Significance: Although acidic conditions favor the misfolding of various proteins in amyloid diseases, the molecular underpinnings of this process are unclear. We used an array of spectroscopic, biochemical, and electron microscopic methods to unravel the effects of pH on serum amyloid A, a protein precursor of reactive amyloidosis, the major complication of chronic inflammation and one of the major human systemic amyloidoses worldwide. We found that at lysosomal pH this protein forms unusually stable proteolysis-resistant soluble oligomers that have solvent-accessible apolar surfaces, disrupt lipid vesicles, and undergo an α-helix to β-sheet transition in the presence of lipids. Such oligomers are likely to escape lysosomal degradation, accumulate in the lysosomes, and disrupt cellular membranes, thereby contributing to the development of amyloid A amyloidosis.

Serum amyloid A (SAA) is an acute-phase protein that circulates mainly on plasma high-density lipoprotein (HDL). SAA interactions with its functional ligands and its pathogenic deposition in reactive amyloidosis depend, in part, on the structural disorder of this protein and its propensity to oligomerize. In vivo, SAA can displace substantial fraction of the major HDL protein, apoA-I, and thereby influence the structural remodeling and functions of acute-phase HDL in ways that are incompletely understood. We use murine SAA1.1 to report the first structural stability study of human plasma HDL that has been enriched with SAA. Calorimetric and spectroscopic analyses of these and other SAA-lipid systems reveal two surprising findings. First, progressive displacement of the exchangeable fraction of apoA-I by SAA has little effect on the structural stability of HDL and its fusion and release of core lipids. Consequently, the major determinant for HDL stability is the non-exchangeable apoA-I. A structural model explaining this observation is proposed, which is consistent with functional studies of acute-phase HDL. Second, we report an alpha-helix folding/unfolding transition in SAA in the presence of lipid at near-physiologic temperatures. This new transition may have potentially important implications for normal functions of SAA and its pathogenic misfolding.

In the acute phase of the inflammatory response, secretory phospholipase A₂ (sPLA₂) reaches its maximum levels in plasma, where it is mostly associated with high density lipoproteins (HDL). Overexpression of human sPLA₂ in transgenic mice reduces both HDL cholesterol and apolipoprotein A-I (apoA-I) plasma levels through increased HDL catabolism by an unknown mechanism. To identify unknown PLA₂-mediated activities on the molecular components of HDL, we characterized the protein and lipid products of the PLA₂ reaction with HDL. Consistent with previous studies, hydrolysis of HDL phospholipids by PLA₂ reduced the particle size without changing its protein composition. However, when HDL was destabilized in the presence of PLA₂ by the action of cholesteryl ester transfer protein or by guanidine hydrochloride treatment, a fraction of apoA-I, but no other proteins, dissociated from the particle and was rapidly cleaved. Incubation of PLA₂ with lipid-free apoA-I produced similar protein fragments in the range of 6–15 kDa, suggesting specific and direct reaction of PLA₂ with apoA-I. Mass spectrometry analysis of isolated proteolytic fragments indicated at least two major cleavage sites at the C-terminal and the central domain of apoA-I. ApoA-I proteolysis by PLA₂ was Ca²⁺-independent, implicating a different mechanism from the Ca²⁺-dependent PLA₂-mediated phospholipid hydrolysis. Inhibition of proteolysis by benzamidine suggests that the proteolytic and lipolytic activities of PLA₂ proceed through different mechanisms. Our study identifies a previously unknown proteolytic activity of PLA₂ that is specific to apoA-I and may contribute to the enhanced catabolism of apoA-I in inflammation and atherosclerosis.