Essential role of NAT1/p97/DAP5 in embryonic differentiation and the retinoic acid pathway.

NAT1/p97/DAP5 is a newly identified protein that shares homology with the translation initiation factor eIF4G. Studies in vitro and in transfected cells indicated that NAT1 might suppress global translation, thereby repressing cellular proliferation. Here we studied the functions of NAT1 in vivo by disrupting its gene in mice. NAT1(-/-) embryos died during gastrulation, indicating a crucial role for NAT1 in embryogenesis. Undifferentiated NAT1(-/-) embryonic stem cells were normal in morphology, proliferation, global translation and gene expression profile. However, NAT1(-/-) cells exhibited an impaired ability to differentiate: they were resistant to differentiation induced by retinoic acid, and teratomas derived from them consisted of undifferentiated and poorly differentiated tissues. The expression of retinoic acid-responsive genes, such as the cell-cycle inhibitor p21(WAF1), was selectively impaired in NAT1(-/-) cells. Transcription from synthetic retinoic acid-responsive elements was also impaired. These data demonstrated that this translation initiation factor homolog controls specific gene expression pathways required for cellular differentiation.

Binding of an antibody mimetic of the human low density lipoprotein receptor to apolipoprotein E is governed through electrostatic forces. Studies using site-directed mutagenesis and molecular modeling.

Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp(52), Glu(53), and Asp(56) are essential for high-affinity binding. Although Asp(31) (CDRH1), Glu(58) (CDRH2), and Asp(97) (CDRH3) did not appear to be critical, the Asp(97) --> Ala variant acquired reactivity with apoE2. A Thr(57) --> Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8.apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.

Low expression of the apolipoprotein B mRNA-editing transgene in mice reduces LDL levels but does not cause liver dysplasia or tumors.

Hepatic expression of apolipoprotein (apo) B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1) has been proposed as a gene therapy approach for lowering plasma low density lipoprotein (LDL) levels. However, high-level expression of APOBEC-1 in transgenic mouse and rabbit livers causes liver dysplasia and hepatocellular carcinoma. To determine the physiological and pathological effects of low-level hepatic expression of APOBEC-1, we used a 52-kb rat APOBEC-1 genomic clone (RE4) to generate transgenic mice expressing low levels of APOBEC-1 (2 to 5 times those in nontransgenic mice). Liver function, liver histology, editing of apoB mRNA at the normal editing site (C6666), and abnormal editing at multiple sites (hyperediting) in these mice were compared with those in transgenic mice expressing intermediate (I-20) or high (I-28) levels of APOBEC-1 in the liver. Hyperediting of mRNA coding for the novel APOBEC-1 target 1 (NAT1) was also examined. In the high-expressing I-28 line, 50% of the mice had palpable tumors at 15 weeks of age, whereas in the intermediate-expressing I-20 line, 50% of the mice had evidence of liver tumors after 1 year. In contrast, low-expressing RE4 mice had normal liver function and histology and did not develop liver tumors when examined at 3 to 17 months of age. Moreover, hyperediting of apoB and NAT1 mRNA in the liver was robust in the I-20 mice but barely detectable in the RE4 mice. The low-level expression resulted in sufficient APOBEC-1 to edit essentially all apoB mRNA at the normal editing site, virtually eliminating apoB-100 and LDL in the plasma of RE4 mice. When RE4 mice were crossed with human apoB transgenic mice, which possess high plasma LDL concentrations, plasma LDL levels in the offspring were reduced to very low levels. These results indicates that long-term hepatic expression of APOBEC-1 at low levels sufficient to eliminate LDL does not cause apparent liver damage or liver tumors in transgenic mice. RE4 APOBEC-1 transgenic mice should prove valuable for studying the roles of apoB-containing lipoproteins in lipid metabolism and atherosclerosis.

Two efficiency elements flanking the editing site of cytidine 6666 in the apolipoprotein B mRNA support mooring-dependent editing.

