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Updated: Jul 15, 2025
Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein
Published on: October 12, 2017
Characterization of novel truncated apolipoprotein A-I in human high-density lipoprotein generated by sequential
Shao-Jui Lai1, Takahiro Kameda1, Maasa Morita2
1Clinical Bioanalysis and Molecular Biology, Field of Applied Laboratory Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan.
Insights
Myeloperoxidase (MPO) and chymase sequentially modify high-density lipoprotein (HDL). This generates novel apolipoprotein A-I (apoA-I) fragments, useful for predicting cardiovascular disease (CVD) risk in patients with normal HDL cholesterol.
Area of Science:
- Biochemistry
- Cardiovascular Research
- Proteomics
Background:
- High-density lipoprotein (HDL) cholesterol is a key cardiovascular disease (CVD) biomarker.
- HDL's anti-atherosclerotic functions can be impaired by protein modifications, independent of HDL-cholesterol (HDL-C) levels.
- Inflammatory enzymes like myeloperoxidase (MPO) and chymase, found in atherosclerotic plaques, can alter HDL structure and function.
Purpose of the Study:
- To investigate the combined effects of MPO and chymase on HDL.
- To identify novel apolipoprotein A-I (apoA-I) fragments generated by sequential MPO and chymase modification.
- To assess the potential of these fragments as biomarkers for CVD in individuals with normal HDL-C.
Main Methods:
- Sequential treatment of HDL with MPO followed by chymase.
- Mass spectrometry to identify and characterize apoA-I cleavage fragments.
- Analysis of fragment properties, including potential for trapping in HDL particles.
Main Results:
- Sequential MPO and chymase treatment generated two novel truncated apoA-I fragments.
- A 16.5 kDa fragment was identified, with chymase cleavage occurring at Tyr100 after MPO modification.
- These fragments may be retained within HDL particles, suggesting potential for detection and quantification.
Conclusions:
- Sequential MPO and chymase modification of HDL yields novel apoA-I fragments.
- These fragments can be quantified using ELISA, offering a potential new biomarker for CVD risk.
- This approach may aid in identifying CVD risk in patients with normal HDL-C levels.
Abstract:
High-density lipoprotein (HDL) cholesterol is a well-known biomarker, which has been associated with reduction in the risk of cardiovascular diseases (CVD). However, some HDL anti-atherosclerotic functions may be impaired without altered HDL-cholesterol (HDL-C) level via its dysfunctional proteins or other physiological reactions in vivo. We previously showed that activated mast cell-derived chymase could modestly cleave apolipoprotein A-I (apoA-I) in HDL3, and further easily cleave lipid-free apoA-I. In contrast, myeloperoxidase (MPO) secreted by macrophages, the main cell type in atherosclerotic plaques, could oxidize HDL proteins, which might modify their tertiary structures, increasing their susceptibility to other enzymes. Here we focused on the co-modification and impact of chymase and MPO, usually secreted during inflammation from cells with possible co-existence in atheromas, on HDL. Only after sequential treatment with MPO and then chymase, two novel truncated apoA-I fragments were generated from HDL. One fragment was 16.5 kDa, and the cleavage site by chymase after MPO modification was the C-terminal of Tyr100 in apoA-I, cross-validated by three different mass spectrometry methods. This novel apoA-I fragment can be trapped in HDL particles to avoid kidney glomerular filtration and has a specific site for antibody generation for ELISA tests. As such, its quantification can be useful in predicting patients with CVD having normal HDL-C levels.
