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Structural and Functional Changes of Reconstituted High-Density Lipoprotein (HDL) by Incorporation of α-synuclein: A Potent Antioxidant and Anti-Glycation Activity of α-synuclein and apoA-I in HDL at High Molar Ratio of α-synuclein
α-synuclein (α-syn) is a major culprit of Parkinson’s disease (PD), although lipoprotein metabolism is very important in the pathogenesis of PD. α-syn was expressed and purified using the pET30a expression vector from an E. coli expression system to elucidate the physiological effects of α-syn on li...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2021
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8707077/ https://www.ncbi.nlm.nih.gov/pubmed/34946565 http://dx.doi.org/10.3390/molecules26247485 |
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author | Cho, Kyung-Hyun |
author_facet | Cho, Kyung-Hyun |
author_sort | Cho, Kyung-Hyun |
collection | PubMed |
description | α-synuclein (α-syn) is a major culprit of Parkinson’s disease (PD), although lipoprotein metabolism is very important in the pathogenesis of PD. α-syn was expressed and purified using the pET30a expression vector from an E. coli expression system to elucidate the physiological effects of α-syn on lipoprotein metabolism. The human α-syn protein (140 amino acids) with His-tag (8 amino acids) was expressed and purified to at least 95% purity. Isoelectric focusing gel electrophoresis showed that the isoelectric point (pI) of α-syn and apoA-I were pI = 4.5 and pI = 6.4, respectively. The lipid-free α-syn showed almost no phospholipid-binding ability, while apoA-I showed rapid binding ability with a half-time (T(1/2)) = 8 ± 0.7 min. The α-syn and apoA-I could be incorporated into the reconstituted HDL (rHDL, molar ratio 95:5:1:1, palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):cholesterol:apoA-I:α-syn with the production of larger particles (92 Å) than apoA-I-rHDL (86 and 78 Å) and α-syn-rHDL (65 Å). An rHDL containing both apoA-I and α-syn showed lower α-helicity around 45% with a red shift of the Trp wavelength maximum fluorescence (WMF) from 339 nm, while apoA-I-HDL showed 76% α-helicity and 337 nm of WMF. The denaturation by urea addition showed that the incorporation of α-syn in rHDL caused a larger increase in the WMF than apoA-I-rHDL, suggesting that the destabilization of the secondary structure of apoA-I by the addition of α-syn. On the other hand, the addition of α-syn induced two-times higher resistance to rHDL glycation at apoA-I:α-syn molar ratios of 1:1 and 1:2. Interestingly, low α-syn in rHDL concentrations, molar ratio of 1:0.5 (apoA-I:α-syn), did not prevent glycation with more multimerization of apoA-I. In the lipid-free and lipid-bound state, α-syn showed more potent antioxidant activity than apoA-I against cupric ion-mediated LDL oxidation. On the other hand, microinjection of α-syn (final 2 μM) resulted in 10% less survival of zebrafish embryos than apoA-I. A subcutaneous injection of α-syn (final 34 μM) resulted in less tail fin regeneration than apoA-I. Interestingly, incorporation of α-syn at a low molar ratio (apoA-I:α-syn, 1:0.5) in rHDL resulted destabilization of the secondary structure and impairment of apoA-I functionality via more oxidation and glycation. However, at a higher molar ratio of α-syn in rHDL (apoA-I:α-syn = 1:1 or 1:2) exhibited potent antioxidant and anti-glycation activity without aggregation. In conclusion, there might be a critical concentration of α-syn and apoA-I in HDL-like complex to prevent the aggregation of apoA-I via structural and functional enhancement. |
format | Online Article Text |
id | pubmed-8707077 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-87070772021-12-25 Structural and Functional Changes of Reconstituted High-Density Lipoprotein (HDL) by Incorporation of α-synuclein: A Potent Antioxidant and Anti-Glycation Activity of α-synuclein and apoA-I in HDL at High Molar Ratio of α-synuclein Cho, Kyung-Hyun Molecules Article α-synuclein (α-syn) is a major culprit of Parkinson’s disease (PD), although lipoprotein metabolism is very important in the pathogenesis of PD. α-syn was expressed and purified using the pET30a expression vector from an E. coli expression system to elucidate the physiological effects of α-syn on lipoprotein metabolism. The human α-syn protein (140 amino acids) with His-tag (8 