-PCSK9. The amounts of LDL internalized by these cells were higher or similar to those of cells expressing C375A-PCSK9 or C378A-PCSK9 (Fig. 4). Thus, we also consider these four to be loss-of-func- tion mutants. Cells expressing R237A-PCSK9 did not show any significant difference in LDL internalization as com- pared with cells expressing WT-PCSK9 (Fig. 4). R237A-PCSK9 is therefore a neutral variant. Cells expressing one of the remaining six PCSK9 mutants AC B Fig. 2. Structural model of human PCSK9 with the conserved protrusion. (A) The structurally disordered loop Gly213–Arg218 (orange) with the furin recognition motif is located on the catalytic domain (green). Also shown is the prodomain (gray) blocking the active site and the CRD, the C-terminal domain (pink). (B) Amino acid residue conservation in 18 vertebrate PCSK9 homologs mapped, employing CONSURF [31], onto the space-filling representation of the PCSK9 model. The spatial orientation is identical to (A) (upper), and rotated 180° around a vertical axis (lower). The color scale extends from cyan (highly variable residues), through white (intermediate) to magenta (highly conserved). Yellow residues are of intermediate variability, but with low statistical confidence [31]. The conserved protrusion is visible in the panel as an extended patch in magenta (upper part, right-hand side). (C) Magnification of the conserved protrusion showing the 13 residues of the cur- rent study giving mutants with a low level of protein secretion (yellow background), loss-of-function mutants (blue), gain-of-function mutants (pink), no change (green), as well as three residues of the disordered loop Gly213–Arg218 previously shown to give rise to gain-of-function mutants (orange). The model is rotated 50° around a vertical axis with respect to (B), upper panel. J. Cameron et al. Conserved protrusion on PCSK9 FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS 4125 S153A-PCSK9, Q190A-PCSK9, D204A-PCSK9, K222A-PCSK9, D374A-PCSK9 and S376A-PCSK9 internalized less LDL than cells expressing WT-PCSK9 (Fig. 4). The amounts of LDL internalized by cells expressing these mutants were in the same range or lower than in cells expressing the gain-of-function mutant S127R-PCSK9. Thus, we consider these mutants to be gain-of-function mutants. The four loss-of-function mutants involving Arg194, Asp238, Thr377 or Phe379 are located close together on the conserved protrusion (Fig. 2C). Four gain-of- function mutants, involving Gln190, Lys222, Asp374 and Ser376, are located together in a separate region between the loss-of-function patch and the disordered loop Gly213–Arg218. The two remaining gain-of-func- tion mutants, involving Ser153 and Asp204, are located on opposing edges of the conserved protrusion (Fig. 2C). Five of the six gain-of-function mutant residues are clustered in the vicinity of the disordered loop consist- ing of residues Gly213–Arg218 (Fig. 2C). This loop contains the furin cleavage site RFHR 218 [26], and cleavage by furin at this site results in PCSK9 that is inactive in degrading the LDLR [26]. To determine whether the gain-of-function mutants had reduced furin cleavage, the amounts of furin-cleaved PCSK9 in the media of HEK293 cells transiently transfected with the different PCSK9 plasmids were determined by wes- tern blot analysis. HEK293 cells were chosen for these analyses because truncated PCSK9 due to cleavage by furin is more prominent than in the medium of HepG2 cells. R215H-PCSK9 was included as a negative con- trol. Furin-cut PCSK9 was present in small amounts in the media of cells transfected with gain-of-function plasmids as well as in the media of cells transfected with WT-PCSK9 plasmid or with loss-of-function Fig. 4. Internalization of LDL by HepG2 cells transiently transfected with mutant PCSK9 plasmids. The effects of mutants S153A-PCSK9, Q190A-PCSK9, R194A-PCSK9, D204A-PCSK9, K222A-PCSK9, R237A-PCSK9, D238A-PCSK9, D374A-PCSK9, C375A-PCSK9, S376A-PCSK9, T377A-PCSK9, C378A-PCSK9 and F379A-PCSK9 on the internalization of fluorescently labeled LDL (10 lgÆmL )1 ) were studied in transiently transfected HepG2 cells by flow