Fluorescence study of domain structure and lipid interaction of human apolipoproteins E3 and E4

Human apolipoprotein E (apoE) isoforms exhibit different conformational stabilities and lipid-binding properties that give rise to altered cholesterol metabolism among the isoforms. Using Trp-substituted mutations and site-directed fluorescence labeling, we made a comprehensive comparison of the conformational organization of the N- and C-terminal domains and lipid interactions between the apoE3 and apoE4 isoforms. Trp fluorescence measurements for selectively Trp-substituted variants of apoE isoforms demonstrated that apoE4 adopts less stable conformations in both the N- and C-terminal domains compared to apoE3. Consistent with this, the conformational reorganization of the N-terminal helix bundle occurs at lower guanidine hydrochloride concentration in apoE4 than in apoE3 as monitored by fluorescence resonance energy transfer (FRET) from Trp residues to acrylodan attached at the N-terminal helix. Upon binding of apoE3 and apoE4 variants to egg phosphatidylcholine small unilamellar vesicles, similar changes in Trp fluorescence or FRET efficiency were observed for the isoforms, indicating that the opening of the N-terminal helix bundle occurs similarly in apoE3 and apoE4. Introduction of mutations into the C-terminal domain of the apoE isoforms to prevent self-association and maintain the monomeric state resulted in great increase in the rate of binding of the C-terminal helices to a lipid surface. Overall, our results demonstrate that the different conformational organizations of the N- and C-terminal domains have a minor effect on the steady-state lipid-binding behavior of apoE3 and apoE4: rather, self-association property is a critical determinant in the kinetics of lipid binding through the C-terminal helices of apoE isoforms.     

Lipolysis-induced degradation of apolipoproteins B and E of human very low density lipoproteins

We have found that in vitro lipolysis of human very low density lipoproteins (VLDL) by purified bovine milk lipoprotein lipase (LpL) promotes degradation of the apolipoprotein (apo) B moiety of VLDL. Analysis by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis showed that lipolysis of VLDL by purified LpL for 1 h at 37 degrees C induced the selective degradation of the high Mr apo-B (apo-B-100) from most hypertriglyceridemic VLDL and from a few normolipidemic VLDL into several small fragments with molecular weights ranging from 90,000-490,000. No detectable degradation of apo-B occurred in control VLDL when incubated without LpL. The apo-E moiety of VLDL from certain individuals was also degraded following lipolysis of VLDL, and the extent of degradation of apo-B and -E in VLDL was varied among the individual VLDL. The major degradation products of apo-E, identified from the gel, were 31,000- and/or 28,000-Da species. In contrast to the apo-E moiety of VLDL, purified apo-E was not degraded when incubated with LpL. Incubation of low density lipoproteins (LDL) with LpL showed only a minimal effect on the apoproteins of LDL. When high density lipoprotein (HDL) was included in the lipolysis mixture as an acceptor of lipolytic surface remnants, the apoproteins of HDL remained unaltered, while the apo-B moiety of VLDL remnants in the mixture was degraded. Inclusion of protease inhibitors in the lipolysis mixture prevented the degradation of apo-B, but the hydrolysis of VLDL-triglyceride was minimally affected. A selective degradation of apo-B in VLDL also occurred during lipolysis of VLDL when VLDL was perfused through rat hearts. These results suggest that conformational changes in apo-B and apo-E caused by VLDL lipolysis may increase the susceptibility of apo-B and apo-E to degradation by the proteases co-isolated with VLDL. The consequences of the lipolysis-induced degradation of apo-B and apo-E on changes in metabolic properties of VLDL remnants remain to be determined.     