amino acids) was expressed and purified to at least 95% purity. Isoelectric focusing gel electrophoresis showed that the isoelectric point (pI) of α-syn and apoA-I were pI = 4.5 and pI = 6.4, respectively. The lipid-free α-syn showed almost no phospholipid-binding ability, while apoA-I showed rapid binding ability with a half-time (T(1/2)) = 8 ± 0.7 min. The α-syn and apoA-I could be incorporated into the reconstituted HDL (rHDL, molar ratio 95:5:1:1, palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):cholesterol:apoA-I:α-syn with the production of larger particles (92 Å) than apoA-I-rHDL (86 and 78 Å) and α-syn-rHDL (65 Å). An rHDL containing both apoA-I and α-syn showed lower α-helicity around 45% with a red shift of the Trp wavelength maximum fluorescence (WMF) from 339 nm, while apoA-I-HDL showed 76% α-helicity and 337 nm of WMF. The denaturation by urea addition showed that the incorporation of α-syn in rHDL caused a larger increase in the WMF than apoA-I-rHDL, suggesting that the destabilization of the secondary structure of apoA-I by the addition of α-syn. On the other hand, the addition of α-syn induced two-times higher resistance to rHDL glycation at apoA-I:α-syn molar ratios of 1:1 and 1:2. Interestingly, low α-syn in rHDL concentrations, molar ratio of 1:0.5 (apoA-I:α-syn), did not prevent glycation with more multimerization of apoA-I. In the lipid-free and lipid-bound state, α-syn showed more potent antioxidant activity than apoA-I against cupric ion-mediated LDL oxidation. On the other hand, microinjection of α-syn (final 2 μM) resulted in 10% less survival of zebrafish embryos than apoA-I. A subcutaneous injection of α-syn (final 34 μM) resulted in less tail fin regeneration than apoA-I. Interestingly, incorporation of α-syn at a low molar ratio (apoA-I:α-syn, 1:0.5) in rHDL resulted destabilization of the secondary structure and impairment of apoA-I functionality via more oxidation and glycation. However, at a higher molar ratio of α-syn in rHDL (apoA-I:α-syn = 1:1 or 1:2) exhibited potent antioxidant and anti-glycation activity without aggregation. In conclusion, there might be a critical concentration of α-syn and apoA-I in HDL-like complex to prevent the aggregation of apoA-I via structural and functional enhancement. MDPI 2021-12-10 /pmc/articles/PMC8707077/ /pubmed/34946565 http://dx.doi.org/10.3390/molecules26247485 Text en © 2021 by the author. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Cho, Kyung-Hyun Structural and Functional Changes of Reconstituted High-Density Lipoprotein (HDL) by Incorporation of α-synuclein: A Potent Antioxidant and Anti-Glycation Activity of α-synuclein and apoA-I in HDL at High Molar Ratio of α-synuclein |
title | Structural and Functional Changes of Reconstituted High-Density Lipoprotein (HDL) by Incorporation of α-synuclein: A Potent Antioxidant and Anti-Glycation Activity of α-synuclein and apoA-I in HDL at High Molar Ratio of α-synuclein |
title_full | Structural and Functional Changes of Reconstituted High-Density Lipoprotein (HDL) by Incorporation of α-synuclein: A Potent Antioxidant and Anti-Glycation Activity of α-synuclein and apoA-I in HDL at High Molar Ratio of α-synuclein |
title_fullStr | Structural and Functional Changes of Reconstituted High-Density Lipoprotein (HDL) by Incorporation of α-synuclein: A Potent Antioxidant and Anti-Glycation Activity of α-synuclein and apoA-I in HDL at High Molar Ratio of α-synuclein |
title_full_unstemmed | Structural and Functional Changes of Reconstituted High-Density Lipoprotein (HDL) by Incorporation of α-synuclein: A Potent Antioxidant and Anti-Glycation Activity of α-synuclein and apoA-I in HDL at High Molar Ratio of α-synuclein |
title_short | Structural and Functional Changes of Reconstituted High-Density Lipoprotein (HDL) by Incorporation of α-synuclein: A Potent Antioxidant and Anti-Glycation Activity of α-synuclein and apoA-I in HDL at High Molar Ratio of α-synuclein |
title_sort | structural and functional changes of reconstituted high-density lipoprotein (hdl) by incorporation of α-synuclein: a potent antioxidant and anti-glycation activity of α-synuclein and apoa-i in hdl at high molar ratio of α-synuclein |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8707077/ https://www.ncbi.nlm.nih.gov/pubmed/34946565 http://dx.doi.org/10.3390/molecules26247485 |
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