cytometry. WT-PCSK9 plasmid, empty plasmid, and the catalytically inactive S386A-PCSK9 plasmid, as well as D374Y-PCSK9 and S127R-PCSK9, were used as controls. The values relative to WT-PCSK9 are given as the mean from three experi- ments (± SEM). The amount of LDL internalized by cells transfected with WT-PCSK9 was assigned a value of 100. Fig. 3. Autocatalytic cleavage of the mutants S153A-PCSK9, Q190A-PCSK9, R194A-PCSK9, D204A-PCSK9, K222A-PCSK9, R237A-PCSK9, D238A-PCSK9, D374A-PCSK9, C375A-PCSK9, S376A-PCSK9, T377A-PCSK9, C378A-PCSK9 and F379A-PCSK9 was determined by western blot analysis of cell lysates from HepG2 cells transiently transfected with the mutant PCSK9 plasmids. WT-PCSK9 plasmid, empty plasmid and the catalytically inactive S386A-PCSK9 plasmid were used as controls. Uncleaved, pro-PCSK9 and mature, cleaved PCSK9 are indicated (upper panel). The lower panel shows the amount of mature, cleaved PCSK9 in the media. Three separate experiments were performed; one representative experiment is shown. Conserved protrusion on PCSK9 J. Cameron et al. 4126 FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS plasmids (Fig. 5). Clearly, the relative levels of full- length mature and furin-cut PCSK9 in the media do not correlate with mutants being gain-of-function or not. R194A-PCSK9 and D204A-PCSK9 are post-translationally modified As can be seen from Figs 3 and 5, abnormal migration of mature, cleaved PCSK9 was observed in lysates and media of HepG2 cells and HEK293 cells transfected with the R194A-PCSK9 plasmid or the D204A-PCSK9 plasmid. However, the corresponding uncleaved pro- PCSK9 (Fig. 3) and the furin-cleaved PCSK9 (Fig. 5) appeared to migrate normally. To study whether the abnormal migration of the mature forms of R194A- PCSK9 and D204A-PCSK9 was due to altered auto- catalytic cleavage, western blot analyses of media from transfected HepG2 cells were performed using an anti- body against the prodomain of PCSK9. The prodo- mains of R194A-PCSK9 and D204A-PCSK9 migrated normally (Fig. 6). Thus, the two mutants were autocat- alytically cleaved in a normal fashion. To study whether the abnormal migration of mature, cleaved R194A-PCSK9 and D204A-PCSK9 was due to abnormal glycosylation, the sensitivities of R194A-PCSK9 and D204A-PCSK9 to an enzyme mix designed to remove all sugars were determined in cell lysates of transiently transfected HepG2 cells. The results showed that the differences in the migration of mature PCSK9 remained after the enzyme treatment (Fig. 7). Thus, an abnormal post-translational modifi- cation other than glycosylation appears to be respon- sible for the abnormal migration of R194A-PCSK9 and D204A-PCSK9. Discussion Vertebrate genome sequencing projects are currently supplying the research community with sequence data from a large number of species that have varying evo- lutionary relationships with humans. The data from these projects make it possible to study in detail the level of evolutionary residue conservation in proteins, including estimates of statistical significance. In the present study, we have extracted protein sequence data from 21 chordate proteomes and mapped the degree of residue conservation onto the surface of a PCSK9 structure model. We found that most of the residues on the surface of this LDLR-degrading protein appear to be tolerant to substitutions. However, a single large protrusion on the catalytic domain contains a number of residues that are highly conserved (Fig. 2B). We have performed site-directed mutagenesis of 13 resi- dues contributing to this protrusion (Fig. 2C), and show that most mutants have either increased or decreased ability to degrade the LDLR and internalize Fig. 5. Amounts of furin-cleaved PCSK9 in the media of HEK293 cells transiently transfected with the mutant PCSK9 plasmids S153A-PCSK9, Q190A-PCSK9, R194A-PCSK9, D204A-PCSK9, K222A-PCSK9, R237A-PCSK9, D238A-PCSK9, D374A-PCSK9, C375A-PCSK9, S376A-PCSK9, T377A-PCSK9, C378A-PCSK9 and F379A-PCSK9 were determined by western blot analysis. WT-PCSK9 plasmid, and the gain-of-function plasmids D374Y-PCSK9 and R215H-PCSK9, were used as controls. R215H-PCSK9 is not cleaved