Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on March 28, 2006
Human Molecular Genetics 2006 15(9):1551-1558; doi:10.1093/hmg/ddl077

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Effect of mutations in the PCSK9 gene on the cell surface LDL receptors

Jamie Cameron, Øystein L. Holla, Trine Ranheim, Mari Ann Kulseth, Knut Erik Berge^* and Trond P. Leren

Medical Genetics Laboratory, Department of Medical Genetics, Rikshospitalet University Hospital, N-0027 Oslo, Norway

^* To whom correspondence should be addressed. Tel: +47 23075580; Fax: +47 23075561; Email: knut.erik.berge{at}rikshospitalet.no

Received December 23, 2005; Revised March 14, 2006; Accepted March 22, 2006

	ABSTRACT

TOP ABSTRACT Introduction RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene is involved in the post-transcriptional regulation of the low-density lipoprotein (LDL) receptors (LDLR). Mutations in the PCSK9 gene have been associated with both hypocholesterolemia and hypercholesterolemia through ‘loss-of-function’ and ‘gain-of-function’ mechanisms, respectively. We have studied the effect of the four loss-of-function mutations R46L, G106R, N157K and R237W and the two gain-of-function mutations S127R and D374Y on the autocatalytic activity of PCSK9, as well as on the amount of the cell surface LDLR and internalization of LDL in transiently transfected HepG2 cells. The two groups of mutations did not differ with respect to autocatalytic activity of PCSK9, but they did differ with respect to the amount of cell surface LDLR and internalization of LDL. The four loss-of-function mutations had a 16% increased level of cell surface LDLR and a 35% increased level of internalization of LDL as compared with WT-PCSK9. The two gain-of-function mutations had a 23% decreased level of cell surface LDLR and a 38% decreased level of internalization of LDL as compared with WT-PCSK9. Our studies have also shown that transfer of media from transiently transfected HepG2 cells to untransfected HepG2 cells, reduces the amount of cell surface LDLR and internalization of LDL in the untransfected cells within 20 min of media transfer. Thus, PCSK9 or a factor acted upon by PCSK9, is secreted from the transfected cells and degrades LDLR both in transfected and untransfected cells.

	Introduction

TOP ABSTRACT Introduction RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene with a cDNA of 3617 bp, encodes a protein of 692 amino acids which is a proprotein convertase of the subtilase family (1,2). PCSK9 is synthesized as a soluble zymogen which undergoes autocatalytic intramolecular cleavage in the endoplasmic reticulum (ER) (1,3). After cleavage of the prosegment, mature PCSK9 exits ER and is efficiently secreted (1).

The promoter region of the PCSK9 gene contains a sterol regulatory element (4) and transcription is regulated by the intracellular cholesterol level (2,5). Overexpression of the wild-type (WT) PCSK9 gene in mice results in hypercholesterolemia because of reduced number of low-density lipoprotein (LDL) receptors (LDLR) (6–8). A reduced number of LDLR has also been observed in cultured cells overexpressing PCSK9 (7,8). As the reduced number of LDLR, by PCSK9, is not accompanied by changes in LDLR mRNA levels (6,7), PCSK9 is apparently involved in the post-transcriptional regulation of the LDLR. Degradation of the LDLR by PCSK9 is dependent on maintained catalytic activity of PCSK9 (7,9) and appears to take place in a post-Golgi compartment or at the cell surface (7,9,10).

The effect of PCSK9 on the LDLR is cell-specific. Overexpression of PCSK9 reduces the number of LDLR in liver and kidney cells (7,8), but not in fibroblasts, the Huh7 human hepatoma cell line or in chinese hamster ovary cells (7). It is therefore possible that PCSK9 may require another cell-specific protein to exert its effect on the LDLR. Thus, the exact mechanism by which PCSK9 degrades the LDLR remains to be determined.

If the normal function of PCSK9 is to reduce the number of LDLR, one would expect mutations in the gene which disrupt the normal function of PCSK9, to result in increased number of LDLR and thereby cause hypocholesterolemia. In line with this notion, hypocholesterolemia has been found in mice which lack the PCSK9 gene (11). Moreover, nonsense mutations Y142X and C679X (12) and missense mutations R46L (13,14), G106R (13), N157K (13), R237W (13), L253F (14) and A443T (14) in the PCSK9 gene have been associated with low levels of plasma cholesterol. These mutations are referred to as ‘loss-of-function’ mutations, but as of yet, no studies of how these mutations affect the LDLR have been performed.

