Flavonoids regulate LDLR through different mechanisms tied to their specific structures.

Flavonoids, polyphenolic compounds found in plant-based diets, are associated with reduced risk of cardiovascular disease and longevity. These components are reported to reduce plasma levels of low-density lipoprotein (LDL) through an upregulation of the LDL receptor (LDLR), but the mechanism is still largely unknown. In this study, we have systematically screened the effect of 12 flavonoids from six different flavonoid subclasses on the effect on LDLR. This paper provides an in-depth analysis on how these flavonoids affect LDLR regulation and functionality. We found that most but not all of the tested flavonoids increased LDLR mRNA levels. Surprisingly, this increase was attributed to different regulatory mechanisms, such as enhanced LDLR promoter activity, LDLR mRNA stabilization, or LDLR protein stabilization, of which specific effectual parts of the flavonoid molecular structure could be assigned. These types of comparative analysis of various flavonoids enhance clarity and deepen the understanding of how the different structures of flavonoids affect LDLR regulation. Our data offer useful insights that may guide future research in developing therapeutic approaches for cardiovascular health.

Functional characterization of missense variants affecting the extracellular domains of ABCA1 using a fluorescence-based assay.

Excess cholesterol originating from nonhepatic tissues is transported within HDL particles to the liver for metabolism and excretion. Cholesterol efflux is initiated by lipid-free or lipid-poor apolipoprotein A1 interacting with the transmembrane protein ABCA1, a key player in cholesterol homeostasis. Defective ABCA1 results in reduced serum levels of HDL cholesterol, deposition of cholesterol in arteries, and an increased risk of early onset CVD. Over 300 genetic variants in ABCA1 have been reported, many of which are associated with reduced HDL cholesterol levels. Only a few of these have been functionally characterized. In this study, we have analyzed 51 previously unclassified missense variants affecting the extracellular domains of ABCA1 using a sensitive, easy, and low-cost fluorescence-based assay. Among these, only 12 variants showed a distinct loss-of-function phenotype, asserting their direct association with severe HDL disorders. These findings emphasize the crucial role of functional characterization of genetic variants in pathogenicity assessment and precision medicine. The functional rescue of ABCA1 loss-of-function variants through proteasomal inhibition or by the use of the chemical chaperone 4-phenylbutyric acid was genotype specific. Genotype-specific responses were also observed for the ability of apolipoprotein A1 to stabilize the different ABCA1 variants. In view of personalized medicine, this could potentially form the basis for novel therapeutic strategies.

Molecular genetic testing and measurement of levels of GPIHBP1 autoantibodies in patients with severe hypertriglyceridemia: The importance of identifying the underlying cause of hypertriglyceridemia.

BACKGROUND: Severe hypertriglyceridemia can be caused by pathogenic variants in genes encoding proteins involved in the metabolism of triglyceride-rich lipoproteins. A key protein in this respect is lipoprotein lipase (LPL) which hydrolyzes triglycerides in these lipoproteins. Another important protein is glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) which transports LPL to the luminal side of the endothelial cells. OBJECTIVE: Our objective was to identify a genetic cause of hypertriglyceridemia in 459 consecutive unrelated subjects with levels of serum triglycerides ≥20 mmol/l. These patients had been referred for molecular genetic testing from 1998 to 2021. In addition, we wanted to study whether GPIHBP1 autoantibodies also were a cause of hypertriglyceridemia. METHODS: Molecular genetic analyses of the genes encoding LPL, GPIHBP1, apolipoprotein C2, lipase maturation factor 1 and apolipoprotein A5 as well as apolipoprotein E genotyping, were performed in all 459 patients. Serum was obtained from 132 of the patients for measurement of GPIHBP1 autoantibodies approximately nine years after molecular genetic testing was performed. RESULTS: A monogenic cause was found in four of the 459 (0.9%) patients, and nine (2.0%) patients had dyslipoproteinemia due to homozygosity for apolipoprotein E2. One of the 132 (0.8%) patients had GPIHBP1 autoantibody syndrome. CONCLUSION: Only 0.9% of the patients had monogenic hypertriglyceridemia, and only 0.8% had GPIHBP1 autoantibody syndrome. The latter figure is most likely an underestimate because serum samples were obtained approximately nine years after hypertriglyceridemia was first identified. There is a need to implement measurement of GPIHBP1 autoantibodies in clinical medicine to secure that proper therapeutic actions are taken.

