LDLRs and Genetic Causes of LDL Elevation

Question 1

Summarise the genetic and molecular structure of the LDLR.

Answer 1

These 860AA, 93kDa proteins that are formed of five domains, from the N-terminal extracellular domain to the C-terminal intracellular ones.

The gene is made up of 18 exons spanning 45kbp, producing a 5.3kbp transcript.

Question 2

Describe domain I of the LDLR.

Answer 2

The N-terminal binding region has 7 40-AA Class A repeats (~50% identical), each with 6 cysteine residues that form disulphide bonds.

The original deletion studies showed that ApoB100 (hence LDL) binding requires only repeats 2-7, while only repeat 5 is needed for ApoE. However, this has now been contested, as it has been shown that deletion of repeats 1 and 2 confers familial hypercholesterolaemia.

Question 3

Describe domain II of the LDLR.

Answer 3

This is a 400AA epidermal growth factor (EGF) precursor homology domain, named due to the ~30% homology with the EGF precursor gene.

This includes three “growth factor” repeats, A, B and C. A and B have binding activity, and are separated from C by a YWTD repeat region that forms a beta-propeller motif important for receptor recycling and releasing bound LDL in response to lower pH in the endosome.

Question 4

Describe domain III of the LDLR.

Answer 4

This domain contains 2 N-linked and 18 O-linked oligosaccharides, which appear to have little function, as attested by its deletion, but may act as a spacer to ensure the binding region is beyond the extracellular matrix.

Question 5

Describe domain IV of the LDLR.

Answer 5

This is the single helical transmembrane domain, comprising a chain of 22 hydrophobic amino acid residues.

Question 6

Describe domain V of the LDLR.

Answer 6

The internal C-terminal domain (50 residues) contains a signal sequence (NPXY) essential for receptor-ligand (LDL) internalization. This interacts with clathrin, concentrating the LDLRs in the coated pits.

Question 7

What triggers release of the LDL from the LDLR?

Answer 7

The release of the LDL when in the acidic endosome is thought to be mediated by the β-propellor region of the EGF precursor domain, which contains a large number of histidine residues that allow it to act as a sensor for the acidity. These have no net charge at physiological pH (7.4) but in the endosome (pH 5.3) they are protonated, leading to a conformational change in the domain.

A crystal structure of the extracellular domain at pH 5.3 showed the β-propellor domain interacting with the N-terminal Binding Region (repeat 4 and 5), hence it is thought that the change in conformation allows it to displace the LDL.

Question 8

What is familial hypercholesterolaemia?

Answer 8

This is a family of genetic disorders characterised by incredibly high levels of plasma LDL, which leads to early onset CVD/CHD/atherosclerosis and xanthomas (deposition of LDL-derived cholesterol under the skin and in tendons).

In 90% of cases this is caused by mutations in the LDLR gene preventing proper uptake of LDLs.

Question 9

What is the frequency and severity of FH?

Answer 9

Those heterozygous for the disorder tend to have 2-4 fold higher blood LDL levels, with 5-10 fold higher in homozygous individuals.

An autosomal dominant disorder, this has incidence (in Europe) of 1 in 500 (htz) and 1 in 106 (hmz); though recent next-gen sequencing studies suggest that it may in fact be as high as 1 in 200 and 160,000 respectively.

The high frequency is thought to be due to Alu repeats nearby and within the gene leading to instability through Alu-mediated recombination.

Question 10

How are LDLR muations described and classified?

Answer 10

Around 90% of these are SNPs or small (

Question 11

What are class 1 LDLR mutations?

Answer 11

No or negligible LDL-R synthesis in the endoplasmic reticulum (ER) – hence no surface expression.

Question 12

What are class 2 LDLR mutations?

Answer 12

Defective transport to the Golgi body for glycosylation and sorting. About 50% of all LDL-R mutations are class 2. Usually, the receptor movement is delayed, rather than halted, and for a few mutants lacking the transmembrane domain the receptor is secreted. When this happens the mutation is labelled subclass 2B.

Question 13

What is an example of a class 2A LDLR mutation?

Answer 13

In the Lebanese Mutation, the receptor is truncated after amino acid 659 - a nonsense mutation cuts off everything after the β-propeller region exposing two cysteine residues from glycan repeat C.