Mutations in the PCSK9 gene not only cause hypocholesterolemia through a loss-of-function mechanism, but they may also cause hypercholesterolemia through a ‘gain-of-function’ mechanism (8,15–18). Such a gain-of-function mechanism could either represent a higher activity of the LDLR-degrading function of PCSK9, or it could represent a qualitatively different property of mutant PCSK9, as is the case for two mutations in the superoxide dismutase 1 gene that cause amyotrophic lateral sclerosis through a novel protein–protein interaction (19). Mutations in the PCSK9 gene which have been associated with hypercholesterolemia, are referred to as gain-of-function mutations.

For the gain-of-function mutation S127R, reduced number of LDLR has been found in Epstein Barr-virus (EBV)-transformed lymphocytes (8), but not in fibroblasts (20) from patients heterozygous for the mutation. For the D374Y gain-of-function mutation, no significant reduction in the number of cell surface LDLR (18,21) or internalization of LDL (21) has been found by studies of EBV-transformed lymphocytes from D374Y heterozygotes. Another mechanism for the hypercholesterolemia caused by gain-of-function mutations has been suggested by the finding of increased secretion of apolipoprotein B-containing lipoproteins in two patients heterozygous for the S127R mutation (22). However, studies of transgenic mice and transfected hepatocyte cell lines have provided conflicting data regarding the effect of gain-of-function mutations on secretion of apolipoprotein B (7,8,10,18). A third mechanism for the hypercholesterolemia caused by gain-of-function mutations has been proposed by Naoumova et al. (23). They found an abnormal composition of LDL isolated from D374Y heterozygotes and decreased binding of LDL from these patients to LDLR on normal fibroblasts (23). Thus, several potential mechanisms by which mutations in the PCSK9 gene cause hypercholesterolemia, have been proposed.

To provide more information on how mutations in the PCSK9 gene affect cholesterol metabolism, we have performed studies of the four loss-of-function mutations R46L, G106R, N157K and R237W (13) and the two gain-of-function mutations S127R and D374Y (15–18) on the LDLR in transiently transfected HepG2 cells. Mutation R496Q identified in a Type III hyperlipoproteinaemia subject, but with unknown effect on cholesterol levels, was also included in the studies.

	RESULTS

TOP ABSTRACT Introduction RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Autocatalytic cleavage and secretion of mutant PCSK9
To study whether the four loss-of-function mutations R46L, G106R, N157K and R237W and the two gain-of-function mutations S127R and D374Y in the PCSK9 gene affect the autocatalytic cleavage of PCSK9, HepG2 cells were transiently transfected with mutant PCSK9 plasmids using WT, empty plasmid and the catalytically inactive S386A mutant (1) PCSK9 plasmid as controls. The amounts of pro-PCSK9 and cleaved PCSK9 in cell lysates were determined by western blot analysis using anti-FLAG antibody. In cells expressing WT-PCSK9, two bands of 73 and 64 kDa were observed, which correspond to pro-PCSK9-FLAG and the cleaved, mature form of PCSK9-FLAG, respectively (Fig. 1).

View larger version (29K): [in this window] [in a new window]   Figure 1. Autocatalytic cleavage and secretion of mutant PCSK9. Mutant gain-of-function and loss-of-function pCMV-PCSK9-FLAG plasmids were transiently transfected in HepG2 cells using WT-PCSK9 plasmid, empty plasmid and the catalytically inactive S386A plasmid, as controls. Two days after transfection cells were lysed and 40 µg of cell lysate were subjected to SDS–PAGE and immunoblotted with anti-FLAG. Uncleaved, pro-PCSK9 and cleaved, mature PCSK9 are indicated. A representative experiment is shown (upper panel). The autocatalytic cleavage of the mutant PCSK9 from this experiment is expressed as percentage of maturation of WT-PCSK9. Cell lysates from HepG2 cells transfected with plamids containing mutations N157K and R237W, were analyzed on a separate gel. The lower panel shows the amount of mature PCSK9 in the medium of HepG2 cells transfected with the different PCSK9 plasmids, as determined by western blot analysis using anti-FLAG antibody after TCA precipitation.

