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J. Biol. Chem., Vol. 281, Issue 41, 30561-30572, October 13, 2006
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From the Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada
Received for publication, July 7, 2006 , and in revised form, August 14, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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. In contrast, the loss-of-function mutations A443T and C679X lead either to the lack of trans-Golgi network/recycling endosome localization and an enhanced susceptibility to furin cleavage (A443T) or to the inability of PCSK9 to exit the endoplasmic reticulum (C679X). Furthermore, we report the presence of both native and furin-like cleaved forms of PCSK9 in circulating human plasma. Thus, we propose that PCSK9 levels are finely regulated by the basic amino acid convertases furin and PC5/6A. The latter may reduce the lifetime of this proteinase and its ability to degrade the cell-surface LDL receptor, thereby regulating the levels of circulating LDL cholesterol. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, in which n = 0, 2, 4, or 6 (2). These proteinases are phylogenetically more closely related to each other and to yeast kexin than to SKI-1/S1P and PCSK9/NARC-1, which belong to the pyrolysin (5) and proteinase K (4) subfamilies, respectively. The latter enzymes recognize the motifs RX(hydrophobic/aliphatic)X (6) and (V/I)FAQ (7, 8), respectively. These enzymes have been implicated in a wide variety of functions regulating cellular homeostasis and a number of pathologies, including cancer, inflammation, neurodegenerative diseases, atherosclerosis, and viral infections. It was recently realized that some of these convertases play critical roles in the regulation of lipids and/or sterols (6) through the inactivation of lipases, e.g. by PC5/6, PACE4, and furin (9); through the activation of specific membrane-bound transcription factors (SREBP-1 and SREBP-2) by SKI-1/S1P (10); or by enhancement of the degradation of the low density lipoprotein (LDL) receptor (LDLR) by PCSK9 (7, 11–13). Indeed, PCSK9 is expressed mostly in adult liver hepatocytes and small intestinal enterocytes together with the LDLR (4). It was discovered recently that point mutations in the PCSK9 gene within its coding exons (14) are associated with either familial hypercholesterolemia (15–19) or hypocholesterolemia (20–22) phenotypes. This led to the identification of the PCSK9 gene as the third chromosomal locus associated with autosomal dominant familial hypercholesterolemia, with the LDLR and apoB comprising the other two loci (15).
It has been suggested that some PCSK9 single point mutations result in a gain or enhanced activity of PCSK9 in the degradation of the LDLR in acidic compartments, likely endosomes (7, 13), whereas others would cause a loss of function (20, 21), and that these mutations would be associated with the development of hypercholesterolemia or hypocholesterolemia, respectively (23). It was thus hypothesized that high levels of active PCSK9 are associated with a faster rate of degradation of the cell-surface LDLR, resulting in increased amounts of circulating LDL cholesterol, as the uptake of the latter in liver hepatocytes by the LDLR will be diminished accordingly and vice versa. Thus, the level of cell-surface LDLR is indirectly proportional to the level of PCSK9 in the liver and presumably small intestine. This hypothesis is reinforced by the in vivo observations that, in mice lacking a functional Pcsk9 gene (Pcsk9 knockout mice), the level of hepatocyte cell-surface LDLR is greatly enhanced, resulting in an 
50% drop in total circulating cholesterol (24), whereas higher levels of circulating LDL cholesterol are observed in mice overexpressing PCSK9 following recombinant adenoviral infections (7, 11, 12, 25).
Regulation of the levels of active PCSK9 could be achieved by various mechanisms, which among others could implicate (i) its transcription, where its mRNA levels are up-regulated by SREBP-2 (26) and hence by hydroxymethylglutaryl-CoA inhibitors known as "statins" (27) and down-regulated by cholesterol (28) via a reduced level of activated nuclear SREBP-2 (26, 27); (ii) the translational efficiency of the PCSK9 mRNA, which may be controlled by specific factors; (iii) post-translational modifications of PCSK9, including its zymogen cleavage and/or activation, glycosylation, and sulfation (4, 29), or possibly other modifications and processing events that modulate its half-life (4); (iv) regulation of the cellular localization and/or sorting of mature PCSK9; and (v) the cell-surface effect of secreted PCSK9 (30) and its possible uptake into endosomes. It was thus plausible that some of the single point mutations of PCSK9 associated with autosomal dominant familial dyslipidemia could enhance or abrogate one or more of these regulatory events (23).