This prevents trafficking from the ER to the Golgi due to recruitment of ER ‘gatekeeper proteins’, resulting in degradation of the receptor protein without stimulation of an immune response.

The mutation is so known because it occurs in the Lebanon in higher frequencies, 1 in 100,000 rather than 1,000,000

Question 14

What are class 3 LDLR mutations?

Answer 14

Mutations in the ligand binding repeats prevent binding of LDL. This can be tested for through the use of antibodies raised to wildtype LDLRs.

Question 15

What are class 4 LDLR mutations?

Answer 15

Here binding of LDL is normal, but the mutant receptors are unable to internalise receptor-ligand complexes due to disruption of the 50-residues cytoplasmic domain (790-839). This prevents the receptors from congregating in clathrin coated pits or from internalising. Mutations that cause this generally occur early, as the first 22 amino acids are sufficient for rapid internalisation

Question 16

Give an example of a class 4 LDLR mutation.

Answer 16

The internalisation signal is conserved across species as NPXY – with the final residue (807) crucially aromatic (W, Y or P).

In humans this motif is specifically FDNPVY, but one mutation that is associated with FH is point mutation of 807 to a cysteine; Y807C.

This prevents phosphorylation and subsequent recognition by LDLRAP1, a protein involved in stimulating internalisation.

Question 17

What are class 5 LDLR mutations?

Answer 17

These receptors cannot recycle properly, which leads to a relatively mild phenotype as LDL-R is still present in the cell surface (though all must be newly synthesized).

Question 18

What are the four known monogenetic causes of hypercholesterolaemia?

Answer 18

FH - familial hypercholesterolaemia
FDB - familial defective ApoB100
ARH - Autosomal recessive hypercholesterolaemia
PCSK9 mutations

Question 19

What is FDB?

Answer 19

Familial defective ApoB100 is a disruption of LDL – LDLR binding due to mutations in the ApoB-100 gene. Over 20 rare mutations have been identified, most of which lead to hypobetalipoproteinaemia due to prevention of lipoprotein synthesis.

Question 20

What is the wildtype ApoB100 structure?

Answer 20

The ApoB-100 protein encircles the protein, with the C-terminal end (the last 11%) forming a triangular loop that bends the end back over the equator at R 3500, crossing shortly after the binding site for LDLRs; Site B – 3359-3369. This is stabilised by the interaction between Arg-3500 and Trp-4369.

Question 21

What is the most common mutation associated with FDB?

Answer 21

R3500Q, which has a frequency of 1 in 1,000.

The R3500Q mutation changes the conformation of the loop as it can no longer cross at the point at which it normally would. Instead, the C-terminal tail crosses back over site B and preventing binding to LDLRs. This mechanism was verified through targeted mutation of W4369 to Y, creating the same effect.

Question 22

What is ARH?

Answer 22

Autosomal Recessive Hypercholesterolaemia creates identical symptoms to FH – elevated LDL levels and resulting xanthoma, but is not caused by the LDLR or ApoB genes.

This is due to mutation of LDLRAP1, an adaptor protein that recruits the LDLRs to clathrin triskelions. Unlike FH patients, the heterozygous have normal LDL levels due to the recessive nature – only homozygous ARH gives symptoms.

Question 23

How does LDLRAP1 lead to LDLR internalisation?

Answer 23

LDLRAP1 binds to LDLR NPXY motifs in the cytosolic domain by a 134-residue PT-binding domain, and to clathrin with its little pentapeptide clathrin box; LLDLE.

Mutations that disrupt these interactions prevent internalisation of the LDL-LDLR, but this effect is largely specific to the liver and lymphocytes, as many cell types (including fibroblasts) also use another adaptor protein, Dab 2, which can compensate.

Question 24

How was PCSK9 discovered? What is indicated about the role of this protein?

Answer 24

Proprotein Convertase Subtilisin Kexin type 9 was first studied upon discovery of two autosomal dominant mutations, S127R and F216L, that lead to hypercholesterolaemia – responsible for 2% of genetic cases.

Overexpression the gene was shown to decrease the level of expressed LDLR, so these mutations were assumed to be gain-of-function.

Question 25

How does PCSK9 regulate LDLR activity?