by furin. Three separate experiments were performed; one representative experiment is shown. WT R194A D204A PCSK9 prodomain Fig. 6. Western blot analysis using an antibody to PCSK9 recogniz- ing the prodomain was used to identify the prodomains of WT-PCSK9, R194A-PCSK9 and D204A-PCSK9 in the media of HepG2 cells. Fig. 7. Western blot analysis was performed for deglycosylated cell lysates. The figure shows a representative western blot of cell lysates of HepG2 cells transiently transfected with WT plasmid or plasmids containing R194A-PCSK9 or D204A-PCSK9 with or with- out prior treatment with the Glycoprotein Deglycosylation Kit. A horizontal dotted line is included to show that all the mature PCSK9s after deglycosylation have increased mobility due to degly- cosylation. J. Cameron et al. Conserved protrusion on PCSK9 FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS 4127 LDL as compared to WT-PCSK9 (Fig. 4). Only one of these residues, Asp374, has previously been associ- ated with hypercholesterolemia in human populations [18,19,33]. Previous studies have described a number of natu- rally occurring loss-of-function mutations in PCSK9 that result in proteins that are not autocatalytically cleaved and ⁄ or not folded properly [4,10,13,23,24]. Apart from the previously studied active site mutant S386A-PCSK9 [23], the only mutants of the present study that clearly have impaired secretion are C375A- PCSK9 and C378A-PCSK9 (Fig. 3). Both residues are absolutely conserved in all chordates (Fig. 1B and sup- plementary Fig. S1), demonstrating their importance for the formation of a disulfide bridge stabilizing the conformation and overall shape of the conserved protrusion (Fig. 2). Four of the mutants, R194A-PCSK9, D238A- PCSK9, T377A-PCSK9, and F379A-PCSK9, were secreted in a similar fashion as WT-PCSK9 (Fig. 3), but nevertheless present as loss-of-function mutants (Fig. 4). The residues involved are located close together on the conserved protrusion, at the far end from both the prodomain and the disordered loop Gly213–Arg218 (Fig. 2C). All four residues are highly conserved in chordates (Fig. 1B and supplementary Fig. S1), and are clearly under strong purifying selec- tive pressure; that is to say, there is substantial nega- tive natural selection against amino acid replacements for these residues. Arg194 is conserved in all verte- brates, but is replaced by Glu in B. floridae, whereas Asp238 is conserved in all vertebrates except for the fish Fugu (T. rubripes), where it is replaced by Glu, a residue with similar properties to Asp. Thr377 is conserved in every single chordate, whereas Phe379 is conserved in all vertebrates except for the rat, where it is substituted by another aromatic residue, Tyr. As discussed above, conservation of these residues is not necessary for protein expression, folding, autocatalysis or secretion, strongly indicating that this patch on the PCSK9 surface is, instead, of importance for the direct physical protein–protein interactions leading to PCSK9-mediated degradation of the LDLR. During the preparation of this manuscript, Kwon et al. [34] published the crystal structure of the protein complex formed between PCSK9 and the EGF-A domain of the LDLR. They found that the interaction between PCSK9 and EGF-A was primarily hydropho- bic, with some additional specific polar interactions. Phe379 was in the center of the hydrophobic surface, and Arg194, Asp238 and Thr377 were involved in the polar interactions with EGF-A [34]. Thus, the surface region of PCSK9 involved in this interaction coincides with the part of the conserved protrusion associated with loss-of-function mutants in the present study. Our findings that mutations R194A, D238A, T377A and F379A were loss-of-function mutations are in agree- ment with the notion that they diminish the binding of PCSK9 to EGF-A. The crystal structure obtained by Kwon et al. [34] shows that the N-terminal amine of mature PCSK9 Ser153 forms a salt bridge with a residue in EGF-A, but that the Ser153 side-chain does not directly contact the binding partner. Correspondingly, our results showed that S153A-PCSK9 is not a