Functional Analysis of Mutant Apolipoprotein AV
Date Issued
2008
Date
2008
Author(s)
Liao, Pei-Jhen
Abstract
Cardiovascular disease (CAD) is one of the major causes of mortality in Taiwan. Hypertriglyceridemia is an independent risk factor involved in CAD. Triglyceride (TG) is a kind of lipid and is associated with lipoprotein in the circulation. Different lipoproteins compose of different lipid. TG is the major composition of VLDL and chylomicron. When lipoproteins circulate to tissue, it would be hydrolyzed by lipoprotein lipase which is attached to vessel wall via heparan sulfate proteoglycan (HSPG) and converted to free fatty acid. Several researchers have discovered that different apolipoproteins may involve in this process. Recently, a novel apolipoprotein, Apolipoprotein AV, has been identified by gene comparison. TG level from human Apolipoprotein a5 transgenic mice and Apolipoprotein a5 knockout mice show that apolipoprotein AV (APOAV) plays a crucial role in triglyceride metabolism. In 2003, we have identified a novel single nucleotide polymorphism (SNP), APOA5 c.553G>T, resulting in the substitution of cysteine for glycine at residue 185. Subjects with this genetic variant have higher serum TG level. Thus, the aim of this study is to investigate the effect of different amino acid at this position on the metabolism of TG. Using site directed mutagenesis, we obtained APOAV185C, APOAV185D, APOAV185R mutant and established respective stable clones. We used HEK293 cell line and selected five stable clones separately, including SC-pCR3-A5WT, SC-pCR3-A5185C, SC-pCR3-A5185R, SC-pCR3-A5185D and SC-pCR3-uni which did not contain APOAV gene and was used as a negative control. Although APOAVWT and mutants could activate LPL activity, the extent of activation by APOA5185C, APOA5185R, APOA5185D were lower than that of wild type. According to ELISA data, the secretion of APOAV from SC-pCR3-A5185C was only 50% of that from SC-pCR3-A5WT. It is likely that reduced APOAV185C secretion resulted in lower LPL activation ability. In physical condition, when LPL released from parenchymal cells, it would circulate to the site of action and attach to vessel wall via HSPG. LPL was added to 96-well plate which had been coated with HSPG to mimic the physical condition, and APOAV enriched VLDL was used as substrate, APOAV-unenriched VLDL as control. In one independent triplicate data, we found that APOAVWT could activate HSPG bound LPL activity (p<0.05), other mutants could also activate LPL activity but there is no statistical significance. In the future, VLDL from apoa5 knockout mice should be useful to rule out the influence of endogenous APOAV, and obtain more significant data.
Subjects
hypertriglyceridemia
apolipoprotein a5
lipoprotein lipase
apolipoprotein A5 c.553G >
SDGs
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