Human apolipoprotein E. Role of arginine 61 in mediating the lipoprotein preferences of the E3 and E4 isoforms.

Li, Dong; Wilson, Charles B.; Wardell, Mark R.; Simmons, T; Mahley, Robert W.; Weisgraber, Karl H.; Agard, David A.

doi:10.1016/s0021-9258(17)31797-0

Cited by 283 publications

(133 citation statements)

References 35 publications

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“…To examine the carboxyl-terminal modulation of the receptor-binding activity of apoE2 and apoE-Leiden, we applied a truncation approach used previously to map the major lipoprotein-binding regions of the carboxyl-terminal domain and the region involved in apoE4 domain interaction (17,28). In contrast to previous studies in which discrete regions containing critical structural elements were identified (17,28), this study shows that the entire carboxyl terminus of apoE2 up to position 272 appears to be required for complete modulation of receptor-binding activity. Sequential restoration of the carboxyl terminus from position 191 to 272 progressively reduced the binding activity to the level of intact apoE2 (less than 0.1% of apoE3's activity).…”

Section: Discussionmentioning

confidence: 99%

“…The glutathione S-transferase fusion expression vector pGEX3X (16) was used for expression of apoE mutants. The carboxylterminal truncations of apoE were created by polymerase chain reaction as previously described (17). Construct sequences were verified by double-stranded DNA sequencing (18).…”

Section: Construction and Expression Of Apoe Mutantsmentioning

confidence: 99%

“…The expressed fusion protein was purified by glutathione-agarose affinity chromatography as previously described (17). The fusion protein was complexed with dimyristoylphosphatidylcholine (DMPC) (Sigma) and then cleaved with Factor Xa (Haematologic Technologies) at a ratio of 200:1 (w:w, fusion protein:Factor Xa) at 0 Њ C overnight.…”

Section: Construction and Expression Of Apoe Mutantsmentioning

confidence: 99%

“…The fusion protein was complexed with dimyristoylphosphatidylcholine (DMPC) (Sigma) and then cleaved with Factor Xa (Haematologic Technologies) at a ratio of 200:1 (w:w, fusion protein:Factor Xa) at 0 Њ C overnight. The truncation variants were purified by heparin-Sepharose affinity chromatography followed by diethylaminoethyl (DEAE) ion exchange high-performance liquid chromatography (HPLC) (17). Intact human apoE2, apoE3, and apoE-Leiden were isolated from the d Ͻ 1.006 g/ml lipoprotein fraction as described previously (19).…”

Section: Construction and Expression Of Apoe Mutantsmentioning

confidence: 99%

“…Apolipoprotein E was iodinated with the Bolton-Hunter reagent (Dupont NEN) (22); specific activities ranged from 150 to 900 dpm/ng. The iodinated protein was reduced with ␤ -mercaptoethanol (0.1% final concentration) and incubated with normal human plasma at 37 Њ C for 2 h as described previously (17). The plasma was fractionated into various lipoprotein classes by Superose 6 column chromatography (10/50 HR, Pharmacia).…”

Section: Determination Of Apoe Distribution Among Plasma Lipoproteinsmentioning

confidence: 99%

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

Innerarity

Arnold

et al. 1998

Journal of Lipid Research

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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 receptorbinding 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. The results suggest that the entire carboxyl terminus up to residue 272, not a discrete smaller segment, is responsible for the modulation in apoE2. Intact apoE-Leiden and the 223 and 244 variants displayed similar activities ( ϳ 25% of apoE3's), but the 191 variant's activity was identical to that of intact apoE3. ApoE-Leiden and its tr uncated variants formed larger DMPC discs than did intact or truncated apoE3 or apoE2. These discs contained more apoE molecules than apoE3 discs, suggesting that the apparently normal binding activity of the apoE-Leiden 191 variant results from an increased number of apoE molecules and that the binding activity is actually defective. Direct comparison in a solidphase assay revealed that the binding activity of the apoE-Leiden fragment was defective (51.4 ؎ 9.4%). Thus, the defective binding of apoE-Leiden results from a direct effect of the seven amino acid repeat on receptor-binding activity rather than from an indirect effect operating through the carboxyl terminus as previously believed.-Dong, L-M., T. L. Innerarity, K. S. Arnold, Y. M. Newhouse, and K. H. Weisgraber. The carboxyl terminus in apolipoprotein E2 and the seven amino acid repeat in apolipoprotein E-Leiden: role in receptor-binding activity.