Normally, apolipoprotein B (apoB) mRNA editing deaminates a single cytidine (C6666) in apoB mRNA. However, when the catalytic subunit of the editing enzyme complex, APOBEC-1, was overexpressed in transgenic mice and rabbits, numerous cytidines in the apoB mRNA and in a novel mRNA, NAT1, were aberrantly hyperedited, and the animals developed liver dysplasia and hepatocellular carcinomas. To identify the RNA motifs in the apoB mRNA that support physiological editing and those that support aberrant hyperediting, we constructed rabbit apoB RNA substrates and tested them in vitro for physiological editing and hyperediting. Three previously unrecognized RNA elements that are critical for efficient physiological editing at C6666 were identified. In concert with the mooring sequence (6671-6681), the 5' efficiency element (6609-6628), an A-rich region (6629-6640), and the 3' efficiency element (6717-6747) increased editing at C6666. The 5' efficiency element was the most potent, elevating physiological editing to wild-type levels in combination with the mooring sequence. The 3' efficiency element was somewhat less important but increased physiological editing to levels approaching wild type. These elements encompass 139 nucleotides on the apoB RNA transcript and are sufficient for editing with the efficiency of full-length apoB mRNA. Furthermore, a distal downstream apoB region (6747-6824) may function as a recognition element in the apoB mRNA. Hyperediting at C6802 in the rabbit apoB mRNA is mediated by RNA elements similar to those required for normal physiological editing at C6666. Similarly sized upstream and downstream flanking regions of C6802 are necessary for hyperediting in combination with a degenerate mooring sequence.

Identification of the low density lipoprotein receptor-binding site in apolipoprotein B100 and the modulation of its binding activity by the carboxyl terminus in familial defective apo-B100.

Familial defective apolipoprotein B100 (FDB) is caused by a mutation of apo-B100 (R3500Q) that disrupts the receptor binding of low density lipoproteins (LDL), which leads to hypercholesterolemia and premature atherosclerosis. In this study, mutant forms of human apo-B were expressed in transgenic mice, and the resulting human recombinant LDL were purified and tested for their receptor-binding activity. Site-directed mutagenesis and other evidence indicated that Site B (amino acids 3,359-3,369) binds to the LDL receptor and that arginine-3,500 is not directly involved in receptor binding. The carboxyl-terminal 20% of apo-B100 is necessary for the R3500Q mutation to disrupt receptor binding, since removal of the carboxyl terminus in FDB LDL results in normal receptor-binding activity. Similarly, removal of the carboxyl terminus of apo-B100 on receptor-inactive VLDL dramatically increases apo-B-mediated receptor-binding activity. We propose that the carboxyl terminus normally functions to inhibit the interaction of apo-B100 VLDL with the LDL receptor, but after the conversion of triglyceride-rich VLDL to smaller cholesterol-rich LDL, arginine-3,500 interacts with the carboxyl terminus, permitting normal interaction between LDL and its receptor. Moreover, the loss of arginine at this site destabilizes this interaction, resulting in receptor-binding defective LDL.

Human low density lipoprotein receptor fragment. Successful refolding of a functionally active ligand-binding domain produced in Escherichia coli.

The low density lipoprotein (LDL) receptor plays a key role in cholesterol homeostasis, mediating cellular uptake of lipoprotein particles by high affinity binding to its ligands, apolipoprotein (apo) B-100 and apoE. The ligand-binding domain of the LDL receptor contains 7 cysteine-rich repeats of approximately 40 amino acids; each repeat contains 6 cysteines, which form 3 intra-repeat disulfide bonds. As a first step toward determining the structure of the LDL receptor, both free and bound to its ligands, we produced in Escherichia coli a soluble fragment containing the ligand-binding domain (residues 1-292) as a thrombin-cleavable, heat-stable thioredoxin fusion. Modest amounts (5 mg/liter) of partially purified but inactive fragment were obtained after cell lysis, heat treatment, thrombin cleavage, and gel filtration under denaturing conditions. We were able to refold the receptor fragment to an active conformation with approximately 10% efficiency. The active fragment was isolated and purified with an LDL affinity column. The refolded receptor fragment was homogeneous, as determined by sodium dodecyl sulfate or non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing. The purified fragment did not react with fluorescein-5-maleimide, indicating that all 42 cysteines were disulfide linked. In addition, the refolded fragment exhibited properties identical to those of the intact native receptor: Ca2+-dependent binding and isoform-dependent apoE binding (apoE2 binding <5% of apoE3). Furthermore, antibodies to the fragment recognized native receptors and inhibited the binding of 125I-LDL to fibroblast LDL receptors. We conclude that we have produced a properly folded and fully active receptor fragment that can be used for further structural studies.

