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.

Kyung-Hyun Cho

doi:10.3390/molecules26247485

Abstract

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 (T1/2) = 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.

Highlights

Parkinson’s disease (PD), which is the second most common neurodegenerative disease in the elderly population, is frequently associated with mutations of α-synuclein (α-syn) [1,2]
While LDL-cholesterol is a major carrier of blood cholesterol, brain cholesterol handling is strongly dependent on the high-density lipoprotein (HDL) metabolism, and HDLlike particles can be synthesized from the brain and glial cells [11]
Because α-syn shows binding ability with lipid using its conserved domain, it can be considered as an apolipoprotein [33]

Summary

Introduction

Parkinson’s disease (PD), which is the second most common neurodegenerative disease in the elderly population, is frequently associated with mutations of α-synuclein (α-syn) [1,2]. Brain cholesterol and lipoprotein are believed to play an important role in the action of α-syn, a major component of Lewy bodies, in the pathogenesis of PD [4]. Low levels of serum apolipoprotein A-I (apoA-I) and HDL-C are correlated with the earlier onset of PD [5,6]. Lower plasma HDL and apoA-I levels have been associated with earlier PD onset [7] and higher PD risk [8,9,10]. There are no LDL-like particles and apo-B containing lipoproteins as cholesterol carriers [12]. Serum HDL can interact with α-syn in the brain [15]. A putative interaction between α-syn and apoA-I might frequently occur to regulate cholesterol homeostasis. There is limited information on α-syn and HDL in the protein level on the neuron system

Methods

Results

Conclusion