Genetic studies of human apolipoproteins

Summary Apolipoprotein H (APO H) has recently been identified as a structural component of chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Although the precise metabolic function of APO H in lipid metabolism is not certain, it has been suggested that APO H may be involved in triglyceride (TG) metabolism. In addition to the previously described quantitative polymorphism, we have recently detected a common qualitative polymorphism at the APO H structural locus. To test the role of APO H genetic variation in determining lipoprotein and lipid levels, we have estimated the allelic effects of APO H variation on TG, VLDL, LDL, HDL, HDL3, and total cholesterol on 356 Nigerian blacks(189 males, 167 females). While no significant effect of phenotype was observed on lipoprotein levels, the effect of interaction between phenotype and gender was significant. Therefore, data on males and females were analyzed separately using analysis of variance after adjusting for age and body mass index. Logarithmic transformation of pertinent variables was done to bring the distribution of the variables closer to normality. A statistically significant effect of phenotype was observed on triglyceride levels in females only (P<0.05). Further analysis of this phenotypic effect revealed that it is due to the impact of the APO H ^* 3 allele, which raises triglycerides by 9.92 mg/dl as compared to the common allele, APO H ^* 2. These findings are in accordance with the postulated role of APO H in triglyceride metabolism. On the basis of its sex-specific effect, we propose a hypothesis that may explain the combined influence of the quantitative and qualitative polymorphisms at the APO H locus on triglyceride levels in females.     

Genetic control of lipid transport in mice. II. Genes controlling structure of high density lipoproteins

Genetic factors controlling the structure of high density lipoproteins (HDL) in mice have been examined. Surveys of inbred strains of mice revealed genetic structural variations of the two major apolipoproteins of mouse HDL, apolipoproteins A-I and A-II. The structural variations alter the charge of the proteins as judged by isoelectric focusing of HDL under denaturing conditions. The structural variations are inherited as single Mendelian genes exhibiting co-dominant expression. The structural gene for mouse apolipoprotein A-II, designated Alp-2, resides on mouse chromosome 1, tightly linked to Ly-m20, a lymphocyte alloantigen locus. Previous studies, as well as our results, suggest that the structural gene for mouse apolipoprotein A-I, designated Alp-1, is on mouse chromosome 9. The genetic structural variation for apo-A-I results in a shift in the charge of the entire family of apo-A-I isoforms, indicating that they are all encoded by a common structural gene. The structure of intact HDL, examined primarily by electrophoretic techniques, exhibits numerous and complex phenotypes among different strains of mice. One variation, controlling the density and possibly the size of HDL, has been studied in two sets of recombinant inbred strains of mice. The results indicate that the variation is controlled by a single major gene that is either tightly linked to or identical with the Alp-2 gene on chromosome 1. In addition to structural variation, inbred strains of mice exhibited considerable quantitative variation of plasma HDL. Thus, the mouse provides a useful model system for examining the genetic control of mammalian HDL structure and regulation.     

Domain structure and lipid interaction in human apolipoproteins A-I and E,a general model

Detailed structural information on human exchangeable apolipoproteins (apo) is required to understand their functions in lipid transport. Using a series of deletion mutants that progressively lacked different regions along the molecule, we probed the structural organization of lipid-free human apoA-I and the role of different domains in lipid binding, making comparisons to apoE, which is a member of the same gene family and known to have two structural domains. Measurements of alpha-helix content by CD in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that deletion of the amino-terminal or central regions disrupts the tertiary organization, whereas deletion of the carboxyl terminus has no effect on stability and induces a more cooperative structure. These data are consistent with the lipid-free apoA-I molecule being organized into two structural domains similar to apoE; the amino-terminal and central parts form a helix bundle, whereas the carboxyl-terminal alpha-helices form a separate, less organized structure. The binding of the apoA-I variants to lipid emulsions is modulated by reorganization of the helix bundle structure, because the rate of release of heat on binding is inversely correlated with the stability of the helix bundle. Based on these observations, we propose that there is a two-step mechanism for lipid binding of apoA-I: apoA-I initially binds to a lipid surface through amphipathic alpha-helices in the carboxyl-terminal domain, followed by opening of the helix bundle in the amino-terminal domain. Because apoE behaves similarly, this mechanism is probably a general feature for lipid interaction of other exchangeable apolipoproteins, such as apoA-IV.     

Genetic variation of apolipoproteins in North Indians

Genetic variation at three apolipoprotein loci (APOA4, APOH, and APOE) has been examined in nine endogamous populations of Punjab, North India. The overall pattern of allele frequency variation at different loci is compatible with that of European populations, but observed microvariation differentiates the populations according to their position in the Indian caste structure. The most common allele at the APOA4 locus was APOA4*1 with a narrow frequency range (89%-92%). APOH*2 allele frequency was highest in these populations (0.852-0.914). APOE*E4 allele frequency was relatively low (6%-10%) in the North Indian populations compared to its frequency in many European populations. The anthropological usage of these polymorphisms was evaluated using multivariate analyses. Genetic distance analysis and principal correspondence analysis showed that the North Indian populations are closest to Europeans, followed by Chinese and African populations. Overall, this study highlights the usefulness of apolipoproteins as genetic markers for clinical, population, and anthropological studies.     