Uptake of chylomicrons by the liver, but not by the bone marrow, is modulated by lipoprotein lipase activity.

We have shown that chylomicrons are catabolized by the liver and bone marrow in rabbits and marmosets. In the present investigation, we studied the role of various apolipoproteins and lipoprotein lipase in the clearance of these particles by the liver and bone marrow in rabbits. Incubation of chylomicrons with purified apolipoprotein (apo) E or C-II resulted in more rapid clearance of these particles from the plasma, whereas incubation of chylomicrons with apoA-I, apoC-I, apoC-III1, or apoC-III2, did not affect their clearance rates. Analysis of tissue uptake revealed that the increased plasma clearance rate of chylomicrons enriched with apoE or apoC-II was primarily due to enhanced uptake by the liver. The uptake of chylomicrons by the bone marrow increased after their enrichment with apoA-I but decreased after their enrichment with apoC-II. Because apoC-II is a cofactor for lipoprotein lipase, we hypothesized that the increased clearance rates were due to faster hydrolysis of chylomicrons and rapid generation of chylomicron remnants. To test this hypothesis, lipoprotein lipase activity was inhibited by injection of an antilipoprotein lipase monoclonal antibody. Inhibition of lipoprotein lipase retarded clearance of chylomicrons from the plasma and decreased their uptake by the liver but did not affect their uptake by the bone marrow. These studies suggest that bone marrow can take up chylomicrons in the absence of lipoprotein lipase activity and provide an explanation for the presence of foam cells in the bone marrow of type I hyperlipoproteinemic patients.

Two distinct TATA-less promoters direct tissue-specific expression of the rat apo-B editing catalytic polypeptide 1 gene.

The species and tissue specificity of apolipoprotein (apo) B mRNA editing is determined by the expression of apoB editing catalytic polypeptide 1 (APOBEC-1), the cytidine deaminase that catalyzes apoB mRNA editing. To understand the molecular mechanisms that regulate the transcription of APOBEC-1, we characterized rat APOBEC-1 cDNA and genomic DNA. cDNA cloning and RNase protection analysis showed two alternative promoters for the tissue-specific expression of APOBEC-1 in the liver and intestine, Pliv and Pint. Both promoters lack a TATA box, and Pint belongs to the MED-1 class of promoters, which initiate transcription at multiple sites. We also identified two allelic forms of the APOBEC-1 gene from the characterization of two rat APOBEC-1 P1 genomic clones, RE4 and RE5. The RE4 allele is 18 kilobases long and contains six exons and five introns, whereas the RE5 allele contains an additional approximately 8 kilobases of intron sequences and an extra exon encoding a 5'-untranslated region; however, the APOBEC-1 transcripts from the two alleles appear to have similar, if not identical, functions. Transgenic mouse studies showed that Pliv was preferentially used in the liver, kidney, brain, and adipose tissues, whereas Pint was preferentially used in the small intestine, stomach, and lung. Our results suggest that the tissue-specific expression of APOBEC-1 is governed by multiple regulatory elements exerting control over a single coding sequence. The presence or absence of these regulatory elements may determine the tissue-specific expression of APOBEC-1 in other mammalian species.

A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA-editing enzyme.

Transgene expression of the apolipoprotein B mRNA-editing enzyme (APOBEC-1) causes dysplasia and carcinoma in mouse and rabbit livers. Using a modified differential display technique, we identified a novel mRNA (NAT1 for novel APOBEC-1 target no. 1) that is extensively edited at multiple sites in these livers. The aberrant editing alters encoded amino acids, creates stop codons, and results in markedly reduced levels of the NAT1 protein in transgenic mouse livers. NAT1 is expressed ubiquitously and is extraordinarily conserved among species. It has homology to the carboxy-terminal portion of the eukaryotic translation initiation factor (eIF) 4G that binds eIF4A and eIF4E to form eIF4F. NAT1 binds eIF4A but not eIF4E and inhibits both cap-dependent and cap-independent translation. NAT1 is likely to be a fundamental translational repressor, and its aberrant editing could contribute to the potent oncogenesis induced by overexpression of APOBEC-1.

