College of Pharmacy, Pharmaceutical Analysis Laboratory, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada.
J Pharm Pharm Sci. 2018;21(1s):309s-324s. doi: 10.18433/jpps30246.
Liver fatty acid binding protein (FABP1) is a cytoplasmic polypeptide that transports substrates throughout the cytosol and functions as an antioxidant. A common polymorphic variant, FABP1 T94A has a minor allele frequency of 26-38%, 8.3±1.9% homozygous in the human population. The purpose of this study was to mutate and isolate recombinant rat FABP1 to the T94A variant to evaluate the mutant's antioxidant activity using in vitro studies.
Site-directed mutagenesis was used to generate a mutation in rat cDNA within a pGEX-6p-2 vector. This plasmid was transformed into competent cells and cultured for expression of FABP1 T94A mutant. The mutated protein was purified using GSTrap Fastflow columns within an ÄKTA FPLC system. A 2,7-dichlorofluorescein (DCF) assay was used to screen the T94A variant antioxidant activity. Additionally, Thiobarbituric Acid Reactive Substances (TBARS) assay was used in determining T94A mutant antioxidant activity in hydrophilic and lipophilic environments through the use of the azo compounds AAPH and MeO-AMVN, respectively and in the presence and absence of the long-chain fatty acid palmitate and α-bromo palmitate.
Although the FABP1 T94A (20 μM) mutant significantly reduced DCF fluorescence compared to control (no protein; P< 0.001), there were no significant difference when compared to the wild-type (WT) FABP1. T94A was able to diminish the formation of malondialdehyde (MDA) in both lipophilic and hydrophilic systems. There were significant differences between T94A mutant and WT FABP1 at concentrations 1 and 10 μM (P< 0.05) in the hydrophilic milieu, however, this was not seen at 20 μM and also not seen in the lipophilic milieu at all concentrations. When T94A was pre-incubated with the long-chain fatty acids palmitate or α -bromo palmitate, MDA formation was decreased in both lipid peroxidation systems. There were no statistical differences between the WT FABP1 and T94A bound with fatty acids in both lipid peroxidation systems, however, there was a slight statistical difference when the T94A and WT FABP1 bound α-Br-PA in the AAPH lipid peroxidation system only.
The T94A has antioxidant activity in both hydrophilic and lipophilic environments. The T94A variant of FABP1 does not have a loss of function in regard to acting as an antioxidant but the extent of function may be influenced by ligand binding. We conclude that populations having the minor T94A allele frequency would have similar ROS scavenging potential as those with nascent FABP1.
肝脂肪酸结合蛋白（FABP1）是一种胞质多肽，可在整个细胞质中运输底物，并作为抗氧化剂发挥作用。常见的多态性变体 FABP1 T94A 的次要等位基因频率为 26-38%，人群中纯合子的频率为 8.3±1.9%。本研究的目的是突变并分离重组大鼠 FABP1 至 T94A 变体，以使用体外研究评估突变体的抗氧化活性。
使用定点突变在 pGEX-6p-2 载体中的大鼠 cDNA 中生成突变。将该质粒转化到感受态细胞中，并进行培养以表达 FABP1 T94A 突变体。使用 GSTrap Fastflow 柱在 ÄKTA FPLC 系统中纯化突变蛋白。使用 2,7-二氯荧光素（DCF）测定法筛选 T94A 变体的抗氧化活性。此外，通过使用偶氮化合物 AAPH 和 MeO-AMVN 分别在亲水和亲脂环境中以及在长链脂肪酸棕榈酸和α-溴代棕榈酸的存在和不存在下，使用硫代巴比妥酸反应物质（TBARS）测定法确定 T94A 突变体的抗氧化活性。
尽管 FABP1 T94A（20 μM）突变体与对照（无蛋白；P<0.001）相比显着降低了 DCF 荧光，但与野生型（WT）FABP1 相比无显着差异。T94A 能够减少亲脂和亲水系统中丙二醛（MDA）的形成。在亲水环境中，T94A 突变体和 WT FABP1 在浓度为 1 和 10 μM 时（P<0.05）有显着差异，但在 20 μM 时没有差异，在所有浓度下在亲脂环境中也没有差异。当 T94A 与长链脂肪酸棕榈酸或α-溴代棕榈酸预孵育时，两种脂质过氧化系统中的 MDA 形成均减少。在两种脂质过氧化系统中，WT FABP1 和与脂肪酸结合的 T94A 之间没有统计学差异，但是当 T94A 和 WT FABP1 在 AAPH 脂质过氧化系统中仅与α-Br-PA 结合时，存在略微的统计学差异。
T94A 在亲水和亲脂环境中均具有抗氧化活性。FABP1 的 T94A 变体在作为抗氧化剂方面没有功能丧失，但功能的程度可能受配体结合的影响。我们得出的结论是，具有较小 T94A 等位基因频率的人群可能具有与新生 FABP1 相似的 ROS 清除潜力。
