PubMed Health⌕ Search

Biomedical subjects

K H Weisgraber

Publications and source records attributed to K H Weisgraber.

At least 19 recordsLinked to original sources
PubMed1995

ApoE3 binding to tau tandem repeat I is abolished by tau serine262 phosphorylation.

The risk of Alzheimer's disease is determined, in part, by inheritance of specific alleles of ApoE. Isoform specific interactions of ApoE have been shown with the microtubule-associated protein tau, which forms the neurofibrillary tangle in this disease. Synthetic peptides representing each of the four microtubule-binding domains of tau more avidly bind ApoE3 than ApoE4. Phosphorylation of serine262 in domain I of tau decreases tau binding to microtubules and also abolishes binding by ApoE3. Understanding the molecular mechanisms of the high avidity, isoform-specific interactions of ApoE with tau may help in developing approaches for disease intervention.

Alzheimer Disease↗
PubMed1995

Interaction of apolipoprotein E with laminin increases neuronal adhesion and alters neurite morphology.

The extracellular matrix protein laminin profoundly affects neuronal adhesion, spreading, differentiation, and growth by binding integrin-type cell surface receptors. Laminin binds other basement membrane components, including heparan sulfate proteoglycans. Apolipoprotein E (apoE) also binds basement membrane and heparan sulfate proteoglycans and colocalizes with s-laminin in the neuromuscular junction. We sought to determine whether apoE interacts with laminin and whether, as a result, apoE alters the regulation of neuronal adhesion and differentiation by laminin. We demonstrate high-avidity interaction between apoE and laminin in vitro. Compared to laminin alone, a laminin-apoE substrate produces increased numbers of live, attached hippocampal neurons in culture. In addition, neurons grown on laminin-apoE substrates have larger growth cones, increased neuritic branching, and flattened cell bodies compared to neurons grown on laminin alone. ApoE may be important in the development and maintenance of neurons in the central nervous system by regulating interactions between the neuron and the extracellular matrix.

Apolipoproteins E↗
PubMed1995

Apolipoprotein B and E basic amino acid clusters influence low-density lipoprotein association with lipoprotein lipase anchored to the subendothelial matrix.

Lipoprotein accumulation in the subendothelial matrix is an important step in atherogenesis. We have previously shown that addition of lipoprotein lipase (LPL) markedly increased binding of apolipoprotein B (apoB)-containing lipoproteins to an endothelial cell-derived matrix, and this enhanced lipoprotein binding was inhibited by apoE. In the present studies we examined the role of various regions of apoB in the binding of LDL to LPL-containing endothelial cell matrix and the ability of various apoE domains to decrease lipoprotein retention. We studied three apoB epitope-specific monoclonal antibodies for their ability to block the binding of 125I-LDL to LPL-containing matrix. Of these, monoclonal antibody 4G3, which recognizes an arginine-containing epitope in apoB, was the most effective in reducing LDL binding. Chemical modification of LDL apoB lysines or arginines markedly reduced the ability of the lipoprotein to block the binding of 125I-LDL to LPL-containing matrix, suggesting that apoB positively charged amino acids are involved in the interaction. Furthermore, polyarginine or polylysine markedly decreased 125I-LDL binding to LPL-containing matrix, whereas polyleucine was ineffective. These data suggest that apoB positively charged regions are important in LDL binding. To explore the role of charge modifications on apoE by single arginine-cysteine interchanges, we examined the effects of the three major human apoE isoforms (apoE2, apoE3, and apoE4). ApoE3 was the most effective in decreasing 125I-LDL retention, followed by apoE4; apoE2 was the least effective. Similarly, apoE2-containing HDL was much less effective than apoE3-containing HDL in decreasing 125I-LDL retention.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1995

Identification and characterization of a novel apolipoprotein E variant, apolipoprotein E3' (Arg136-->His): association with mild dyslipidemia and double pre-beta very low density lipoproteins.

