Search

Human apolipoprotein A-I (apoA-I) has an important role in the efflux of cholesterol from peripheral cells, the first step in reverse cholesterol transport. Deletion of amino acids Glu146-->Arg160 in apoA-I (apoA-ISeattle) removes a large section of a lipid binding helix and is associated in vivo with an atherogenic lipoprotein profile characterized by a deficiency in high-density lipoproteins (HDL). In the present study, we asked whether apoA-ISeattle had normal ability to recruit lipids from cells and to form nascent high-density lipoprotein (HDL) particles and whether the altered secondary structure affected lecithin:cholesterol acyltransferase (LCAT) activity. Wild-type apoA-I and apoA-ISeattle expressed in transfected Chinese hamster ovary cells formed nascent HDL particles with similar density distribution and protein-to-lipid ratio. Phospholipid subclass distribution of apoA-ISeattle nascent HDL demonstrated a significant increase in sphingomyelin and phosphatidylethanolamine compared to wild type. ApoA-ISeattle nascent HDL had a unique size distribution compared to wild-type nascent HDL; large (9-20 nm) particles predominated while there were virtually no small (7.5 nm) particles. LCAT reactivity was impaired by apoA-ISeattle nascent HDL where cholesterol esterification was only half that of wild-type complexes. The apoA-ISeattle conformation on nascent HDL was studied with a panel of monoclonal antibodies (Mabs) specific for apoA-I. Mabs that recognize the putative LCAT activation site, residues 95-122, had normal reactivity. As expected, the Mabs that recognized residues 141-164 were unreactive because of the 146-160 deletion; in addition, there was low reactivity with a Mab that recognizes residues 220-242. The data suggest that apoA-I residues 146-160 and/or 220-242 partake in normal LCAT activation and that cooperative interactions between helices may be important for maximal cholesterol esterification.

Equilibrium structures of Li 2 CO 3 and K 2 CO 3 were calculated using ab initio molecular orbital calculations carried out at the Hartree-Fock (HF) level. Of the four structures considered for Li 2 CO 3 and K 2 CO 3 , the most stable was a structure with all five atoms in a plane. The harmonic frequencies were also calculated and found to be in agreement with the present Raman measurements. Structure factors, calculated from the ab initio data for each of the four structures considered, are compared with existing X-ray results.

Electronic couplings in long‐range electron transfer have been calculated using ab initio molecular orbital theory to investigate the effect of the binding energy of the electron on the decay of through‐space and through‐bond couplings. Through‐space and through‐bond couplings for anions and cations of the CF3 dimer and of CnH2n and CnF2n chains were calculated by ab initio molecular orbital theory. The anions and cations provide systems for which the electron binding energies, Be, differ by about a factor of 10. Through‐space couplings decay exponentially, exp(−βR), with increasing distance, R, between the donor/acceptor carbon atoms. The decay coefficient β varies approximately as B1/2e. In contrast, the decay coefficients for through‐bond coupling in CnF2n and CnH2n chains are not affected significantly by the binding energy of the electron.

Ab initio molecular orbital theory at the G2 level has been used to study the energy of N2H2 and related compounds. Overall, the agreement between theory and experiment is good. The G2 enthalpy of formation ΔH0f0(N2H2) of 49.6 kcal/mol supports the experimental estimate of ≥46.6 kcal/mol derived by Ruscic and Berkowitz [J. Chem. Phys. 95, xxx4 (1991)] in a recent photoionization study. Predicted dissociation energies are D0(HN■NH)=122.8 kcal/mol, D0(HNNH–H)=43.6 kcal/mol, and D0(H2N■NH–H)=82.1 kcal/mol. The G2 value for the proton affinity (PA) of N2 at 298 K is PA298=118.1 kcal/mol. The G2 ionization potential of N2H3 of 7.54 eV is in agreement with the new value of ≤7.61 eV reported by Ruscic and Berkowitz. The G2 results for the ionization potential of N2H4 and the appearance potential of N+2 from N2H2 are in disagreement with experimental results suggesting that detection of the origins in these cases are thwarted by large geometry changes and significant Franck–Condon effects.