Differences in receptor binding of LDL subfractions.

Differences in low density lipoprotein (LDL) receptor-binding affinity among LDL particles of different size were examined in competitive binding assays in human skin fibroblasts and LDL (d = 1.020 to 1.050 g/mL) from subjects with a predominance of large (> or = 272 A), medium (259 to 271 A), and small (< or = 257 A) LDL. Among 57 normolipidemic subjects with LDL cholesterol (-C) levels < 160 mg/dL, binding affinity was reduced by 16% in those with predominantly large LDL and by 14% in those with small LDL compared with most subjects who had a predominance of medium-size LDL and in all LDL size subgroups in 66 subjects with LDL-C > or = 160 mg/dL. Differences in LDL receptor-binding affinity were further investigated by using LDL density subfractions (I, d = 1.026 to 1.032 g/mL; II, d = 1.032 to 1.038 g/mL; and III, d = 1.038 to 1.050 g/mL) from three subjects with predominantly large (pattern A) and small (pattern B) LDL particles. The binding affinity (Kd) of LDL-II was similar for patterns A and B (9.2 +/- 1.4 and 9.4 +/- 0.7, respectively) and 30% lower in LDL-III from both groups (P < .05). The binding affinity of LDL-I in pattern A (12.6 +/- 1.5 micrograms/mg) was lower (P < .05) than that in LDL-II and LDL-I from pattern B (8.0 +/- 2.4 micrograms/mg). After incubation with a monoclonal antibody that specifically blocked the LDL receptor-binding domain of apoE, LDL-I from two pattern B subjects showed substantially lower binding affinity (Kd = 20.0 and 19.2 micrograms/mg) than in pattern A (Kd = 13.2 and 14.2 micrograms/mg), a result consistent with our finding of a higher apoE content in pattern B LDL-I (P < .001). Thus, factors associated with variations in particle size and apoE content in LDL subclasses in normolipidemic subjects contribute to the differences in LDL receptor binding that may result in differing metabolic behavior in vivo.

Hyperediting of multiple cytidines of apolipoprotein B mRNA by APOBEC-1 requires auxiliary protein(s) but not a mooring sequence motif.

An RNA-binding cytidine deaminase (APOBEC-1) and unidentified auxiliary protein(s) are required for apolipoprotein (apo) B mRNA editing. A sequence motif on apoB mRNA ("mooring sequence," nucleotides 6671-6681) is obligatory for the editing of cytidine 6666 (C6666), the only cytidine on apoB mRNA converted to uridine in normal animals. Transgenic animals with hepatic overexpression of APOBEC-1 develop liver tumors, and other non-apoB mRNAs are edited, suggesting a loss of the normally precise specificity. In this study, we examined apoB mRNA from these transgenic animals to determine if cytidines aside from C6666 are edited. Multiple cytidines downstream from C6666 in apoB mRNA were edited extensively by the overexpressed APOBEC-1. This pathophysiological "hyperediting" could be mimicked in vitro by incubating a synthetic apoB RNA substrate with the transgenic mouse liver extracts. Multiple cytidines in the synthetic apoB RNA were edited by recombinant APOBEC-1 but only with supplementation of the auxiliary protein(s). Mutations in the mooring sequence markedly decreased the normal editing of C6666 but, surprisingly, increased the hyperediting of downstream cytidines. Furthermore, cytidines in an apoB RNA substrate lacking the mooring sequence were also edited in vitro. These results indicate that the hyperediting of apoB mRNA by overexpressed APOBEC-1 depends upon auxiliary protein(s) but is independent of the mooring sequence motif. These results suggest that hyperediting may represent the first step in a two-step recognition model for normal apoB mRNA editing.

Apolipoprotein B mRNA-editing protein induces hepatocellular carcinoma and dysplasia in transgenic animals.