Characterization of two lipoproteins containing apolipoproteins B and E from lesion-free human aortic intima

Lesion-free areas of aortic intimas from seven men, 30 to 49 years old, were extracted with aqueous buffer within a few hours after an accidental or sudden death. Two lipoprotein fractions could be isolated by density gradient ultracentrifugation from all cases. The mean composition of fraction I (d less than 1.012 g/ml) resembled that reported for the cholesteryl ester-rich, beta-migrating very low density lipoprotein (beta-VLDL); the composition of fraction II (d 1.021-1.046 g/ml) resembled that of plasma low density lipoprotein (LDL). Mean diameter of the particles was 35 +/- 8 nm in fraction I and 25 +/- 5 nm in fraction II (22 +/- 2 nm in plasma LDL). Both fractions contained apolipoproteins B (apoB) and E (apoE), and had increased electrophoretic mobilities and reduced contents of linoleic acid. The immunoreactivity of apoB to a polyclonal and two monoclonal antibodies in both fractions was not different from that of plasma lipoproteins. The apoE isoform patterns in both fractions were similar to those obtained from the respective postmortem plasmas. When incubated with mouse peritoneal macrophages, fractions I and II enhanced the incorporation of radioactive oleate into cholesteryl esters by 10- to 20-fold and 3- to 4-fold, respectively, in comparison to plasma LDL. In conclusion, our results indicate that lesion-free human aortic intima contains two types of apoB- and apoE-containing lipoprotein particles, both of which might be potentially atherogenic.     

Genetic structure of hereditary catalepsy in mice

Catalepsy or pronounced freezing is a natural passive defense strategy in animals and a syndrome of some mental disorders in human. Hereditary catalepsy was shown to be associated with depressive-like features in rats and mice. The loci underlying the difference in predisposition to catalepsy between catalepsy-prone CBA/lacJ and catalepsy-resistant AKR/J mice were mapped using congenic line and selective breeding approaches. Three congenic mouse lines (AKR.CBA-D13Mit76C, AKR.CBA-D13Mit76A and AKR.CBA-D13Mit78) carrying the 59- to 70-, 61- to 70- and 71- to 75-c m fragments of chromosome 13 transferred from the CBA to the AKR genome were created by nine successive backcrossing of (CBA × AKR)F₁ on AKR strain. Because catalepsy was found only in the AKR.CBA-D13Mit76C and AKR.CBA-D13Mit76A mice, the major gene of catalepsy was mapped on the fragment of 61–70 c m . Selective breeding of the (CBA × (CBA × AKR))BC backcross generation for high predisposition to catalepsy showed numerous genome-wide distributed CBA-derived alleles as well as the AKR-derived alleles mapped on chromosome 17 and on the proximal parts of chromosomes 10 and 19 that increased the cataleptogenic effect of the major gene.     

Inhibition of lymphocyte proliferation by synthetic peptides homologous to human plasma apolipoproteins B and E

Apolipoproteins B and E of the human plasma lipoproteins are known inhibitors of lymphocyte proliferation. In this report, two synthetic peptide amides, apoB3358-3372 and apoE141-155, showed a dose-dependent inhibition of both the murine mixed lymphocyte culture reaction and the anti-T3 induced proliferation of lymphocytes. Their structures and antiproliferative potencies were similar to that of the heptadecapeptide CKS-17, a consensus peptide of a highly conserved region among HTLV-I, -II and C-type human retroviral proteins. SP-9-2-amide, a peptide homologous to the amino-terminal half of CKS-17, also suppressed lymphocyte activation. In contrast, a peptide homologous to the gp41 protein of HTLV-III that is sequence related to CKS-17 (approximately 35% homology) showed little antiproliferative activity. Neurotensin, a control peptide, showed no activity. The results suggest that a basic tetrapeptide sequence common to CKS-17-amide, SP-9-2, apoB3358-3372 and apoE141-155, but not HTLV-III-amide may account, in part, for the antiproliferative activities of these peptides.     