Ab initio molecular orbital theory at the G2 level has been used to study the energy of N2H2 and related compounds. Overall, the agreement between theory and experiment is good. The G2 enthalpy of formation ΔH0f0(N2H2) of 49.6 kcal/mol supports the experimental estimate of ≥46.6 kcal/mol derived by Ruscic and Berkowitz [J. Chem. Phys. 95, xxx4 (1991)] in a recent photoionization study. Predicted dissociation energies are D0(HN■NH)=122.8 kcal/mol, D0(HNNH–H)=43.6 kcal/mol, and D0(H2N■NH–H)=82.1 kcal/mol. The G2 value for the proton affinity (PA) of N2 at 298 K is PA298=118.1 kcal/mol. The G2 ionization potential of N2H3 of 7.54 eV is in agreement with the new value of ≤7.61 eV reported by Ruscic and Berkowitz. The G2 results for the ionization potential of N2H4 and the appearance potential of N+2 from N2H2 are in disagreement with experimental results suggesting that detection of the origins in these cases are thwarted by large geometry changes and significant Franck–Condon effects.

Familial defective apoB-100 is a genetic mutation that is characterized by abnormal low density lipoprotein (LDL) and moderate hypercholesterolemia. Heterozygotes for this disorder possess two populations of LDL. One has normal receptor binding, and the other, which can be isolated by monoclonal antibody 19 immunoaffinity chromatography, has almost no binding activity. The mutation that disrupts binding is a Gln for Arg substitution of apoB-100 residue 3500. NMR spectra of LDL containing (13CH3)2Lys residues show that chemically modified Lys exist in two microenvironments. In normal human LDL, there are about 50 Lys with pK 8.9 and 170 Lys with pK 10.5; an upper limit of 10 pK 8.9 Lys may be particularly involved in binding to the LDL receptor. Examination of the mixture of normal LDL and mutant LDL from five patients shows that the latter have fewer pK 8.9 Lys. In purified defective LDL at least seven Lys are redistributed from the active to normal pool. The CD spectra of mutant and normal LDL are identical. Therefore, substitution of Gln for Arg at position 3500 induces a change in local conformation which disrupts the receptor-binding domain of apoB-100.

Two- and three-body interaction energies are calculated by using ab initio molecular orbital theory for tetrahedral, octahedral, and cubic arrangements of H 2 O molecules around Cu + and Cu 2+ ions in [Cu(OH 2 ) n ] q+ clusters to assess the nonadditivity of interactions in hydrated transition-metal-ion clusters. The results are compared to cull many-body results for the clusters. For Cu 2+ there appears to be a slow convergence of the interaction terms to the full many-body result

Ab initio molecular orbital theory (Mo/ller–Plesset theory to fourth order with a series of extended basis sets) has been used to calculate the total energies of GeH+n (n=1–4), AsH+n (n=1–3) and SeH+n (n=1–2). In combination with previously published results on the neutral hydride molecules, the data are used to derive ionization energies of the neutral species and appearance potentials of the ions with an expected accuracy of ±0.15 eV. Experimental data are reviewed in light of these results. Theoretical predictions of the structures of several cations are discussed including the Jahn–Teller distortion in GeH+4.

CD11b is an alpha chain of the leukocyte beta(2)-integrin, Mac-1, which mediates binding and extravasation of leukocytes. Because this event is critical in atherosclerosis, we examined the role of CD11b in lesion formation. Atherosclerosis-susceptible, low density lipoprotein receptor-deficient (LDL-R(-/)-) mice were irradiated and repopulated with bone marrow cells from CD11b-deficient (CD11b(-/)-) mice. After 4 weeks,2% of the peripheral blood leukocytes of the CD11b(-/)- bone marrow-transplanted LDL-R(-/)- mice expressed CD11b, whereas approximately 25% of the CD11b(+/)+ bone marrow-transplanted LDL-R(-/)- mice expressed CD11b. After consuming a high-fat diet for 16 weeks the mean lesion aortic valve area, cholesterol accumulation in the aorta, and the degree of intimal macrophage infiltration were similar in mice reconstituted with either CD11b(+)(/+) or CD11b(-/)- bone marrow cells. The studies confirm that CD11b expression of bone marrow-derived cells does not influence the development of atherosclerosis in hypercholesterolemic LDL-R(-/)- mice.

