MUTATIONS AND VARIANTS OF APOLIPOPROTEIN B TIIAT AFFECf PLASMA CHOLESTEROL LEVELS
INTRODUCTION
In the United States and other Western societies, heart disease is the leading cause of death. Numerous epidemiological studies have demonstrated that increased levels of plasma cholesterol, particularly low density lipoprotein (LDL) cholesterol, are associated with atherosclerosis (1, 2). One of the main areas of research on atherosclerosis is the identification of genetic factors in humans that increase the probability of developing pre mature atherosclerosis. Certainly, one of the dominant genes codes for the LDL receptor. Individuals who are homozygous for familial hypercholesterolemia possess few if any functional LDL receptors, have a six- to eightfold elevation of LDL cholesterol, and usually die of heart disease before the age of 20 (3). Three other genes that have been shown to be important for lipoprotein metabolism are the apo-B, the apo-E, and the li poprotein( a) genes (4-6).
This brief review will focus on the importance of the apo-B gene in controlling plasma cholesterol levels and on the role of mutations in apo-B in causing either hypocholester olemia or hypercholesterolemia. Apolipoprotein B is an essential part of a number of plasma lipoproteins: LDL, intermediate density lipoproteins (IDL), very low density lipo proteins (VLDL), chylomicrons, and chylomicron remnants. About two-thirds of plasma cholesterol in humans is transported in plasma LDL, high levels of which are positively correlated with coronary heart disease. In humans, apo-B 100 is synthesized by the liver and is required for the assembly and secretion of VLDL. Very low density lipoproteins are converted by lipoprotein lipase to IDL and then to LDL. The LDL are cleared from the circulation by the LDL receptor, with apo-B 100 serving as the ligand.
Apolipoprotein B is also of interest because of a unique mRNA editing process that enables the apo-B gene to make two structurally related but different-size apo-B pro teins that have different functions: apo-B 100 (4536 amino acids) and apo-B48 (2152 amino acids). In humans, apo-B48 is synthesized in the intestine. It plays a role analo gous to that of apo-B 100 in the liver, in that it is necessary for the assembly and secretion of another major class of lipoproteins, chylomicrons. Chylomicrons transport dietary triglycerides absorbed from the intestinal lumen and are converted to chylomicron remnants by lipoprotein lipase. The remnants are rapidly cleared from the circulation by the liver, with apo-E serving as the ligand. Because apo-B48 contains only the amino terminal 2152 amino acids of apo-B100, it lacks the carboxy-terminal receptor-binding domain and therefore does not bind to LDL receptors. In addition, apo-B48-containing lipoproteins are not converted to LDL but are completely cleared from the plasma. Plas ma from fasted subjects contains very little apo-B48 (for a review of apo-B, see Refs. 7 and 8).
STRUCTURE OF APOLIPOPROTEIN B
A number of laboratories determined the structure of apo-B 100 by cloning and se quencing its eDNA. Human apo-B mRNA is 14.5 kilobases (kb) in length and codes for a mature protein consisting of 4536 amino acids that have a molecular weight of about 550,000 (about 10% of which is accounted for by carbohydrates) (7) (Fig. 1) The apo-B gene is 43 kb in length, contains 28 introns and 29 exons, and is located in the short arm of chromosome 2. The distribution of the introns within the gene is unusual in that 24 of the 28 introns occur in the 5'-terminal one-third of the gene. Over half of the protein is encoded by the extremely long exon 26, whose 7572 base pairs make it one of the largest exons yet reported in the human genome (13).
APOLIPOPROTEIN B MUTATIONS
The two genetic abnormalities attributed to mutations in the apo-B gene are familial hypobetalipoproteinemia, which is associated with low plasma cholesterol levels, and familial defective apo-B 100 (FDB), which is characterized by high plasma cholesterol levels.