 
Quantitation of the amount of mature PCSK9 from the WT and mutant PCSK9 constructs revealed a wide variation in the autocatalytic cleavage of the different mutant constructs (Fig. 1). The loss-of-function mutant G106R seemed to abolish the autocatalytic cleavage in a manner similar to that of the catalytically inactive S386A mutant. The autocatalytic cleavage of the three other loss-of-function mutants was similar to WT-PCSK9 plasmid. The gain-of-function mutant D374Y appeared to have almost normal autocatalytic activity, whereas the autocatalytic cleavage of the gain-of-function mutant S127R was reduced by 66% when compared with WT-PCSK9. Thus, no consistent difference in the autocatalytic activity between loss-of-function and gain-of-function mutations could be observed. The R496Q mutation did not appear to affect the autocatalytic activity of PCSK9.

Western blot analyses using antibody against FLAG were performed to determine the amount of mature PCSK9 secreted from HepG2 cells transfected with the different PCSK9 constructs (Fig. 1). The results showed that mature PCSK9 was found only in the media from cells transfected with constructs leading to mature PCSK9. Neither the empty plasmid, the autocatalytically inactive mutant S386A nor the loss-of-function mutant G106R had PCSK9 detectable in the media.

Effect of mutations in the PCSK9 gene on the amount of LDLR and internalization of LDL
To study whether the loss-of-function and gain-of-function mutations in the PCSK9 gene cause hypocholesterolemia or hypercholesterolemia by affecting the amount of cell surface LDLR and internalization of LDL, the amount of cell surface LDLR and internalization of LDL were studied by flow cytometry in transiently transfected HepG2 cells.

The loss-of-function mutant G106R had the highest amount of cell surface LDLR and was comparable to that of the autocatalytically inactive S386A mutant (Fig. 2). When compared with the WT-PCSK9, the S386A and G106R mutants had a 43% and 32% increased amount of LDLR, respectively. The corresponding level of empty plasmid was 28%. These findings are in agreement with previous findings that maintained autocatalytic activity of PCSK9 is required to degrade LDLR (7,9). The other three loss-of-function mutants had more cell surface LDLR than WT-PCSK9 as well. In contrast, the gain-of-function mutants had lower amounts of cell surface LDLR than WT-PCSK9. The D374Y mutant had a 36% reduction in the cell surface LDLR compared with WT-PCSK9, and the S127R mutation had a corresponding 10% reduction compared with WT-PCSK9. For the four loss-of-function mutants, the mean increase in the cell surface LDLR was 16% compared with WT-PCSK9, whereas the mean reduction in the cell surface LDLR for the two gain-of-function mutants was 23%. The findings of markedly increased levels of cell surface LDLR in HepG2 cells transfected with the S386A or G106R PCSK9 constructs when compared with HepG2 cells transfected with WT-PCSK9, as well as the markedly decreased levels of LDLR in HepG2 cells transfected with the D374Y PCSK9 construct, were confirmed by western blot analysis (Fig. 2).

View larger version (30K): [in this window] [in a new window]   Figure 2. Amount of cell surface LDLR and internalization of LDL in HepG2 cells transiently transfected with mutant PCSK9 plamids. Flow cytometry was used to study the amount of cell surface LDLR and internalization of LDL in HepG2 cells transiently transfected with the gain-of-function mutants S127R and D374Y, the R496Q mutant and the loss-of-function mutants R46L, G106R, N157K and R237W PCSK9. WT-PCSK9, empty plasmid and the catalytically inactive mutant S386A were used as controls. Prior to studies the cells were incubated for 24 h in medium containing 5 mg/ml LPDS. The cells were labelled with the IgG-C7 LDLR antibody and counterstained with Alexa Fluor® 488 anti-IgG antibody to determine the amount of cell surface LDLR. To determine the amount of LDL internalized, the cells were incubated with fluorescently labelled LDL (10 µg/ml) for 2 h at 37°C. Values are given as mean±SEM from two to four experiments (upper panel). Lower panel shows the amount of LDLR in cell lysates from HepG2 cells transfected with the different PCSK9 plasmids, as determined by western blot analysis.

