Lipid clearance assay and native gel analyses of rHDL formation.

<p>(A) ApoA-I WT and A164S were combined with DMPC at a 1:100 molar ratio and lipid binding measured by absorbance at 325nm at indicate times. Readings were fitted to one-way decay of non-linear regression. (B) Bar graph shows the lipid clearance rate described as t1/2 values calculated from panel A. Data are mean ±SEM (* = p<0.05, n = 3). (C) ApoAI-A164S and apoA-I WT were incubated with DMPC lipids at 37°C for indicated times and analyzed by native gel. <i>Arrows</i> indicate apoA-I WT and apoA-I A164S rHDL particles of diameters of approximately 10 nm. Lipid-free (LF) proteins at time 0 h prior to mixing with DMPC lipids are included as controls. (D) Formation of HDL upon incubation of apoA-I WT and A164S with rat serum. 0.75mg/ml of WT or A164S apoA-I was incubated with 200 μl rat serum. Samples were collected at indicated times and equal amounts of protein (0.56μg) separated by blue native PAGE and western blotting for human apoA-I was performed. Lipid-free (LF) proteins (not incubated with serum) and serum without protein (ctrl) are included as controls.</p

Cholesterol efflux from macrophages.

<p>J774 macrophages enriched with ³H-cholesterol were incubated with apoA-I WT or A164S and cholesterol efflux quantified as a function of protein concentration (A) or time (B) by scintillation counting of the resulting treatment media. An ACAT inhibitor and CPT-cAMP were used to prevent formation of cholesteryl esters of the ³H-cholesterol and to induce expression of ABCA1, respectively. Data from concentration gradient experiments were fitted using the Michaelis-Menten equation. Time dependent efflux treatments were performed with 50 μg/ml apoA-I WT or A164S. Each figure represents 3 independent experiments and displays mean±SD. (C) ApoA-I WT or A164S were incubated with rat serum for 2 h followed by incubation with J774 macrophages enriched with ³H-cholesterol for 2 h (10 μg/ml apoA-I). Rat serum was used as control for background efflux. Data is mean ±SEM (**** = p<0.0001, n = 3).</p

Limited proteolysis analysis.

<p>ApoA-I WT, A164S and L178H (5μg) were incubated for indicated times at 37°C in the presence of chymotrypsin. The cleaved products from limited proteolysis from different time points were separated by SDS-PAGE and visualized by coomassie staining. <i>Arrows</i> indicate migration distances of full-length proteins.</p

Analysis of alpha-helical content and thermal stability of apoA-I A164S.

<p>(A) CD analysis of apoA-I WT and A164S. Alpha-helical content of 0.2 mg/ml of apoA-I was calculated from molar ellipticity at 222 nm (n = 3). (B) Thermal stability of 0.2 mg/ml of apoA-I was assessed from normalized sigmoidal decrease of molar ellipticity at 222 nm. The results are mean ± SEM (n = 3). (C) Stopped-flow coupled to CD: apoA-I WT and A164S (0.5 mg/ml final concentration) were mixed with 100 mM SDS at the volume ratio 1:5 and molar ellipticity at 222 nm was measured. (D). Bar graph shows the t1/2 of apoA-I WT and A164S. Data is mean ±SEM (* = p<0.05, n = 6). (E) Near CD analysis of apoA-I WT and A164S (1.25 mg/ml).</p

Limited proteolysis of apoA-I proteins to assay for structure accessibility.