Fig. 1. Schematic structure of human apo-BlOO. Thrombin cleaves apo-BlOO at resi dues 1297 and 3249, resulting in thrombolytic fragments T4, T3, and T2. Mono clonal antibodies 4G3, 3A10, SEll, and MB47 have been mapped to the regions shown (9, 10). Each of these antibodies completely inhibits the binding of LDL to the LDL receptor. 3500 denotes the point mutation (CGG CAG, which causes an Arg Gin substitution) that disrupts the binding of LDL to the LDL receptor. The cross-hatched rectangle designates a best estimate of the receptor-binding region based on the evidence from the natural mutation that disrupts receptor binding (11) and from the monoclonal antibody studies (9, 10). The B25 through B89 notations denote the points of truncation of apo-B species associat ed with familial hypobetalipoproteinemia. (Reproduced, with permission, from Innerarity (12).)
Work from several laboratories has demonstrated that hypobetalipoproteinemia can be caused by mutations in the coding region of the apo-B gene. As a consequence, affect ed subjects who are heterozygous for this gene have only 25% to 50% of the normal lev els of VLDL and LDL, and subjects with the homozygous phenotype have few if any apo BlOO-containing lipoproteins in their plasma (7, 8, 13). Investigations using either apo B gene-associated DNA polymorphisms or protein polymorphisms in association with antibody polymorphisms demonstrated that the disorder is linked to the apo-B gene (14, 15). More recently, a large number of mutations in the apo-B gene that cause a prema ture termination of translation have been identified (for a review, see Refs. 8 and 13). Examination of apo-B-containing lipoproteins from these subjects revealed truncated forms of apo-BlOO. From the analysis of several of these mutations, it appears that a minimum length of apo-B is necessary before any truncated apo-B-containing lipopro teins are detected in the plasma. Apparently, full-length apo-B is required for normal VLDL secretion (16).
The second lipoprotein disorder due to mutations of apo-B is FDB. This was first detected by an in vitro assay that measured the ability of LDL from hypercholesterolemic subjects to compete with normal 1251-labeled LDL for binding to the LDL receptor. The LDL from subjects with this disorder had about 32% of normal receptor-binding activity (17). To identify the mutation responsible for the defect in apo-BlOO's ability to bind to its receptor, the mutant apo-BlOO allele of the original proband was sequenced between nucleotides 7500 and 11916. This region, which codes for amino acids 2488 to 3901, includes the receptor-binding domain. Only one mutation was found in this domain: CGG was changed to CAG in codon 3500, causing a glutamine-for-arginine substitution at this site (11).
Because a single copy of apo-BlOO is present on each LDL particle, LDL from heterozygotes with FDB are a mixture of normal and defective-binding LDL. The defective-binding LDL have very little receptor-binding activity. Thus, a single amino acid substitution at residue 3500 virtually abolishes the receptor binding of these LDL. Inter estingly, this mutation is at or very near the epitope of MB47, a monoclonal antibody that effectively inhibits the receptor binding of LDL. MB47 binds to the LDL from various mammals, suggesting that it binds to an evolutionarily conserved sequence of apo-B 100 (18). In addition, MB47 binds to the LDL from subjects with FDB with a higher affinity than to LDL from normal individuals (19).
The main clinical consequence of FDB is hypercholesterolemia. To determine the im pact of this mutation on plasma cholesterol levels, we screened 1100 subjects for familial defective apo-B 100 and uncovered 11 probands with this disorder. Family studies identi fied another 30 individuals, for a total of 41 heterozygotes for this mutation (20). In our study, FDB heterozygotes had an average plasma cholesterollevel81 mg/dl higher than age- and sex-matched controls. In two other studies the investigators found even higher levels of plasma LDL. Tybjaerg-Hansen et al. (21) found that 10 FDB heterozygotes had plasma cholesterol levels 163 mg/dl higher than the 50th percentile of the Lipid Re search Clinic's age- and sex-matched controls. Schuster et al. (22) identified 18 sub jects with this disorder and found they had levels of total cholesterol 134 mg/dl higher than the control subjects. No other known genetic mutation, with the exception of mutations in the LDL receptor gene, causes such a large increase in LDL plasma cholesterol (20).