Apolipoprotein (apo) E mediates the removal of chylomicron and VLDL remnants from plasma. In a proband with mild hyperlipidemia and a family history of premature coronary artery disease, we have identified a new mutant of apoE with an isoelectric point close to but distinct from that of apoE3. Sequencing of the apoE gene from this subject (JB) revealed that the subject was heterozygous for a G to A substitution in codon 136, resulting in the substitution of histidine for arginine; therefore, we have designated this isoform apoE3' (Arg136-->His). Examination of the proband's kindred revealed that the nine carriers (all heterozygotes) of the variant isoform displayed a twofold elevation in the concentration of very low density lipoprotein (VLDL) cholesterol (40 +/- 8 mg/dl) and triglyceride (109 +/- 19) compared to the nine noncarriers (19 +/- 3 and 55 +/- 13, respectively). In all carriers, the VLDL displayed an abnormal double pre-beta pattern upon electrophoresis. The low density lipoprotein receptor-binding activity of purified apoE3' (Arg136-->His) when complexed with DMPC was slightly defective (80% of the activity of normal apoE). The mutant apoE also displayed a reduced affinity for heparin compared to apoE3. As both of these biochemical parameters are known to be important in VLDL clearance, the defects associated with this variant are likely responsible for the increase in VLDL observed in carriers. None of the carriers displayed clinical features of type III hyperlipoproteinemia, suggesting that the relatively mild dyslipoproteinemic phenotype associated with this variant might be associated with recessive expression of this disorder. However, the abnormal VLDL phenotype appears to be dominantly expressed.

Adult↗
PubMed1992

Hypocholesterolaemic effects of lovastatin in familial defective apolipoprotein B-100.

Familial defective apolipoprotein B-100 (FDB) is an autosomal dominant disorder associated with hypercholesterolaemia in which an aminoacid substitution in apoprotein B-100 leads to low-density lipoprotein (LDL) particles which have defective binding to the LDL receptor. All known patients are heterozygous, and their plasma contains normal and poorly binding LDL particles. 12 hypercholesterolaemic patients from 10 unrelated families with FDB were treated with lovastatin. In 6 patients treated with 20 mg lovastatin daily, LDL cholesterol decreased by 21.5% from 6.23 to 4.89 mmol/l (95% confidence interval 0.74, 1.96 mmol/l), whereas it fell by 32.1%, from 6.99 to 4.81 mmol/l (95% CI 1.55, 2.70 mmol/l), in 9 patients who received 40 mg daily. These results indicate that the hypercholesterolaemia of FDB may respond to treatment with statins.

Adult↗
PubMed1992

Role of apolipoprotein E in hepatic lipase catalyzed hydrolysis of phospholipid in high-density lipoproteins.

We reported earlier that hepatic lipase (HL)-catalyzed hydrolysis of phospholipid monolayers is activated by apolipoprotein (apo) E [Thuren et al. (1991b) J. Biol. Chem. 266, 4853-4861]. On the basis of these studies, it was postulated that apoE-rich high-density lipoproteins (HDL) were preferred substrates for HL. In the present study, we tested this hypothesis, as well as further characterizing the activation of HL hydrolysis of phospholipid by apoE. The apoE-rich HDL, referred to as HDL-I, were isolated by heparin-Sepharose chromatography, and the phospholipid hydrolysis by HL was compared to an apoE-poor HDL, designated HDL-II. The hydrolysis of HDL-I phosphatidylcholine was approximately 3-fold higher than HDL-II, supporting the hypothesis that HL preferably hydrolyzes the phospholipids in apoE-rich HDL. In order to gain additional insight into the nature of the activation, we used phospholipid monolayers as model systems. Comparison of the ability of the two thrombolytic fragments of apoE (22 kDa, residues 1-191; 12 kDa, residues 192-299) revealed that only the 12-kDa fragment was capable of activating the hydrolysis of phospholipid by HL (1.75-fold). However, activation was less than with the intact protein (2.8-fold for apoE3), suggesting that the intact protein was required for full activation. The fact that the 12-kDa fragment, which represents a major lipid region of the protein, did activate HL suggests that activation occurs at the lipid-water interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1992

The functional characteristics of a human apolipoprotein E variant (cysteine at residue 142) may explain its association with dominant expression of type III hyperlipoproteinemia.

Type III hyperlipoproteinemia typically is associated with homozygosity for apolipoprotein (apo) E2(Arg158----Cys). Dominant expression of type III hyperlipoproteinemia associated with apoE phenotype E3/3 is caused by heterozygosity for a human apoE variant, apoE3(Cys112----Arg, Arg142----Cys). However, this apoE3 variant was not separable from the normal apoE3 in these patients' plasma because the two proteins have identical amino acid composition, charge, and molecular weight. Therefore, to determine the functional characteristics of this protein, we used recombinant DNA techniques to produce this apoE variant in bacteria. We also produced a non-naturally occurring variant, apoE(Arg142----Cys), that had only the cysteine substituted at residue 142. These two apoE variants were purified from cell lysates of the transfected Escherichia coli by ultracentrifugal flotation in the presence of phospholipid, by gel filtration chromatography, and by heparin-Sepharose chromatography. Both Cys142 apoE variants bound to lipoprotein receptors on human fibroblasts with only about 20% of normal binding activity. Therefore, cysteine at residue 142, not arginine at residue 112, is responsible for the decreased receptor binding activity of the variants. Cysteamine treatment and removal of the carboxyl-terminal domain had little effect on the binding activity, whereas both modulate the receptor binding activity of apoE2(Arg158----Cys). The mutation at residue 142 decreased the binding activity of apoE to both heparin and the monoclonal antibody 1D7 (this antibody inhibits receptor binding of apoE), whereas apoE2(Arg158----Cys), which is associated with recessive expression of type III hyperlipoproteinemia, binds normally to both. The Arg112, Cys142 variant predominantes 3:1 over normal apoE3 in the very low density lipoproteins of plasma from an affected subject, as assessed by differential reactivity with the antibody 1D7. The unique combination of functional properties of the Arg112, Cys142 variant provides a possible explanation for its association with dominant expression of type III hyperlipoproteinemia.