Hepatic apolipoprotein E expression promotes very low density lipoprotein-apolipoprotein B production in vivo in mice

In addition to its role in the uptake of apolipoprotein B (apoB)-containing lipoproteins, apoE promotes hepatic very low density lipoprotein-triglyceride (VLDL-TG) production in animal models. However, it is not known if apoE increases the amount of TG per VLDL particle or the number of VLDL particles secreted. VLDL-apoB production is a measure of the rate of VLDL particle secretion. We determined the effects of apoE deficiency and apoE overexpression on VLDL-apoB production in mice. [(35)S]methionine was injected into endogenously label VLDL-apoB and Triton WR-1339 was simultaneously injected to block the catabolism of VLDL. Compared with wild-type mice, the VLDL-apoB production rate was decreased by 33% in apoE-deficient mice. Conversely, VLDL-apoB production was increased by 48% in mice overexpressing apoE compared with controls. Nascent VLDL, obtained from post-Triton plasma, had a decreased, not increased, content of TG per apoB in the apoE-overexpressing group compared with the control group. This study demonstrates that hepatic apoE expression increases the output of VLDL triglyceride by increasing the production rate of VLDL-apoB, suggesting that hepatic apoE influences the number of VLDL particles secreted by the liver.     

Dietary cholesterol does not affect the synthesis of apolipoproteins B and E by rat hepatocytes.

To examine the unproved hypothesis that dietary cholesterol affects the synthesis of apolipoprotein B and E, we fed rats a cholesterol-rich diet that has been shown to alter dramatically the serum concentrations of these apolipoproteins. Rats fed for 4 weeks on a cholesterol-rich diet accumulate increased concentrations of low Mr apolipoprotein B (+2.7-fold) and decreased concentrations of apolipoprotein E (-40%) in their serum. Hepatocytes obtained from similarly treated rats were placed in monolayer culture and the rate of synthesis de novo of apolipoproteins was determined. Although cells from cholesterol-fed rats remained filled with lipid droplets throughout the experimental period, there was no difference in plating efficiency or viability, compared with cells obtained from chow-fed control rats. Both groups of cells synthesized and secreted immunoprecipitable apolipoproteins B and E at similar rates throughout the 18 h experiment. Thus there was a discordance between the effects of dietary cholesterol on serum apolipoprotein concentrations and hepatocyte synthesis and secretion. The data indicate that altered hepatic apolipoprotein synthesis cannot account for the changes in serum apolipoprotein concentrations caused by dietary cholesterol.     

Role of apolipoproteins E and C in type V hyperlipoproteinemia

Type V hyperlipoproteinemia is characterized by elevations of chylomicron (CM) and very low density lipoprotein (VLDL) triglycerides. The development of this lipid disorder involves a multitude of metabolic derangements including deficient clearance of triglycerides and/or their increased output aggravated by obesity, diabetes, alcohol intake, or use of some hormones. Some studies have suggested that the apolipoprotein E4 phenotype is involved in this dyslipoproteinemia but this concept is still a matter of controversy. Therefore, we determined the apoE phenotype in 21 patients with severe hypertriglyceridemia classified as type V. Their apoE4 gene frequency was 0.595 which is 2.6-fold higher (P less than 0.001) than that in the Finnish population. Correspondingly, their apoE3 gene frequency was lower than that in the normal population. No differences were noted in plasma lipoproteins of the apoE4 phenotypes and the other type V subjects. The apolipoprotein C-II and C-III distribution was similar to that in normolipidemic subjects. The results suggest that apoE4 may be involved in the development of type V hyperlipoproteinemia.     