Apolipoprotein (apo-) B mRNA editing is the deamination of cytidine that creates a new termination codon and produces a truncated version of apo-B (apo-B48). The cytidine deaminase catalytic subunit [apo-B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1)] of the multiprotein editing complex has been identified. We generated transgenic rabbits and mice expressing rabbit APOBEC-1 in their livers to determine whether hepatic expression would lower low density lipoprotein cholesterol concentrations. The apo-B mRNA from the livers of the transgenic mice and rabbit was extensively edited, and the transgenic animals had reduced concentrations of apo-B100 and low density lipoproteins compared with control animals. Unexpectedly, all of the transgenic mice and a transgenic rabbit had liver dysplasia, and many transgenic mice developed hepatocellular carcinomas. Many of the mouse livers were hyperplastic and filled with lipid. Other hepatic mRNAs with sequence motifs similar to apo-B mRNA were examined for this type of editing (i.e., cytidine deamination). One of these, tyrosine kinase, was edited in livers of transgenic mice but not of controls. This result demonstrates that other mRNAs can be edited by the overexpressed editing enzyme and suggests that aberrant editing of hepatic mRNAs involved in cell growth and regulation is the cause of the tumorigenesis. Finally, these findings compromise the potential use of APOBEC-1 for gene therapy to lower plasma levels of low density lipoproteins.

Cloning and mutagenesis of the rabbit ApoB mRNA editing protein. A zinc motif is essential for catalytic activity, and noncatalytic auxiliary factor(s) of the editing complex are widely distributed.

Apolipoprotein (apo) B mRNA editing is the specific deamination of cytidine (nucleotide 6666) to uridine in apoB mRNA. We isolated a full-length cDNA clone encoding the rabbit apoB mRNA editing protein (REPR), a subunit of the editing complex. Rabbit REPR is analogous to a rat enterocyte 27-kDa protein that has been shown to have cytidine deaminase activity. Like rat REPR, rabbit REPR edited synthetic apoB RNA when mixed with chicken enterocyte extract. Surprisingly, the REPR also acquired editing activity when mixed with extracts from various organs of the rabbit (liver, gallbladder, stomach, intestine, adrenals, thyroid, testes, spleen, kidney, and lung) or the chicken (kidney and liver). In contrast, the rabbit REPR mRNA was found only in the small and large intestine. Thus, the auxiliary protein(s) of the apoB mRNA editing complex, which are essential for editing activity, exist in organs devoid of significant apoB mRNA editing or apoB synthesis. REPR requires zinc for its catalytic activity. We mutated putative zinc-coordinating residues (His61, Cys93, Cys96) and 2 additional residues (Glu63, Pro92) of the rabbit REPR that are conserved in other cytidine or deoxycytidylate deaminases and in rat REPR. The wild-type and mutant REPR cDNAs each produced 28-kDa proteins when transcribed and translated in vitro. Compared with the wild-type editing activity, the mutations of His61-->Ala, Glu63-->Ala, Cys93-->Ala, and Cys96-->Ala abolished or greatly reduced editing activity, whereas the mutations of His61-->Cys (which also can coordinate zinc) and Pro92-->Ala had a lesser effect. These results indicate that His61, Cys93, and Cys96 are essential for editing activity, probably because they coordinate zinc, whereas Glu63 also is essential, because it may be involved in the deaminase reaction. In addition, the widespread distribution of the auxiliary factor(s) portends their involvement in other RNA editing reactions.

A unique haplotype of the apolipoprotein B-100 allele associated with familial defective apolipoprotein B-100 in a Chinese man discovered during a study of the prevalence of this disorder.

