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

alpha-synuclein (alpha-syn) is a major culprit of Parkinson's disease (PD), although lipoprotein metabolism is very important in the pathogenesis of PD. alpha-syn was expressed and purified using the pET30a expression vector from an E. coli expression system to elucidate the physiological effects of alpha-syn on lipoprotein metabolism. The human alpha-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 alpha-syn and apoA-I were pI = 4.5 and pI = 6.4, respectively. The lipid-free alpha-syn showed almost no phospholipid-binding ability, while apoA-I showed rapid binding ability with a half-time (T-1/2) = 8 & PLUSMN; 0.7 min. The alpha-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:alpha-syn with the production of larger particles (92 & ANGS;) than apoA-I-rHDL (86 and 78 & ANGS;) and alpha-syn-rHDL (65 & ANGS;). An rHDL containing both apoA-I and alpha-syn showed lower alpha-helicity around 45% with a red shift of the Trp wavelength maximum fluorescence (WMF) from 339 nm, while apoA-I-HDL showed 76% alpha-helicity and 337 nm of WMF. The denaturation by urea addition showed that the incorporation of alpha-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 alpha-syn. On the other hand, the addition of alpha-syn induced two-times higher resistance to rHDL glycation at apoA-I:alpha-syn molar ratios of 1:1 and 1:2. Interestingly, low alpha-syn in rHDL concentrations, molar ratio of 1:0.5 (apoA-I:alpha-syn), did not prevent glycation with more multimerization of apoA-I. In the lipid-free and lipid-bound state, alpha-syn showed more potent antioxidant activity than apoA-I against cupric ion-mediated LDL oxidation. On the other hand, microinjection of alpha-syn (final 2 mu M) resulted in 10% less survival of zebrafish embryos than apoA-I. A subcutaneous injection of alpha-syn (final 34 mu M) resulted in less tail fin regeneration than apoA-I. Interestingly, incorporation of alpha-syn at a low molar ratio (apoA-I:alpha-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 alpha-syn in rHDL (apoA-I:alpha-syn = 1:1 or 1:2) exhibited potent antioxidant and anti-glycation activity without aggregation. In conclusion, there might be a critical concentration of alpha-syn and apoA-I in HDL-like complex to prevent the aggregation of apoA-I via structural and functional enhancement.

Automated summary

The study demonstrates the physiological effects of alpha-synuclein on lipoprotein metabolism, showing its weaker phospholipid-binding ability compared to apoA-I. Incorporation of alpha-syn in reconstituted HDL affects the alpha-helicity and functionality of apoA-I, with potential destabilization of the secondary structure.