loss-of-function mutant. Instead, S153A-PCSK9 appears to lead to decreased internalization of LDL as compared to WT- PCSK9. One might speculate that this could be due to a slight change in the ability of residue 153 to form a salt bridge to EGF-A, e.g. through an inductive effect. All the other residues that were associated with gain-of-function mutations in the present study were located either between the EGF-A binding patch and the disordered loop Gly213–Arg218 (Gln190, Lys222, Asp374, and Ser376), or between the disordered loop and the prodomain (Asp204) (Fig. 2C). These residues are under selective pressure, with Asp204 and Asp374 being conserved in all vertebrates. Lys222 is conserved in nonfish vertebrates, whereas the conservation of Ser376 appears to be slightly lower (Fig. 1B and sup- plementary Fig. S1). Mutations of the disordered loop residues, Arg215 [24], Phe216, and Arg218 [26], located in this part of the conserved protrusion, have previ- ously been shown to be associated with resistance to furin cleavage. We therefore investigated whether the gain-of-function mutants of the present study showed reduced cleavage by furin. However, we did not find any difference in the amounts of the furin-cleaved bands when investigating the media of cells transfected with loss-of-function mutants as compared with gain- of-function mutants (Fig. 5.). Some caution should be exercised when interpreting these data, as in our study overexpressed PCSK9 has to be cut by endogenous furin, and these conditions may not be physiologically relevant for the in vivo situation. Interestingly, the basic residues in the furin recognition sequence RFHR 218 of the disordered loop Gly213–Arg218 are conserved in all vertebrates, but not in the pufferfish T. rubripes and T. nigroviridis or in the opossum (Fig. 1B). The cephalochordate homo- logs have a deletion of four residues in this loop as compared with the human PCSK9, and appear to completely lack the disordered loop. It appears that PC regulation of PCSK9 is a vertebrate invention and that this level of regulation has subsequently been lost in some vertebrate subgroups. Conserved protrusion on PCSK9 J. Cameron et al. 4128 FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS If the gain-of-function character of the mutants of the present study is not due to reduced furin affinity, another possibility is that a different, unknown, mac- romolecule is interacting in a specific manner with the relevant part of the conserved protrusion of PCSK9. This macromolecule may be competing with EGF-A binding or may inhibit the PCSK9-mediated degrada- tion of the LDLR by another mechanism. Fan et al. [35] have recently suggested that multimerization of PCSK9 is important for its LDLR-regulating activity. They found that mutation of Asp374, a residue in the conserved protrusion, affected PCSK9 self-association. It is, however, not obvious that PCSK9 self-association is important in vivo when PCSK9 is secreted at low concentrations. Previous studies have found no indica- tions of multimerization for mature PCSK9 [3]. Earlier studies have shown that the naturally occur- ring mutant D374Y-PCSK9 binds LDLR more effi- ciently than WT-PCSK9 [8,10,25], and Kwon et al. [34] suggested that this was due to an additional hydrogen bond between PCSK9 Tyr374 and His306 of the EGF- A. We now show that D374A-PCSK9, which results in a residue Ala374 that clearly cannot form any hydro- gen bonds with its side-chain, is also a gain-of-function mutant, although it is only half as potent as D374Y- PCSK9. This may indicate that the naturally occurring D374Y-PCSK9 is a gain-of-function mutant due to two different mechanisms: one is to strengthen the interac- tion between PCSK9 and EGF-A, and the other is to disrupt the binding to PCSK9 of a putative inhibitory macromolecule. The sequence data that were gathered for the present study reveal interesting phylogenetic relationships in addition to the identification of the conserved protru- sion discussed above. Homologs of full-length PCSK9 were found in a single copy in a number of verte- brates, and at least in duplicate in the cephalochordate