In this study, we present evidence that the level of mature PCSK9 is indeed under the control of proteolysis by some members of the basic aa-specific PC family, including furin and/or PC5/6A, and that the cleavage by these PCs may provide a rationale behind the hypercholesterolemia phenotype associated with the French (F216L and R218S) (15, 18) and Anglo-Saxon (D374Y) (16, 17, 19) mutations and those associated with hypocholesterolemia in African Americans (C679X and A443T) (21).
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Anti-PCSK9 Antibodies—The antibodies used were as follows: anti-V5 epitope monoclonal antibody (mAb) (Invitrogen), rabbit anti-human LDLR-(184–196) polyclonal antibody (pAb) (Research Diagnostics), rabbit anti-
-actin pAb (Sigma), purified rabbit anti-human PCSK9-(490–502) pAb (Cayman Chemical, catalog no. 10007185), and goat anti-human PCSK9-(679–692) pAb (Imgenex, catalog no. IMX-3786). An in-house pAb directed against human PCSK9 (Ab1-hPC9) was obtained from rabbits injected with affinity-purified pro-PCSK9 (aa 31–454) expressed in Escherichia coli BL21 (see Fig. 3).5
Transfections and Biosynthetic Analyses—Transfections were done with 3 x 105 HEK293 cells using Effectene (Qiagen Inc.) and a total of 0.5 µg of cDNAs. Alternatively, 5 x 105 HuH7 or 6 x 105 HepG2 cells were transfected with Lipofectamine 2000 (Invitrogen) and a total of 4 µg of cDNAs. Two days post-transfection, the cells were washed and then incubated for various times with either 250 µCi/ml [35S]Met/Cys or 400 µCi/ml Na2 35SO4 (PerkinElmer Life Sciences). Some experiments were done in the presence of A23187 [GenBank] (2 µM), tunicamycin (5 µg/ml), or NaClO3 (30 mM). The cells were lysed in modified radioimmune precipitation assay buffer (150 mM NaCl and 50 mM Tris-HCl, pH 7.5), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and proteinase inhibitor mixture (Roche Applied Science), after which the lysates and media were prepared for immunoprecipitation (31). The antibodies used were anti-V5 mAb (1:500), Ab1-hPC9 (1:200), rabbit anti-human PCSK9-(490–502) pAb (1:200), and goat anti-human PCSK9-(679–692) pAb (1:200). Immunoprecipitates were resolved by SDS-PAGE on 8% Tricine gels and autoradiographed. Most of these experiments were repeated at least three times. Quantitation was performed on a Storm imager (Amersham Biosciences) using ImageQuant Version 5.2 software.
Protein Solubilization, Western Blotting, and Immunodetection—Cells in 35-mm plates were incubated for 24 h in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. The cells were washed, incubated for 30 min with radioimmune precipitation assay buffer with proteinase inhibitors on ice, and homogenized, and the lysates were clarified (32). Protein content was assayed with Bradford reagent. Fifty micrograms of proteins (or 10 µl of medium) were analyzed on 8% Tricine gels, followed by Western blotting for LDLR (160 kDa), -actin (42 kDa), and PCSK9-V5. The membranes were successively incubated with primary antibodies (anti-human LDLR-(184–196), 1:2000; anti-V5-horse radish peroxidase and anti--actin, 1:5000; and Ab1-hPC9, 1:3000) and an appropriate secondary antibody if required and revealed using ECL Plus (Amersham Biosciences). For experiments with human plasma, 30 µl of the latter were immunoprecipitated with Ab1-hPC9 (1:200) and then analyzed by SDS-PAGE on 8% glycine gels. Following transfer to a polyvinylidene difluoride membrane, PCSK9 was detected with the same antibody (1:3000), followed by rabbit TrueBlot (eBioscience) as a secondary antibody. Affinity removal of IgGs and albumin from plasma was performed using a ProteoSeek removal kit (Pierce).