Variants in the CETP gene affect levels of HDL cholesterol by reducing the amount, and not the specific lipid transfer activity, of secreted CETP.

BACKGROUND: Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters in plasma from high density lipoprotein (HDL) to very low density lipoprotein and low density lipoprotein. Loss-of-function variants in the CETP gene cause elevated levels of HDL cholesterol. In this study, we have determined the functional consequences of 24 missense variants in the CETP gene. The 24 missense variants studied were the ones reported in the Human Gene Mutation Database and in the literature to affect HDL cholesterol levels, as well as two novel variants identified at the Unit for Cardiac and Cardiovascular Genetics, Oslo University Hospital in subjects with hyperalphalipoproteinemia. METHODS: HEK293 cells were transiently transfected with mutant CETP plasmids. The amounts of CETP protein in lysates and media were determined by Western blot analysis, and the lipid transfer activities of the CETP variants were determined by a fluorescence-based assay. RESULTS: Four of the CETP variants were not secreted. Five of the variants were secreted less than 15% compared to the WT-CETP, while the other 15 variants were secreted in varying amounts. There was a linear relationship between the levels of secreted protein and the lipid transfer activities (r = 0.96, p<0.001). Thus, the secreted variants had similar specific lipid transfer activities. CONCLUSION: The effect of the 24 missense variants in the CETP gene on the lipid transfer activity was mediated predominantly by their impact on the secretion of the CETP protein. The four variants that prevented CETP secretion cause autosomal dominant hyperalphalipoproteinemia. The five variants that markedly reduced secretion of the respective variants cause mild hyperalphalipoproteinemia. The majority of the remaining 15 variants had minor effects on the secretion of CETP, and are considered neutral genetic variants.

Missense mutation Q384K in the APOB gene affecting the large lipid transfer module of apoB reduces the secretion of apoB-100 in the liver without reducing the secretion of apoB-48 in the intestine.

BACKGROUND: Molecular genetic testing of patients with hypobetalipoproteinemia may identify a genetic cause that can form the basis for starting proper therapy. Identifying a genetic cause may also provide novel data on the structure-function relationship of the mutant protein. OBJECTIVE: To identify a genetic cause of hypobetalipoproteinemia in a patient with levels of low density lipoprotein cholesterol at the detection limit of 0.1 mmol/l. METHODS: DNA sequencing of the translated exons with flanking intron sequences of the genes adenosine triphosphate-binding cassette transporter 1, angiopoietin-like protein 3, apolipoprotein B, apolipoprotein A1, lecithin-cholesterol acyltransferase, microsomal triglyceride transfer protein and proprotein convertase subtilisin/kexin type 9. RESULTS: The patient was homozygous for mutation Q384K (c.1150C>A) in the apolipoprotein B gene, and this mutation segregated with hypobetalipoproteinemia in the family. Residue Gln384 is located in the large lipid transfer module of apoB that has been suggested to be important for lipidation of apolipoprotein B through interaction with microsomal triglyceride transfer protein. Based on measurements of serum levels of triglycerides and apolipoprotein B-48 after an oral fat load, we conclude that the patient was able to synthesize apolipoprotein B-48 in the intestine in a seemingly normal fashion. CONCLUSION: Our data indicate that mutation Q384K severely reduces the secretion of apolipoprotein B-100 in the liver without reducing the secretion of apolipoprotein B-48 in the intestine. Possible mechanisms for the different effects of this and other missense mutations affecting the large lipid transfer module on the two forms of apoB are discussed.

Bone morphogenetic protein 1 cleaves the linker region between ligand-binding repeats 4 and 5 of the LDL receptor and makes the LDL receptor non-functional.