Amino Acid Sequence↗
PubMed1992

Effects of apolipoprotein E, beta-very low density lipoproteins, and cholesterol on the extension of neurites by rabbit dorsal root ganglion neurons in vitro.

Previous studies suggest that during nerve regeneration apoE acts as a lipid transport protein that assists in the rapid initial extension of axons and then in their myelination. To determine whether apoE and/or apoE-containing lipoproteins can modulate axon growth, we assessed their effect on the out-growth of neurites from neurons in mixed cultures of fetal rabbit dorsal root ganglion cells in vitro. Incubation with beta-very low density lipoprotein (beta-VLDL) particles, which are rich in apoE and cholesterol, increased neurite outgrowth and branching. Unesterified cholesterol added to the cultures had a similar, but less pronounced, effect. These data suggest that cholesterol might be the component responsible for the enhanced neurite growth. In contrast, purified, lipid-free apoE added to the cultures reduced neurite branching. Neurite branching was also reduced when purified apoE was added along with beta-VLDL or cholesterol; however, the striking finding was that under these conditions the neurites extended farther from the neuronal cell body. Dorsal root ganglion cells were examined for the presence of receptors for native and apoE-enriched beta-VLDL. Immunocytochemistry, ligand blots, 45Ca2+ blots, and studies of the interaction of the cells with fluorescent lipoproteins provided evidence of two types of receptors for apoE-containing lipoproteins on neurons: the low density lipoprotein (LDL) receptor, which binds native beta-VLDL, and the LDL receptor-related protein, which binds apoE-enriched beta-VLDL. These findings indicate that apoE may play two complementary roles in neurite outgrowth. When complexed with lipoproteins, apoE stimulates neurite growth by the receptor-mediated delivery of cholesterol and perhaps other components necessary for neurite outgrowth. When apoE as a free protein is added together with apoE-containing lipoproteins, apoE decreases neurite branching and promotes neurite extension away from the cell body. These actions, which would be complementary in promoting target-directed nerve growth in vivo, provide the first direct evidence that apoE and apoE-containing lipoproteins can modulate the outgrowth of neuronal processes.

Animals↗
PubMed1991

Three-dimensional structure of the LDL receptor-binding domain of human apolipoprotein E.

Human apolipoprotein E, a blood plasma protein, mediates the transport and uptake of cholesterol and lipid by way of its high affinity interaction with different cellular receptors, including the low-density lipoprotein (LDL) receptor. The three-dimensional structure of the LDL receptor-binding domain of apoE has been determined at 2.5 angstrom resolution by x-ray crystallography. The protein forms an unusually elongated (65 angstroms) four-helix bundle, with the helices apparently stabilized by a tightly packed hydrophobic core that includes leucine zipper-type interactions and by numerous salt bridges on the mostly charged surface. Basic amino acids important for LDL receptor binding are clustered into a surface patch on one long helix. This structure provides the basis for understanding the behavior of naturally occurring mutants that can lead to atherosclerosis.

Amino Acid Sequence↗
PubMed1991

Identification of the disulfide-linked homodimer of apolipoprotein E3 in plasma. Impact on receptor binding activity.