<p>ApoA-I proteins (0.3 mg/ml) were incubated with chymotrypsin at 37°C for indicated times followed by SDS-PAGE separation and coomassie staining of the gel. <i>Arrows</i> indicate migration distance of full-length proteins.</p

Secondary Structure Changes in ApoA-I Milano (R173C) Are Not Accompanied by a Decrease in Protein Stability or Solubility

<div><p>Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein (HDL) and a principal mediator of the reverse cholesterol transfer pathway. Variants of apoA-I have been shown to be associated with hereditary amyloidosis. We previously characterized the G26R and L178H variants that both possess decreased stability and increased fibril formation propensity. Here we investigate the Milano variant of apoAI (R173C; apoAI-M), which despite association with low plasma levels of HDL leads to low prevalence of cardiovascular disease in carriers of this mutation. The R173C substitution is located to a region (residues 170 to 178) that contains several fibrillogenic apoA-I variants, including the L178H variant, and therefore we investigated a potential fibrillogenic property of the apoAI-M protein. Despite the fact that apoAI-M shared several features with the L178H variant regarding increased helical content and low degree of ThT binding during prolonged incubation in physiological buffer, our electron microscopy analysis revealed no formation of fibrils. These results suggest that mutations inducing secondary structural changes may be beneficial in cases where fibril formation does not occur.</p></div

Electron microscopy (negative stain) analysis shows no fibril formation of apoA-I-M after four weeks of incubation at 37°C.

<p>ApoAI-Milano (R173C), apoAI-WT and apoAI-Iowa (G26R) proteins were incubated at 37°C for four weeks followed by negative stain EM analysis. While the positive control apoAI-Iowa formed elongated fibrils (right panel), neither apoAI-WT (middle panel) nor apoAI-M (left panel) displayed any fibril formation. Size bars are 100 nm.</p

The amyloidophilic dye ThT does not bind to the apoAI-Milano protein.

<p>The binding of thioflavin T (ThT) to apoAI-M was assessed and compared to apoAI-WT. No significant binding of ThT to apoAI-WT and apoAI-M was observed over a four-week period of incubation at 37°C. This result is in contrast to apoAI-Iowa protein, known for its propensity to form cross-beta amyloids, which had higher ThT binding than both apoAI-WT and apoAI-M, and thus higher beta sheet content. *p<0.05, n = 3; **p<0.01, n = 3.</p

Covalent Cys-Cys binding and dimer formation of ApoAI-M.

<p><i>A</i>, Purified apoAI-M (M) and apoAI-WT (WT) proteins (2 µg) were analyzed by SDS-PAGE (4–15% Tris-glycine) in the presence or absence of the reducing agent DTT. Formed apoAI-M dimers are indicated (<i>arrow</i>). <i>B</i>, Western blot analysis of apoAI-M (M) and apoAI-WT (WT) proteins in plasma samples from mice treated with the respective apoA-I protein. The SDS-PAGE separation was performed in the presence or absence of the reducing agent DTT to distinguish protein in covalently attached Cys-Cys dimers (<i>arrow</i>). Analysis of mouse plasma from control animals treated with saline (NaCl) was included to show specificity of the antibodies for human apoA-I protein. Data shown is representative of three experiments/animals.</p

Structural transitions of apoA-I proteins assayed by CD spectroscopy.

<p><i>A</i>, The alpha helical content was calculated from the value of molar ellipticity at the wavelength 222 nm at the time point 0 days for apoAI-WT and apoAI-M (25°C). <i>B</i>, Circular dichroism spectroscopy was used to analyze secondary structure changes over time. Scans ranging from 200 nm to 260 nm of apoAI-Iowa (G26R), apoAI-Milano (R173C), apoAI-L178H and apoAI-WT proteins (at concentrations of 0.2 mg/ml) incubated at 37°C for up to 21 days (0, 7, 14 and 21 days of incubation) are shown. While the secondary structure of apoAI-WT protein is unchanged during the time course, the spectral changes of the apoAI-Milano and the apoAI-L178H proteins indicate increased alpha-helical content (as indicated by an increase in molar ellipticity at 222 nm), whereas the amyloidogenic apoAI-Iowa displays a reduction in alpha helical secondary structure with time. <i>C,</i> The percentage of alpha helix was measured during 16 days of incubation at 37°C at different time points (0, 4, 8, 12 and 16 days). Boltzmann function was used to determinate the transition time of the apoAI-Milano variant. ApoAI-WT did not exhibit any significant changes in the alpha helical content when incubated at identical conditions during same time period. **p<0.01, n = 3.</p