Hepatic Clearance of HDLs and CM Remnants

Question 1

What is responsible for the hepatocytic clearance of CM remnants?

Answer 1

LRP Receptors (LDLR-Related Protein) recognise ApoE.

This means it is also able to take up IDLs and HDLs.

LDLRs are also capable of clearing the lipoprotein remnants.

Question 2

What is the structure of LRP?

Answer 2

The largest known membrane protein, LRP is the equivalent of 4 LDLRs, with 31 ligand binding repeats, 22 EGF repeats and two NPXY motifs, contrasting with the respective 7, 3 and 1 found on LDLRs.

Question 3

What is the secretion capture model?

Answer 3

Hepatocytes secrete large amounts of ApoE, displyaing it on Heparin Sulphate Proteoglycans (HSPGs) to enrich lipoprotein remnants with it, allowing for uptake by LRP.

Question 4

Where does the liver take up lipoproteins from?

Answer 4

Clearance occurs in the perisinusoidal space (AKA space of Disse), which is located between a hepatocyte and a sinusoid (small blood vessel).

This acts to trap or prolong the residence time of the lipoproteins, allowing additional action of hepatic lipase and acquisition of ApoE molecules.

Question 5

What controls entry into the space of Disse?

Answer 5

The endothelial cells lining the sinusoid are fenestrated with openings of ~100 nm diameter, and hence are often called ‘sieve-plates’. This prevents entry (and hence uptake) or larger/mature CMs and VLDLs, while allowing CM remnants, LDLs and HDLs through.

Hepatocyte microvilli do tend to extend into this space to allowing interaction with/absorption of plasma proteins.

Question 6

What is responsible for HDL clearance?

Answer 6

The HDL receptor has now been identified as scavenger receptor class B type 1 (SR-B1)

This is a CD36 related receptor that is part of the family involved in atherosclerotic uptake of oxLDL (indeed B1 is capable of both)

Question 7

How does SR-B1 take up HDLs?

Answer 7

Through a retroendocytosis action, in which the SR-B1, perhaps localised to caveolae (non-clathrin coated invaginations), bind the CE rich HDL2 and stimulate endocytosis.

Question 8

What happens to the HDL and receptor after internalisation.

Answer 8

Endocytosis into the early endosome allows the cholesteryl esters to be extracted from the HDL2, which remains bound to SR-B1.

The resulting HDL3-like particle and the SR-B1 are then recycled back out into the plasma, where it can continue to sequester excess cholesterol, and the cholesteryl esters extracted from it are used by the cell.

Question 9

What mutations have been identified in SR-B1 receptors and where?

Answer 9

Three human SR-BI mutations have been identified, all occurring in the large extracellular loop that is characteristic of the CD36 superfamily (Cluster of Differentiation – genes commonly involved in the immune system), indicating the critical function of this in the mechanism.

Question 10

Describe two SR-B1 mutations.

Answer 10

S112F and T175A are at highly conserved spots across all vertebrates – the 175 residue only varying in zebrafish (Danio rerio)

Question 11

What is the structure of the extracellular loop of SR-B1?

Answer 11

The loop possesses six cysteine residues – the middle four (280, 321, 323 and 334) forming two disulphide bridges while the outer ones (251, 384) remain reduced.

Site-directed mutagenesis of the cysteine 384 reduces activity greatly, implying an important role

Question 12

Where is SR-B1 glycosylated?

Answer 12

SR-B1 is also glycosylated at N-109 and N-173, loss of which also impairs function and trafficking to the cell-surface.

Question 13

Other than HDL clearance, what else is SR-B1 involved in?

Answer 13

oxLDL uptake
Cell Signalling
Cell infiltration by hepatitis C, plasmodium and mycobacteria.

Question 14

What is hepatic lipase responsible for?

Answer 14

Extraction and hydrolysis of TG from the core of lipoproteins, primarily IDLs, in the space of Disse to produce free fatty acids and 2-monoglyceride that can be taken up by the cells.

Question 15

Where is HL found?

Answer 15

It is expressed by hepatocytes and steroidogenic tissues.

It is found bound to the hepatocytes and sinusoid endothelial cells on heparin sulphate proteoglycans (HSPGs).

Question 16

How does HL interact with the lipoproteins?

Answer 16

Transiently, but because multiple LPLs are attached to each HSPG tree, around 40 LPLs can be attached to each structure allowing each capture of a lipoprotein to provide large quantities of TG.

Question 17

What is the structure of HL?

Answer 17

Similar to that of LPL, which is associated more with lipolysis of large, TG rich lipoproteins.

