Introduction
Lipoproteins
Many lipids, such as cholesterol, cholesterol esters, phospholipids, and triacylglycerols, and lipid-soluble substances, such as fat-soluble vitamins, are transported through the aqueous circulation by lipoprotein particles. Plasma lipoproteins can be divided into six major classes: very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), high density lipoprotein (HDL), chylomicrons, and chylomicron remnants. The first four classes, VLDL, IDL, LDL, and HDL, are derived from the liver and they can be found in the plasma of both fasted and nonfasted subjects. The last two classes, chylomicrons and chylomicron remnants, are derived from the small intestine and they can only be found after a fatty meal in the plasma. LDL, which is the end product from lipolysis of VLDL and IDL processing by lipoprotein lipase, is the major cholesterol-transporting lipoprotein in humans, carrying about two-thirds of the total plasma cholesterol. The lipid composition of LDL is ~35% cholesteryl ester, ~12%
cholesterol, ~8% triglycerides, and 20% phospholipids. The cholesterol delivered by LDL is being endocytosed followed by hydrolysis within the endosome (Brown and Goldstein, 1986).
Cholesterol metabolism
The cholesterol pool of the human body comes from two sources: the absorption of dietary cholesterol and the de novo biosynthesis of cholesterol in all tissues (Mayes, 1998). All mammalian cells can synthesize the required cholesterol for themselves, but only some are synthesized locally. Most cholesterol used by peripheral tissues are originated from the liver (Spady and Dietschy, 1983). The de novo synthesis of cholesterol from acetyl-CoA in liver and other body tissues provides about three quarters of the cholesterol in the body. The rate-limiting step in the de novo synthesis is the conversion of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA reductase) to mevalonate, which is catalyzed by HMG-CoA reductase. The inhibition of this
reductase is located in the endoplasmic reticulum and the enzyme is regulated by several mechanisms: the feedback inhibition from high cholesterol level acting on the activity of preexisting HMG-CoA reductase, degradation of existing HMG-CoA reductase accelerated by high cholesterol level, decreasing the amount of mRNA coding for HMG-CoA reductase by high cholesterol level, and inhibition of the enzyme by phosphorylation of HMG-CoA reductase and activation of the enzyme by dephosphorylation of HMG-CoA reductase (Champe and Harvey, 1994). The membrane level of free cholesterol in peripheral cells is tightly regulated by esterification or sequestration in lipoproteins. Most cells can esterify and store excess cholesterol via the acyl CoA:cholesterol acyltransferase (ACAT) reaction (Suckling and Stange, 1985).
Hyperlipidemia and atherosclerosis
Elevated plasma cholesterol concentrations, especially LDL, are strongly correlated with atherosclerotic coronary heart disease (CHD) (Freedman et al., 1993;
Toshima et al., 2000). A high level of plasma LDL usually originates from excessive lipoprotein secretion from the liver or defects in plasma LDL clearance process (Lougheed and Steinbrecher, 1996). The elevated plasma LDL would increase the risk of LDL being trapped in the intima and oxidized by free radicals generated by adjacent cells, including endothelial cells, smooth muscle cells, and isolated macrophages. Since the oxidized LDL is able to impair endothelium-dependent relaxation and act as a chemoattractant substance for monocytes, it is highly atherogenic. When endothelial cells are being stimulated by oxidized LDL, they secrete cytokines and cause accumulations of monocytes and macrophages, leading to the formation of foam cells. The foam cells form the core of the atherosclerotic plaque, which is originated from macrophages and smooth muscle cells through the cholesterol loading process. The scavenger receptors in macrophages can recognize oxidized LDL but not native LDL (Cotran et al. 1994). The internalization of oxidized LDL by scavenger receptors in macrophages is not subject to feedback regulation and the uptake is not saturated, thus, large amounts of modified LDL would accumulate to form foam cells.