The nature of disulfide-linked structures of apolipoprotein (apo) E3 in the plasma of E3/3 subjects was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under nonreducing conditions followed by immunoblotting with apoE-specific antibodies. In addition to the expected presence of the heterodimer apoE3-A-II and monomeric apoE3, a band with an apparent Mr approximately 100,000 was also observed in plasma that had been treated with sulfhydryl-trapping reagents. This band and apoE3-A-II were both eliminated by disulfide reduction, which produced a corresponding increase in monomeric apoE3. Both bands were absent in plasma from a subject with the E4/4 phenotype. In spite of its apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the high molecular weight band was demonstrated to represent the disulfide-linked homodimer of apoE3. It was isolated from purified apoE3 preparations that had undergone oxygen-mediated dimerization and shown to elute from a Sephacryl S-300 column in a position with the expected molecular weight of a homodimer. The apoE3 dimer displayed a preference for high density lipoproteins, as determined by agarose chromatography of E3/3 plasma but was stripped from high density lipoproteins by ultracentrifugation. Quantitation of the relative ratios of homodimer, apoE3-A-II, and monomer in the plasma of 22 normolipidemic E3/3 subjects by immunoblotting revealed that the disulfide-linked structures accounted for the majority (approximately 55%) of plasma apoE. Both the homodimer and apoE3-A-II displayed a reduced ability to compete with low density lipoproteins for fibroblast low density lipoprotein receptors (20 and 30% of monomeric apoE3 binding activity, respectively). These results raise the possibility that the amount or availability of receptor-active apoE3 in E3/3 subjects may be rate limiting for metabolic events involving the low density lipoprotein receptor.

Apolipoproteins E↗
PubMed1991

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-100↗
PubMed1991

Two apolipoprotein E5 variants illustrate the importance of the position of additional positive charge on receptor-binding activity.

Apolipoprotein (apo) E polymorphism has a significant effect on plasma cholesterol and low density lipoprotein cholesterol concentrations. The association of two apoE5 isoforms with elevated plasma low density lipoprotein cholesterol levels in two unrelated subjects led us to investigate the primary structures and receptor-binding properties of their apoE. Cysteamine modification and isoelectric focusing demonstrated that the apoE5 isoform from subject 1 did not contain cysteine but that the apoE5 isoform from subject 2 contained one residue of cysteine. The structural mutation in the apoE5 isoform of subject 1 was determined by peptide sequencing. Like apoE4, this variant had arginine at position 112 but differed from apoE4 by the substitution of arginine for proline at position 84. When purified and subjected to a competitive binding assay, this apoE5(84 Pro----Arg, 112 Cys----Arg) variant had the same receptor-binding activity as normal apoE3. Because subject 2 was of Japanese descent and her apoE5 contained one cysteine residue, we suspected that it would contain the lysine-forglutamic acid mutation at position 3 that has been described previously in Japanese subjects. This was confirmed by directly sequencing the first 10 amino acid residues of her apoE. When subjected to the competitive binding assay, the total apoE from subject 2, which consisted of approximately equal amounts of normal apoE3 and apoE5(3 Glu----Lys), had a binding activity of 188%, confirming the previously reported enhanced binding of this variant. These results demonstrate that the enhancement of receptor-binding activity of more basic isoforms of apoE depends on the position at which additional positively charged amino acids are incorporated.

Amino Acids↗
PubMed1990

Apolipoprotein C-I modulates the interaction of apolipoprotein E with beta-migrating very low density lipoproteins (beta-VLDL) and inhibits binding of beta-VLDL to low density lipoprotein receptor-related protein.

The binding of native rabbit beta-very low density lipoproteins (beta-VLDL) to the low density lipoprotein receptor-related protein (LRP) requires incubation with exogenous apolipoprotein (apo) E. Inclusion of a mixture of the C apolipoproteins in the incubation inhibits this binding. In the present study, the ability of the individual C apolipoproteins (C-I, C-II, and C-III) to block binding of beta-VLDL to the LRP was examined by measuring cholesteryl ester formation in mutant fibroblasts that lack low density lipoprotein receptors or by measuring binding to the LRP using ligand blotting. In each assay, both apoC-I and apoC-II inhibited binding; apoC-I was the more effective inhibitor. Apolipoprotein C-III had no effect on binding activity, regardless of its sialylation level. Binding of human apoE to rabbit beta-VLDL in the absence or presence of human apoC-I, apoC-II, and monosialo-apoC-III was also determined, by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of these studies are consistent with a mechanism in which exogenous human apoE displaces the endogenous apoE and the beta-VLDL particle becomes enriched with apoE (by 4.2-fold in this study). At this higher apoE content, the beta-VLDL bound to the LRP. Inclusion of apoC-I, apoC-II, or apoC-III in the incubation mixture resulted in a differential displacement of apoE from the beta-VLDL; however, at the concentrations examined, only apoC-I and apoC-II were capable of displacing sufficient apoE to abolish binding to LRP.

Amino Acid Sequence↗
PubMed1990

In the absence of a downstream element, the apolipoprotein E gene is expressed at high levels in kidneys of transgenic mice.