B. floridae. PCSK9 thus appears to be restricted to chordates, and possibly limited to the Cephalochordata and Vertebrata. On the basis of the presumed diver- gence of the chordate subphyla, one might speculate on a Cambrian or late Proterozoic origin of PCSK9. Interestingly, we were unable to find any bovine PCSK9 homolog that appears to be functional, but the Bos taurus genome does appear to contain a PCSK9- like pseudogene. One might speculate that the cow, with its diet of mainly grasses and plant material, might be thriving without PCSK9, as do some human individuals without functional PCSK9 [13,14]. The CRD, whose function still appears to be a mystery, is the C-terminal PCSK9 domain. It does not appear to occur in any vertebrate protein apart from PCSK9 itself, but was detected in sequence data from two mollusk proteins of unknown function. Although the available data are limited, these mollusk CRDs do not appear to be present in proteins that contain pro- tease domains (see supplementary Doc. S1). This might indicate that mollusks employ the CRD for a different purpose than vertebrates do in PCSK9. It is possible that investigations on mollusk CRD-containing pro- teins might give indications on the function of the PCSK9 CRD. The multiple sequence alignments of the PCSK9 homologs (Fig. 1 and supplementary Figs S1 and S2) clearly show the catalytic domain to be more con- served than the CRD. This is also the case for PCSK9 conservation within the group of primates [36]. Resi- due identities between human and opossum are 76% and 53% for the catalytic domain and CRD, respec- tively. The prodomain is also fairly well conserved, apart from the structurally disordered region compris- ing the N-terminal 30 residues (Fig. 1A). This segment is very rich in acidic residues, with seven of 10 N-ter- minal residues of human PCSK9 being Asp or Glu. This is immediately followed by five small aliphatic residues and a segment with five more acidic residues. The N-terminal region of the prodomain will clearly interact strongly and nonspecifically with a positively charged moiety. The signal sequence is not conserved, except for a Leu-rich segment. The three catalytic residues are absolutely conserved in all PCSK9 homologs (Fig. 1B), as is the last residue of the prodomain, Gln152, supporting the notion that these residues are essential for autocatalysis and effi- cient secretion of PCSK9. The 18 Cys residues of the PCSK9 CRD are conserved in all chordates, as well as in the mollusk CRDs (Fig. 1C and supplementary Fig. S2). This clearly demonstrates that the nine disul- fide bridges covalently stabilizing the three modules of this domain are essential for its processing and func- tion. The CRD also contains a number of conserved Ser, Thr and small aliphatic residues. These are mainly located deep in the structure, and are most likely essential for correct folding of the CRD. There is a single patch of conserved residues on the surface of the CRD, comprising Arg458, Thr459, Trp461, and Glu481. These residues all contribute to the part of the CRD surface that is interacting with the catalytic domain. The evolutionary conservation of these resi- dues indicates that although this interaction might be weak [16], it appears to be of functional importance. Piper et al. [16] have noted a large number of His residues in the CRD. With a pK a value for His between 6 and 7, it is likely that the net charge of the CRD will become substantially more positive at endosomal pH 5–5.5 than at pH 7.4 at the plasma J. Cameron et al. Conserved protrusion on PCSK9 FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS 4129 membrane. It is tempting to speculate that this could result in an altered interaction with the strongly acidic N-terminal region of the prodomain. This might be the reason why PCSK9 binds more strongly to the LDLR in the endosomal ⁄ lysosomal compartments than in the plasma [7,10]. However, the positions and number of His residues are not particularly conserved in the CRD (Fig. 1C). Whereas human PCSK9 has 15 His residues out of a total of 245 CRD residues (6.1%), the propor- tions are 5.3% for