Familial defective apolipoprotein B-100 (FDB)
Both VLDL and its product, LDL, contain a copy of a large structural apolipoprotein B-100 (apo B-100) (Elovson et al., 1988). The C-terminal segment of apo B-100 on the surface of these particles can be recognized by hepatic and extrahepatic LDL receptors. This interaction is responsible for ~75% of the clearance of LDL in the plasma, primarily through the liver (Lund et al., 1989). Increased LDL concentrations resulting from inefficient clearance of LDL particles by the receptors may cause by the defect in apo B-100. This defect in the ligand, familial defective apolipoproteinB-100 (FDB), is a dominantly inherited genetic disease. Three forms of FDB have been described genetically: a CGC-to-CAG change in apo B-100 codon 3500 (R3500Q), a CGC-to-TGC change in codon 3531 (R3531C), and a CGG-to-TGG change in codon 3500 (R3500W). These three mutations affect the function of apo B-100 by decreasing the binding affinity of LDL receptors (Innerarity et al., 1987; Gaffney et al., 1995; Pullinger et al., 1995).
Familial hypercholesterolemia (FH)
Another reason for inefficient clearance of LDL particles is that a defect has occurred in the LDL receptor. This class of genetic disorder is an autosomal dominant familial hypercholesterolemia (FH), and it is characterized by increased LDL cholesterol levels in plasma, which will then cause cholesterol deposition in tissues in the forms of tendon xanthomas and atheroma, leading to premature arteriosclerosis and CHD (Goldstein and Brown, 1989). Heterozygous FH is one of the most common inborn errors of metabolism, with a frequency of ~ 1/500 in most populations.
Heterozygous carriers may have up to three-fold elevations in plasma cholesterol, ranging from 300 to 600 mg/dl. The homozygous FH occurs in about one in a million individuals and is more severely affected than the heterozygote carrier, having circulating LDL level six to eight times higher than normal (from 600 — >1,200 mg/dl) and usually have heart attacks before the age of 20 (Goldstein et al, 2001).
The LDL receptor gene: relation of exons to protein domains
chromosome 19p13 (Figure 1) (Sudhof et al., 1985). Half of the 5.3 kb mRNA comprises a long 3' untranslated region, which encodes a protein with 860 amino acids. At the N terminal end, 21 hydrophobic amino acids comprise the signal sequence, which is cleaved from the protein during the translocation process into the endoplasmic reticulum (ER). The resultant 839-amino acid protein (120 kDa precursor receptor) contains 1~2 N-linked and 9~18 O-linked carbohydrate chains, which undergo several carbohydrate processing reactions in the Golgi apparatus en route to the cell surface, resulting in a 160 kDa mature receptor (Cummings et al., 1983).
Many of the LDL receptor exons share an evolutionary history with exons from other genes; an observation suggested that the LDL receptor gene was assembled by exon shuffling. Figure 2 shows how exons of the gene are correlated with the functional domains of the mature protein. The signal sequence is encoded by exon 1 and the ligand-binding domains encoded by exons 2 ~ 6 are made by 7 ~ 40 amino acid repeats (R1 ~ R7), which form two loops joined by disulfide bonds. The domain contains clusters of negatively charged amino acids, which bind the lipoprotein particles to ligands apo B-100 and apo E. Exons 7 ~ 14 encode EGF-precursor homology domain, which is 33% identical to the human epidermal growth factor precursor, containing three EGF-like repeats: tandem pairs A-B and C, which form two pairs of short antiparallel β strands. This domain is involved in positioning the ligand-binding domain on the cell surface, and plays an important role in acid dependant dissociation of the lipoprotein after the endocytosis process. Exon 15 encodes 58 amino acids, which are riched in serine and threonine residues; many of which serve as attachment sites for O-linked carbohydrate chains. The membrane spanning domain, encoded by exons 16 and 17, anchors the protein in the cellular membrane. The cytoplasmic domain, encoded by exons 17 and 18, is critical to the ligand uptake and signaling functions. The tetra-amino-acid NPXY motif within the cytoplasmic domain forms a tight hairpin conformation to mediate the interaction between the receptor tails and the endocytosis mechanism, and to serve as a docking site for cytoplasmic adaptor and scaffold proteins (Yamamoto et al., 1984).