 
The loss-of-function mutant G106R internalized 71% more LDL than WT-PCSK9 (Fig. 2). This amount was comparable to that of empty plasmid and the autocatalytically inactive mutant S386A. The three other loss-of-function mutants internalized more LDL than WT-PCSK9 plasmid as well. In contrast, the gain-of-function mutants internalized less LDL than WT-PCSK9 plasmid, of which the D374Y mutant had the largest reduction of 68%. The gain-of-function mutant S127R had only a 7% reduction in the amount of LDL internalized when compared with WT-PCSK9. For the four loss-of-function mutants, the mean amount of LDL internalized was 35% higher than for WT-PCSK9, whereas the two gain-of-function mutants internalized 38% less LDL than WT-PCSK9. Our findings therefore indicate that the loss-of-function mutations cause hypocholesterolemia by reduced ability of PCSK9 to degrade LDLR, whereas the gain-of-function mutations cause hypercholesterolemia by increased ability of PCSK9 to degrade LDLR. The R496Q mutant had amounts of cell surface LDLR and LDL internalized that were comparable to that of WT-PCSK9.

Effect of transfer of medium from transfected HepG2 cells on the LDLR in untransfected HepG2 cells
Flow cytometric analysis of transfected HepG2 cells with respect to the amount of cell-associated PCSK9-FLAG using antibodies against the FLAG tag on permeabilized cells, revealed one major peak of untransfected cells and a minor, flattened peak of cells which had been transfected to a variable degree. This is shown for transfection with the D374Y mutant PCSK9 plasmid in Figure 3, and reflects a transfection efficiency of transiently transfected HepG2 cells of 10–20%. However, with respect to the cell surface LDLR and internalization of LDL in HepG2 cells transfected with the D374Y mutant, the entire population of cells was shifted towards lower amounts when compared with HepG2 cells transfected with empty plasmid. This is shown by the amount of LDL internalized in Figure 3.

View larger version (35K): [in this window] [in a new window]   Figure 3. Distribution of HepG2 cells transiently transfected with D374Y PCSK9 plasmid with respect to amount of LDL internalized using flow cytometric analysis. The figure shows the distribution of HepG2 cells transiently transfected with the D374Y-PCSK9 (upper panels) or empty plasmid (lower panels) as determined by flow cytometry. The amount of fluorescence per cell is shown on a logarithmic scale on the X-axis, and the number of cells is shown on the Y-axis. The amount of cell-associated PCSK9-FLAG of the transfected cells was determined in permeabilized cells using a FLAG antibody stained with Alexa 488 (left panels). The amount of LDL internalized was determined by incubating the transfected cells with DiD-labelled LDL (right panel).

 
If PCSK9 acted solely on the LDLR in the cell in which it has been transfected, one would have expected two populations of cells to be discernible with respect to the amount of LDL internalized. The cells expressing the transfected PCSK9 would constitute one population with a low amount of LDL internalized and the untransfected cells would constitute another population with a high amount of LDL internalized. Our observation that the entire population of transiently transfected HepG2 cells was shifted towards lower amounts LDL internalized, indicates that PCSK9 reduces the number of LDLR in transfected as well as in untransfected cells.

To further test this hypothesis, cultured HepG2 cells were transiently transfected with G106R or D374Y PCSK9 plasmids to study the effect of media from these cells on untransfected HepG2 cells after media transfer. Empty plasmid, WT-PCSK9 and the S386A mutant were used as controls. Twenty-four hours after transfection, the transfection solution was removed and the cells were washed once with phosphate-buffered saline (PBS). The cells were then given growth media containing 10% lipoprotein-deficient serum (LPDS). After a further 24 h, the media was removed and transferred to untransfected HepG2 cells. The amount of cell surface LDLR and internalization of LDL in untransfected HepG2 cells incubated with medium that had been transferred from transfected HepG2 cells, were determined by flow cytometry after 24 h of incubation (Fig. 4). In untransfected HepG2 cells incubated with medium from D374Y-PCSK9-transfected HepG2 cells, the amount of cell surface LDLR was 54% lower than in HepG2 cells incubated in medium from empty plasmid-transfected HepG2 cells. Similarly, internalization of LDL was reduced by 88% in HepG2 cells incubated in medium from D374Y-PCSK9-transfected HepG2 cells, when compared with cells incubated in medium from empty plasmid-transfected HepG2 cells. Incubation of untransfected HepG2 cells with medium from HepG2 cells transfected with the G106R mutant, reduced the amount of cell surface LDLR and internalization of LDL by 10% and 21%, respectively when compared with medium from HepG2 cells transfected with empty plasmid. The corresponding values for transfection with WT-PCSK9 were 24% and 34%, respectively. As can be seen from Figures 2 and 4, the effects of the different mutant PCSK9 plasmids on the amount of cell surface LDLR and internalization of LDL, were similar in transfected HepG2 cells (Fig. 2) and in untransfected HepG2 cells receiving medium from HepG2 cells transfected with the different PCSK9 plasmids (Fig. 4).