Hence it is found as a head to tail homodimer, so has two N-terminal active sites - serine protease triads contained in a hydrophobic groove protected from solvent by a lid region.

Question 18

What is the structure of ApoE?

Answer 18

Secreted APOE is a 34-kDa (299 residues) glycoprotein with an O-glycan chain at Thr194.

ApoE has a very different structure in aqueous solution compared to the extensive conformational changes that takes place when it binds lipid in the surface of a lipoprotein particle.

In solution it is composed of an N-terminal four helix bundle and a C-terminal helical lipid binding domain. Upon binding the lipoprotein, the conformation changes to a large horseshoe shape – albeit one too small to bind to a mature CM.

Question 19

What is notable about human ApoE genetics?

Answer 19

ApoE is polymorphic due to single base mutations, which give rise to amino acid substitutions.

Hence it has severl isoforms, the three common ones being ApoE2, 3 and 4.

Question 20

What are the differences in sequence of ApoE2, 3 and 4?

Answer 20

The three common isoforms of ApoE differ in two amino acid positions:

ApoE2 (Cys-112 and Cys-158; ~8% of the population)
ApoE3 (Cys-112 and Arg-158; ~77%), the most prevalent protein (often noted as the wild-type one)
ApoE4 (Arg-112 and Arg-158; 15%)

Question 21

What diseases are the different ApoE isoforms associated with?

Answer 21

Heart disease & stroke, Alzheimer’s disease and viral infections (HIV, herpes simplex, and hepatitis C).

These associations reflect the disparate roles that ApoE carries out, some related to lipid transport and others to cell signalling functions.

Question 22

How does the lipoprotein binding domain vary between isoforms?

Answer 22

The binding region of ApoE is centred around amino acids 140-150, which contains 7 positively-charged residues (Arg, Lys & His).

ApoE2, which lacks an Arginine residue (Arg158Cys), has defective binding, while ApoE4, which gains one (Cys112Arg), has normal or increased binding.

Question 23

Why does ApoE2 have decreased lipoprotein binding affinity?

Answer 23

Although the ApoE2 isoform’s amino acid substitution is outside the 140-150 domain (R158C), this still has a disruptive influence.

In wild-type ApoE3, a salt bridge is formed between R158 and N154 an interaction not possible in ApoE2; hence a bridge tends to form between R150 and D154 which has a knock-on effect within the 140-150 domain to disrupt binding affinity

Question 24

What diseases are associates with ApoE2?

Answer 24

A small proportion of individuals homozygous for ApoE2 have a disease called type III hyperlipoproteinaemia, which is characterised by the accumulation of remnant lipoproteins due to lack of ApoE activity.

However most of those homozygous for E2 in fact have hypolipidaemia, because the lack of CM remnant uptake in the liver leads to overexpression of LDLR and hence greater uptake of plasma LDL.

Question 25

How are VLDLs made in the liver?

Answer 25

The current model is that of cotranslational deposition into the ER of the ApoB protein, throughout which microsomal triglyceride transfer protein (MTP) adds small amounts of lipids taken from the cytosolic pool of TG (i.e. a lipid droplet) to the nascent chain, moving the TG droplet into the ER lumen for later addition.

This process is thought to be the same as that used to produce chylomicrons in the intestine.

Question 26

What is produced by MTP lipidation of ApoB?

Answer 26

This produces only a small lipoprotein; chylomicrons and VLDLs are formed by addition of more TG by an MTP-independent mechanism.

Question 27

How is VLDL production prevented when hepatocyte lipid levels are low?

Answer 27

If insufficient lipid is available for ApoB to assume a stable conformation, the poorly lipidated particles are degraded in the cytosol by the proteasome and/or by lumenal proteases.

Question 28

How are the VLDLs made in the ER lumen trafficked for secretion?

Answer 28

In the liver, the small precursors are termed pre-VLDLs.

These are trafficked to the Golgi, and are lipidated during transit, meaning that they become VLDL2s by the time they reach the Golgi, where they merge with the tips of the cisternae and transfer through the trans-golgi network before being secreted.

Question 29

What alternative pathway can VLDL2s take upon reaching the trans-golgi plate?

Answer 29

They can be secreted or further lipidated to form VLDL1 which lead to production of small, dense LDL particles which are particularly susceptible to oxidation and hence strongly pro-atherogenic.

Question 30

How is MTP targeted in therapy?

Answer 30

Lomitapibe MTP inhibition was approved by the FDA in 2012 as an FH treatment, as it prevents the production/secretion of VLDLs and chylomicrons.

It inhibits by binding to MTP and directly preventing it from transferring TG to ApoB.