rat, 5.0% for opossum, 3.0% for medaka fish, and 1.3% for the mollusk Aplysia. Asn533, which is glycosylated in human PCSK9, has been shown not to be essential for PCSK9 secretion [13,26]. It is conserved in placental mammals only. The corresponding residue is not likely to be glycosylated in other vertebrates. In conclusion, there is a single, large, evolutionarily conserved protrusion on the surface of the catalytic domain of PCSK9. The lack of other residue conserva- tion on the PCSK9 surface makes it less likely that there are other parts of PCSK9 that interact with high specificity with other macromolecules as part of the PCSK9-mediated degradation of the LDLR. A cluster of residues on the conserved protrusion is involved in the binding of PCSK9 to the EGF-A domain of the LDLR, and mutations of these residues lead to loss of function, as found in our study and in the study of Kwon et al. [34]. The part of the protrusion located around the disordered loop Gly213–Arg218 contains a number of conserved residues for which site-directed mutagenesis produced gain-of-function mutants. These residues appear to be involved in some form of inhibi- tion of the PCSK9-mediated degradation of the LDLR. However, our data do not clearly support a model that solely involves reduced cleavage by furin. Thus, further studies are needed to clarify whether these residues are involved in the binding of a different macromolecule that inhibits the degradation of the LDLR by PCSK9. Experimental procedures Data collection and bioinformatics analysis Database resources provided by the NCBI [28], uniprot [29], the ensembl project [30], the DOE Joint Genome Institute (http://genome.jgi-psf.org), the Baylor College of Medicine Human Genome Sequencing Center (http:// www.hgsc.bcm.tmc.edu/projects/bovine), the Aplysia EST project (http://aplysia.cu-genome.org) and the B. glabrata Genome Initiative (http://biology.unm.edu/biomphalaria- genome) were searched for homologs of human PCSK9. A major proportion of the extracted 24 PCSK9 homologs from 23 species is due to automatic gene searching in genomic data from early-stage sequencing projects. This necessitated some manual trimming and manipulation of the sequences (see supplementary Doc. S1 and supplementary Table S1). Multiple sequence alignments were generated with mus- cle [37], and the multiple sequence alignments were viewed and manipulated with jalview [38]. A PCSK9 structural model was generated from a published experimental struc- ture [10] as described previously [24]. Amino acid conserva- tion in all vertebrate PCSK9 homologs, but excluding the two pufferfish species, was mapped onto the PCSK9 model employing consurf [31,32]. The protein structure illustra- tions were generated with pymol [39]. Cell cultures HepG2 cells and HEK293 cells, obtained from the Euro- pean Collection of Cell Cultures (Porton Down, UK), were cultured in MEM (Gibco, Carlsbad, CA, USA) containing penicillin (50 UÆ mL )1 ), streptomycin (50 lgÆmL )1 ), l-gluta- mine (2 mm) and 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), in a humidified atmosphere (37 °C, 5% CO 2 ). Mutagenesis, cloning and expression of PCSK9 Mutations S153A, Q190A, R194A, D204A, K222A, R237A, D238A, D374A, C375A, S376A, T377A, C378A or F379A were introduced into a pCMV–PCSK9–FLAG plas- mid kindly provided by J. D. Horton (University of Texas Southwestern Medical Center, Dallas, TX, USA), using QuickChange XL Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions. The primer sequences used for the mutagenesis are given in supplementary Table S2. The resulting mutant plasmids are referred to as S153A-PCSK9, Q190A-PCSK9, R194A- PCSK9, D204A-PCSK9, K222A-PCSK9, R237A-PCSK9, D238A-PCSK9, D374A-PCSK9, C375A-PCSK9, S376A- PCSK9, T377A-PCSK9, C378A-PCSK9, and F379A- PCSK9. The integrity of each plasmid was confirmed by DNA sequencing. An empty plasmid, pcDNA3.1 ⁄ myc his-c (Invitrogen), as well as four previously published mutant PCSK9 plasmids containing mutations S386A, S127R, R215H or D374Y [23,24], were used as controls in the transfection experiments together with WT-PCSK9 plasmid. Transient transfections