Lipoprotein clearance mechanisms in LDL receptor-deficient "Apo-B48-only" and "Apo-B100-only" mice.

The role of the low density lipoprotein receptor (LDLR) in the clearance of apo-B48-containing lipoproteins and the role of the LDLR-related protein (LRP) in the removal of apo-B100-containing lipoproteins have not been clearly defined. To address these issues, we characterized LDLR-deficient mice homozygous for an "apo-B48-only" allele, an "apo-B100-only" allele, or a wild-type apo-B allele (Ldlr-/- Apob48/48, Ldlr-/-Apob100/100, and Ldlr-/-Apob+/+, respectively). The plasma apo-B48 and LDL cholesterol levels were higher in Ldlr-/-Apob48/48 mice than in Apob48/48 mice, indicating that the LDL receptor plays a significant role in the removal of apo-B48-containing lipoproteins. To examine the role of the LRP in the clearance of apo-B100-containing lipoproteins, we blocked hepatic LRP function in Ldlr-/-Apob100/100 mice by adenoviral-mediated expression of the receptor-associated protein (RAP). RAP expression did not change apo-B100 levels in Ldlr-/-Apob100/100 mice. In contrast, RAP expression caused a striking increase in plasma apo-B48 levels in Apob48/48 and Ldlr-/-Apob48/48 mice. These data imply that LRP is important for the clearance of apo-B48-containing lipoproteins but plays no significant role in the clearance of apo-B100-containing lipoproteins.

Apolipoprotein B-100: conservation of lipid-associating amphipathic secondary structural motifs in nine species of vertebrates.

Development of a computer program called LOCATE allowed us to show that human apolipoprotein B-100 is composed of five domains, NH2-alpha1-beta1-alpha2-beta2-alpha3-COOH, enriched, alternately, in amphipathic alpha helixes and amphipathic beta strands. Using updated versions of this program, here we compare the complete sequence of human apolipoprotein B-100 with partial sequences from eight additional species of vertebrates (chicken, frog, hamster, monkey, mouse, pig, rat, and rabbit). The lipid-associating amphipathic alpha helixes cluster in domains alpha2 (between residues 2075 +/- 25 and 2575 +/- 25) and alpha3 (between residues 4100 +/- 100 and 4550 +/- 50) in all species for which those regions have been sequenced but with little conservation of individual helixes. Lipid-associating amphipathic beta strands cluster in domains beta1 (approximately residues 827-2000) and beta2 (approximately residue 2571 to residue 4000 +/- 50) in all species for which these regions have been sequenced, with conservation of several individual amphipathic beta strands. Hydrophobic segments are present in apolipoprotein B-100 sequences of all nine species but the frequency of occurrence is no greater than generally found in beta sheet-containing proteins. We conclude that four alternating lipid-associating domains, -beta1-alpha2-beta2-alpha3-COOH, are common supramolecular features of apolipoprotein B-100 in nine vertebrate species.

Human apolipoprotein B transgenic mice generated with 207- and 145-kilobase pair bacterial artificial chromosomes. Evidence that a distant 5'-element confers appropriate transgene expression in the intestine.

We reported previously that approximately 80-kilobase pair (kb) P1 bacteriophage clones spanning either the human or mouse apoB gene (clones p158 and p649, respectively) confer apoB expression in the liver of transgenic mice, but not in the intestine. We hypothesized that the absence of intestinal expression was due to the fact that these clones lacked a distant DNA element controlling intestinal expression. To test this possibility, transgenic mice were generated with 145- and 207-kb bacterial artificial chromosomes (BACs) that contained the human apoB gene and more extensive 5'- and 3'-flanking sequences. RNase protection, in situ hybridization, immunohistochemical, and genetic complementation studies revealed that the BAC transgenic mice manifested appropriate apoB gene expression in both the intestine and the liver, indicating that both BACs contained the distant intestinal element. To determine whether the regulatory element was located 5' or 3' to the apoB gene, transgenic mice were generated by co-microinjecting embryos with p158 and either the 5'- or 3'-sequences from the 145-kb BAC. Analysis of these mice indicated that the apoB gene's intestinal element is located 5' to the structural gene. Cumulatively, the transgenic mouse studies suggest that the intestinal element is located between -33 and -70 kb 5' to the apoB gene.