Immunofluorescence and Confocal Microscopy—HuH7 cells were seeded on chamber slides (Nunc, Rochester, NY) and transfected the following day. Forty-eight hours post-transfection, the cells were fixed with 3% paraformaldehyde for 30 min at 4 °C and incubated for 1 h at 4 °C with 5% normal goat serum containing 0.1% Triton X-100 (blocking buffer), followed by overnight incubation at 4 °C with anti-V5 mAb (1:1000), rabbit anti-V5 pAb (1:1000) (Sigma), or Ab1-hPC9 in blocking buffer with or without the endosomal marker antibody (anti-MPR300, Abcam). The cells were then incubated for 60 min with Alexa Fluor 647-conjugated goat anti-rabbit IgG and with Alexa Fluor 555-conjugated goat anti-mouse IgG (10 µg/ml; Molecular Probes) and mounted in glycerol and 1,4-diazabicyclo[2.2.2]octane (Sigma). Immunofluorescence analyses were performed with a Zeiss LSM 510 confocal microscope. Images were processed with Adobe Photoshop CS2 (Version 9.0).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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70–80% loss of total LDLR immunoreactivity as quantified by Storm imaging, representative of three independent experiments) (Fig. 2), suggesting that, in HuH7 cells endogenously expressing substantial amounts of LDLR, these modifications are not critical for overexpressed PCSK9 activity in this receptor.
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75 kDa) autocatalytically cleaves its N-terminal prosegment, resulting in a 1:1 tight binding complex of PCSK9 and the prosegment (aa 31–152, 15 kDa) (4). The latter complex then exits the ER and is secreted constitutively as a mature 60-kDa protein, originally called N1 (Fig. 3B, WT lane) (4). However, in many cell lines, we also observed the presence of an N-terminally truncated form of N1 of 53 kDa, called N2 (Fig. 3B) (4, 7). The loss of 7 kDa from the N1 product also occurs in a number of natural mutants (Fig. 3, A and B), including S127R (15), R237W (7), R46L and A53V (15), and N157K (22), but not in the active-site mutant H226A, which remains in the ER as pro-PCSK9 (7) (Fig. 3B). Interestingly, either this N2 form is absent or its levels are substantially reduced in two natural mutants (repeated at least in three independent experiments), i.e. the French mutants R218S (18) and F216L (15), respectively (Fig. 3B, arrows). This suggested that Arg218 may be critical for the production of the N2 form. Sequence alignment of vertebrate PCSK9 showed a complete conservation of Arg218, which, in most cases, is found within an RXXR or a KXXXXR sequence (Fig. 4), typical of a basic aa-specific PC recognition motif recognized by furin and/or PC5/6-like enzymes (2). The mutation R218S would completely disrupt this motif, as it eliminates the P1 Arg, and the mutation F216L would affect the P3 position of this motif (Fig. 4, lower). These observations fit with our repeated inability to obtain an N-terminal sequence of N2 using Edman degradation, as the N-terminal Gln219 of human PCSK9 would be expected to cyclize on the sequencer into pyroglutamic acid and block the sequencing reaction (data not shown), as was observed for the N-terminal sequence of pro-PCSK9 that starts at Gln31 (4).
Accordingly, we tested the hypothesis that cleavage at Arg218 is performed by one or more basic aa-specific PCs. Preliminary pulse-chase experiments revealed that (i) the intracellular processing of the N1 form of PCSK9 into N2 occurs only on the form that reaches the Golgi (endoglycosidase H (endo H)-resistant form), (ii) such processing occurs late along the secretory pathway, and (iii) the products are rapidly secreted and not stored in the HEK293 cells (data not shown). To define the enzymes implicated in this cleavage, we coexpressed WT PCSK9 (tagged with V5 at the C terminus) with all the convertases in HEK293 cells. Biosynthetic labeling of these cells with [35S]Met/Cys for 4 h followed by immunoprecipitation and SDS-PAGE analysis revealed that only membrane-bound furin (not soluble furin, lacking the transmembrane cytosolic tail (33)) and, to a lesser extent, soluble PC5/6A are capable of processing the N1 form of PCSK9 into the N2 form, with the concomitant loss of the co-immunoprecipitated 15-kDa PCSK9 prosegment (Fig. 5, lower left panel) (4). Notice that the form of PCSK9 that is competent for this processing is visible in small amounts in cell extracts as an endo H-resistant protein just above the major ER-localized endo H-sensitive PCSK9 form (Fig. 5, upper right panel, arrowhead). This intracellular form was lost upon furin digestion, with the concomitant appearance of secreted PCSK9-