View larger version (12K): [in this window] [in a new window]   Figure 4. Effect of transfer of media from HepG2 cells transiently transfected with PCSK9 plasmids to untransfected HepG2 cells on the amount of cell surface LDLR and internalization of LDL. HepG2 cells were transiently transfected with empty plasmid, the catalytically inactive mutant S386A, WT-PCSK9, G106R or the D374Y mutant PCSK9 plasmids. After 24 h the transfection solution was removed and the cells were washed once in PBS before the cells were cultured in medium containing 5 mg/ml LPDS for another 24 h. The media were removed and placed on untransfected HepG2 cells. After 24 h incubation the untransfected cells were harvested and analyzed by flow cytometry to check the amount of cell surface LDLR and the amount of LDL internalized by the cells. The values are expressed relative to the amount of cell surface LDLR in untransfected cells incubated in medium from HepG2 cells incubated with empty plasmid. The results from a representative experiment are shown.

 
Experiments were performed to determine how fast the LDLR of untransfected HepG2 cells are degraded after transfer of medium from HepG2 cells transiently transfected with PCSK9 constructs. For these studies the D374Y mutant which we have shown to cause a greater reduction in the LDLR than WT-PCSK9 was compared with empty plasmid. The amount of cell surface LDLR on the untransfected HepG2 cells receiving medium from transfected cells was determined by flow cytometry. As can be seen from Figure 5, a reduction in the amount of cell surface LDLR was observed within 20 min and reached a maximum within 3 h. After 3 h of incubation, a 50% lower amount of cell surface LDLR was observed in untransfected HepG2 cells which had received medium from HepG2 cells transfected with the D374Y mutant, as compared with untransfected cells which had received medium from HepG2 cells transfected with empty plasmid.

View larger version (6K): [in this window] [in a new window]   Figure 5. Degradation of LDLR in untransfected HepG2 cells after transfer of the media from HepG2 cells transiently transfected with the D374Y PCSK9 plasmid. HepG2 cells were transiently transfected with the D374Y mutant PCSK9 plasmid or empty plasmid. After 24 h the transfection solution was removed and the cells were washed once in PBS before the cells were cultured in medium containing 5 mg/ml LPDS for another 24 h. The media was removed and placed on untransfected HepG2 cells in duplicate. After incubation for 20 min, 1, 3, 6 or 10 h, the untransfected cells incubated with medium from transfected cells were harvested and analyzed by flow cytometry to check the amount of cell surface LDLR. The values are expressed relative to the amount of cell surface LDLR in untransfected HepG2 cells incubated in fresh, unconditioned medium.

 

	DISCUSSION

TOP ABSTRACT Introduction RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
In order to identify the mechanism by which naturally occurring mutations in the PCSK9 gene affect cholesterol metabolism, we have performed studies of HepG2 cells transiently transfected with mutant PCSK9 constructs. These studies have shown that the four loss-of-function mutants R46L, G106R, N157K and R237W had a mean increase in the number of cell surface LDLR and in the internalization of LDL of 16% and 35%, respectively when compared with WT-PCSK9. The two gain-of-function mutations S127R and D374Y had a mean decrease in the number of cell surface LDLR and the internalization of LDL of 23% and 38%, respectively when compared with WT-PCSK9. Thus, as compared with WT-PCSK9, loss-of-function mutations increase the number of LDLR and gain-of-function mutations decrease the number of LDLR. However, a wide variation in the effect of the mutant PCSK9s on the LDLR was observed for mutations within the two mutation categories. These data which were supported by the results of western blot analysis of cells lysates of transfected HepG2 cells, indicate that mutations in the PCSK9 gene affect the LDLR-degrading property of PCSK9 to a varying extent, with the two extremes being the G106R mutation and the D374Y mutation.