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Section: Discussionmentioning

confidence: 99%

Section: Construction and Expression Of Apoe Mutantsmentioning

confidence: 99%

Section: Construction and Expression Of Apoe Mutantsmentioning

confidence: 99%

Section: Construction and Expression Of Apoe Mutantsmentioning

confidence: 99%

Section: Determination Of Apoe Distribution Among Plasma Lipoproteinsmentioning

confidence: 99%

See 3 more Smart Citations

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

Innerarity

Arnold

et al. 1998

Journal of Lipid Research

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Association of human, rat, and rabbit apolipoprotein E with ?-amyloid

et al. 1997

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In humans, apolipoprotein E (apoE) has three major isoforms, E2 (Cys112, Cys158), E3 (Cys112, Arg158), and E4 (Arg112, Arg158). While epsilon4 is a genetic risk factor for Alzheimer's disease (AD), epsilon2 may protect against late-onset AD. Using native preparations of apoE from conditioned tissue culture media or plasma lipoproteins, we have previously shown that when equivalent amounts of apoE3 or E4 were incubated with beta-amyloid (A beta), apoE3 formed 20 times as much SDS-stable complex with the peptide as apoE4. This preferential binding of A beta to apoE3 was abolished when apoE was purified by a process which includes delipidation and denaturation. Here we expand these observations to include A beta binding to lipoprotein-associated and purified apoE2. Lipoproteins isolated from the plasma of individuals homozygous for either epsilon2 or epsilon3 were incubated with A beta(1-40). SDS-stable complex formation was analyzed by a non-reducing gel shift assay, followed by immunoblotting with either A beta or apoE antibodies. ApoE2:A beta complex formation was comparable to apoE3:A beta in both native and purified preparations of apoE. In addition, lipoprotein-associated rat apoE (Arg112, Arg158), like human apoE4, did not form complex with A beta, while lipoprotein-associated rabbit apoE (Cys112, Arg158) did bind the peptide. These binding studies provide one possible explanation for protective effects of both apoE2 and E3 against the development of Alzheimer's disease.

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Lipoprotein effects on aβ accumulation and degradation by microglia in vitro

et al. 1999

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An inflammatory response involving activated microglia in neuritic beta-amyloid plaques is found in Alzheimer's disease (AD) brain. Because HDL lipoproteins have been shown to carry the beta-amyloid peptide (Abeta) in plasma and CSF, we have investigated the influence of plasma high-density lipoprotein (HDL) and lipidated ApoE and ApoJ particles on the interaction of cultured rat microglia with Abeta1-42. Microglia degraded Abeta via a pathway sensitive to cytochalasin D and the scavenger receptor inhibitor, fucoidan. HDL increased the degradation of Abeta and the ratio of multimeric/monomeric Abeta in a dose-dependent manner. In contrast, lipidated ApoJ and ApoE decreased the degradation of Abeta, and the effects were ApoE isoform-dependent. Immuno-electron microscopy revealed internalized Abeta in endosomes and lysosomes as well as cell-associated Abeta in deep invaginations, which may be related to caveolae and surface-connected compartments. These data suggest that lipoprotein-dependent Abeta trafficking to microglia could be relevant to plaque pathogenesis in AD.

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Human apolipoprotein E. Role of arginine 61 in mediating the lipoprotein preferences of the E3 and E4 isoforms.

Cited by 283 publications

References 35 publications

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

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

Association of human, rat, and rabbit apolipoprotein E with ?-amyloid

Lipoprotein effects on aβ accumulation and degradation by microglia in vitro

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