Answer 25

PCSK9 facilitates a decrease in LDLR through secretion into the blood (after autocatalytic cleavage) where it binds to the N-terminal region of the EGF-A domain in a Ca2+ dependent manner by its catalytic domain. Mutations here have been found to inhibit the binding PCSK9.

Binding to the LDLR is thought to direct it to the lysosome, stimulating degradation indirectly and hence reducing the LDL uptake.

The S127R and F216L mutations are thought to increase the autocatalysis, upregulating the secretion and so activity of the enzyme.

Question 26

What therapies target PCSK9?

Answer 26

PCSK9 is the target of many pharmaceutical projects, through inhibition of the autocatalysis, disruption of the PCSK9-LDLR interaction through small molecules, peptides or antibodies (recently approved treatments include alirocumab and evolocumab), RNAi treatments (in phase I trials, using a lipid nanoparticle suspension vector) and even CRISPR-Cas9 gene therapy.

Question 27

What is SR-A? Describe its genetic structure.

Answer 27

The macrophage-specific scavenger receptor responsible for foam cell formation.

Found on chromosome 8, the gene produces two isoforms are due to alternative splicing.

Type I has the common exons 1-8 and Type I-specific exons 10 and 11, while the Type II protein consists of exons 1-8 and Type II-specific exon 9.

The principal binding region is encoded by exons 6-8 (collagenous domain) and so both receptors can clear oxidized LDL.

Question 28

What is the domain structure of SR-A?

Answer 28

(I) N-terminal cytosol domain 
(II) Transmembrane domain
(III) Spacer domain
(IV) α-helical coiled-coil motif
(V) Collagenous domain
(VI) C-terminal cysteine-rich motif

Question 29

How do SR-A receptors promote atherosclerosis?

Answer 29

Unlike LDL-Rs, their numbers are not downregulated as the cells take in large amounts of cholesterol and so there is continuous uptake of oxidized LDL, leading to fatty streak lesions.

However, this can be reversed by mechanisms such as reverse cholesterol transport.

Question 30

What is the primary effect of statins?

Answer 30

Statins inhibit HMG-CoA Reductase through substrate mimicry, thus lowering the cellular cholesterol content and hence reducing the inhibition of cholesterol uptake – ultimately lowering the blood LDL content. HMG CoA-Reductase was targeted as it is the rate-limiting step in the 27-enzyme pathway for de novo synthesis of cholesterol from acetate – a process which occurs primarily in the liver.

Question 31

What are secondary ways in which statins are thought to be anti-atherosclerotic?

Answer 31

Statins are also thought to prevent atherosclerosis through improving endothelial function, supressing inflammation, plaque stabilisation and anti-thrombotic effects, through the mechanisms for all of these remain unclear – but may be mediated by stimulation of NO production.

Question 32

How have statins been rationally designed?

Answer 32

Statins competitively inhibit HMG CoA-Reductase by mimicking HMG-CoA with bulky hydroxyl acid groups attached to it in such a way that prevent catalysis.

Several rounds of rational drug design have improved the design of the added groups. Some, such as Lovastatin and Mevastatin are administered in Lactone form and converted to hydroxyl acids in the liver, whereas others such as Atorvastatin are already hydroxyl acid salts.

Question 33

What effect of statins is not related to lipid metabolism?

Answer 33

Mevalonic acid production by HMG-CoA reductase is not only rate-limiting for cholesterol biosynthesis, but also for geranylgeranylpyrophosphate and farnesyl pyrophosphate, which are important metabolic regulators.

Post-translational modification of proteins by these compounds (prenylation) regulates the subcellular location of G-proteins, influencing many signalling cascades within the cell.

Additionally, oxysterols and intermediates found later in the cholesterol biosynthetic pathway also affect the activity of nuclear orphan receptors, such as liver X receptors and farnesoid X activated receptors (LXR and FXR).

Question 34

What are the overall pros and cons of statins use?

Answer 34

The JUPITER Trial, the largest statins trial to date, clearly showed that they reduce LDL levels significantly, and decrease CVD mortality by 0.2-0.6%. Although statins have also been linked to cancer through the important role of cholesterol in cell proliferation, but no study has detected an increase in cancer mortality for statins patients, and one study even found a reduction in cancer incidence.

Statins are diabetogenic though – however the reduction in CVD mortality is generally seen to outweigh the increased risk of diabetes.