Genetic factors controlling wool shedding in a composite Easycare sheep flock

Historically, sheep have been selectively bred for desirable traits including wool characteristics. However, recent moves towards extensive farming and reduced farm labour have seen a renewed interest in Easycare breeds. The aim of this study was to quantify the underlying genetic architecture of wool shedding in an Easycare flock. Wool shedding scores were collected from 565 pedigreed commercial Easycare sheep from 2002 to 2010. The wool scoring system was based on a 10‐point (0–9) scale, with score 0 for animals retaining full fleece and 9 for those completely shedding. DNA was sampled from 200 animals of which 48 with extreme phenotypes were genotyped using a 50‐k SNP chip. Three genetic analyses were performed: heritability analysis, complex segregation analysis to test for a major gene hypothesis and a genome‐wide association study to map regions in the genome affecting the trait. Phenotypes were treated as a continuous or binary variable and categories. High estimates of heritability (0.80 when treated as a continuous, 0.65–0.75 as binary and 0.75 as categories) for shedding were obtained from linear mixed model analyses. Complex segregation analysis gave similar estimates (0.80 ± 0.06) to those above with additional evidence for a major gene with dominance effects. Mixed model association analyses identified four significant (P < 0.05) SNPs. Further analyses of these four SNPs in all 200 animals revealed that one of the SNPs displayed dominance effects similar to those obtained from the complex segregation analyses. In summary, we found strong genetic control for wool shedding, demonstrated the possibility of a single putative dominant gene controlling this trait and identified four SNPs that may be in partial linkage disequilibrium with gene(s) controlling shedding.     

Allotypes associated with B apolipoproteins in rabbits

Western blot analysis of the alloantisera (i.e., anti-Lpq1, anti-Lpq2, anti-Lpq3, and anti-Lpq4) which defined the three lpq genes of rabbit linkage group VIII showed that they reacted strongly with an apolipoprotein of molecular weight 320,000. They also cross-reacted with an apolipoprotein of molecular weight 220,000. The two apolipoproteins that reacted with the alloantisera were found by SDS-polyacrylamide gel electrophoresis to be present in very low density (VLDL), intermediate density (IDL), and low density (LDL) lipoprotein fractions and by Western blot analysis to react with an anti-apolipoprotein B antiserum. These results support the conclusion that the alloantisera react with allotypes associated with the B apolipoproteins. The distribution of the four allotypes among different lipoprotein fractions, however, differed. The quantitative competitive Enzyme Linked Immunosorbant Assay (ELISA) showed that the Lpq1, Lpq2, and Lpq4 allotypes were found in the highest concentration in VLDL, IDL, and LDL, and in significantly lower concentrations in plasma chylomicrons. The concentrations of these allotypes in high density lipoproteins (HDL) as measured in the ELISA were about 1% of the concentrations found in LDL. The Lpq3 allotype, on the other hand, was present in the highest concentrations only in IDL and LDL and in significantly lower concentrations in VLDL and plasma chylomicrons. Surprisingly, the concentration of the Lpq3 allotype in HDL was 20% of the level found in LDL.     

Binding of cholesterol by apolipoproteins A-I and E]

It was shown that cholesterol can interact with some guanidine group-containing compounds (guanidine proper, arginine, metformine and dodecylguanidine bromide) as well as with the arginine-rich proteins--apoproteins A-1 and E. In the latter case this interaction results in the formation of cholesterol-apoprotein complexes. Analysis of such complexes revealed that one apo-A-1 molecule binds 17-22, whereas one apo-E molecule--30-35 sterol molecules, which approximately correspondence to the amount of arginine residues in these proteins. The formation of cholesterol-apoprotein complexes seems to be due to: (1) formation of hydrogen bonds and ion-dipole interactions between the hydroxyl groups of cholesterol and the guanidine groups of the apoprotein arginine residues and, presumably, the carboxylic groups of aspartic or glutamic acids, eventually resulting in the production of chelate complexes; (2) hydrophobic interaction of the cholesterol aliphatic chain with the nonpolar side chains of the amino acids occupying the third position from arginine in the protein molecule.     

Genetic studies of human apolipoproteins. VII. Population distribution of polymorphisms of apolipoproteins A-I, A-II, A-IV, C-II, E, and H in Nigeria.

Familial porphyria cutanea tarda (PCT) results from a generalized deficiency of uroporphyrinogen decarboxylase (URO-D) activity. The molecular defect responsible for this disorder has not been characterized. To determine whether decreased levels of URO-D mRNA are responsible for subnormal URO-D activity, steady-state levels of URO-D mRNA in lymphoblastoid cells were determined. Northern blots were hybridized with a URO-D cDNA probe and quantified by densitometry. No difference in the levels of URO-D mRNA was detected between affected individuals and their normal relatives. Thus, the deficiency of URO-D activity in two familial PCT pedigrees characterized here does not arise from a deficiency of URO-D mRNA.