2001 Fiscal Year Final Research Report Summary
Characterization and measurement of high-density lipoprotein (HDL)-binding proteins present in human peripheral monocytes
| Project/Area Number |
12672241
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory medicine
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| Research Institution | Shinshu University |
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Principal Investigator |
KATSUYAMA Tsutomu Department of Laboratory Medicine, Shinshu University School of Medicine, Professor, 医学部・医学科臨床検査医学, 教授 (90020809)
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| Co-Investigator(Kenkyū-buntansha) |
OTA Hiroyoshi Department of Biomedicai Laboratory Sciences, Shinshu University School of Medicine, Professor, 医学部・保健学科生体情報検査学, 教授 (50273107)
NAKAYAMA Jun Department of Pathology, Shinshu University School of Medicine, Professor, 医学部・医学科病理学, 教授 (10221459)
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| Project Period (FY) |
2000 – 2001
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| Keywords | monocytes / HDL-binding proteins / high-density lipoprotein (HDL) / mass spectrometry / flow cytometry / affinity chromatography / reverse-phase high performance liquid chromatography |
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| Research Abstract |
Plasma levels of high-density lipoprotein (HDL) are inversely correlated with the risk of atherosclerotic coronary artery disease. HDL is believed to play a role in the process of reverse cholesterol transport (RCT). Although the interaction of HDL with binding sites on the surface of peripheral cells appears to be an important first step in RCT, the nature of this association is not fully understood. We previously identified a binding site for HDL3 on the surface of human peripheral blood monocytes.
In this study, we found that pre-β-HDL purified by ion-exchange chromatography from very high density lipoprotein (VHDL, 1.21 < d < 1.25 g/ml) was susceptible to degradation if isolated in the absence anti-proteases, resulting in the smaller lyso-pre-β-HDL The mass of the larger fragment from lyso-pre-β-HDL was confirmed using MALDI-TOF mass spectrometrical analysis, which showed a fragment of approximately 22625.3Da. The ability of pre-β-HDL and lyso-pre-β-HDL to compete for HDL binding to
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human monocytes was determined using a flow cytometry-based assay. Pre-β-HDL competed efficiently for binding whereas lyso-pre-β-HDL was significantly less effective. We propose that in vivo degradation of pre-β-HDL would affect the ability of these particles to bind to human monocytes.
The purification of HDL-binding proteins was achieved by chromatography using DEAE ion-exchange, wheat germ lectin and apoHDL3 affinity columns. Analysis of purified HDL-binding proteins by reverse-phase high performance liquid chromatography showed the presence of two main proteins at 100k and 120kDa. In order to measure the amount of HDL-binding protein present in CHAPS-solubilized human mononuclear cells a 96-well plate assay was developed that utilized similar principles as the purification method. The present study detailing the purification and measurement of HDL-binding proteins in mononuclear cells provides significant information likely to further our understanding of the physiological function and fate of HDL. Less
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