The cell-surface low-density lipoprotein receptor (LDLR) internalizes low-density lipoprotein (LDL) by receptor-mediated endocytosis and plays a key role in the regulation of plasma cholesterol levels. The ligand-binding domain of the LDLR contains seven ligand-binding repeats of approximately 40 residues each. Between ligand-binding repeats 4 and 5, there is a 10-residue linker region that is subject to enzymatic cleavage. The cleaved LDLR is unable to bind LDL. In this study, we have screened a series of enzyme inhibitors in order to identify the enzyme that cleaves the linker region. These studies have identified bone morphogenetic protein 1 (BMP1) as being the cleavage enzyme. This conclusion is based upon the use of the specific BMP1 inhibitor UK 383367, silencing of the BMP1 gene by the use of siRNA or CRISPR/Cas9 technology and overexpression of wild-type BMP1 or the loss-of-function mutant E214A-BMP1. We have also shown that the propeptide of BMP1 has to be cleaved at RSRR120↓ by furin-like proprotein convertases for BMP1 to have an activity towards the LDLR. Targeting BMP1 could represent a novel strategy to increase the number of functioning LDLRs in order to lower plasma LDL cholesterol levels. However, a concern by using BMP1 inhibitors as cholesterol-lowering drugs could be the risk of side effects based on the important role of BMP1 in collagen assembly.

Strategies to prevent cleavage of the linker region between ligand-binding repeats 4 and 5 of the LDL receptor.

A main strategy for lowering plasma low-density lipoprotein (LDL) cholesterol levels is to increase the number of cell-surface LDL receptors (LDLRs). This can be achieved by increasing the synthesis or preventing the degradation of the LDLR. One mechanism by which an LDLR becomes non-functional is enzymatic cleavage within the 10 residue linker region between ligand-binding repeats 4 and 5. The cleaved LDLR has only three ligand-binding repeats and is unable to bind LDL. In this study, we have performed cell culture experiments to identify strategies to prevent this cleavage. As a part of these studies, we found that Asp193 within the linker region is critical for cleavage to occur. Moreover, both 14-mer synthetic peptides and antibodies directed against the linker region prevented cleavage. As a consequence, more functional LDLRs were observed on the cell surface. The observation that the cleaved LDLR was present in extracts from the human adrenal gland indicates that cleavage of the linker region takes place in vivo. Thus, preventing cleavage of the LDLR by pharmacological measures could represent a novel lipid-lowering strategy.

Characterization of the mechanisms by which missense mutations in the lysosomal acid lipase gene disrupt enzymatic activity.

Hydrolysis of cholesteryl esters and triglycerides in the lysosome is performed by lysosomal acid lipase (LAL). In this study we have investigated how 23 previously identified missense mutations in the LAL gene (LIPA) (OMIM# 613497) affect the structure of the protein and thereby disrupt LAL activity. Moreover, we have performed transfection studies to study intracellular transport of the 23 mutants. Our main finding was that most pathogenic mutations result in defective enzyme activity by affecting the normal folding of LAL. Whereas, most of the mutations leading to reduced stability of the cap domain did not alter intracellular transport, nearly all mutations that affect the stability of the core domain gave rise to a protein that was not efficiently transported from the endoplasmic reticulum (ER) to the Golgi apparatus. As a consequence, ER stress was generated that is assumed to result in ER-associated degradation of the mutant proteins. The two LAL mutants Q85K and S289C were selected to study whether secretion-defective mutants could be rescued from ER-associated degradation by the use of chemical chaperones. Of the five chemical chaperones tested, only the proteasomal inhibitor MG132 markedly increased the amount of mutant LAL secreted. However, essentially no increased enzymatic activity was observed in the media. These data indicate that the use of chemical chaperones to promote the exit of folding-defective LAL mutants from the ER, may not have a great therapeutic potential as long as these mutants appear to remain enzymatically inactive.

Prevalence of cholesteryl ester storage disease among hypercholesterolemic subjects and functional characterization of mutations in the lysosomal acid lipase gene.

