High-level lipoprotein [a] expression in transgenic mice: evidence for oxidized phospholipids in lipoprotein [a] but not in low density lipoproteins.

Efforts to elucidate the role of lipoprotein [a] (Lp[a]) in atherogenesis have been hampered by the lack of an animal model with high plasma Lp[a] levels. We produced two lines of transgenic mice expressing apolipoprotein [a] (apo[a]) in the liver and crossed them with mice expressing human apolipoprotein B-100 (apoB-100), generating two lines of Lp[a] mice. One had Lp[a] levels of approximately 700 mg/dl, well above the 30 mg/dl threshold associated with increased risk of atherosclerosis in humans; the other had levels of approximately 35 mg/dl.

Immunochemical evidence that human apoB differs when expressed in rodent versus human cells.

LDL from human apolipoprotein B-100 (apoB-100) transgenic (HuBTg+/+) mice contains more triglyceride than LDL from normolipidemic subjects. To obtain novel monoclonal antibody (MAb) probes of apoB conformation, we generated hybridomas from HuBTg+/+ that had been immunized with LDL isolated from human plasma. One apoE-specific and four anti-apoB-100-specific hybridomas were identified.

Regulatable liver expression of the rabbit apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1) in mice lacking endogenous APOBEC-1 leads to aberrant hyperediting.

Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which results in translation of the apoB-48 protein instead of the genomically encoded apoB-100. ApoB-48-containing lipoproteins are cleared more rapidly from plasma and are less atherogenic than apoB-100-containing low-density lipoproteins (LDLs). In humans, the intestine predominantly produces apoB-48 whereas the liver secretes apoB-100 only.

DGAT1 promoter polymorphism associated with alterations in body mass index, high density lipoprotein levels and blood pressure in Turkish women.

Triglyceride synthesis is catalyzed by acyl CoA:diacylglycerol acyltransferases (DGAT), microsomal enzymes that use diacylglycerol and fatty acyl CoAs as substrates. Because DGAT1 expression is up-regulated during adipocyte differentiation and DGAT1 deficiency is associated with leanness in mice, we hypothesized that alterations in DGAT1 expression may affect human body weight. We identified five polymorphisms in the human DGAT1 promoter and 5' non-coding sequence in a random Turkish population.

Subendothelial retention of atherogenic lipoproteins in early atherosclerosis.

Complications of atherosclerosis are the most common cause of death in Western societies. Among the many risk factors identified by epidemiological studies, only elevated levels of lipoproteins containing apolipoprotein (apo) B can drive the development of atherosclerosis in humans and experimental animals even in the absence of other risk factors. However, the mechanisms that lead to atherosclerosis are still poorly understood.

Secreted forms of the amyloid-beta precursor protein are ligands for the class A scavenger receptor.

Upon activation, platelets secrete a 120-kDa protein that competes for the binding and internalization of acetyl low density lipoproteins (AcLDL) by macrophages. From the amino-terminal amino acid sequence, amino acid composition, and immunoblot analysis, we identified the active factor in platelet secretion products as sAPP, an alpha-secretase cleavage product of the beta-amyloid precursor protein (APP), that contains a Kunitz-type protease inhibitor (KPI) domain. We showed that both sAPP751 (also called Nexin II) and sAPP695, which does not contain a KPI domain, are ligands for the class A scavenger receptor (SR-A).

The molecular mechanism for the genetic disorder familial defective apolipoprotein B100.

Familial defective apolipoprotein B100 (FDB) is a genetic disorder in which low density lipoproteins (LDL) bind defectively to the LDL receptor, resulting in hypercholesterolemia and premature atherosclerosis. FDB is caused by a mutation (R3500Q) that changes the conformation of apolipoprotein (apo) B100 near the receptor-binding site. We previously showed that arginine, not simply a positive charge, at residue 3500 is essential for normal receptor binding and that the carboxyl terminus of apoB100 is necessary for mutations affecting arginine 3500 to disrupt LDL receptor binding.

Role of extracellular retention of low density lipoproteins in atherosclerosis.

The pathogenesis for atherosclerosis is still unclear, and several hypotheses have been articulated to explain the initiating events in atherogenesis. Although these hypotheses are by no means mutually exclusive, there is a growing body of recent evidence that has led to the concept that subendothelial retention of apolipoprotein B100-containing lipoproteins is the initiating event in atherogenesis. Subsequently, a series of biological responses to this retained material leads to specific molecular and cellular processes that promote lesion formation.

