Low Density Lipoprotein Receptor gene LDLR : Structure and function

LDLR codes for the Low Density Lipoprotein Receptor (LDLR) and this gene

is mainly expressed in the in liver. LDLR is an ~860-amino acid cell surface

glycoprotein (51). The most important physiological ligand for the LDLR is LDL,

which carries ~70% of cholesterol in humans (51) (LDL has been described in Section

1.2.5). LDLR plays an important role in cholesterol homeostasis because its main function is to clear LDL from plasma.

LDLR has five domains, namely: (i) Ligand Binding Domain, (ii) EGF

Precursor Homology, (iii) O-linked Sugar Domain, (iv) Membrane Spanning Domain and (v) Cytoplasmic Domain. The schematic figure of mature LDLR (i.e. LDLR with

the 21 amino acid signal peptide sequence removed) is shown in Figure 1.9 (39)

shows the structure of cholesterol. Figure 1.3 shows how LDLR is involved in clearance of LDL from blood.

The first 21 amino acids of LDLR is the signal peptide sequence which gets cleaved soon after the protein is translated. The 21 amino acid signal directs location of translation on the membrane of the Endoplasmic Reticulum (ER) as the protein is

getting translated (39).

The ligand binding domain as its name implies, is the domain that binds LDL. It is a cysteine rich domain and many disulphide bonds are present in this domain. This extensive disulphide structure gives this domain stability. This domain is

negatively charged and this negative charge is complementary to the charge of regions

of apo E (39). The second domain is the Epidermal Growth Factor (EGF) precursor

homology domain and the name was given because this domain of LDLR resembles

the part of the extracellular domain of EGF (39). The third domain is called O-linked

sugar domain because of the clustering of O-linked sugar chains (39). The fourth

domain, which is the membrane spanning domain, is rich in hydrophobic amino acid

residues so that it can interact with the hydrophobic cell membrane (39). The fifth

domain is the cytoplasmic domain and this domain is important for the clustering of LDLR in clathrin-coated pits that occurs in LDL clearance by LDLR. LDLR clusters

and then internalizes LDL. This clathrin is important for this clustering of LDLR (39).

In clearance of LDL from blood (Figure 1.3), receptor-ligand complexes

occurs in clathrin coated pits. LDLR clusters in clathrin coated pits. The receptor- ligand complex is internalized into the cell within the clathrin coated pits. This complex is delivered to the endosomes where the pH is low (i.e. acidic). At this low pH, the receptor dissociates from the ligand; the receptor gets recycled back to the cell surface and the ligand moves from the endosome to the lysosome. In the lysosome,

LDL is hydrolysed and cholesterol is released into the cell (51). This entire process is

A loss-of-function mutation in the LDLR gene leads to less or no clearance of LDL, thus resulting in hypercholesterolemia. For the LDLR, mutations have been

classified into 4 classes (39). In Class I mutations, no receptor is synthesized due to a

major deletion mutation that results in no protein product expressed. In Class II mutations, LDLR is synthesized but does not undergo its normal post translational modification, which occurs in the ER. So, LDLR does not get to the cell surface; LDLR remains in ER until it is degraded. In Class III mutations, LDLR is synthesized and reaches the cell surface but fails to bind its ligand. In Class IV mutations, LDLR is synthesized, reaches cell surface, binds to LDL but fails to cluster and clustering of LDLR (which occurs in clathrin coated pits), is vital for receptor mediated

endocytosis. So, for LDLR, the mutations are classified based on the aspect of receptor mediated endocytosis it is affecting and not the type of mutation. The

downstream effect of all the classes is reduced clearance of LDL. LDLR mutations are

Figure 1.9 Structure of LDLR (a) The multidomain LDLR. LDLR has five domains. The Ligand binding domain is for binding of LDL and the cytoplasmic domain is important for internalization of LDLR –LDL complex into the cell. The membrane spanning domain is rich is hydrophobic residues so that it can interact with the hydrophobic layer of the cell membrane. The EGF (Epidermal Growth Factor) precursor homology domain is

homologous to part of EGF. Figure 1.9 was modified from (39). Figure 1.3 shows the mechanism of LDLR action. The main function of LDLR is to clear LDL from the blood. LDLR clears LDL from the blood through receptor mediated endocytosis. In receptor mediated endocytosis, LDLR binds LDL. The LDLR-LDL complex gets internalized into the cell. The LDLR-LDL complex enters the endosome. In the endosome the pH drops which dissociates the LDLR-LDL complex. LDLR gets recycles back to the cell surface, while LDL is transported to the lysosome and gets metabolized in the lysosome. In the lysosome, cholesterol is released from LDL.

1.4.2.2 Apolipoprotein B-100 gene APOB: Structure and function

APOB codes for both Apolipoprotein B-48 (ApoB-48) and Apolipoprotein B- 100 (ApoB-100). The difference between the two isoforms of the protein is that the ApoB-48 isoform, which is 48% of the length of the full-length ApoB-100 isoform, is expressed in the small intestine. In contrast, the ApoB-100 isoform is expressed in

the liver. ApoB is the ligand that LDLR recognizes in LDL (52) (Apolipoproteins

and LDL were discussed in Section 1.2). ApoB, which is one of the largest

monomeric proteins known (53) is the apolipoprotein of LDL (52). Because of the

insoluble nature of ApoB, it has been difficult to completely study its tertiary structure

(26) (53). So researchers have used experimental and in silico data to predict the three

dimensional structure of ApoB. Figure 1.6b is also a diagram for ApoB structure.

Some cases of FH result from a perfectly normal LDLR but a mutation in APOB;

these mutations disrupt the ApoB structure such that it cannot bind with LDLR and

thus clearance of LDL from plasma gets disrupted (27). APOB mutation is the second

most common cause of FH as alluded to earlier.

Figure 1.6b (Top) was modified from (26) and Figure 1.6 b (Bottom) was

modified from (27.) ApoB, which is made up of 4536 amino acids, is believed to

wrap around the spherical lipid core The Receptor binding region of the ApoB protein

is believed to span amino acid residues 3000 to 4000 (27) .