Lysosomal acid lipase hydrolyzes cholesteryl esters and triglycerides contained in low density lipoprotein. Patients who are homozygous or compound heterozygous for mutations in the lysosomal acid lipase gene (LIPA), and have some residual enzymatic activity, have cholesteryl ester storage disease. One of the clinical features of this disease is hypercholesterolemia. Thus, patients with hypercholesterolemia who do not carry a mutation as a cause of autosomal dominant hypercholesterolemia, may actually have cholesteryl ester storage disease. In this study we have performed DNA sequencing of LIPA in 3027 hypercholesterolemic patients who did not carry a mutation as a cause of autosomal dominant hypercholesterolemia. Functional analyses of possibly pathogenic mutations and of all mutations in LIPA listed in The Human Genome Mutation Database were performed to determine the pathogenicity of these mutations. For these studies, HeLa T-REx cells were transiently transfected with mutant LIPA plasmids and Western blot analysis of cell lysates was performed to determine if the mutants were synthesized in a normal fashion. The enzymatic activity of the mutants was determined in lysates of the transfected cells using 4-methylumbelliferone-palmitate as the substrate. A total of 41 mutations in LIPA were studied, of which 32 mutations were considered pathogenic by having an enzymatic activity <10% of normal. However, none of the 3027 hypercholesterolemic patients were homozygous or compound heterozygous for a pathogenic mutation. Thus, cholesteryl ester storage disease must be a very rare cause of hypercholesterolemia in Norway.

Mutations affecting the transmembrane domain of the LDL receptor: impact of charged residues on the membrane insertion.

Familial hypercholesterolemia (FH) is caused by mutations in the low density lipoprotein receptor (LDLR) gene. To study the impact of mutations affecting the hydrophobic transmembrane domain of the LDLR, each of the 22 amino acids of the transmembrane domain was individually mutated to arginine. The more centrally in the transmembrane domain an arginine was located, the lower amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum were observed. This led to lower amounts of the 160 kDa mature LDLR on the cell surface. For the mutants V797R-LDLR, L798R-LDLR and L799R-LDLR a proportion of full-length receptors including the transmembrane and cytoplasmic domains, was secreted into the endoplasmic reticulum lumen to appear in the culture medium. When the transmembrane domain of the epidermal growth factor receptor (EGFR) was replaced by that of the mutant L799R-LDLR, similar effects were observed for the EGFR as for L799R-LDLR. Introducing arginines in the transmembrane domain of the LDLR also affected metalloproteinase cleavage of the ectodomain and γ-secretase cleavage within the transmembrane domain. The most likely explanation for the low amounts of the 120 kDa precursor is that a basic residue in the hydrophobic transmembrane domain prevents the mutant LDLR from being inserted in the endoplasmic reticulum membrane from the Sec61 translocon complex. As a consequence, quality control systems could be activated. However, our data indicate that proteasomal degradation, lysosomal degradation, autophagy or ectodomain cleavage were not the underlying mechanism for degradation of these mutant LDLRs.

Variable phenotypic expression of nonsense mutation p.Thr5* in the APOE gene.

Subjects with hypercholesterolemia who do not carry a mutation in the low density lipoprotein receptor gene, in the apolipoprotein B gene or in the proprotein convertase subtilisin/kexin type 9 gene, could possible carry a mutation in the apolipoprotein E (APOE) gene. DNA from 844 unrelated hypercholesterolemic subjects who did not carry a mutation in any of the three above mentioned genes, was subjected to DNA sequencing of the APOE gene. Two subjects were found to be heterozygous for mutation p.Thr5*. This mutation which generates a stop codon in the signal peptide, is assumed to prevent the synthesis of APOE. Family studies revealed that the mutation was carried on an APOE4 allele in both families. In one of the families only those who had an APOE2 allele as the second allele, had hypercholesterolemia. These were functionally hemizygous for APOE2 and presented with a Type III hyperlipoproteinemia phenotype. However, in the second family, hypercholesterolemia was observed in the index patient who had APOE3 as the second allele, but not in four heterozygous family members who also had APOE3 as the second allele. These findings underscore that the phenotypic expression of mutations in the APOE gene is variable and that the trait exhibits reduced penetrance.