The LDL receptor pathway
The LDL receptor sequential pathway is summarized in Figure 3. The receptor begins its life in the endoplasmic reticulum, and then it travels to the Golgi complex, cell surface, coated pit, endosome, and back to the surface. On the cell surface, LDL receptor binds to LDL particles that contain apo B-100. The LDL particles are internalized via clathrin-coated pit on the plasma membrane. The coated pits “pinch off” from the membrane to form coated vesicles, and then they fuse together to lose their clathrin protein coat and become endosomes. Proton pumps acidify the endosomes, causing lipoprotein release from the receptor. Within a few minutes, the receptor is recycled to the cell surface, where it can be used for another round of endocytosis. The lipoproteins are delivered to lysosomes, where enzymes degrade the apolipoproteins into their constituent amino acids and hydrolyze the cholesteryl esters to free cholesterol. The released cholesterol is used in the synthesis of cell membranes, and some specialized cells, and it is also a precursor for sterol end products.
Regulation of LDL receptor synthesis
The amount of LDL receptor synthesis in cells is strongly regulated by the need or availability of cholesterol. When intracellular cholesterol raised, the expression of LDL receptors is down-regulated; excess cholesterol also down-regulates the expression of HMG-CoA reductase, which is the major rate limiting enzyme in cholesterol biosynthesis and the primary target of modern lipid-lowering drugs, such as statines. Also, the activity of ACAT, a lipid droplet enzyme that re-esterifies cholesterol into cholesteryl esters in the cytoplasm, is increased when intracellular cholesterol increased. By these mechanisms, cells are able to maintain sufficient amount of intracellular cholesterol for various functions (Figure 3).
LDL receptor mutations
At the present, more than 840 mutations, including gross deletions, minor deletions, insertions, point mutations, and splice-site mutations, have been reported to
et al., 2003). Among these, Alu sequences are always involved in deletions or insertions of the LDL receptor gene (Lehrman et al., 1985, 1987). The mutations characterized at the molecular level can be divided into five different classes (Figure 4) (Hobbs et al, 1992). Class 1 mutations (null alleles) affect the receptor synthesis, which means that no receptor can be detected in the affected subjects. Class 2 mutations (transport defective alleles) are the most common LDL receptor gene mutations. Patients in this class have a relative LDL receptor deficiency at the cellular surface, because the intracellular transport between endoplasmic reticulum and Golgi apparatus is either completely blocked (class 2A) or delayed (class 2B). Class 3 mutations (binding defective alleles) prevent receptors from binding LDL through ligands apo B/E. Class 4 mutations (internalization defective alleles) still conserve the binding ability on LDL particles, but fail to internalize the bound LDL. Class 4 can be either complete (4A) or partial (4B).Class 5 mutations (recycling defective alleles) produce receptors that bind and internalize LDL normally, but fail to discharge the LDL in endosomes and recycle them to the cellular surface.
Aims
Previously, 170 unrelated hyperlipidemic Chinese and two clinically diagnosed familial hypercholesterolemia (FH) patients were screened by multiplex polymerase chain reaction (PCR), long PCR, and single strand conformation polymorphism (SSCP) analyses to detect mutations in the LDL receptor gene. Eight point mutations (W-18X, D69N, R94H, E207K, C308Y, I402T, A410T, and A696G), two gross deletions (Del e3-5 and Del e6-8), and one polymorphism (I602V) were found (Table 1) (Huang, 2001; Lin et al., 2002). In this study, the effect of seven point mutations and two gross deletions on processing and stability of LDL receptor was characterized by transient expression in COS-7 cells.