Apolipoprotein (apo)E is synthesized in atherosclerotic lesions by macrophages, however, its role in lesions is not known. Whereas apoE could exacerbate atherosclerosis by promoting macrophage uptake of cholesterol-rich lipoproteins or modulating protective inflammatory responses, it could also restrict lesion formation by facilitating cholesterol efflux out of lesions. The role of apoE was examined in lethally irradiated male C57BL/6J wild-type (WT) mice that were repopulated with bone marrow cells (BMT) from either identical C57BL/6J mice (WT+WT BMT) or C57BL/6J apoE-deficient mice (WT+E-/- BMT). This enabled us to compare normal mice with mice possessing macrophages that did not express apoE. The participation of macrophage-derived apoE in atherosclerosis was assessed by placing the mice on an atherogenic diet. Male WT+E-/- BMT mice had significantly reduced lesion area in the aortic valves (P0.01) compared with male WT+WT BMT mice ( approximately 22,000 vs. approximately 49,000 microm2/section, respectively). Further evaluation revealed that plasma cholesterol, lipoprotein cholesterol distribution, and plasma apoE were similar between the two groups, indicating that these known risk factors did not account for the differences in lesion area. However, the two groups were distinguished by the amount of apoE found in the lesions. ApoE antigen was expressed abundantly in WT+WT BMT lesions, whereas WT+E-/- BMT lesions contained little apoE. These findings indicate that the majority of apoE in lesions is synthesized locally by resident macrophages, and suggest that locally produced apoE can promote diet-induced atherosclerosis in male wild-type mice.

Low density lipoprotein (LDL) receptor-deficient (LDLR-/-) mice consuming a high fat diet were used to assess the effect of endogenous and exogenous estradiol (E2) on atherosclerosis. Sexually mature female mice were ovariectomized (OVX) and implanted with subcutaneous, slow-release pellets designed to release 6 microg/day of exogenous 17beta-estradiol (17beta-E2 ), 17alpha-estradiol (17alpha-E2 ), or placebo (E2- deficient). Sham-operated control female (endogenous E2 ) and male mice were studied as controls. Aortic atherosclerotic lesion area was reduced by physiologic amounts of both endogenous and exogenous E2 compared to E2-deficient female mice. Although plasma cholesterol levels were reduced by exogenous E2 despite the absence of the LDL receptor, endogenous E2 was not associated with any cholesterol changes. In contrast, only 17alpha-E2 was associated with decreased fasting triglyceride. In subgroup analyses matched for time-averaged plasma total cholesterol, aortic lesion area was reduced by the presence of estradiol (E2 ). E2 protected LDLR-/- female mice from atherosclerosis and this protection was independent of changes in plasma cholesterol levels.