Studies of the autoinhibitory segment comprising residues 31-60 of the prodomain of PCSK9: Possible implications for the mechanism underlying gain-of-function mutations.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and is internalized as a complex with the LDLR. In the acidic milieu of the sorting endosome, PCSK9 remains bound to the LDLR and prevents the LDLR from folding over itself to adopt a closed conformation. As a consequence, the LDLR fails to recycle back to the cell membrane. Even though it is the catalytic domain of PCSK9 that interacts with the LDLR at the cell surface, the structurally disordered segment consisting of residues 31-60 and which is rich in acidic residues, has a negative effect both on autocatalytic cleavage and on the activity of PCSK9 towards the LDLR. Thus, this unstructured segment represents an autoinhibitory domain of PCSK9. One may speculate that post-translational modifications within residues 31-60 may affect the inhibitory activity of this segment, and represent a mechanism for fine-tuning the activity of PCSK9 towards the LDLR. Our data indicate that the inhibitory effect of this unstructured segment results from an interaction with basic residues of the catalytic domain of PCSK9. Mutations in the catalytic domain which involve charged residues, could therefore be gain-of-function mutations by affecting the positioning of this segment.

Mutation p.L799R in the LDLR, which affects the transmembrane domain of the LDLR, prevents membrane insertion and causes secretion of the mutant LDLR.

Mutations in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolemia (FH). The mechanism by which mutations in the LDLR affecting the transmembrane domain of the receptor cause FH has not been thoroughly investigated. In this study, we have selected 12 naturally occurring mutations affecting the transmembrane domain and studied their effect on the LDLR. The main strategy has been to transiently transfect HepG2 cells with mutant LDLR plasmids and to study the mutant LDLRs in cell lysates and in media by western blot analysis. The most striking finding was that mutation p.L799R led to secretion of the entire 160 kDa mature L799R-LDLR. Residue 799Leu is in the middle of the 22-residue transmembrane domain, and introduction of a basic residue in the hydrophobic core of the transmembrane domain could prevent L799R-LDLR from being correctly recognized and integrated in the membrane by the Sec61 translocon complex. This would then lead to translocation of the entire L799R-LDLR into the lumen of the endoplasmic reticulum. Mutation p.L799R should be considered a member of a separate class of FH-causing mutations that affects the insertion of the LDLR in the cell membrane.

Mutation G805R in the transmembrane domain of the LDL receptor gene causes familial hypercholesterolemia by inducing ectodomain cleavage of the LDL receptor in the endoplasmic reticulum.

More than 1700 mutations in the low density lipoprotein receptor (LDLR) gene have been found to cause familial hypercholesterolemia (FH). These are commonly divided into five classes based upon their effects on the structure and function of the LDLR. However, little is known about the mechanism by which mutations in the transmembrane domain of the LDLR gene cause FH. We have studied how the transmembrane mutation G805R affects the function of the LDLR. Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum. This led to reduced amounts of the mature 160 kDa LDLR at the cell surface. However, significant amounts of a secreted 140 kDa G805R-LDLR ectodomain fragment was observed in the culture media. Treatment of the cells with the metalloproteinase inhibitor batimastat largely restored the amounts of the 120 and 160 kDa forms in cell lysates, and prevented secretion of the 140 kDa ectodomain fragment. Together, these data indicate that a metalloproteinase cleaved the ectodomain of the 120 kDa precursor G805R-LDLR in the endoplasmic reticulum. It was the presence of the polar Arg805 and not the lack of Gly805 which led to ectodomain cleavage. Arg805 also prevented γ-secretase cleavage within the transmembrane domain. It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

PCSK9 acts as a chaperone for the LDL receptor in the endoplasmic reticulum.

