Novel polymorphisms of the APOA2 gene and its promoter region affect body traits in cattle

Apolipoprotein A-II (APOA2) is one of the major constituents of high-density lipoprotein and plays a critical role in lipid metabolism and obesity. However, similar research for the bovine APOA2 gene is lacking. In this study, polymorphisms of the bovine APOA2 gene and its promoter region were detected in 1021 cows from four breeds by sequencing and PCR-RFLP methods. Totally, we detected six novel mutations which included one mutation in the promoter region, two mutations in the exons and three mutations in the introns. There were four polymorphisms within APOA2 gene were analyzed. The allele A, T, T and G frequencies of the four loci were predominant in the four breeds when in separate or combinations analysis which suggested cows with those alleles to be more adapted to the steppe environment. The association analysis indicated three SVs in Nangyang cows, two SVs in Qinchun cows and the 9 haplotypes in Nangyang cows were significantly associated with body traits (P < 0.05 or P < 0.01). The results of this study suggested the bovine APOA2 gene may be a strong candidate gene for body traits in the cattle breeding program.     

An ABCA1 truncation shows no dominant negative effect in a familial hypoalphalipoproteinemia pedigree with three ABCA1 mutations

The ATP binding cassette transporter (ABCA1) A1 is a key determinant of circulating high density lipoprotein cholesterol (HDL-C) levels. Mutations in ABCA1 are a major genetic contributor to low HDL-C levels within the general population. Following the finding of three different ABCA1 mutations, p.C978fsX988, p.T1512M and p.N1800H in a subject with hypoalphalipoproteinemia, we aimed to establish whether the p.C978fsX988 truncation exerted a dominant negative effect on the full-length ABCA1 alleles within family members as has been reported for other ABCA1 truncations. Characterisation of the p.C978fsX988 mutant in transfected HEK 293 cells showed it to be expressed as a GFP fusion protein but lacking in cholesterol efflux function. This was in keeping with results from cholesterol efflux assays in the fibroblasts of p.C978fsX988 carriers which also showed impaired efflux. Allele- specific quantification of p.C978fsX988 mRNA and analysis of ABCA1 protein levels in the fibroblasts of p.C978fsX988 heterozygotes showed negligible levels of mRNA and protein expression. There was no evidence of a dominant negative effect on wildtype or p.N1800H protein levels. We conclude that in the case of the p.C978fsX988 truncated mutant a lack of expression precludes it from having a dominant negative effect.     

Species differences of macrophage very low-density-lipoprotein (VLDL) receptor protein expression

Triglyceride-rich lipoproteins (TGRLs) and low-density-lipoprotein (LDL) cholesterol are independent risk factors for coronary artery disease. We have previously proposed that the very low-density-lipoprotein (VLDL) receptor is one of the receptors required for foam cell formation by TGRLs in human macrophages. However, the VLDL receptor proteins have not been detected in atherosclerotic lesions of several animal models. Here we showed no VLDL receptor protein was detected in mouse macrophage cell lines (Raw264.7 and J774.2) or in mouse peritoneal macrophages in vitro. Furthermore, no VLDL receptor protein was detected in macrophages in atherosclerotic lesions of chow-fed apolipoprotein E-deficient or cholesterol-fed LDL receptor-deficient mice in vivo. In contrast, macrophage VLDL receptor protein was clearly detected in human macrophages in vitro and in atherosclerotic lesions in myocardial infarction-prone Watanabe-heritable hyperlipidemic (WHHLMI) rabbits in vivo. There are species differences in the localization of VLDL receptor protein in vitro and in vivo. Since VLDL receptor is expressed on macrophages in atheromatous plaques of both rabbit and human but not in mouse models, the mechanisms of atherogenesis and/or growth of atherosclerotic lesions in mouse models may be partly different from those of humans and rabbits.     

Acid Lipase Inhibitor in Chicken Plasma Identified as Apolipoprotein A-I

We have reported a inhibitor of acid lipases in liver lysosomes and erythrocytes from chickens [M. Fujii et al., Int. J. Biochem., 22, 895–898 (1990)]. In this paper, the properties of the inhibitor were described in comparison with those of apo A-I of chicken.