Lipoprotein(a) [Lp(a)] is associated with atherosclerosis and with disease processes involving thrombosis. Lp(a) contains apoprotein (a) [apo(a)], which has a sequence highly homologous to plasminogen. Hence, Lp(a) binds directly to extracellular matrix, cellular plasminogen receptors and fibrin(ogen) and competes for the binding of plasminogen to these regulatory surfaces. These interactions may contribute to the proatherothrombogenic consequences of high Lp(a) levels. These interactions are mediated by lysine binding sites (LBS). Therefore, we examined the role of apo(a) kringle IV-10 [the only apo(a) kringle demonstrated to exhibit lysine binding activity in the intact lipoprotein] in the interaction of Lp(a) with these regulatory molecules. We have compared directly apo(a) KIV-10 with plasminogen K4 to examine whether these highly structurally homologous kringle modules are also functionally homologous. Futhermore, because the plasminogen K5-protease domain (K5-PD) binds directly to fibrin, we have also examined the ability of this plasminogen fragment to inhibit the interaction of Lp(a) with these regulatory molecules and with extracellular matrix. Apo(a) KIV-10 competed effectively for the binding of 125I-Lp(a) to these surfaces but was less effective than either intact Lp(a), plasminogen K4 or plasminogen. Plasminogen KS-PD was a better competitor than apo(a) KIV-10 for 125I-Lp(a) binding to the representative extracellular matrix, Matrigel, and to plasmin-treated fibrinogen. In contrast, plasminogen K5-PD did not compete for the interaction of Lp(a) with cells, although it effectively competed for plasminogen binding. These results suggest that Lp(a) recognizes sites in all of the regulatory molecules that are also recognized by apo(a) KIV-10 and that Lp(a) recognizes sites in extracellular matrix and in plasmin-modified fibrinogen that also are recognized by plasminogen K5-PD. Thus, the interaction of Lp(a) with cells is clearly distinct from that with extracellular matrix and with plasmin-treated fibrinogen and the recognition sites within Lp(a) and plasminogen for these regulatory molecules are not identical.

The effects of polyunsaturated fatty acids (PUFA) on the structure of recombinant high density lipoprotein (rHDL) was investigated using homogeneous particles containing phosphatidylcholine (PC), [3H]cholesterol, and apolipoprotein A-I (apoA-I). The PC component of the rHDL contained sn -1 16:0 and sn -2 18:1 (POPC), 18:2 (PLPC), 20:4 (PAPC), 20:5 n-3 (PEPC), or 22:6 n-3 (PDPC). The concentration of guanidine HCl (D1/2) required to denature one-half of the apoA-I on rHDL containing long chain PUFA was reduced (1.57-1.70 m) compared to those containing POPC (2.83 m). Intrinsic apoA-I tryptophan fluorescence emission intensity and lifetimes were decreased for rHDL containing long chain PUFA compared to POPC and PLPC rHDL. Monoclonal antibody binding studies demonstrated that apoA-I had decreased immunoreactivity with monoclonal antibodies spanning amino acid residues 115-147 in rHDL containing long chain PUFA. PC lipid fluidity, measured as diphenylhexatriene (DPH) fluorescence polarization, was increased in PUFA rHDL compared to POPC rHDL. There also was a strong correlation between the number of sn -2 double bonds in rHDL and DPH fluorescence lifetime (r 2 = 0. 89). LCAT reactivity of the homogeneous size rHDL was ordered POPC = PLPCPAPCPEPCPDPC. We conclude that rHDL with long chain PUFA in the sn -2 position of PC contain apoA-I that is less stable and in a different conformation than that in POPC rHDL and have a fatty acyl region that is more fluid and hydrated. The weaker interaction of apoA-I with PC containing PUFA may lead to hypercatabolism of apoA-I in plasma explaining, in part, the decreased plasma HDL and apoA-I concentrations seen with PUFA diets.

Ovarian theca/interstitial cells produce androgens in response to luteinizing hormone (LH) stimulation and apolipoprotein (apo) E exerts a selective effect on the type of steroid product made by these cells. We have identified an apoE synthetic peptide containing the low density lipoprotein (LDL) receptor binding domain, acetyl-Y(LRK LRKRLLRDADDL)2C, that mimics the activity of native apoE. Depending on the concentration, the apoE synthetic peptide either enhanced or inhibited the LH-stimulated production of androstenedione with concomitant changes in the mRNA for its synthetic enzyme, P450 17alpha-hydroxylase, C17-20 lyase, without any changes in progesterone production or the mRNA for its synthetic enzyme, P450 cholesterol side-chain cleavage. The apoE synthetic peptide caused changes in the rate of transcription of the mRNA for P450 17alpha-hydroxylase, C17-20 lyase without altering its stability. Pretreatment of the theca/interstitial cells with receptor-associated protein, which blocks apoE binding to members of the LDL receptor superfamily, prevented the apoE synthetic peptide-mediated stimulation of androstenedione and mRNA for P450 17alpha-hydroxylase, C17-20 lyase, but did not attenuate the inhibitory activity of the peptide. Thus, apolipoprotein E selectively altered the type of steroid made by ovarian theca/interstitial cells by regulating the transcription of mRNA for the gene for P450 17alpha-hydroxylase, C17-20 lyase, in part through its interaction with apolipoprotein E-specific receptors of the LDL receptor superfamily.