N218 (Fig. 5, Furin lane). Because PCs act only on substrates that reach the TGN or beyond (usually endo H-resistant forms) and because the major intracellular PCSK9 forms observed are endo H-sensitive (as evidenced by the smaller size of both pro-PCSK9 and PCSK9 by 1.5 kDa) (Fig. 5, upper right panel, arrows), hence residing mostly in the ER, these would be expected to be resistant to furin processing (29). Such a cleavage of mature PCSK9 would be expected to release two fragments, a 66-aa N-terminal V5-negative peptide (aa 153–218) and a 474-aa C-terminal V5-immunoreactive 53-kDa V5-positive N2 form (aa 219–692). Using Ab1-hPC9, we detected an additional 7-kDa immunoreactive protein in the medium. This fragment possibly corresponds to the N-terminal segment of the catalytic subunit (aa 153–218) produced by furin cleavage. Because Ab1-hPC9 recognized all PCSK9 fragments in the medium, the data also show that furin did not affect the level of the 15-kDa prosegment in the medium (Fig. 5, lower right panel). Aside from its molecular mass, the tentative identity of this 7-kDa fragment was further tested by its [35S]Met labeling (likely due to Met201) but absence of its 35SO4 or [35S]Cys labeling (data not shown). Thus, from now on, we will refer to the N-terminally truncated N2 product as PCSK9-N218 (PCSK9 lacking the N-terminal 218 aa) (Fig. 5). The data also show that coexpression of the serpin 1-PDX (1-antitrypsin Portland), which inhibits most of the basic aa-specific PCs (34, 35), completely blocked the formation of PCSK9-N218 (Fig. 5). To further confirm that Arg218 is the furin/PC5A-processing site, we coexpressed the R218S and F216L mutants with all the PCs and, as a control, with membrane-bound 
-secretase (BACE-1) (31). The data show that furin and PC5/6A can no longer process the R218S mutant and that both cleave the F216L mutant to a lesser extent (compare Figs. 5 and 6).
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N218 and is partially resistant to cleavage by endogenous convertases (Fig. 6, right panels, compare pIRES lanes) or by overexpressed furin (compare Furin lanes).
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(E/D)L. This led us to design mutants of PCSK9 that should greatly enhance the ability of furin to process this molecule. Thus, upon replacement of the WT RFHR218QA sequence with an optimal furin recognition sequence (RRRR218EL), we noted that endogenous furin in HEK293 cells could completely process PCSK9 at Arg218, whereas the motifs RFHR218EA and RFHR218EL resulted in intermediate furin cleavability (Fig. 7). Notice the virtual absence of prosegment co-immunoprecipitation with PCSK9-N218 produced (Fig. 7), which would be predicted because such a cleavage would remove PCSK9 segment 153–218, which contains the active-site Asp186. This also suggests that PCSK9-N218 is inactive and hence unable to tightly bind the prosegment, which, in all PCs, binds only to the catalytically active convertase (37–39).
Effect of PCSK9 Cleavage by Furin/PC5/6A on Its Ability to Enhance the Degradation of the LDLR and Its Subcellular Localization—Previous studies revealed that overexpressed PCSK9 co-localizes with the LDLR5 and results in an enhanced degradation of the LDLR in a number of cell lines and in vivo (7, 12, 13). Accordingly, we analyzed the activity of WT PCSK9, mutant R218S, the RRRR218EL variant, the active-site mutant H226A, and the Cys/His-rich domain (CHRD) of PCSK9 (aa 405–692) on the degradation of the LDLR in HuH7 cells (Fig. 8A). The results revealed that only WT PCSK9 and its R218S mutant are active in enhancing the degradation of the LDLR compared with the pIRES control empty vector, whereas the RRRR218EL variant and the CHRD are inactive, similar to the active-site mutant H226A (Fig. 8A). Therefore, PCSK9-N218 is an inactive form of PCSK9 that is secreted from cells compared with the active-site mutant H226A, which results in uncleaved zymogen pro-PCSK9 that remains in the ER (7). Thus, the RRRR218EL variant of PCSK9 provides an ideal control for the activity of PCSK9 toward the LDLR within the secretory pathway, as opposed to active-site mutants that do not exit from the ER and hence do not co-traffic with the LDLR to the cell surface/endosomes (4, 7).
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90% of the LDLR was lost at 100 µM (Fig. 8B). Under these conditions, we did not observe loss of cellular viability (data not shown). This result independently confirms the critical role played by furin in HepG2 cells in regulating the level of the LDLR. Finally, we also compared the ability of the S127R and D374Y mutants to enhance the degradation of the LDLR. Western blots of HuH7 cells showed that, although the autocatalytic processing of D374Y is similar to that of WT PCSK9, the former is more active in enhancing the degradation of the LDLR compared with either S127R or WT PCSK9 (Fig. 8C). This result agrees with the observed reduced susceptibility of the D374Y mutant to furin processing (Fig. 6, right panels).