Susceptibility to atherosclerosis in mice expressing exclusively apolipoprotein B48 or apolipoprotein B100.

All classes of lipoproteins considered to be atherogenic contain apo-B100 or apo-B48. However, there is a distinct paucity of data regarding whether lipoproteins containing apo-B48 or apo-B100 differ in their intrinsic ability to promote the development of atherosclerosis. To address this issue, we compared the extent of atherosclerosis in three groups of animals: apo-E-deficient mice (apo-B+/+apo-E-/-) and apo-E-deficient mice that synthesize exclusively either apo-B48 (apo-B48/48apo-E-/-) or apo-B100 (apo-B100/100apo-E-/-). Mice (n = 25 in each group) were fed a chow diet for 200 days, and plasma lipid levels were assessed throughout the study. Compared with the levels in apo-B+/+apo-E-/- mice, the total plasma cholesterol levels were higher in the apo-B48/48apo-E-/- mice and were lower in the apo-B100/100apo-E-/- mice. However, the ranges of cholesterol levels in the three groups overlapped. Compared with those in the apo-B+/+apo-E-/- mice, atherosclerotic lesions were more extensive in the apo-B48/48apo-E-/- mice and less extensive in the apo-B100/100apo-E-/- mice. Once again, however, there was overlap among the three groups. The extent of atherosclerosis in each group of mice correlated significantly with plasma cholesterol levels. In mice from different groups that had similar cholesterol levels, the extent of atherosclerosis was quite similar. Thus, susceptibility to atherosclerosis was dependent on total cholesterol levels. Whether mice synthesized apo-B48 or apo-B100 did not appear to have an independent effect on susceptibility to atherosclerosis.

Reading-frame restoration by transcriptional slippage at long stretches of adenine residues in mammalian cells.

We previously characterized a mutant apoB allele (the apoB86 allele) that produces both a truncated apoB (apoB86) and a full-length apoB100. The mutant allele contained a deletion of a single cytosine in exon 26, creating a stretch of eight consecutive adenines in the -1 reading frame. The altered reading-frame allele was restored, with approximately 10% efficiency, by the transcriptional insertion of an extra adenine into the stretch of eight consecutive adenines, thereby accounting for the synthesis of the full-length apoB100. Here, we demonstrate that this reading-frame restoration does not occur when the long stretch of adenines is interrupted by a cytosine. To assess whether reading-frame restoration is unique to a single site in the apoB gene, the same mutation (eight consecutive adenines in the -1 reading frame) was inserted into another site within the apoB gene. Reading-frame restoration occurred at the second site and was abrogated when the stretch of adenines was interrupted by another base. Of note, a computerized analysis of human cDNA sequences revealed that long stretches of adenines in protein-coding sequences occur at a lower than predicted frequency, suggesting that evolution has selected against these sequences.

Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100.

Apolipoprotein (apo)-B is found in two forms in mammals: apo-B100, which is made in the liver and the yolk sac, and apo-B48, a truncated protein made in the intestine. To provide models for understanding the physiologic purpose for the two forms of apo-B, we used targeted mutagenesis of the apo-B gene to generate mice that synthesize exclusively apo-B48 (apo-B48-only mice) and mice that synthesize exclusively apo-B100 (apo-B100-only mice). Both the apo-B48-only mice and apo-B100-only mice developed normally, were healthy, and were fertile. Thus, apo-B48 synthesis was sufficient for normal embryonic development, and the synthesis of apo-B100 in the intestines of adult mice caused no readily apparent adverse effects on intestinal function or nutrition. Compared with wild-type mice fed a chow diet, the levels of low density lipoprotein (LDL)-cholesterol and very low density lipoprotein- and LDL-triacylglycerols were lower in apo-B48-only mice and higher in the apo-B100-only mice. In the setting of apo-E-deficiency, the apo-B100-only mutation lowered cholesterol levels, consistent with the fact that apo-B100-lipoproteins can be cleared from the plasma via the LDL receptor, whereas apo-B48-lipoproteins lacking apo-E cannot. The apo-B48-only and apo-B100-only mice should prove to be valuable models for experiments designed to understand the purpose for the two forms of apo-B in mammalian metabolism.