To specify and localize carboxyl-terminal domain functions of human hepatic lipase (HL) and human lipoprotein lipase (LPL), two subdomain chimeras were created in which portions of the carboxyl-terminal domain were exchanged between the two lipases. The first chimera (HL-LPLC1) was composed of residues 1-344 of human HL, residues 331-388 of human LPL, and residues 415-476 of human HL. The second chimera (HL-LPLC2) consisted of just two segments, residues 1-414 of human HL and residues 389-448 of human LPL. These chimeric constructs effectively divided the HL C-terminal domain into halves, with corresponding LPL sequences either in the first or second portion of that domain. Both chimeras were lipolytically active and hydrolyzed triolein emulsions to a similar extent compared with native HL and LPL. Heparin-Sepharose chromatography demonstrated that HL-LPLC1 and HL-LPLC2 eluted at 0.80 and 1.3 M NaCl, respectively, elution positions that corresponded to native HL and LPL. Hence, substitution of LPL sequences into the HL carboxyl-terminal domain resulted in the production of functional lipases, but with distinct heparin binding properties. In addition, HL-LPLC2 trioleinase activity was responsive to apoC-II activation, although the -fold stimulation was less than that observed with native LPL. Moreover, an apoC-II fragment (residues 44-79) was specifically cross-linked to LPL and HL-LPLC2, but not to HL or HL-LPLC1. Finally, both chimeras hydrolyzed phospholipid with a specific activity similar to that of HL, which was unaffected by the presence of apoC-II. These findings indicated that in addition to a region found within the amino-terminal domain of LPL, apoC-II also interacted with the last half of the carboxyl-terminal domain (residues 389-448) to achieve maximal lipolytic activation. In addition, the relative heparin affinity of HL and LPL was determined by the final 60 carboxyl-terminal residues of each enzyme.

Proteinuria in passive Heymann nephritis is primarily caused by reactive oxygen species that are produced by glomerular cells. Reactive oxygen species apparently exert their damaging effects on the glomerular filter by lipid peroxidation and subsequent adduct formation on matrix proteins of glomerular basement membranes. This raised the question as to the source of polyunsaturated fatty acids required as substrates for lipid peroxidation. Here we have localized by immunocytochemistry rat apolipoprotein E and apolipoprotein B within subepithelial immune deposits. Moreover, apolipoprotein B extracted from isolated glomeruli of proteinuric passive Heymann nephritis rats shows degradation and lipid peroxidation adduct formation, similar to apoproteins of oxidized lipoproteins in atherosclerotic lesions. These data provide evidence that lipoproteins accumulate within immune deposits and suggest that their lipids generate lipid-peroxidation-derived reactive compounds.

Lp(a) competes with plasminogen for binding to cells but it is not known whether this competition is due to the ability of Lp(a) to interact directly with plasminogen receptors. In the present study, we demonstrate that Lp(a) can interact directly with plasminogen binding sites on monocytoid U937 cells and endothelial cells. The interaction of Lp(a) with these sites was time dependent, specific, saturable, divalent ion independent and temperature sensitive, characteristics of plasminogen binding to these sites. The affinity of plasminogen and Lp(a) for these sites also was similar (Kd = 1-3 microM), but Lp(a) bound to fewer sites (approximately 10-fold less). Both gangliosides and cell surface proteins with carboxy-terminal lysyl residues, including enolase, a candidate plasminogen receptor, inhibited Lp(a) binding to U937 cells. Additionally, Lp(a) interacted with low affinity lipoprotein binding sites on these cells which also recognized LDL and HDL. The ability of Lp(a) to interact with sites on cells that recognize plasminogen may contribute to the pathogenetic consequences of high levels of circulating Lp(a).