The loss-of-function mutant G106R completely or almost completely failed to undergo autocatalytic cleavage in a manner similar to that of the autocatalytically inactive mutant S386A. This was surprising as the G106R mutation is in the prosegment and does not affect the catalytic triad Asp-186, His-226 and Ser-386, nor the oxyanion hole Asn-317 (1). However, other mutations in the prosegment of PCSK9 have previously been shown to reduce the autocatalytic activity of PCSK9 by more than 80% (8). Moreover, for other proproteins it has been shown that mutations in the prosegment may result in abolished enzyme activity because of abnormal folding of the propeptide (24,25). Consistent with the finding that the G106R mutant failed to undergo autocatalytic cleavage, no mature G106R mutant could be found in media of transfected HepG2 cells. The G106R mutant was also the mutant with the greatest increase in the amount of cell surface LDLR and in the amount of LDL internalized, of 32 and 68%, respectively when compared with WT-PCSK9. Furthermore, of the four loss-of-function studied, mutation G106R was the mutation with the most distinctive co-segregation with hypocholesterolemia (13). Thus, the G106R mutation seems to be the loss-of-function mutation with the most marked cholesterol-lowering effect which is apparently because of the mutant protein being retained in ER.

The gain-of-function mutant D374Y had a decrease in the amount of cell surface LDLR and the amount of LDL internalized of 36% and 68%, respectively when compared with WT-PCSK9. The corresponding figures for the S127R mutation were 10% and 7%, respectively. Thus, on the basis of the effect on the amount and function of LDLR in transiently transfected HepG2 cells, the D374Y mutation may cause a more severe hypercholesterolemia than the S127R mutation. This notion is supported by the findings of higher levels of total serum cholesterol before lipid-lowering drugs were started, in studies of D374Y-mutation carriers when compared with studies of S127R-mutation carriers. For D374Y-mutation carriers mean levels of total serum cholesterol of 13.6 (±2.9) mmol/l (23), 10.9 (±2.31) mmol/l (17) and 9.0 mmol/l (16) have been reported. For S127R-mutation carriers mean levels of total serum cholesterol of 9.7 (±0.93) mmol/l (20) and 9.4 (±1.75) mmol/l (15) have been reported. We have ourselves identified two S127R-mutation carriers with a mean level of total serum cholesterol of 9.6 (±2.26) mmol/l.

Conflicting data have been reported with respect to how gain-of-function mutations affect the amount of cell surface LDLR and the amount of LDL internalized by the LDLR in cell and animal studies. With respect to the S127R mutation, western blot analyses of cell lysates have shown reduced amounts of LDLR in stably transfected HepG2 cells when compared with WT-PCSK9 (8). In contrast, no difference in the amount of LDLR between S127R and WT-PCSK9 has been found in HepG2 cells or in mouse livers in studies where adenovirus-containing mutant S127R-PCSK9 has been used (7). Consistent with the reduced amount of LDLR in stably transfected HepG2 cells (8), decreased internalization of LDL was also observed (8). However, no significant reduction in the amount of LDLR was observed by S127R-PCSK9 constructs in stably transfected McArdle RH7777 cells when compared with WT-PCSK9 (18). For the D374Y mutation, analysis of the total amount of LDLR protein in McArdle RH7777 cells by western blot analysis, revealed only slightly reduced amounts of LDLR in transfected cells when compared with WT-PCSK9 (18).

The explanation for the conflicting data with respect to how gain-of-function mutations affect the LDLR, could partly be due to differences in the methods used. The use of adenovirus may possibly cause an extreme overexpression of WT-PCSK9 that may mask a further reduction of the LDLR by mutant gain-of-function PCSK9s. Moreover, the use of western blot analysis to determine the amount of LDLR of cell lysates may have a lower sensitivity to detect differences in LDLR than the use of flow cytometry to measure the amount of cell surface LDLR and internalization of LDL, which was used in our study. The effect of mutant PCSK9s on the LDLR could also differ between different cell types.

The effect on the autocatalytic cleavage of PCSK9 by gain-of-function and loss-of-function mutations, was variable. No marked difference between the two groups of mutations was observed. This is illustrated by a near normal autocatalytic cleavage of the gain-of-function mutation D374Y and the loss-of-function mutations R46L and R237W. Thus, except from the apparent autocatalytically inactive loss-of-function mutation G106R, it is impossible to determine how mutant PCSK9s affect degradation of the LDLR on the basis of their ability to undergo autocatalytic cleavage. Thus, different residues may be involved in cleavage of the different substrates for PCSK9.