The purified inhibitor migrated with the same mobility on SDS–PAGE as apo A-I, and had a molecular weight of 27,000. The peptide map from the lipase inhibitor was similar to that of apo A-I. Antibodies to the acid lipase inhibitor also reacted with apo A-I. Apo A-I inhibited the acid lipase activities of liver lysosomes and erythrocytes from chickens as strongly as the lipase inhibitor. The N-terminal amino acid sequence of lipase inhibitor was identical to that of apo A-I as far as residue 20. The amino acid sequence of peptides obtained from the inhibitor by cleavage with CNBr corresponded to internal sequence of apo A-I, and so the CNBr-peptides were derived by cleavage after the methionine residues in apo A-I. The findings showed that the inhibitor of the acid lipases in liver lysosomes and erythrocytes from chickens was identical to apo A-I.     

Longer action means better drug: tuning up protein therapeutics

An increasing number of proteins are currently available on the market as therapeutics and this branch of the pharmaceutical industry will expand substantially during the coming years. As many diseases result from dysfunction of proteins forming multicomponent complexes, protein drugs with their inherent high specificity and affinity seem to be optimal medical agents. On the other hand, proteins are often highly instable and sensitive to degradation, which questions their applicability as effective therapeutics. Therefore, redesign and engineering of proteins is usually a required step in the present day drug development.Several approaches have been applied to optimize the protein properties central to their pharmaceutical use. This review focuses on different strategies that improve two crucial factors influencing protein drug efficiency: protein stability and its in vivo half-life. We provide examples of successful genetic and chemical modifications applied in the design of effective protein therapeutics.     

Impact of self-association on function of apolipoprotein A-I

Self-association is an inherent property of the lipid-free forms of several exchangeable apolipoproteins, including apolipoprotein A-I (apoA-I), the main protein component of high density lipoproteins (HDL) and an established antiatherogenic factor. Monomeric lipid-free apoA-I is believed to be the biologically active species, but abnormal conditions, such as specific natural mutations or oxidation, produce an altered state of self-association that may contribute to apoA-I dysfunction. Replacement of the tryptophans of apoA-I with phenylalanines (ΔW-apoA-I) leads to unusually large and stable self-associated species. We took advantage of this unique solution property of ΔW-apoA-I to analyze the role of self-association in determining the structure and lipid-binding properties of apoA-I as well as ATP-binding cassette A1 (ABCA1)-mediated cellular lipid release, a relevant pathway in atherosclerosis. Monomeric ΔW-apoA-I and wild-type apoA-I activated ABCA1-mediated cellular lipid release with similar efficiencies, whereas the efficiency of high order self-associated species was reduced to less than 50%. Analysis of specific self-associated subclasses revealed that different factors influence the rate of HDL formation in vitro and ABCA1-mediated lipid release efficiency. The α-helix-forming ability of apoA-I is the main determinant of in vitro lipid solubilization rates, whereas loss of cellular lipid release efficiency is mainly caused by reduced structural flexibility by formation of stable quaternary interactions. Thus, stabilization of self-associated species impairs apoA-I biological activity through an ABCA1-mediated mechanism. These results afford mechanistic insights into the ABCA1 reaction and suggest self-association as a functional feature of apoA-I. Physiologic mechanisms may alter the native self-association state and contribute to apoA-I dysfunction.     

N-Glycosylation regulates endothelial lipase-mediated phospholipid hydrolysis in apoE- and apoA-I-containing high density lipoproteins

Endothelial lipase (EL) is a member of the triglyceride lipase gene family with high phospholipase and low triacylglycerol lipase activities and a distinct preference for hydrolyzing phospholipids in HDL. EL has five potential N-glycosylation sites, four of which are glycosylated. The aim of this study was to determine how glycosylation affects the phospholipase activity of EL in physiologically relevant substrates. Site-directed mutants of EL were generated by replacing asparagine (N) 62, 118, 375, and 473 with alanine (A). These glycan-deficient mutants were used to investigate the kinetics of phospholipid hydrolysis in fully characterized preparations of spherical reconstituted high density lipoprotein (rHDL) containing apolipoprotein E2 (apoE2) [(E2)rHDL], apoE3 [(E3)rHDL], apoE4 [(E4)rHDL], or apoA-I [(A-I)rHDL] as the sole apolipoprotein. Wild-type EL hydrolyzed the phospholipids in (A-I)rHDL, (E2)rHDL, (E3)rHDL, and (E4)rHDL to similar extents. The phospholipase activities of EL N118A, EL N375A, and EL N473A were significantly diminished relative to that of wild-type EL, with the greatest reduction being apparent for (E3)rHDL. The phospholipase activity of EL N62A was increased up to 6-fold relative to that of wild-type EL, with the greatest enhancement of activity being observed for (E2)rHDL. These data show that individual N-linked glycans have unique and important effects on the phospholipase activity and substrate specificity of EL.     