Lipoprotein lipase (LPL) increases the cellular uptake and degradation of LDL by fibroblasts and macrophages via a heparin-sensitive process. The roles of the LDL receptor, LDL receptor-related protein (LRP), and proteoglycans in this process were studied. In up-regulated human fibroblasts, LPL increased degradation of 125I-low density lipoprotein (LDL) (5 micrograms/ml) only 30% during a 6-h incubation at 37 degrees C. Monoclonal antibody 47 (which interacts with the receptor binding region of apoB) decreased LDL degradation 93% in the absence of LPL, but did not reduce the LPL-mediated increase in degradation. In contrast, addition of the 39-kDa receptor-associated protein (RAP) caused a 43% decrease in the LPL-dependent LDL degradation in non-up-regulated fibroblasts. Monoclonal antibody 47 did not decrease LDL degradation by THP-1 macrophages and RAP caused a13% decrease in LPL-mediated LDL degradation. LPL also increased the association of acetyl LDL with the surface of the macrophages but did not increase acetyl LDL degradation. The kinetics of LPL-mediated LDL metabolism in macrophages was then compared with that in fibroblasts. The half-lives of cell surface LDL and LPL during a subsequent 37 degrees C incubation were approximately 1 h in THP-1 cells versus 6 h in fibroblasts. In addition, 50% of the 125I-LDL and 30% of the 125I-LPL were degraded within 3 h. After metabolic labeling of THP-1 proteoglycans with 35SO4,30% of pericellular heparan sulfate was lost between 2-4 h of the chase period. Therefore, some of the LPL-mediated LDL degradation in the THP-1 cells could be accounted for by internalization of cell surface proteoglycans. We conclude that LRP, but not the LDL receptor, is involved in LPL-mediated degradation of LDL in fibroblasts. This process is much more rapid in THP-1 cells and in addition to LRP may involve other receptors and internalization of proteoglycans.

The inverse correlation between plasma high density lipoprotein (HDL) levels and the risk for cardiovascular disease has been attributed in part to the role of HDL in facilitating the transport of cholesterol to the liver for catabolism. One component of this reverse cholesterol transport is removal of excess cholesterol from peripheral cells. An immunochemical approach was employed to evaluate the role of human apolipoprotein (apo) A-I in cellular cholesterol efflux and to test the hypothesis that discrete structural domains of the molecule mediate this function. Two apoA-I-specific monoclonal antibodies (AI-11 and AI-14) inhibited in vitro cellular cholesterol efflux from THP-1 monocytic cells to HDL or apoA-I proteoliposomes by approximately 50%. Six other antibodies had no effect although three of these bound significant proportions of the apoA-I proteoliposomes. Antibody AI-11 binds apoA-I amino acid residues 96-111 (Banka, C. L., Bonnet, D. J., Black, A. S., Smith, R. S., and Curtiss, L. K. (1991) J. Biol. Chem. 266, 23886-23892). The AI-14 epitope was localized to residues 74-105. Therefore, the two antibodies that inhibited HDL promotion of cellular cholesterol efflux bound overlapping but distinct regions of the apoA-I molecule.