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N218; and in media, PCSK9 and PCSK9-N218. In contrast, rabbit antihuman PCSK9-(490–502) antibody recognized only pro-PCSK9 and PCSK9-N218 but not PCSK9 (Fig. 9A). This unexpected observation could be rationalized by the hypothesis that, in the native conformation of PCSK9, segment 490–502 may interact with some amino acids in N-terminal sequence 153–218 of PCSK9 or its prosegment (aa 31–152), which remains noncovalently complexed with the catalytic subunit (Figs. 5 and 9A), hence masking its recognition by rabbit anti-human PCSK9-(490–502) pAb. This is further supported by the fact that this antibody recognized all denatured PCSK9 forms on Western blots (data not shown). Interestingly, the Italian mutation R496W (which results in a hypercholesterolemia phenotype) was reported in segment 490–502 (recognized by rabbit anti-human PCSK9-(490–502) pAb) (18, 41).
Evidence is now available to suggest that the PCSK9-enhanced degradation of the LDLR occurs in acidic endosomes/lysosomes (7, 13). We therefore tested the ability of PCSK9 and its RRRR218EL variant to enter the TGN/recycling endosome pathway of HuH7 cells using anti-V5 or rabbit anti-human PCSK9-(490–502) antibody (Fig. 9B). Indeed, although anti-V5 mAb showed the presence of PCSK9 in the TGN/recycling endosome pathway co-localizing with the cation-independent mannose 6-phosphate receptor (measured with anti-MPR300 antibody), rabbit anti-human PCSK9-(490–502) antibody did not, as expected, because the latter could recognize only inactive pro-PCSK9 (ER-localized) and PCSK9-N218 (Fig. 9, A and B). In agreement, neither antibody detected the presence of the PCSK9 RRRR218EL variant in endosomes (Fig. 9B). Because the latter is expected to generate PCSK9-N218 via furin/PC5/6A cleavage, this result suggests that the inability of PCSK9-N218 to enhance the degradation of the LDLR (Fig. 8) may be due to its the combined loss of catalytic potential and absence from endosomes/lysosomes, the compartment where such degradation is believed to take place (7, 13). We conclude that the natural mutations R218S, F216L, and D374Y associated with hypercholesterolemia are correlated with the complete or partial loss of convertase degradation by furin and/or PC5/6A. This would result in an enhanced function of PCSK9, likely due to an increased level of the convertase within endosomes co-localizing with the LDLR.
Circulating Forms of PCSK9 in Human Plasma—To substantiate the physiological relevance of the ex vivo observation of the cleavage of human PCSK9 by furin/PC5/6A into secretable PCSK9-N218, we characterized the forms of PCSK9 that are found in the normal plasma of two individuals, one female and one male, as well as in lipoprotein-deficient serum prepared from a commercial pool of normal human plasma (Bioreclamation Inc.). After multiple trials with various antibodies, we selected our in-house human antibody (Ab1-hPC9) for immunoprecipitation, followed by Western blotting. We used, as a secondary antibody, rabbit TrueBlot anti-IgG, which does not recognize denatured IgGs and hence would not interfere with the immunodetection of PCSK9 on Western blots. The data show that, in all cases, PCSK9 and its PCSK9-N218 product are clearly the circulating forms in both male and female plasma, the ratio of which could vary between individuals (Fig. 10). Furthermore, a similar result was obtained with pooled human sera. We used preimmune rabbit serum as a control. Taking into account the slightly higher molecular mass of V5-tagged PCSK9 compared with untagged PCSK9 (Fig. 10, left panel), we also show that the plasma forms comigrated with markers obtained from the medium of HEK293 cells overexpressing the uncleavable R218S or fully processed RRRR218EL variant. Interestingly, PCSK9 immunoreactivity was lost when the lipoprotein-deficient serum was subjected to preclearing using an affinity column that removes IgGs and albumin (Fig. 10), and hence, special care must be taken when sera are fractionated. We conclude that furin/PC5/6A processing of PCSK9 is physiological and that, because some full-length protein circulates, it is likely that PCSK9 could enhance the degradation of the LDLR at sites distal from the liver.