PCSK9 (proprotein convertase subtilisin/kexin type 9) binds to the LDLR (low-density lipoprotein receptor) at the cell surface and disrupts recycling of the LDLR. However, PCSK9 also interacts with the LDLR in the ER (endoplasmic reticulum). In the present study we have investigated the role of PCSK9 for the transport of the LDLR from the ER to the cell membrane. A truncated LDLR consisting of the ectodomain (ED-LDLR) was used for these studies to avoid PCSK9-mediated degradation of the LDLR. The amount of secreted ED-LDLR was used as a measure of the amount of ED-LDLR transported from the ER. From co-transfection experiments of various PCSK9 and ED-LDLR plasmids, PCSK9 increased the amount of WT (wild-type) ED-LDLR in the medium, but not of an ED-LDLR lacking the EGF (epidermal growth factor)-A repeat or of a Class 2a mutant ED-LDLR which fails to exit the ER. Mutant PCSK9s which failed to undergo autocatalytic cleavage or failed to exit the ER, failed to increase the amount of WT-ED-LDLR in the medium. These mutants also reduced the amount of WT-ED-LDLR intracellularly, which could partly be prevented by the proteasome inhibitor lactacystine. WT-ED-LDLR promoted autocatalytic cleavage of pro-PCSK9. The findings of the present study indicate that the binding of WT-ED-LDLR to pro-PCSK9 in the ER promotes autocatalytic cleavage of PCSK9, and autocatalytically cleaved PCSK9 acts as a chaperone to promote the exit of WT-ED-LDLR from the ER.

Mutations in the SORT1 gene are unlikely to cause autosomal dominant hypercholesterolemia.

OBJECTIVE: To study whether mutations in the SORT1 gene could be a cause of autosomal dominant hypercholesterolemia and to study the effect of sortilin on the binding and internalization of low density lipoprotein (LDL). METHODS: 842 unrelated hypercholesterolemic subjects without mutations in genes known to cause autosomal dominant hypercholesterolemia, were screened for mutations in the SORT1 gene by DNA sequencing. Transfections of wild-type or mutant SORT1 plasmids in HeLa T-REx cells and the use of siRNA were used to study the effect of sortilin on the number of cell-surface LDL receptors and on the binding and internalization of LDL. RESULTS: A total of 45 mutations in the SORT1 gene were identified of which 15 were missense mutations. Eight of these were selected for in vitro studies, of which none had a major impact on the amount of LDL bound to the cell surface. There was a positive correlation between the amount of sortilin on the cell surface and the amount of LDL bound. The observation that a mutant sortilin which is predominantly found on the cell surface rather than in post-Golgi compartments, bound very high amounts of LDL, indicates that sortilin does not increase the binding of LDL through an intracellular mechanism. Rather, our data indicate that sortilin binds LDL on the cell surface. CONCLUSION: Even though sortilin binds and internalizes LDL by receptor-mediated endocytosis, mutations in the SORT1 gene are unlikely to cause autosomal dominant hypercholesterolemia and may only have a marginal effect on plasma LDL cholesterol levels.

Serum levels of proprotein convertase subtilisin/kexin type 9 in subjects with familial hypercholesterolemia indicate that proprotein convertase subtilisin/kexin type 9 is cleared from plasma by low-density lipoprotein receptor-independent pathways.

Secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low-density lipoprotein receptor (LDLR) at the cell surface and disrupts the normal recycling of the LDLR. When human PCSK9 is injected into LDLR-deficient mice, PCSK9 is still rapidly cleared by the liver. This finding may suggest that PCSK9 is physiologically also cleared by receptors other than the LDLR. An alternative explanation could be that PCSK9 has undergone modifications during purification and is cleared by scavenger receptors on liver endothelial sinusoidal cells when injected into mice. If the only mechanism for clearing PCSK9 in humans is through the LDLR, one would expect that differences in the number of LDLRs would affect the plasma levels of low-density lipoprotein cholesterol (LDLC) and PCSK9 in a similar fashion. In this study, levels of LDLC and PCSK9 were measured in familial hypercholesterolemia (FH) homozygotes, FH heterozygotes, and normocholesterolemic subjects. The ratio between the levels of LDLC and PCSK9 was 1.7-fold higher in FH heterozygotes and 3-fold higher in FH homozygotes than in the normocholesterolemic subjects. Thus, defective LDLRs have a greater impact on the levels of LDLC than on the levels of PCSK9. By assuming that the rate of PCSK9 synthesis is similar in the 3 groups, this finding suggests that in humans, plasma PCSK9 is also cleared by LDLR-independent mechanisms.