The prevalence of familial defective apolipoprotein (apo) B-100 (FDB) was determined by sampling 5,160 volunteer subjects from among 14,058 eligible employees of a bank in California. The sample was ethnically diverse (44.6% of the population was non-Caucasian). The prevalence of FDB in the study population was 0.08% with a 90% confidence interval of 0.01-0.14%. Four subjects were found to have the apoB 3500 codon mutation by mutagenic polymerase chain reaction, which creates an MspI site at the 3500 codon of normal alleles but not alleles coding for the Arg-->Gln mutation of FDB. Three of these were Caucasian and born in North America. The fourth was a native of China. Haplotype analysis of the affected allele of the Chinese subject using 10 markers described by Ludwig and McCarthy (1990. Am. J. Hum. Genet. 47: 712-720) revealed a unique haplotype that differed from the haplotype of all other subjects with FDB. This unique allele had 30 repeats of a 3' hypervariable element instead of 48 as was found in the allele associated with FDB in other subjects, and in the 3' region there was an EcoRI site that was also not present in the allele most commonly found in association with FDB. We conclude that the prevalence of FDB in our ethnically diverse population is lower than that reported in previous studies of predominantly Caucasian populations and that the Chinese subject represents either an independent mutation or possibly recombination at the 3' end of the apoB gene, an event not previously described.

Role of heparan sulfate proteoglycans in the binding and uptake of apolipoprotein E-enriched remnant lipoproteins by cultured cells.

Addition of apolipoprotein (apo) E to rabbit beta-very low density lipoproteins (beta-VLDL) has been shown to result in a marked enhancement of their binding and uptake by various cell types. Apolipoprotein E binds to lipoprotein receptors and proteoglycans. To distinguish between apoE binding to these sites, cells were treated with heparinase. Heparinase treatment of receptor-negative familial hypercholesterolemic (FH) fibroblasts and human hepatoma cells (HepG2) released 30-40% of newly synthesized cell surface 35S-labeled proteoglycans and decreased the binding of beta-VLDL+apoE to FH and normal fibroblasts and HepG2 cells by more than 80%. Furthermore, heparinase treatment significantly decreased the uptake of fluorescently labeled beta-VLDL+apoE by HepG2 cells and decreased cholesteryl ester synthesis in FH fibroblasts by 75%. Likewise, canine chylomicron remnants enriched in apoE demonstrated enhanced binding that was 80% inhibited by heparinase treatment of HepG2 cells. Heparinase treatment did not affect beta-VLDL (without added apoE) or low density lipoprotein (LDL) binding to these cells or the binding activity of beta-VLDL+apoE to the LDL receptor-related protein (LRP) or to the LDL receptor on ligand blots. Chinese hamster ovary (CHO) mutant cells lacking the synthesis of either heparan sulfate (pgsD-677) or all proteoglycans (pgsA-745) did not display any enhanced binding of the beta-VLDL+apoE. By comparison, wild-type CHO cells demonstrated enhanced binding of beta-VLDL+apoE that could be abolished by treatment with heparinase. These mutant cells and wild-type CHO cells possessed a similar amount of LRP, as determined by ligand blot analyses and by alpha 2-macroglobulin binding, and possessed a similar amount of LDL receptor activity, as determined by LDL binding. Therefore, we would interpret these data as showing that heparan sulfate proteoglycan may be involved in the initial binding of the apoE-enriched remnants with the subsequent involvement of the LRP in the uptake of these lipoproteins. It remains to be determined whether the heparan sulfate proteoglycan can function by itself in both the binding and internalization of the apoE-enriched remnants or whether the proteoglycan is part of a complex with LRP that mediates a two-step process, i.e. binding and subsequent internalization by the receptor.

The influence of particle size and multiple apoprotein E-receptor interactions on the endocytic targeting of beta-VLDL in mouse peritoneal macrophages.