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Fig. 11 shows the results from the analysis of the biosynthesis of the A443T mutant, which is normally processed and secreted. However, upon coexpression with furin (but much less so with PC5/6A), a new cleavage product appeared, migrating as a fuzzy doublet 10 kDa smaller than PCSK9-N218. Thus, the A443T mutation results in a higher susceptibility of PCSK9 to furin cleavage into a new protein that would lose a larger segment of the catalytic chain and hence would likely be inactive. Finally, immunofluorescence analysis by confocal microscopy in HuH7 cells transiently transfected with either WT PCSK9 or its A443T mutant revealed that the latter did not sort to TGN/recycling endosomes compared with WT PCSK9, which co-localized with the cation-independent mannose 6-phosphate receptor (anti-MPR300-positive) (Fig. 11B). In conclusion, the two hypocholesterolemia-associated mutations result in loss of function due either to the mislocalization of PCSK9 in the ER (C679X) or at least in part to a combination of higher degradation by furin and an inability to enter the recycling endosome pathway (A443T).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In this study, we have presented data to support the novel notion that PCSK9 is under the control of proteolysis by other members of the PC family (2, 3). Up until now, it was thought that, with the exception of PC2, all other PC family members were autonomous in that their zymogen processing and activation were not dependent on other convertases or other proteins. In the case of PC2, although its zymogen processing is autocatalytic (46), its productive folding and hence activation depend on a protein called 7B2, which acts both as an inhibitor and a partner protein (47) and which is processed by furin (48). When we first reported the biosynthesis of WT PCSK9 (originally called NARC-1), we had noticed the presence of two secreted forms: a major one of 60 kDa (called N1) and a minor one of 53 kDa (called N2). Because we could not sequence the latter form, we concluded that its N terminus was blocked and could possibly be a Gln that cyclized in the Edman degradation reaction. Careful analysis of the forms secreted by some of the mutants revealed that, in these, N2 was either absent (R218S) or substantially reduced (F216L and D374Y) (Figs. 3 and 6). The presence of R218S in the motif RXXR218 (Fig. 4) suggested that processing at Arg218 could take place by a furin-like convertase (2). Biosynthetic analyses supported this hypothesis and showed that only membrane-bound furin and, to a lesser extent, PC5/6A are capable of processing PCSK9 into the N2 product, herein called PCSK9-N218 (Fig. 5). Because soluble furin is inefficient in performing such cleavage (Fig. 5) and because soluble furin does not bind to the cell surface (38), it is believed that such late intracellular processing occurs in proximity to membranes either at the cell surface or in a recycling compartment. Notably, PC5/6A binds to the cell surface via its complex formation with tissue inhibitors of metalloproteinases, e.g. TIMP2 and heparan sulfate proteoglycans (38), and accordingly inactivates heparan sulfate proteoglycan-bound proteins such as lipases (9). However, membrane binding or association is not sufficient to ensure processing because the membrane-bound convertases PC5/6B and PC7 (49) and SKI-1/S1P (5) do not process WT PCSK9 (Fig. 5). It is intriguing that membrane-bound furin, but not PC5/6B, can process PCSK9. The difference may be in the subcellular localization of these two enzymes and their recycling pathways, which are not identical. Thus, it has been shown that PC5/6B is localized to a paranuclear, brefeldin A-dispersible, BaCl2-responsive, post-Golgi network (TGN) compartment distinct from furin and TGN38 (50). Interestingly, R218S completely abrogated this processing by furin, whereas F216L and D374Y severely impaired it (Figs. 5 and 6). The bulkier aromatic Phe216 in PCSK9 may be optimal for folding and stabilization of the enzyme. The F216L mutation of the P3 residue in the motif RFHR218 also suggests that this position may be important for furin recognition. Although the P3 requirements have not been systematically analyzed, it has been reported that furin tolerates variations at this position and that the presence of Leu at P3 has a small negative impact on kcat/Km (51). Furthermore, the crystal structure of furin in complex with the inhibitor decanoyl-RVKR-chloromethyl ketone containing an aliphatic Val at P3 suggests that this residue extends into the bulk solvent and that positive charges at this position would be better, as they would make favorable contacts with the carboxylate of Glu257 in furin (36). We have exploited this information in our design of the RRRR218EL mutant (Fig. 7). The unexpected partial resistance of D374Y to furin digestion (Fig. 6) led us to speculate that Asp374 plays an important role in the final conformation adopted by PCSK9, possibly via salt bridges to positively charged amino acids. The observed higher activity of the D374Y mutant in the enhancement of the degradation of the LDLR (Fig. 8) may be related in part to the structural properties of this mutant that could modulate the activation of PCSK9 and its resistance to furin digestion. However, we cannot presently exclude that, aside from the relative resistance to furin processing, the hypercholesterolemia phenotype associated with the F216L and D374Y mutations could also be caused by other as yet undefined properties of these particular mutants.