Human apolipoprotein (apo) E gene constructs with 30 or 5 kilobases of 5'-flanking and 1.5 kilobases of 3'-flanking regions were used to create transgenic mice. High levels of human apoE mRNA were present in the transgenic kidney, but none was detected in the liver, which is normally the major source of apoE. When a construct with 5 kilobases of 5'- and 23 kilobases of 3'-flanking regions was used, only trace levels of human apoE mRNA were detected in the kidney, whereas high levels were found in the liver. These results indicated that regulatory elements downstream of the human apoE gene interacted with the transcription initiation complex to stimulate gene expression in the liver while suppressing expression in the kidney. In each case, human apoE was secreted into the plasma. The source of human apoE in the transgenic kidney was the epithelial cells lining the proximal tubule and Bowman's capsule.

Animals↗
PubMed1990

Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein (LRP) from rat liver membranes binds apoprotein E (apoE)-enriched rabbit beta-migrating very low density lipoproteins (beta-VLDL) in a ligand blotting assay on nitrocellulose membranes. Binding was markedly activated when the beta-VLDL was preincubated with recombinant human apoE-3, native human apoE-3 or E-4, or native rabbit apoE. Human apoE-2, which binds poorly (1-2% of apo E-3 binding) to low density lipoprotein receptors, was approximately 40% as effective as apoE-3 or apoE-4 in binding to LRP. Stimulation of apoE-dependent binding to LRP was blocked by the inclusion of a mixture of human apoC proteins, but not apoA-I or A-II, in the preincubation reaction. High concentrations of apoE did not overcome the apoC inhibition. The effects of apoE and apoC on the ligand blotting assay were paralleled by similar effects in the ability of beta-VLDL to stimulate cholesteryl ester synthesis in mutant human fibroblasts that lack low density lipoprotein receptors. These properties of LRP are consistent with the known effects of apoE and apoC on uptake of chylomicron and very low density lipoprotein remnants in the liver and raise the possibility that LRP functions as a receptor for apoE-enriched forms of these lipoproteins in intact animals.

Animals↗
PubMed1990

Atherogenic lipoproteins resulting from genetic defects of apolipoproteins B and E.

Accelerated atherosclerosis occurs in patients with type III hyperlipoproteinemia and familial hypercholesterolemia. These genetic disorders focus attention on specific types of lipoproteins as being responsible for the development of accelerated coronary artery heart disease. The accumulation of chylomicron remnants of intestinal origin and of VLDL remnants or IDL of hepatic origin observed in type III hyperlipoproteinemia appears to correlate with coronary disease. The presence of defective forms of apo E prevents normal receptor-mediated catabolism of these lipoproteins. Patients with familial hypercholesterolemia have an elevation of plasma LDL (and to a lesser extent an increase in VLDL remnants and IDL) secondary to defective LDL receptors that impair normal catabolism. Familial defective apo B100 is secondary to an abnormality of apo B100 that prevents the normal interaction of LDL with the LDL receptor and increases plasma LDL. However, it has not yet been established that familial defective apo B100 predisposes affected individuals to accelerated atherosclerosis. Animals fed diets high in saturated fat and cholesterol have an accumulation of beta-VLDL, IDL, and LDL that resembles the changes in lipoproteins observed in patients with these genetic disorders. Macrophages (which are presumably derived from circulating monocytes) have emerged as a likely key component in atherogenesis because they appear to be progenitors of foam cells in arterial lesions. Macrophages in the arterial wall express receptors that recognize chylomicron remnants and VLDL remnants (beta-VLDL) and chemically modified LDL. Thus, in the presence of these specific lipoproteins, macrophages are converted to cells that resemble foam cells. The precise stimulus that causes monocyte-derived macrophages to enter specific regions of the arterial wall remains to be determined.

Animals↗
PubMed1990

Familial defective apolipoprotein B-100: a mutation of apolipoprotein B that causes hypercholesterolemia.

Familial defective apolipoprotein B-100 is a genetic disorder of apolipoprotein B-100 that causes moderate to severe hypercholesterolemia. A single amino acid mutation in apolipoprotein B diminishes the ability of low density lipoproteins to bind to the low density lipoprotein receptor. Low density lipoproteins accumulate in the plasma because their efficient receptor-mediated catabolism is disrupted. This mutation has been identified in the United States, Canada, and Europe and is estimated to occur at a frequency of approximately 1/500 in these populations. Thus, it appears that this newly described disorder may be a significant genetic cause of hypercholesterolemia in Western societies.

Apolipoprotein B-100↗