Both we and others (1) have shown that PCSK9 is secreted, but it is not clear where PCSK9 or a factor acted upon by PCSK9, degrades the LDLR. The finding that PCSK9 degrades LDLR in transgenic mice lacking ARH (7), which encodes an adaptor protein required for internalization of the LDLR in liver (26), suggests internalization of LDLR is not required for PCSK9 to degrade the LDLR. Moreover, PCSK9 does not affect glycosylation of the LDLR which takes place in the Golgi apparatus (7,9). Thus, degradation of the LDLR may take place in a secretory compartment between the Golgi apparatus and the cell surface or at the cell surface, as has been suggested by Park et al. (7). Our studies have shown that medium from HepG2 cells transiently transfected with WT or the D374Y mutant PCSK9 plasmid, reduces the amount of cell surface LDLR and internalization of LDL when transferred to untransfected HepG2 cells. Thus, PCSK9 or a factor acted upon by PCSK9, is secreted from the transfected cells and degrades LDLR both in transfected and untransfected cells. As this effect was observed within 20 min of media transfer, degradation of LDLR by PCSK9 or by a factor acted upon by PCSK9, may predominantly take place on the cell surface.

The novel mutation R496Q in the PCSK9 gene was identified in a subject homozygous for apolipoprotein E-2 who presented with Type III hyperlipoproteinaemia. As only 2% of apolipoprotein E-2 homozygotes develop Type III hyperlipoproteinaemia (27), additional genetic or environmental factors are required to develop the full phenotype. It is possible that the R496Q mutation could represent such a genetic factor. Another mutation in codon 496 of PCSK9, R496W, has previously been observed in hypercholesterolaemic subjects who were also heterozygous for mutations in the LDLR gene (28,29). In those studies it was suggested that the R496W mutation in the PCSK9 gene causes a particularly severe phenotype in familial hypercholesterolemia heterozygotes. However, our studies indicate that the R496Q mutation does not affect the amount of cell surface LDLR or internalization of LDL.

We conclude that naturally occurring mutations in the PCSK9 gene cause hyper- or hypocholesterolemia by increased or decreased ability to degrade the LDLR, respectively. However, one cannot exclude the possibility that other mechanisms may be operating as well. Moreover, media from HepG2 cells transfected with the PCSK9 plasmid containing the D374Y gain-of-function mutation, reduce the number of LDLR after transfer to untransfected HepG2 cells. Thus, PCSK9 or a factor acted upon by PCSK9, is secreted from the cells and degrades the LDLR. As this effect could be observed within 20 min of media transfer, degradation of LDLR may predominantly take place on the cell membrane. However, further studies are needed to determine the exact mechanism by which PCSK9 degrades the LDLR.

	MATERIALS AND METHODS

TOP ABSTRACT Introduction RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Cell culture
HepG2 cells (ECACC, Wiltshire, UK) were cultured in Modified eagle media (MEM) (Gibco, Carlsbad, CA, USA), containing penicillin (50 U/ml), streptomycin (50 µg/ml), L-glutamine (2 mM) and 10% fetal calf serum (FCS) (Invitrogen, Carlsbad, CA, USA) in a humidified atmosphere (37°C, 5% CO₂).

Mutagenesis, cloning and expression of PCSK9
The WT-PCSK9 plasmid (pCMV-PCSK9-FLAG) containing the sequence for the FLAG epitope tag fused to the 3' end of the PCSK9 coding sequence, was a generous gift from Dr Jay D. Horton, University of Texas Southwestern Medical Center, Dallas, TX, USA. With respect to the reported normal genetic variation in the PCSK9 gene, the PCSK9 sequence of the pCMV-PCSK9-FLAG construct did not contain 287_289dupCTG in the leucine stretch in exon 1. With respect to the other reported normal genetic variants of the PCSK9 gene, codon 53 encoded alanine, codon 474 encoded isoleucine and codon 670 encoded glutamic acid. Mutations were introduced in pCMV-PCSK9-FLAG by oligonucleotide-directed mutagenesis using QuickChange XL Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. In addition to constructs containing the four loss-of-function mutations R46L, G106R, N157K or R237W and the two gain-of-function mutations S127R and D374Y, mutation R496Q identified in a Type III hyperlipoproteinaemia subject, but with unknown effect on cholesterol levels, was also included in the studies. All seven mutations have been identified in the Norwegian population. The primer sequences used for the mutagenesis are shown in Table 1. The integrity of each construct was confirmed by DNA sequencing. An empty plasmid, pcDNA3.1/myc his-c (Invitrogen) was used as a control in the transfection experiments.