The RRRR218EL variant of PCSK9 is completely processed by endogenous furin, resulting in a well secreted product (PCSK9-N218) that has lost the N-terminal segment of its catalytic structure and is no longer sorted to endosomes (Figs. 7 and 9). This provided an ideal mutant to test whether the enzyme activity of PCSK9 may be needed for its enhancement of the degradation of the LDLR (Fig. 8), as active-site mutants such as H226A do not exit the ER (7). The fact that overexpression of RRRR218EL does not affect the levels of LDLR, whereas that of WT PCSK9 and the R218S mutant does, suggests but does not prove that PCSK9 activity requires an intact catalytic subunit. However, the role of the complexed prosegment in WT PCSK9 has yet to be clarified. So far, we know very little about the zymogen activation of PCSK9. Thus, although it is autocatalytically processed at the VFAQ152 site, it is secreted as a complex with its prosegment (7). It is still a matter of speculation whether, upon re-uptake by the cells, the PCSK9-prosegment complex could dissociate at the acidic pH of endosomes and, similar to other PCs, lead to enzyme activation, possibly following a second cleavage event of the prosegment. Definitive proof of this mechanism is still lacking, as is the finding of other PCSK9 target proteins.
The physiological relevance of the furin processing would have been best demonstrated by the analysis of plasma either from dyslipidemic patients or from conditional knock-out mice lacking liver furin (52). However, the plasma of such mice is not available, and the colony is being currently expanded in our group (in collaboration with Dr. John Creemers, Catholic University of Leuven, Belgium). Until these data become available, the best physiological criterion used in this work is the detection in human plasma of both PCSK9 and PCSK9-N218 (Fig. 10). This suggests that PCSK9 may act on the LDLR at sites away from hepatocytes and intestinal enterocytes. An example would be the adrenal gland, where it was reported that adenoviral expression of PCSK9 can lower the levels of the endogenous adrenal LDLR (12). The detection of plasma PCSK9 opens the door to the future measurement of the circulating levels of this enzyme in dyslipidemia patients with either gain- or loss-of-function PCSK9 mutations and those treated with statins or other cholesterol-lowering drugs as well as in obese and type II diabetic patients.
PCSK9 is Tyr-sulfated in its prosegment (Tyr38) and contains one N-glycosylation site (Asn533) (Fig. 1) (4, 7). These modifications do not affect the folding and secretion of PCSK9 (Fig. 1) or its activity toward the LDLR (Fig. 2). Targeting of the enzyme to both early and late endosomes depends on the presence of the LDLR because, in its absence, PCSK9 cannot enter endosomes.5 Whether this fine-tuning would mean that the secreted complex between the Tyr-sulfated prosegment and N-glycosylated PCSK9 is needed for internalization and endosome sorting will require further studies. Preliminary studies suggested that the presence of a clathrin coat is required for the sorting of PCSK9 into endosomes.6 Interestingly, a similar paired interaction was recently reported to explain the mechanism of endosome/lysosome sorting of the membrane-bound CIC-7 chloride channel, which requires the membrane-bound gray lethal protein (also known as Ostm1) (53) to enter lysosomes. Here, in the absence of the dominant partner Ostm1, the protein CIC-7 is stuck in the ER (54). In the case of PCSK9, in the absence of the LDLR, it is secreted, and whatever is left in the cell is still in the ER. In the presence of the LDLR, both proteins co-localize at the cell surface and in endosomes.5
With respect to the mutations associated with hypocholesterolemia, the Y142X mutation (20) results in a truncated protein ending in the prosegment (data not shown). Our data also show that, although the C679X mutant is stuck in the ER and hence cannot degrade the LDLR in endosomes, the A443T mutant is oversensitive to furin digestion. Thus, not only can furin process A443T into its derivative PCSK9-N218, but it can also further digest it into a smaller protein migrating on SDS-polyacrylamide gel as a fuzzy doublet at 40 kDa (Fig. 11). The fact that this mutation transforms Ala into Thr (PNLVAA443LPP to PNLVAT443LPP) and that the latter is surrounded by three Pro residues in the sequence suggests that it might create an O-glycosylation site, which often occurs at Ser/Thr in Pro-rich regions (55), possibly rationalizing the fuzziness of the second furin product. In the homozygous state, this human mutation is associated with hypocholesterolemia. Our data suggest that this loss-of-function mutation will create an O-glycosylation site that makes PCSK9 a better target for furin degradation (Fig. 11). In this context, it is interesting to note Ala443 in human PCSK9 is Thr in both mouse and rat PCSK9. However, the susceptibility of these rodent PCSK9 proteins to furin digestion has yet to be defined.