Lipoprotein lipase (LPL), the principal enzyme which hydrolyzes triglycerides in circulating plasma lipoproteins, functions while bound to the luminal surface of endothelial cells. LPL is a heparin-binding protein and has been assumed to associate with endothelial cell heparan sulfate proteoglycans (HSPG). Recently, using ligand blotting and affinity chromatography we identified a 116-kDa heparin-releasable LPL-binding protein (hrp-116) from endothelial cells which was not a HSPG (Sivaram, P., Klein, M. G., and Goldberg, I. J. (1992) J. Biol. Chem. 267, 16517-16522). This suggested that, like a number of other heparin-binding proteins, LPL binding to cells also involves non-HSPG proteins. Using heparin-agarose affinity chromatography, a 116-kDa LPL-binding protein was purified from endothelial cell extracts. Microsequencing of peptides generated by Lys-C protease digestion revealed complete homology with four different regions in the NH2-terminal part of human apolipoprotein B (apoB). Western blots using anti-apoB monoclonal antibodies (mAb) that recognize the NH2-terminal region of apoB confirmed that a 116-kDa fragment of apoB was present on endothelial cell membranes. Further evidence that LPL associates with the NH2-terminal region of apoB was obtained by showing 1) that an NH2-terminal fragment of apoB obtained from apoB-transfected CHO cells bound LPL on ligand blots and 2) that NH2-terminal fragments of apoB generated by thrombin digestion of low density lipoprotein bind LPL. Evidence that the NH2-terminal region of apoB mediates LPL interaction with endothelial cells was obtained using monoclonal antibodies. mAb3 and mAb19, which recognize epitopes near the NH2 terminus of apoB, inhibited 125I-LPL binding to cells by 60-65%. In contrast, mAb47, which has determinants at the COOH-terminal end of apoB, inhibited LPL binding by only about 10%. The inhibitory effects of mAb3 and mAb19 were abolished following treatment of cells with heparin, which removes the 116-kDa LPL-binding protein. Furthermore, incubation of 125I-LPL in medium containing an NH2-terminal apoB fragment reduced LPL binding to cells. These data suggest that an NH2-terminal fragment of apoB that binds to endothelial surfaces facilitates LPL binding to cells.

Human plasma apolipoprotein E (apoE) is a low density lipoprotein (LDL) receptor ligand. It targets cholesterol-rich lipoproteins to LDL receptors on both hepatic and peripheral cells. The region of apoE responsible for its binding to the LDL receptor has been localized to amino acids 140-160. An apoE 141-155 monomeric peptide and a dimeric 141-155 tandem peptide were synthesized and tested for their inhibition of 125I-LDL degradation by human fibroblasts and human monocytic-like cells, THP-1. The monomer had no activity at 250 microM, but the dimer inhibited 125I-LDL degradation by 50% at 5 microM. The inhibition was specific for the LDL receptor because the dimer did not inhibit the degradation of 125I-acetylated LDL by scavenger receptors expressed by phorbol ester-stimulated THP-1 cells. As reported for native apoE, amino acid substitutions of Lys-143----Ala, Leu-144----Pro, and Arg-150----Ala decreased the inhibitory effectiveness of the dimer. Furthermore, a trimer of the 141-155 sequence had a 20-fold greater inhibitory activity than the dimer. Studies with a radioiodinated dimer indicated that some of the inhibitory activity could be a result of the interaction of the dimer with LDL. However, direct binding of the 125I-dimeric peptide to THP-1 cells was observed as well. This binding was time-dependent, linear with increasing cell number, Ca(2+)- but not Mg(2+)-dependent, saturable, inhibited by lipoproteins, and increased by preculture of the cells in lipoprotein-depleted medium. Therefore, a synthetically prepared dimeric repeat of amino acid residues 141-155 of apoE binds the LDL receptor.

Plasma apolipoprotein E (apoE) is a ligand for the cellular uptake of cholesterol-rich plasma lipoproteins. ApoE also inhibits mitogen-stimulated lymphocyte proliferation and gonadotropin-stimulated ovarian theca/interstitial cell androgen production. To address the mechanism(s) by which apoE is active and to understand its interaction with the target cells, we prepared and examined the inhibitory activity of a series of apoE synthetic peptides. ApoE peptides representing amino acid residues 93-112, 141-155, 161-171, 172-182, and 174-193 were not active in either bioassay. However, specific inhibition of both lymphocyte proliferation and ovarian androgen production was observed with a self-conjugate of peptide-(141-155). Furthermore, a synthesized dimeric peptide representing two repeats of sequence-(141-155) (i.e. (141-155)-(141-155] was active as well. In both bioassays, the inhibition observed was not a result of direct cell killing. Furthermore, these apoE peptides exhibited activities with characteristics that were shared with those of native apoE. The results indicate that amino acid residues 141-155 of apoE are responsible for the biological activity of apoE. Furthermore, the results suggest that dimers or multimers of native apoE may be a biologically important species.

