Abstract
It is well accepted that high levels of high density lipoproteins (HDL) reduce the risk of atherosclerosis in humans. Apolipoprotein A-I (apoA-I) and apoA-II are the first and second most common protein constituents of HDL. Unlike apoA-I, detailed structural models for apoA-II in HDL are not available. Here, we present a structural model of apoA-II in reconstituted HDL (rHDL) based on two well established experimental approaches: chemical cross-linking/mass spectrometry (MS) and internal reflection infrared spectroscopy. Homogeneous apoA-II rHDL were reacted with a cross-linking agent to link proximal lysine residues. Upon tryptic digestion, cross-linked peptides were identified by electrospray mass spectrometry. 14 cross-links were identified and confirmed by tandem mass spectrometry (MS/MS). Infrared spectroscopy indicated a beltlike molecular arrangement for apoA-II in which the protein helices wrap around the lipid bilayer rHDL disc. The cross-links were then evaluated on three potential belt arrangements. The data clearly refute a parallel model but support two antiparallel models, especially a "double hairpin" form. These models form the basis for understanding apoA-II structure in more complex HDL particles.
Highlights
High density lipoproteins (HDL)3 have received a great deal of attention in recent years due to postulated roles in atheroprotective processes, such as reverse cholesterol transport, anti-inflammation, and anti-oxidation
Generation and Characterization of A-II-POPC-reconstituted high density lipoproteins (HDL) (rHDL) Particles—We hypothesized that apoA-II, like Apolipoprotein A-I (apoA-I), adopts a specific conformation in discoidal rHDL and exhibits sequence-specific interactions with itself and with other apoA-II molecules on the particle [19, 20, 25]
We elected to generate apoA-II-containing rHDL particles that contain POPC, a synthetic lipid with an acyl chain composition commonly found in cellular membranes and lipoproteins
Summary
ApoA-II Purification and Preparation of rHDL Particles— Human apoA-II isolation and purification from normolipidemic subjects was carried out as reported previously for apoA-I [9, 18]. The rHDL particles that were subjected to infrared spectroscopic analysis were prepared, incorporating the anionic analog of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3(phospho-L-serine), in a 1:9 molar ratio with POPC as required by the technique (Table 1). Cross-linking and Tryptic Digestion of ApoA-II—Freshly prepared bis(sulfosuccinimidyl) suberate (BS3) cross-linker (6.5 mg/ml in phosphate-buffered saline, pH 7.8) (Pierce) was added to homogeneous A-II-POPC-rHDL with 1 mg/ml protein concentration, at a protein/BS3 molar ratio of 1:10. The protein/total lipid ratio for these particles (A-II-DMPC/PS-rHDL) was 1:78 with 10% 1,2-dimyristoyl-sn-glycero-3-(phosphor-L-serine) in the lipid mixture as for other particles that were subjected to PATIR-FTIR analysis. These dichroic ratios were converted to order parameters S(Rz) as described
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