Synergy between serum amyloid A and secretory phospholipase A2
- Boston University School of Medicine, United States
- Ulm University, Germany
Figures
Figure 1
SAA remodels phospholipid bilayers into small particles that form substrates for sPLA2.
(A) POPC MLV (~200 nm) were incubated with SAA at 25°C, and the time course of MLV remodeling into smaller particles was monitored by turbidty at 350 nm. Protein:lipid molar ratios were 1:10, 1:50 …
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Figure 1—source data 1
Free fatty acid analysis of SAA:POPC complexes hydrolysed by sPLA2.
- https://doi.org/10.7554/eLife.46630.004
Figure 2 with 1 supplement
Effect of SAA on the lipolysis of HDL and LDL by sPLA2-III or sPLA2-IIa.
(A, B) FFA were generated upon lipolysis of either native HDL or SAA-HDL. For SAA-HDL, the SEC fraction containing HDL-bound proteins (Sec Fr) was isolated from the total incubation mixtures, which …
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Figure 2—source data 1
Free fatty analysis of HDL and LDL hydrolysed by sPLA2.
- https://doi.org/10.7554/eLife.46630.007
Figure 2—figure supplement 1
Characterization of protein-containing complexes formed upon incubation of SAA with human plasma lipoproteins.
(A) Non-denaturing PAGE of incubation mixtures of SAA and single-donor human HDL (SAA-HDL). Molar ratios of SAA to apoA-I, the main HDL protein, are 1:1 or 4:1 (as indicated on the lanes). Fractions …
Figure 3 with 1 supplement
SAA forms complexes with hydrolyzed model or plasma lipids.
(A) SAA was incubated with either unmodified POPC to form SAA-POPC complexes or with hydrolyzed POPC to form SAA + hydPOPC complexes (see 'Materials and methods' for details). Protein:POPC molar …
Figure 3—figure supplement 1
Non-denaturing PAGE monitors remodeling of SAA-containing model and plasma lipoproteins upon their hydrolysis by sPLA2.
(A) SAA-POPC complexes were prepared by incubating SAA with POPC SUV using SAA:POPC molar ratios of 1:1, 1:10 or 1:100 (indicated in the lanes). Lanes for intact SAA-POPC particles are shown, as …
Figure 4 with 1 supplement
Biochemical analysis of the ~7 nm complexes formed by SAA and lipolytic products.
SAA-containing complexes, which were obtained upon lipolysis of model (SAA-POPC) or plasma lipoproteins (HDL and LDL) by sPLA2, were isolated at 1.17–1.20 g/mL density (Figure 4—figure supplement 1).…
Figure 4—figure supplement 1
Gel electrophoresis of isolated SAA complexes with the products of phospholipid hydrolysis.
All gels were stained with Denville Blue protein stain. (A) Non-denaturing PAGE of model SAA-POPC complexes, which were prepared as described in 'Materials and methods' using 1:10 SAA:POPC mol:mol …
Figure 5 with 2 supplements
Structure and stability of SAA complexes with the products of POPC hydrolysis.
Intact SAA-POPC complexes (prepared using 1:10 protein:lipid molar ratio) were hydrolyzed with sPLA2-III, and the 1.17–1.20 g/ml density fraction containing ~7 nm particles was isolated (for …
Figure 5—figure supplement 1
Structure and stability of the 7–7.5 nm complexes formed by SAA and the products of LDL hydrolysis.
SAA was incubated with human plasma LDL (SAA-hydLDL) that had been hydrolyzed either with sPLA2-III or with sPLA2-IIa. The ~7–7.5 nm complexes, which were isolated from the incubation mixtures in …
Figure 5—figure supplement 2
Structure and stability of SAA complexes with lysoPC and POPC.
Complexes of SAA with either lysoPC (SAA-lysoPC) or POPC (SAA-POPC) were prepared using 1:10 SAA:PC molar ratio and analyzed as follows. (A) Non-denaturing PAGE of these SAA-POPC and SAA-lysoPC …
Figure 6 with 2 supplements
FFA removal by SAA and by albumin as a function of pH.
Single-donor LDL were hydrolyzed with sPLA2-III and incubated at pH 4.5–7.5 either alone (control), with albumin (LDL + albumin) or with SAA (LDL + SAA). Next, LDL were re-isolated by density …
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Figure 6—source data 1
Free fatty acid analysis of LDL at different pH.
- https://doi.org/10.7554/eLife.46630.020
Figure 6—figure supplement 1
SAA and albumin sequester naturally occurring FFA from human plasma LDL at pH 7.5.
LDL were obtained from pooled plasma of diabetic and from healthy normolipidemic patients as previously described (Jayaraman et al., 2017b). Diabetic LDL had 20% higher content of endogenous FFA …
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Figure 6—figure supplement 1—source data 1
Free fatty analysis of normal and diabetic LDL.
- https://doi.org/10.7554/eLife.46630.017
Figure 6—figure supplement 2
Non-denaturing PAGE shows that human SAA 1.1 (hSAA1.1) sequesters the products of phospholipid hydrolysis from model and plasma lipoproteins.
(A) Complexes of hSAA1.1 with POPC, which were prepared using 1:10 protein-lipid molar ratio, were hydrolyzed with sPLA2-IIa or sPLA2-III (as indicated on the lanes). Intact non-hydrolyzed complexes …
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Figure 6—figure supplement 2—source data 1
Free fatty acid analysis of LDL incubated with human and mouse SAA.
- https://doi.org/10.7554/eLife.46630.019
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.46630.021