Apolipoprotein B (apoB) was mapped using electron microscopy to visualize pairs of monoclonal antibodies binding to the low density lipoprotein (LDL) surface. The sites at which these monoclonals bind the apoB polypeptide sequence had already been established. The angular distances between all possible pairs of binding sites except one allowed the relative placement of six epitopes on the LDL sphere. We conclude that apoB extends over at least a hemisphere of the LDL surface since four epitopes are located in the Northern Hemisphere at sites arbitrarily designated as the North Pole, the Aleutian Islands, Bogotá, and in the Atlantic Ocean, while two are found in the Southern Hemisphere at Buenos Aires and at Madagascar. ApoB appears to possess a restricted flexibility, since these relative epitope locations show a substantial standard deviation in latitude and longitude. Mapping of additional epitopes may provide an answer to the question of whether apoB circumnavigates the LDL sphere.

Familial defective apoB-100 is a genetic mutation that is characterized by abnormal low density lipoprotein (LDL) and moderate hypercholesterolemia. Heterozygotes for this disorder possess two populations of LDL. One has normal receptor binding, and the other, which can be isolated by monoclonal antibody 19 immunoaffinity chromatography, has almost no binding activity. The mutation that disrupts binding is a Gln for Arg substitution of apoB-100 residue 3500. NMR spectra of LDL containing (13CH3)2Lys residues show that chemically modified Lys exist in two microenvironments. In normal human LDL, there are about 50 Lys with pK 8.9 and 170 Lys with pK 10.5; an upper limit of 10 pK 8.9 Lys may be particularly involved in binding to the LDL receptor. Examination of the mixture of normal LDL and mutant LDL from five patients shows that the latter have fewer pK 8.9 Lys. In purified defective LDL at least seven Lys are redistributed from the active to normal pool. The CD spectra of mutant and normal LDL are identical. Therefore, substitution of Gln for Arg at position 3500 induces a change in local conformation which disrupts the receptor-binding domain of apoB-100.

Low density lipoprotein (LDL) has previously been demonstrated to be a potent inhibitor of human inflammatory cell activation by monosodium urate (MSU) crystals in vitro. As suppression of cellular responses to crystals by LDL is known to be dependent on the binding of LDL to urate crystals we further evaluated the mechanism and regulation of LDL binding to urate crystals in vitro. Using nonlinear, least squares methodology to analyze binding, we found LDL to saturably and reversibly bind with high affinity (Kd 9.3 x 10(-9) M) to MSU crystals. LDL binding was competitively inhibited by LDL but not by a variety of other positively charged moieties. Glycosaminoglycans (GAG) (heparin, heparan sulfate, hyaluronate and chondroitin sulfate), in diminishing order of potency, inhibited the binding of LDL to crystals. This inhibitory activity was dose dependent, sensitive to digestion with glycosidases and appeared to be specific for polymerized GAG, as glucuronic acid, dextran, dextran sulfate, as well as isolated amino sugar constituents of GAG were either weakly inhibitory or inactive. GAG also promoted dissociation of bound LDL from the urate crystal surface. The results indicate that LDL binds saturably and reversibly to urate crystals and that polymerized, but not depolymerized, hyaluronate and other GAG inhibit LDL binding to urate crystals. This suggests that the amount of LDL coating intraarticular urate crystals could vary during the course of a gouty paroxysm.

The early fatty streak lesions of atherosclerosis are characterized by the presence of cholesteryl ester-loaded macrophages or "foam cells." Platelets are also present in the early lesions of atherosclerosis and are often found in close association with foam cells. We have investigated the hypothesis that platelets contribute to foam cell formation by inducing macrophage cholesteryl ester accumulation. Using an in vitro culture system of human monocyte-derived macrophages and autologous platelets, we have demonstrated a platelet-dependent stimulation of macrophage cholesterol esterification and cholesteryl ester accumulation. The response is specific to platelets and is dependent upon activation of the platelets. An active fraction can be isolated from the releasates of thrombin-stimulated platelets that contain large cholesterol-rich platelet membrane vesicles. The results suggest that platelet-derived free cholesterol is required for platelet-induced macrophage foam cell formation.