Transgenic mice that overexpress mouse apolipoprotein B. Evidence that the DNA sequences controlling intestinal expression of the apolipoprotein B gene are distant from the structural gene.

An 87-kilobase (kb) P1 bacteriophage clone (p649) spanning the mouse apolipoprotein (apo) B gene was used to generate transgenic mice that express high levels of mouse apoB. Plasma levels of apoB, low density lipoprotein cholesterol, and low density lipoprotein triglycerides were increased, and high density lipoprotein cholesterol levels were decreased in the transgenic mice, compared with nontransgenic littermate controls. Although p649 contained 33 kb of 5'-flanking sequences and 11 kb of 3'-flanking sequences, the tissue pattern of transgene expression was different from that of the endogenous apoB gene. RNA slot blots and RNase protection analysis indicated that the transgene was expressed in the liver but not in the intestine, whereas the endogenous apoB gene was expressed in both tissues. To confirm the absence of transgene expression in the intestine, the mouse apoB transgenic mice were mated with the apoB knockout mice, and transgenic mice that were homozygous for the apoB knockout mutation were obtained. Because of the absence of transgene expression in the intestine, those mice lacked all intestinal apoB synthesis, resulting in a marked accumulation of fats within the intestinal villus enterocytes. The current studies, along with prior studies of human apoB transgenic animals, strongly suggest that the DNA sequence element(s) controlling intestinal expression of the apoB gene is located many kilobases from the structural gene.

Transgenic mice expressing high levels of human apolipoprotein B develop severe atherosclerotic lesions in response to a high-fat diet.

We previously generated transgenic mice expressing human apolipoprotein (apo-) B and demonstrated that the plasma of chow-fed transgenic animals contained markedly increased amounts of LDL (Linton, M. F., R. V. Farese, Jr., G. Chiesa, D. S. Grass, P. Chin, R. E. Hammer, H. H. Hobbs, and S. G. Young 1992. J. Clin. Invest. 92:3029-3037). In this study, we fed groups of transgenic and nontransgenic mice either a chow diet or a diet high in fat (16%) and cholesterol (1.25%). Lipid and lipoprotein levels were assessed, and after 18 wk of diet, the extent of aortic atherosclerotic lesions in each group of animals was quantified. Compared with the female transgenic mice on the chow diet, female transgenic mice on the high-fat diet had higher plasma levels of cholesterol (312 +/- 17 vs 144 +/- 7 mg/dl; P < 0.0001) and human apo-B (120 +/- 8 vs 84 +/- 3 mg/dl; P < 0.0001). The higher human apo-B levels were due to increased plasma levels of human apo-B48; the human apo-B100 levels did not differ in animals on the two diets. In mice on the high-fat diet, most of the human apo-B48 and apo-B100 was found in LDL-sized particles. Compared with nontransgenic mice on the high-fat diet, the transgenic animals on the high-fat diet had significantly increased levels of total cholesterol (312 +/- 17 vs 230 +/- 19 mg/dl; P < 0.0001) and non-HDL cholesterol (283 +/- 17 vs 193 +/- 19 mg/dl; P < 0.0001). The extent of atherosclerotic lesion development within the ascending aorta was quantified by measuring total lesion area in 60 progressive sections, using computer-assisted image analysis. Neither the chow-fed transgenic mice nor the chow-fed nontransgenic mice had significant atherosclerotic lesions. Nontransgenic animals on the high-fat diet had relatively small atherosclerotic lesions (< 15,000 microns 2/section), almost all of which were confined to the proximal 400 microns of the aorta near the aortic valve. In contrast, transgenic animals on the high-fat diet had extensive atherosclerotic lesions (> 160,000 microns 2/section) that were widely distributed throughout the proximal 1,200 microns of the aorta. Thus, human apo-B expression, in the setting of a diet rich in fats, causes severe atherosclerosis in mice.