APOLIPOPROTEIN B48
A unique physiological process produces a variant form of apo-B known as apo-B48. The complete amino acid sequence of apo-B48 is identical to the 2152 amino-terminal amino acids of apo-B100, and the protein possesses approximately 48% (240 kDa) of the molecular mass of apo-B100 (23-26). Both apo-B100 and apo-B48 are produced from the same gene; the latter is produced by an mRNA editing process that converts nucle otide 6666, a cytosine, to a uracil. The editing changes codon 2153 from a CAA (glutamine) codon to a premature UAA (stop) codon, which terminates the translation of apo-B48 mRNA (23, 25, 26).
Thus far, this apo-B mRNA editing has been found in intestine from the human, rab bit, and rat (23, 27). It has also been found in the rat liver (27) and in the cell lines CaCo- 2 (human intestinal adenocarcinoma) (28), BNL CL.2 (mouse liver) (29), and McArdle 7777 (rat hepatoma) (30), all of which secrete apo-B48. Thus, the apo-B mRNA editing appears to be tissue-specific. However, recently we have found evidence for apo-B48 editing activity in a number of tissue-culture cell lines that do not synthesize apo-B (29).
The target for the apo-B mRNA editing, cytosine 6666, is part of a 26-nucleotide se quence that is completely conserved across several species: mice, rats, rabbits, and hu mans. This conserved sequence is believed either to be a part of or to constitute the rec ognition sequence for the editing machinery. At this point, the exact recognition se quence is not known. Using a series of deletion mutants around cytosine 6666 that were transfected into McArdle 7777 cells, Davies et al. found that as little as the 26 conserved nucleotides sufficed for efficient editing (30). However, when Driscoll et al. used an in vitro system prepared from McArdle 7777 cells, synthetic RNA of 55, 483, and 2383 nu cleotides, but not 26 nucleotides, was edited (31).
Fig. 2A. Schematic representation of expression vectors for c imeric apo-EB proteins.
Apolipoprotein B nucleotide sequences of 63, 186, and 354 bp, all centered around the target base for the rnRNA editing (cytosine 6666), were inserted into an apo-E expression vector. The resulting pHEB vectors were transfected into CaCo-2 cells, a human intestinal adenocarcinoma cell line that secretes apo B48. LTR, Moloney murine leukemia virus long terminal repeat. ATG, translational start site; TGA, translational stop site.
Fig. 2B. Immunoblot of chimeric apo-EB proteins secreted from CaCo-2 cells transfected with pHEB vectors. From CaCo-2 cells transfected with pHEB-354 or pHEB-186, both full-length (EB-354 and EB-186) and truncated proteins (EB-354T and EB-186T) were secreted and then detected on immunoblots with antipeptide 2140-2151. This apo-B antibody has a very high affinity for the car boxy terminus of apo-B48 and a very low affinity for apo-B100. The full-length EB-63 is not detected by antipeptide 2140-2151, but is readily detected with anti-apo-E antibodies (data not shown). No truncated EB-63 could be detect ed. This suggests that only the EB-354 mRNA and the EB-186 mRNA are rec ognized by the mRNA editing mechanism in CaCo-2 cells.
We inserted apo-B sequences of 354, 186, and 63 nucleotides into an apo-E expres sion vector and transfected the resulting vectors into CaCo-2, BNL CL.2, and McArdle 7777 cells. The chimeric apo-EB mRNA containing 354 and 186 nucleotides of apo-B mRNA was edited as efficiently as the endogenous apo-B mRNA in all three cell lines.
By mutation analysis, Chen et al. (32) showed that the recognition mechanism might be somewhat tolerant: 20 mutations immediately flanking the cytosine 6666 had only a mar ginal effect on editing, whereas two other mutations abolished editing.
The apo-B48 mRNA editing seems to be regulated both on a developmental level and on a hormonal level. For example, during fetal life there is a progressive conversion in the intestine from the exclusive synthesis of apo-BlOO to the exclusive synthesis of apo B48 (33). Furthermore, Davidson et al. treated rats with thyroxine and found that the liv er of the hyperthyroid rats produced only apo-B48 (34). The regulatory mechanisms for these developmental and hormonal changes are not known.
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