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.

Apolipoprotein E;-low density lipoprotein receptor interaction. Influences of basic residue and amphipathic alpha-helix organization in the ligand.

Conserved lysines and arginines within amino acids 140-150 of apolipoprotein (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions.

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.

Effect of arginine 172 on the binding of apolipoprotein E to the low density lipoprotein receptor.

The region of apolipoprotein E (apoE) that interacts directly with the low density lipoprotein (LDL) receptor lies in the vicinity of residues 136-150, where lysine and arginine residues are crucial for full binding activity. However, defective binding of carboxyl-terminal truncations of apoE3 has suggested that residues in the vicinity of 170-183 are also important. To characterize and define the role of this region in LDL receptor binding, we created either mutants of apoE in which this region was deleted or in which arginine residues within this region were sequentially changed to alanine.

Phylogenetic analysis of the apolipoprotein B mRNA-editing region. Evidence for a secondary structure between the mooring sequence and the 3' efficiency element.

Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which changes the codon at position 2153 from a genomically encoded glutamine (CAA) to an in-frame stop codon (UAA). The apoB mRNA-editing enzyme complex recognizes the editing region of the apoB pre-mRNA with exquisite precision. Four sequence elements spanning 139 nucleotides (nt) on the apoB mRNA have been identified that specify this precision.

Bacterial expression and purification of the Fab fragment of a monoclonal antibody specific for the low-density lipoprotein receptor-binding site of human apolipoprotein E.

We report the bacterial expression and the purification of a monoclonal antibody (mAb) specific for an epitope that coincides with the LDL receptor (LDLr)-binding domain of human apolipoprotein E (apoE). This antibody resembles the LDLr in its primary structure and its specificity for apoE variants. The recombinant Fab (rFab) fragment of mAb 2E8, consisting of the entire light chain and the Fd portion of the heavy chain, was expressed in Escherichia coli and purified to homogeneity.

The carboxyl terminus in apolipoprotein E2 and the seven amino acid repeat in apolipoprotein E-Leiden: role in receptor-binding activity.

Both apolipoprotein (apo) E2 and apoE-Leiden (tandem repeat of amino acids 121-127) are associated with type III hyperlipoproteinemia and bind defectively to low density lipoprotein receptors. Removing the carboxyl terminus of both variants (residues 192-299) increases receptor-binding activity, suggesting that the carboxyl terminus modulates activity. To identify the region(s) that modulated binding activity, we produced carboxyl-terminal truncations in apoE2 and apoE-Leiden (terminating at positions 191, 223, 244, and 272) and in apoE3 (terminating at positions 191, 223, and 244) and compared their receptor-binding activities as dimyristoylphosphatidylcholine (DMPC) discs.

Identification of the principal proteoglycan-binding site in LDL. A single-point mutation in apo-B100 severely affects proteoglycan interaction without affecting LDL receptor binding.

The subendothelial retention of LDLs through their interaction with proteoglycans has been proposed to be a key process in the pathogenesis of atherosclerosis. In vitro studies have identified eight clusters of basic amino acids in delipidated apo-B100, the protein moiety of LDL, that bind the negatively charged proteoglycans. To determine which of these sites is functional on the surface of LDL particles, we analyzed the proteoglycan-binding activity of recombinant human LDL isolated from transgenic mice.

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).

Increased expression of apolipoprotein E in transgenic rabbits results in reduced levels of very low density lipoproteins and an accumulation of low density lipoproteins in plasma.

Transgenic rabbits expressing human apo E3 were generated to investigate mechanisms by which apo E modulates plasma lipoprotein metabolism. Compared with nontransgenic littermates expressing approximately 3 mg/dl of endogenous rabbit apo E, male transgenic rabbits expressing approximately 13 mg/dl of human apo E had a 35% decrease in total plasma triglycerides that was due to a reduction in VLDL levels and an absence of large VLDL. With its greater content of apo E, transgenic VLDL had an increased binding affinity for the LDL receptor in vitro, and injected chylomicrons were cleared more rapidly by the liver in transgenic rabbits.

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.

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.

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.

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.

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.