Transgenic mice expressing human apoB100 and apoB48.

Transgenic mice that express human apolipoprotein (apo)B have been developed by microinjecting fertilized mouse oocytes with an 80 kb genomic DNA fragment that encompasses the entire human APOB gene. In the transgenic mice expressing the largest amounts of human apoB, the concentration of human apoB100 in the plasma is nearly as high as the levels observed in normolipidemic humans (50 mg/dl). Virtually all of the human apoB100 in the transgenic plasma is located in the LDL fraction, resulting in substantially increased levels of LDL cholesterol. These human apoB-transgenic mice should be useful animal models for understanding various aspects of lipoprotein metabolism and for further delineating the role of LDL in atherogenesis.

Reading-frame restoration with an apolipoprotein B gene frameshift mutation.

We examined a mutant human apolipoprotein B (apoB) allele that causes hypobetalipoproteinemia and has a single cytosine deletion in exon 26. This frameshift mutation was associated with the synthesis of a truncated apoB protein of the predicted size; however, studies in human subjects and minigene expression studies in cultured cells indicated that the mutant allele also yielded a full-length apoB protein. The 1-base-pair deletion in the mutant apoB allele created a stretch of eight consecutive adenines. To understand the mechanism whereby the mutant apoB allele yielded a full-length apoB protein, the cDNA from cells transfected with the mutant apoB minigene expression vector was examined. Splicing of the mRNA was normal; however, 11% of the cDNA clones had an additional adenine within the stretch of eight adenines, yielding nine consecutive adenines. The insertion of the extra adenine, presumably during apoB gene transcription, is predicted to restore the correct apoB reading frame, thereby permitting the synthesis of a full-length apoB protein.

A truncated species of apolipoprotein B, B-83, associated with hypobetalipoproteinemia.

Familial hypobetalipoproteinemia, a syndrome associated with low plasma cholesterol levels, can be caused by apoB gene mutations. We identified a healthy 42-year-old man whose total plasma cholesterol level was 80 mg/dl. His plasma very low density lipoprotein (VLDL) contained a unique truncated apoB species, apoB-83, in addition to the normal B apolipoproteins, apoB-100 and apoB-48. Virtually no apoB-83 was detectable in his low density lipoprotein (LDL). From the subject's kindred, we identified nine other hypocholesterolemic subjects whose VLDL contained apoB-83. A tendency for cholelithiasis was noted in the apoB-83 heterozygotes, particularly in the older individuals. From the apparent size of apoB-83 on SDS-polyacrylamide gels and its reactivity with apoB-specific monoclonal antibodies, we estimated that it would contain approximately 3700-3800 amino acids. DNA sequencing of apoB genomic clones from two affected individuals revealed that apoB-83 was caused by a C----A transversion in exon 26 of the apoB gene (apoB cDNA nucleotide 11458). This mutation converts Ser-3750 (TCA) into a premature stop codon (TAA) and creates a unique MseI restriction endonuclease site. Thus, a single nucleotide transversion in the apoB gene results in a unique truncated apoB species, apoB-83, and the clinical syndrome of familial hypobetalipoproteinemia.

Lipoproteins in pinnipeds: analysis of a high molecular weight form of apolipoprotein E.

