Dataset Information

Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH.

ABSTRACT: APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer conditions. We hypothesized that the yield and solubility of recombinant APOA5 could be increased by: i) engineering a fusion protein construct in a codon optimized expression vector, ii) optimizing an efficient refolding protocol, and iii) screening buffer systems at physiological pH. The result was a high-yield (25 mg/l) bacterial expression system that produces lipid-free APOA5 soluble at concentrations of up to 10 mg/ml at a pH of 7.8 in bicarbonate buffers. Physical characterization of lipid-free APOA5 indicated that it exists as an array of multimers in solution, and far UV circular dichroism analyses show differences in total ?-helicity between acidic and neutral pH buffering conditions. The protein was functional in that it bound and emulsified multilamellar dimyristoyl-phosphatidylcholine vesicles and could inhibit postprandial plasma TG accumulation when injected into C57BL/6J mice orally gavaged with Intralipid.

SUBMITTER: Castleberry M

PROVIDER: S-EPMC6997596 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH.

Castleberry Mark M Davis Xenia X Liu Min M Thompson Thomas B TB Tso Patrick P Davidson W Sean WS

Journal of lipid research 20191212 2

APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer ...[more]

PMID: 31831525

Similar Datasets

Project description:The association of single nucleotide polymorphisms (SNPs) in the apolipoprotein (Apo) A1/C3/A4/A5 gene cluster and serum lipid profiles is inconsistent. The present study was undertaken to detect the association between the ApoA1/C3/A5 gene polymorphisms and their haplotypes with serum lipid levels in the general Chinese population.A total of 1030 unrelated subjects (492 males and 538 females) aged 15-89 were randomly selected from our previous stratified randomized cluster samples. Genotyping of the ApoA1 -75 bp G>A, ApoC3 3238C>G, ApoA5 -1131T>C, ApoA5 c.553G>T and ApoA5 c.457G>A was performed by polymerse chain reaction and restriction fragment length polymorphism combined with gel electrophoresis, and then confirmed by direct sequencing. Pair-wise linkage disequilibria and haplotype analysis among the five SNPs were estimated.The levels of high-density lipoprotein cholesterol (HDL-C) and ApoA1 were lower in males than in femailes (P < 0.05 for each). The allelic and genotypic frequencies of the SNPs were no significant difference between males and females except ApoC3 3238C>G. There were 11 haplotypes with a frequency >1% identified in the cluster in our population. At the global level, the haplotypes comprised of all five SNPs were significantly associated with all seven lipid traits. In particular, haplotype G-G-C-C-A (6%; in the order of ApoA5 c.553G>T, ApoA5 c.457G>A, ApoA5 -1131T>C, ApoC3 3238C>G, and ApoA1 -75bp G>A) and G-A-T-C-G (4%) showed consistent association with total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), ApoA1, ApoB, and the ApoA1/ApoB ratio. In addition, carriers of haplotype G-G-T-C-G (26%) had increased serum concentration of HDL-C and ApoA1, whereas carriers of G-G-C-G-G (15%) had high concentrations of TC, triglyceride (TG) and ApoB. We also found that haplotypes with five SNPs explain much more serum lipid variation than any single SNP alone, especially for TG (4.4% for haplotype vs. 2.4% for -1131T>C max based on R-square) and HDL-C (5.1% for haplotype vs. 0.9% for c.553G>T based on R-square). Serum lipid parameters were also correlated with genotypes and several environment factors.Several common SNPs and their haplotypes in the ApoA1/C3/A5 gene cluster are closely associated with modifications of serum lipid parameters in the general Chinese population.

Project description:Abstract Background Both antiretroviral therapy (ART) and polymorphism in genes involved in lipoprotein metabolism contribute to the development of ART-induced dyslipidemia. Apolipoprotein A5 (APOA5)gene variant, which vary by race/ethnicity, influence plasma lipid concentrations. We explored the association of candidate gene effect on plasma lipid levels with food in HIV-infected children initiating ART in south India. Methods HIV-infected children, between 2 and 12 years of age initiating non-nucleoside reverse transcriptase inhibitor (NNRTI)-based ART during 2010–2014 were included. They were assessed for their dietary intake by 24-hour dietary recall. Fasting blood was drawn for serum triglycerides (TGL), total cholesterol and high-density cholesterol along with CD4 cell count, and HIV-1 viral load. APOA5 gene polymorphisms rs662799, rs3135506 were determined by Taqman single-nucleotide polymorphism (SNP) genotyping assays. The General Linear Model (GLM) was applied to explore the risk of hypertriglyceridemia for SNP’s rs662799 and rs3135506 with food interaction in children. Results Three hundred and ninety HIV-infected children {median age (IQR): 9 (5–11) years; and median viral load: 141,000 (25,876–436,000) copies/mL} were started on NNRTI-based ART. The frequency of c allele was 20% and 3.0% in rs662799, rs3135506, respectively. The GLM model suggested that the SNP rs662799 has significant association with TGL(P < 0.01); there was, however no interaction between gene polymorphisms and food. Also, the prediction model revealed that those who had a carrier allele (C) had higher TGL level as compared with those with wild type (165 vs. 138 mg/dL) and girls had higher TGL levels compared with boys. Conclusion Children with carrier allele of APOA5 are prone to hypertriglyceridemia, irrespective of diet or drugs. These children require periodic monitoring of lipid parameters and effective interventions to prevent the development of atherogenic or metabolic complications. Disclosures All authors: No reported disclosures.

Project description:BackgroundLittle is known about the interactions of apolipoprotein (Apo) A5 gene polymorphisms and alcohol consumption on serum lipid profiles. The present study was undertaken to detect the interactions of ApoA5-1131T>C, c.553G>T and c.457G>A polymorphisms and alcohol consumption on serum lipid levels.Methodology/principal findingsA total of 516 nondrinkers and 514 drinkers were randomly selected from our previous stratified randomized cluster samples. Genotyping was performed by polymerase chain reaction and restriction fragment length polymorphism. The levels of serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), ApoA1 and ApoB were higher in drinkers than in nondrinkers (P<0.05-0.001). The genotypic and allelic frequencies of three loci were not different between the two groups. The interactions between -1131T>C genotypes and alcohol consumption on ApoB levels (P<0.05) and the ApoA1/ApoB ratio (P<0.01), between c.553G>T genotypes and alcohol consumption on low-density lipoprotein cholesterol (LDL-C) levels (P<0.05) and the ApoA1/ApoB ratio (P<0.05), and between c.457G>A genotypes and alcohol consumption on TG levels (P<0.001) were detected by factorial regression analysis after controlling for potential confounders. Four haplotypes (T-G-G, C-G-G, T-A-G and C-G-T) had frequencies ranging from 0.06 to 0.87. Three haplotypes (C-G-G, T-A-G, and C-G-T) were significantly associated with serum lipid parameters. The -1131T>C genotypes were correlated with TG, and c.553G>T and c.457G>A genotypes were associated with HDL-C levels in nondrinkers (P<0.05 for all). For drinkers, the -1131T>C genotypes were correlated with TC, TG, LDL-C, ApoB levels and the ApoA1/ApoB ratio (P<0.01 for all); c.553G>T genotypes were correlated with TC, TG, HDL-C and LDL-C levels (P<0.05-0.01); and c.457G>A genotypes were associated with TG, LDL-C, ApoA1 and ApoB levels (P<0.05-0.01).ConclusionsThe differences in some serum lipid parameters between the drinkers and nondrinkers might partly result from different interactions of the ApoA5 gene polymorphisms and alcohol consumption.

Dataset Information

Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH.

Publications

Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets