Fig 2: 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 and 1:100, as indicated. Protein-free POPC MLV were used as a control. The data for POPC MLV alone (light gray) closely superimposed similar data using POPC MLV with either apoA-I or apoA-II at a 1:100 protein:lipid weight ratio (not shown to avoid overlap). (B) Non-denaturing PAGE of the POPC mixtures with SAA, performed after 6 hr of incubation, shows the formation of SAA-POPC complexes. SAA:POPC molar ratios are indicated; lipid-free SAA is shown for comparison. Gels in this and other figures were stained with Denville Blue protein stain. (C) SAA-POPC particles shown in panel (B) were isolated by size-exclusion chromatography (SEC) and used as substrates for sPLA2-III (left) or sPLA2-IIa (right). Free fatty acids produced per nm of lipid are shown as average values of three independent measurements with standard errors of mean. The protein:lipid molar ratios in the initial incubation mixtures are indicated; sPLA2-free particles were used as controls. FFA produced in the presence and in the absence of sPLA2 were compared using the t-test; ***p<0.001. In SAA-POPC particles, approximately 70% of the total lipid was hydrolyzed by sPLA2-III and 60% by sPLA2-IIa. (D) Thin-layer chromatography analysis of POPC MLV before (intact) or after their incubation with sPLA2-III (+ sPLA2-III). The PC band is indicated; the absence of the lysoPC band underneath the PC band indicates the absence of significant hydrolysis.10.7554/eLife.46630.004Figure 1—source data 1.Free fatty acid analysis of SAA:POPC complexes hydrolysed by sPLA2.

Fig 3: 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). These isolated complexes, marked SAA + hydHDL (A), SAA + hydLDL (B) or SAA + sPLA2-hydrolyzed HDL, LDL or POPC (E), were analyzed for protein (A–D) and lipid composition (E). SDS PAGE (A) and matrix-assisted laser desorption ionization – time of flight mass spectrometry (B) of SAA + hydHDL revealed SAA and apoA-I; the protein mass detected by mass spectrometry was 11,606 Da for SAA and 28,086 for apoA-I (B). Similar analyses of SAA + hydLDL complexes showed only SAA (C, D). (E) Thin-layer chromatography showed the presence of PC and lysoPC in the SAA-containing ~7 nm complexes that were obtained from all hydrolyzed lipoproteins (HDL, LDL) or model lipids (POPC). Lipid-free SAA and the 1.17–1.20 g/mL density fraction isolated from SAA mixtures with non-hydrolyzed HDL or LDL (SAA + HDL in panel (A) and SAA + LDL in (C)) are shown for comparison.

Fig 4: 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 ratios were 1:1, 1:10 or 1:100 as indicated; sPLA2-III or sPLA2-IIa was used as indicated. (B, C) Human plasma lipoproteins including HDL (B) and LDL (C) were hydrolyzed with sPLA2 group-III or -IIa to form hydHDL or hydLDL, respectively. In samples marked SAA + hydLDL (B) or hydHDL (C), the hydrolyzed lipoproteins were incubated with SAA using protein weight ratios of 1:1 SAA:apoA-I (for HDL) or 1:1 SAA:apoB (for LDL) as described in 'Materials and methods'. Similar incubation mixtures of SAA with non-hydrolyzed HDL (SAA+HDL) or LDL (SAA+LDL) are shown for comparison. Figure 3—figure supplement 1 shows non-denaturing PAGE that monitors the remodeling of SAA-containing model and plasma lipoproteins upon their hydrolysis by sPLA2.