Low density lipoprotein (LDL) and beta-very low density lipoprotein (beta-VLDL) are internalized by the same receptor in mouse peritoneal macrophages and yet their endocytic patterns differ; beta-VLDL is targeted to both widely distributed and perinuclear vesicles, whereas LDL is targeted almost entirely to perinuclear lysosomes. This endocytic divergence may have important metabolic consequences since beta-VLDL is catabolized slower than LDL and is a more potent stimulator of acyl-CoA/cholesterol acyl transferase (ACAT) than LDL. The goal of this study was to explore the determinants of beta-VLDL responsible for its pattern of endocytic targeting. Fluorescence microscopy experiments revealed that large, intestinally derived, apoprotein (Apo) E-rich beta-VLDL was targeted mostly to widely distributed vesicles, whereas small, hepatically derived beta-VLDL was targeted more centrally (like LDL). Furthermore, the large beta-VLDL had a higher ACAT-stimulatory potential than the smaller beta-VLDL. The basis for these differences was not due to fundamental differences in the means of uptake; both large and small beta-VLDL were internalized by receptor-mediated endocytosis (i.e., not phagocytosis) involving the interaction of Apo E of the beta-VLDL with the macrophage LDL receptor. However, large beta-VLDL was much more resistant to acid-mediated release from LDL receptors than small beta-VLDL. Furthermore, partial neutralization of the multiple Apo Es on these particles by immunotitration resulted in a more perinuclear endocytic pattern, a lower ACAT-stimulatory potential, and an increased sensitivity to acid-mediated receptor release. These data are consistent with the hypothesis that the interaction of the multivalent Apo Es of large beta-VLDL with multiple macrophage LDL receptors leads to a diminished or retarded release of the beta-VLDL from its receptor in the acidic sorting endosome which, in turn, may lead to the widely distributed endocytic pattern of large beta-VLDL. These findings may represent a physiologically relevant example of a previously described laboratory phenomenon whereby receptor cross-linking by multivalent ligands leads to a change in receptor targeting.

Genetic defects in lipoprotein metabolism. Elevation of atherogenic lipoproteins caused by impaired catabolism.

Certain proteins (called apolipoproteins B and E) on the surface of lipoprotein particles are responsible for mediating the binding of cholesterol-rich particles to specific lipoprotein receptors on the surface of cells and represent a major pathway controlling blood cholesterol levels. Three important disorders of lipoprotein metabolism, which provide insights into the molecular mechanisms responsible for the elevation of specific atherogenic lipoproteins, are the following: (1) Type III hyperlipoproteinemia results from specific mutations in apolipoprotein E that prevent the normal binding of chylomicron remnants and very-low-density lipoprotein remnants to lipoprotein receptors. Patients with this disorder who have elevated levels of these remnant lipoproteins develop atherosclerosis. (2) Familial defective apolipoprotein B-100 results from a single amino acid substitution in apolipoprotein B that prevents low-density lipoprotein from binding normally to the low-density lipoprotein receptor and elevates plasma cholesterol levels. (3) Familial hypercholesterolemia, which results in elevated levels of plasma low-density lipoprotein and premature atherosclerosis, is caused by a variety of mutations in the low-density lipoprotein receptor that interfere with the normal binding of lipoproteins to this receptor. These observations not only provide insights into the mechanisms responsible for normal lipoprotein metabolism, but also highlight the potential role of specific lipoproteins in atherogenesis.

Apolipoprotein B mRNA editing. Direct determination of the edited base and occurrence in non-apolipoprotein B-producing cell lines.

In humans, apolipoprotein (apo) B48 is synthesized in the intestine as an obligatory constituent of chylomicrons. Apolipoprotein B48 is identical to the amino-terminal 2152 amino acids (240 kDa) of apoB100 and is translated from an edited apoB mRNA in which codon 2153 has been converted from glutamine (CAA) to what is recognized as a premature stop codon (UAA). To determine whether the apoB mRNA editing in fact converts cytosine 6666 in codon 2153 to uracil, we incubated a synthetic apoB RNA containing 32P-labeled cytosines in an in vitro editing system prepared from rabbit enterocytes. The in vitro edited RNA was purified and digested to nucleoside 5'-monophosphates, which were analyzed on two-dimensional thin-layer chromatography. We found that the edited base co-migrated with authentic uridine 5'-monophosphate. Thus, cytosine 6666 is converted to uracil, most likely by a nucleotide-specific cytosine deaminase. To determine whether apoB mRNA editing occurs in cell lines that do not synthesize apoB, we stably transfected a high expression vector containing 354 base pairs of apoB sequence into 18 different cell lines. We found apoB mRNA editing activity in five osteosarcoma cell lines and one epidermoid cell line, none of which synthesizes any detectable apoB. Thus, apoB mRNA editing occurs in cell lines that do not synthesize apoB, which suggests that mRNA editing may be a common biological phenomenon in eukaryotic cells.