So far, our studies on selected natural mutants of PCSK9 have revealed that those mutants that are resistant to furin digestion (R218S, F216L, and D374Y) are associated with hypercholesterolemia, whereas those that are missorted (C679X) or more extensively degraded by furin (A443T) are associated with hypocholesterolemia. This suggests that the level of PCSK9 is finely regulated by the basic aa-specific convertases furin and PC5/6A and those mutations that affect the processing of this enzyme and hence its half-life would tilt the balance toward a dyslipidemia phenotype either up or down.
A mutation in another hepatic protein that creates a de novo furin site that causes a loss of function has been reported for fibrinogen Canterbury (56). However, different from the beneficial A443T mutation of PCSK9, the creation of such a furin site in the prosegment of fibrinogen results in a loss of function associated with pathology involving prolonged blood coagulation time. In contrast, loss of furin processing has also been reported in the development of Marfan syndrome and diabetes because of mutations in the processing sites of fibrillin (57) and the insulin receptor (58), respectively. We can now add PCSK9 to this list, whereupon mutations leading to the loss of susceptibility to furin digestion could be associated with the development of hypercholesterolemia.
PCSK9 is the only proprotein convertase for which genetic variants are associated with a dominant disease. Except for the C679X mutations, all other mutations studied here affect the processing of PCSK9 by furin and, to a lesser extent, by PC5/6A. Interestingly, the Italian mutation R496W occurs in a basic aa-rich region within the CHRD (aa 405–692), which, under nondenaturing conditions, is detectable by an anti-C terminus antibody, but is not recognized by rabbit anti-human PCSK9-(490–502) antibody unless it is processed by furin (Fig. 9), suggesting that it may be masked by another N-terminal sequence. However, many other mutations have been reported, some of which may also be associated with increased response to statin therapy (21, 22). Therefore, this study is just the start of their systematic categorization in terms of their processing, subcellular trafficking, and localization, as well as ability to enhance the degradation of the LDLR. The existence of modifier genes that affect the phenotype associated with each mutation is likely, as some mutations can cause either hypercholesterolemia or hypocholesterolemia in different individuals (21, 22). Because the loss of one functional allele of PCSK9 has long-term benefits in protection against cardiovascular related mortality (21), it may be advantageous to have low levels of circulating PCSK9, which is now amenable to quantification using specific enzyme-linked immunosorbent assays. This conclusion should pave the way toward the identification of selective pharmacological PCSK9-lowering agents that could be used in conjunction with statins or other cholesterol-lowering drugs.
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1 Recipient of a postdoctoral scholarship from the Natural Sciences and Engineering Research Council of Canada.
2 Recipient of a Fonds Québécois de la Recherche sur la Nature et les Technologies postdoctoral fellowship.
3 To whom correspondence should be addressed: Laboratory of Biochemical Neuroendocrinology, Clinical Research Inst. of Montreal, 110 Pine Ave. West, Montreal, Quebec H2W 1R7, Canada. Tel.: 514-987-5609; Fax: 514-987-5542; E-mail: seidahn{at}ircm.qc.ca.
4 The abbreviations used are: PC, proprotein convertase; aa, amino acid(s); LDL, low density lipoprotein; LDLR, low density lipoprotein receptor; mAb, monoclonal antibody; pAb, polyclonal antibody; Ab1-hPC9, rabbit antihuman PCSK9 polyclonal antibody; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TGN, trans-Golgi network; WT, wild-type; ER, endoplasmic reticulum; endo H, endoglycosidase H; CHRD, Cys/His-rich domain.
5 N. Nassoury, D. Blasiole, A. T. Oler, J. Hamelin, A. Prat, A. Attie, and N. G. Seidah, submitted for publication.
6 D. Rhainds and N. G. Seidah, manuscript in preparation.
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