Characterization of a naturally occurring degradation product of the LDL receptor.

In this study we have characterized a naturally occurring truncated form of the low density lipoprotein receptor (LDLR). Western blot analysis of transfected cells indicated that the truncated form (∆N-LDLR) is a degradation product of the full-length LDLR generated by cleavage in the linker region between ligand-binding repeats 4 and 5 of the ligand-binding domain. The cleavage of the linker was not caused by components of the culture media, as heat inactivation of the media did not prevent cleavage. Rather, it is assumed that cleavage was caused by an enzyme secreted from the cells. Biotinylation experiments showed that ∆N-LDLR is located on the cell surface and is detectable approximately 5 h after synthesis of the full-length LDLR. Flow cytometric analysis showed that ∆N-LDLR was not able to bind and internalize low density lipoprotein (LDL). ∆N-LDLR appeared to be equally stable as the full-length LDLR. Thus, generation of ∆N-LDLR does not appear to be the first signal for degradation of the LDLR. The existence of two functionally different populations of LDLRs on the cell surface, of which ∆N-LDLR constitutes 28%, must be taken into account when interpreting results of experiments to study LDLRs on the cell surface. Furthermore, if the cleavage of the linker between ligand-binding repeats 4 and 5 could be prevented by an enzyme inhibitor, this could represent a novel therapeutic strategy to increase the number of functioning LDLRs and thereby decrease the levels of plasma LDL cholesterol.

Interaction between the ligand-binding domain of the LDL receptor and the C-terminal domain of PCSK9 is required for PCSK9 to remain bound to the LDL receptor during endosomal acidification.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the epidermal growth factor homology domain repeat A of the low-density lipoprotein receptor (LDLR) at the cell surface and disrupts recycling of the internalized LDLR. As a consequence, the LDLR is rerouted to the lysosomes for degradation. Although PCSK9 may bind to an LDLR lacking the ligand-binding domain, at least three ligand-binding repeats of the ligand-binding domain are required for PCSK9 to reroute the LDLR to the lysosomes. In this study, we have studied the binding of PCSK9 to an LDLR with or without the ligand-binding domain at increasingly acidic conditions in order to mimic the milieu of the LDLR:PCSK9 complex as it translocates from the cell membrane to the sorting endosomes. These studies have shown that PCSK9 is rapidly released from an LDLR lacking the ligand-binding domain at pH in the range of 6.9-6.1. A similar pattern of release at acidic pH was also observed for the binding to the normal LDLR of mutant PCSK9 lacking the C-terminal domain. Together these data indicate that an interaction between the negatively charged ligand-binding domain of the LDLR and the positively charged C-terminal domain of PCSK9 is required for PCSK9 to remain bound to the LDLR during the early phase of endosomal acidification as the LDLR translocates from the cell membrane to the sorting endosome.

Characterization of residues in the cytoplasmic domain of the LDL receptor required for exit from the endoplasmic reticulum.

Newly synthesized low density lipoprotein receptors (LDLRs) exit the endoplasmic reticulum (ER) as the first step in the secretory pathway. In this study we have generated truncating deletions and substitutions within the 50 amino acid cytoplasmic domain of the LDLR in order to identify residues required for the exit from the ER. Western blot analysis was used to determine the relative amounts of the 120 kDa precursor form of the LDLR located in the ER and the 160 kDa mature form that has exited the ER. These studies have shown that the exit of an LDLR lacking the cytoplasmic domain, is markedly reduced. Moreover, the longer the cytoplasmic domain, the more efficient is the exit from the ER. At least 30 residues were required for the LDLR to efficiently exit the ER. Mutations in the two di-acidic motifs ExE(814) and/or ExD(837) had only a small effect on the exit from the ER. The requirement for a certain length of the cytoplasmic domain for efficient exit from the ER, could reflect the distance needed to interact with the COPII complex of the ER membrane or the requirement for the LDLR to undergo dimerization.