Dynamics of dense electronegative low density lipoproteins and their preferential association with lipoprotein phospholipase A(2)

Small, dense, electronegative low density lipoprotein [LDL(-)] is increased in patients with familial hypercholesterolemia and diabetes, populations at increased risk for coronary artery disease. It is present to a lesser extent in normolipidemic subjects. The mechanistic link between small, dense LDL(-) and atherogenesis is not known. To begin to address this, we studied the composition and dynamics of small, dense LDL(-) from normolipidemic subjects. NEFA levels, which correlate with triglyceride content, are quantitatively linked to LDL electronegativity. Oxidized LDL is not specific to small, dense LDL(-) or lipoprotein [a] (i.e., abnormal lipoprotein). Apolipoprotein C-III is excluded from the most abundant LDL (i.e., that of intermediate density: 1.034 < d < 1.050 g/ml) but associated with both small and large LDL(-). In contrast, lipoprotein-associated phospholipase A(2) (LpPLA(2)) is highly enriched only in small, dense LDL(-). The association of LpPLA(2) with LDL may occur through amphipathic helical domains that are displaced from the LDL surface by contraction of the neutral lipid core.     

Inability of HDL from abdominally obese subjects to counteract the inhibitory effect of oxidized LDL on vasorelaxation

Abdominal obesity is associated with a decreased plasma concentration of HDL cholesterol and with qualitative modifications of HDL, such as triglyceride enrichment. Our aim was to determine, in isolated aorta rings, whether HDL from obese subjects can counteract the inhibitory effect of oxidized low density lipoprotein (OxLDL) on endothelium-dependent vasodilation as efficiently as HDL from normolipidemic, lean subjects. Plasma triglycerides were 74% higher (P < 0.005) in obese subjects compared with controls, and apolipoprotein A-I (apoA-I) and HDL cholesterol concentrations were 12% and 17% lower (P < 0.05), respectively. HDL from control subjects significantly reduced the inhibitory effect of OxLDL on vasodilation [maximal relaxation (E(max)) = 82.1 +/- 8.6% vs. 54.1 +/- 8.1%; P < 0.0001], but HDL from obese subjects had no effect (E(max) = 47.2 +/- 12.5% vs. 54.1 +/- 8.1%; NS). In HDL from abdominally obese subjects compared with HDL from controls, the apoA-I content was 12% lower (P < 0.05) and the triglyceride-to-cholesteryl ester ratio was 36% higher (P = 0.08)). E(max)(OxLDL + HDL) was correlated with HDL apoA-I content and triglyceride-to-cholesteryl ester ratio (r = 0.36 and r = -0.38, respectively; P < 0.05). We conclude that in abdominally obese subjects, the ability of HDL to counteract the inhibitory effect of OxLDL on vascular relaxation is impaired. This could contribute to the increased cardiovascular risk observed in these subjects.     

Structure of eukaryotic DNA binding sites for steroid hormone-apolipoprotein A-I complexes

High affinity for DNA and synthetic oligonucleotides was detected for apolipoprotein A-I (ApoA-I) by affinity chromatography, affinity modification, and enzymatic analysis. Competitive inhibition and Southern hybridization showed that the tetrahydrocortisol (THC)-ApoA-I complex specifically bound to high-molecular-weight DNA in regions containing GCC/CGG sequences. The CC(GCC)₃ · GG(CGG)₃ duplex was found to be sensitive to nuclease S1 under the action of the THC-ApoA-I complex. The eukaryotic DNA binding sites for steroid (THC, androsterone)-ApoA-I complexes were found to be involved in the initiation of DNA copying in vitro.