of HepG2 cells and HEK293 cells with WT-PCSK9 plasmid or mutant PCSK9 plasmids were performed as described by Cameron et al. [24]. Western blot analysis of transfected HepG2 and HEK293 cells Western blot analyses of cell lysates and culture media of transiently transfected HepG2 cells or HEK293 cells were Conserved protrusion on PCSK9 J. Cameron et al. 4130 FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... J, Jasmin SB, Stifani S, Basak A, Prat A & Chretien M (2003) The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regenera- 12 13 14 tion and neuronal differentiation Proc Natl Acad Sci USA 100, 928–933 Benjannet S, Rhainds D, Essalmani R, Mayne J, Wickham L, Jin W, Asselin M-C, Hamelin J, Varret M, Allard D et al (2004) NARC-1 ⁄ PCSK9 and its natural mutants... Cameron et al performed as previously described [23] A rabbit antiPCSK9 IgG (Cayman Chemical Company, Ann Arbor, MI, USA) that recognizes the epitope spanning residues 490–502 was used to detect pro -PCSK9 and mature, cleaved PCSK9 A custom-made rabbit polyclonal antibody directed against residues 46–62 of human PCSK9 (Bethyl Laboratories, Montgomery, TX, USA) was used to identify the prodomain of PCSK9. .. following supplementary material is available online: Doc S1 Supplementary materials and methods: sequence data collection Fig S1 Multiple sequence alignment of the signal sequence, the prodomain and the catalytic domain of PCSK9 homologs Conserved protrusion on PCSK9 Fig S2 Multiple sequence alignment of the C-terminal domain for PCSK9 homologs Table S1 Sequence data for PCSK9 homologs Table S2 Primer... used to generate mutant PCSK9 plasmids This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008)... Tumanut C, Gavigan J -A, Huang W-J, Hampton EN, Tumanut R, Suen KF, Trauger JW, Spraggon G, Lesley SA et al (2007) Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity Biochem J 406, 203–207 Zhao Z, Tuakli-Wosornu Y, Lagace TA, Kinch L, Grishin NV, Horton JD, Cohen JC & Hobbs HH (2006) Molecular characterization of loss -of- function mutations in PCSK9 and identification... identification of a compound heterozygote Am J Hum Genet 79, 514–523 Hooper AJ, Marais AD, Tanyanyiwa DM & Burnett JR (2007) The C679X mutation in PCSK9 is present FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS 4131 Conserved protrusion on PCSK9 15 16 17 18 19 20 21 22 23 24 25 26 J Cameron et al and lowers blood cholesterol in a Southern African population Atherosclerosis... Sequence variations in PCSK9, low LDL, and protection against coronary heart disease N Engl J Med 354, 1264–1272 Berge KE, Ose L & Leren TP (2006) Missense mutations in the PCSK9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy Arterioscler Thromb Vasc Biol 26, 1094–1100 Cameron J, Holla ØL, Ranheim T, Kulseth MA, Berge KE & Leren TP (2006) Effect of mutations... alignment with high accuracy and high throughput Nucleic Acids Res 32, 1792–1797 Clamp M, Cuff J, Searle SM & Barton GJ (2004) The Jalview Java alignment editor Bioinformatics 20, 426–427 FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS J Cameron et al 39 Delano WL (2002) The PyMOL Molecular Graphics System DeLano Scientific, Palo Alto, CA Supplementary material The. .. Zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol J Biol Chem 279, 48865–48875 Maxwell KN, Fisher EA & Breslow JL (2005) Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment Proc Natl Acad Sci USA 102, 2069–2074 Rashid S, Curtis DE, Garuti R, Anderson NN, Bashmakov Y, Ho YK, Hammer RE, Moon Y -A & Horton JD... 445–448 Hampton EN, Knuth MW, Li J, Harris JL, Lesley SA & Spraggon G (2007) 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 104, 14604–14609 Piper DE, Jackson S, Liu Q, Romanow WG, Shetterly S, Thibault ST, Shan B & Walker NPC (2007) The crystal structure of PCSK9: a regulator of plasma LDLcholesterol . Investigations on the evolutionary conservation of PCSK9 reveal a functionally important protrusion Jamie Cameron 1 , Øystein L. Holla 1 , Knut. large, evolutionarily conserved protrusion on the surface of the catalytic domain of PCSK9. The lack of other residue conserva- tion on the PCSK9 surface