We have examined the apolipoprotein content of the lipoproteins of several marine mammals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their apolipoprotein (apo) B-100, apoB-48, and apoA-I migrated to virtually the same position as the comparable human apolipoproteins. In cetaceans (bottlenose dolphins and killer whales), the molecular mass of the apoE was identical to that of human apoE (35 kDa). In contrast, in the lipoproteins of pinnipeds (harbor seals, sea lions, and walrus) there was no protein comparable in size to human apoE; however, there were two proteins in the 40- to 44-kDa range. The protein with the higher apparent molecular weight (44 kDa) was apoA-IV, as determined by NH2-terminal amino acid sequencing. Sequencing of the NH2-terminal 15 amino acids of the lower molecular weight protein (40-42 kDa) revealed no obvious homology with human apoE. However, a human apoE-specific monoclonal antibody, 1D7, bound to the 40- to 42-kDa protein, allowing us to identify that protein as apoE. Sequencing of sea lion apoE cDNA clones demonstrated that sea lion apoE is 311 amino acids in length, 12 residues longer than human apoE. All 12 additional residues are located in the NH2-terminal 31 amino acids, a region that has extremely low homology with the NH2-terminal portion of human apoE. The remainder of the sea lion apoE sequence is 74% homologous to human apoE. The sea lion apoE cDNA was expressed in Chinese hamster ovary (CHO) cells as well as CHO ldlD cells, a cell line that is deficient in O-glycosylation of proteins. The size of the sea lion apoE secreted by these two cell lines, compared with the apoE in sea lion plasma, indicated that the predominant form of apoE in sea lion plasma must be posttranslationally modified. Thus, our studies have demonstrated that the higher apparent molecular weight of pinniped (sea lion) apoE is due to a longer polypeptide chain as well as posttranslational modification of the protein.

A truncated species of apolipoprotein B (B67) in a kindred with familial hypobetalipoproteinemia.

We describe a kindred in which the proband and 6 of his 12 children have hypobetalipoproteinemia. The plasma lipoproteins of the affected subjects contained a unique species of apolipoprotein (apo) B, apo B67, in addition to the normal species, apo B100 and apo B48. The size of apo B67 and immunochemical studies with a panel of apo B-specific antibodies indicated that apo B67 was a truncated species of apo B that contained approximately the amino-terminal 3,000-3,100 amino acids of apo B100. Sequencing of genomic apo B clones revealed that affected family members were heterozygous for a mutant apo B allele containing a single nucleotide deletion in exon 26 (cDNA nucleotide 9327). This frameshift mutation is predicted to result in the synthesis of a truncated apo B containing 3,040 amino acids. Apo B67 is present in low levels in the plasma but is easily detectable within the very low density lipoprotein and low density lipoprotein fractions. Examination of the proband's immediate family revealed seven normolipidemic subjects and seven subjects with hypobetalipoproteinemia. In the affected subjects, the mean total and low density lipoprotein cholesterol levels were 120 and 42 mg/dl, respectively. A significantly higher mean high density lipoprotein cholesterol level was found in the affected subjects (75 vs. 55 mg/dl). We hypothesize that the elevated high density lipoprotein cholesterol levels in subjects heterozygous for the apo B67 mutation may be metabolically linked to the low levels of apo B-containing lipoproteins in their plasma.

DNA sequence of the human apolipoprotein B gene.

The sequence of the human apolipoprotein B gene comprises 43 kb divided into 29 exons, one of which is unusually long and contains 7572 bp. Comparison of the gene sequence with four complete and three partial cDNA sequences published elsewhere reveals a total of 60 nucleotide substitutions and 39 amino acid substitutions and one small deletion in the signal peptide.

Structure of the human apolipoprotein B gene.

Human apolipoprotein B100 cDNA is 14 kilobases in length and encodes a 4563-amino acid precursor protein. The corresponding human gene has been isolated as a series of overlapping lambda clones and extends over 43 kilobases. The gene comprises 29 exons and 28 introns. The distribution of introns is extremely asymmetrical, most of them appearing in the 5'-terminal one-third of the gene. Although most of the exons fall within the normal size limits for mammalian genes, two are unusually long: 1906 and 7572 base pairs. The latter exon is by far the longest reported for a vertebrate gene.