View this table: [in this window] [in a new window]   Table 1. Sequences of primers used in the construction of the mutant PCSK9 plasmids

 
HepG2 cells were seeded in 25 cm² collagen-coated flasks (BD Biosciences, San Diego, CA, USA) or in 6 well collagen-coated plates (BD Biosciences) and grown to 80% confluency. They were then transiently transfected with the different PCSK9 constructs using Fugene-6 Reagent (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. A ratio of 6:1 reagent to plasmid DNA was used during transfection. Transfection efficiency was in the range of 10–20%. Cells were used for experiments 2 days after transfection.

Western blot analysis of transfected HepG2 cells
The HepG2 cells were grown and transfected in 25 cm² collagen-coated flasks. Twenty-four hours after transfection, the HepG2 cells were washed with PBS and the media was replaced with MEM containing 5 mg/ml LPDS for 24 h to increase the expression of the LDLR. The cells were then harvested by scraping, pelleted by centrifugation, lysed using in-house lysis buffer (0.1% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl, pH 7.4) containing Complete Protease Inhibitor Cocktail (Roche Diagnostics), vortexed for 30 min at 4°C and centrifuged at 14 000 rpm for 15 min at 4°C to remove cell debris. Protein concentration in the supernatant was determined by BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) using bovine serum albumin (BSA) as a standard. Cell lysate equivalent to 40 µg protein was applied in each well and subjected to SDS–PAGE on a 4–20% Tris–HCl Criterion Precast Gel (Bio-Rad, Hercules, CA, USA). After separation by electrophoresis, the proteins were electrophoretically transferred to an Immun-Blot PVDF Membrane for Protein Blotting (Bio-Rad). Non-specific binding sites were blocked in 5% Blotting Grade Blocker Non-Fat Dry Milk (Bio-Rad) for 1 h or overnight, and the membrane was immunostained with rabbit IgG anti-LDLR (1:1000, PROGENE) or mouse IgG anti-FLAG (1.5 µg/ml, Sigma–Aldrich Corp., St Louis, MO, USA) for 1 h for detection of LDLR and PCSK9-FLAG, respectively. The membrane was then washed three times in Tris-buffered saline with 0.2% Tween-20 (Sigma–Aldrich Corp.) and incubated for 1 h with sheep anti-rabbit IgG or sheep anti-mouse IgG conjugated with horseradish peroxidase (1:10 000, Amersham Biosciences, Little Calfont, UK). After three more washing steps, the bands were visualized with SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology) and chemiluminescence was detected on a ChemiDoc XRS (Bio-Rad). For western blot analysis of PCSK9 from medium of HepG2 cells transfected with PCSK9 constructs, 200 µl of medium was precipitated by trichloroacetic acid (TCA) (10%) before loading.

Analyses of cell surface LDLR, internalization of LDL and cell-associated PCSK9-FLAG
A FACS Canto flow cytometer (BD Biosciences) was used to study the amounts of cell surface LDLR, the internalization of LDL by the LDLR and the amount of cell-associated PCSK9-FLAG. Gates were set to include only live singlet cells. Dead cells were identified by propidium iodide staining (1 µg/µl) (Sigma–Aldrich Corp.). To study the number of cell surface LDLR, the cells were incubated with anti-LDLR IgG-C7 mouse monoclonal antibody (1:20, Progen Biotechnik GmbH, Heidelberg, Germany) for 1 h at 4°C. Cells were then washed three times in PBS with 0.5% BSA and incubated with Alexa Fluor^® 488 goat anti-mouse IgG (H+L) (1:400, Molecular Probes, Eugene, OR, USA) for 30 min at 4°C. The washing steps were repeated twice. To study the LDLR-dependent internalization of LDL, LDL (10 µg/ml) fluorescently labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD) (Molecular Probes) was added to each well and cells were incubated for 2 h at 37°C. After incubation, cells were washed three times with PBS containing 0.5% BSA before flow cytometric analysis was performed.

To study the amount of cell-associated PCSK9-FLAG, cells were permeabilized in 3 ml of 70% ethanol for 30 min at –20°C. The cells were then incubated with mouse anti-FLAG monoclonal antibody (2.5 µg/ml) (Sigma–Aldrich Corp.) for 1 h at 4°C. Cells were washed three times in PBS with 0.5% BSA and incubated with Alexa Fluor^® 488 goat anti-mouse IgG (H+L) (1:400, Molecular Probes) for 30 min at 4°C. Cells were washed as described above and analyzed by flow cytometry.

Conflict of Interest statement. There are no conflicts of interest.

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TOP ABSTRACT Introduction RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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