Abstract 426: Selective Proteolysis of Apolipoprotein A-I by Secretory Phospholipase A2: New Insights Into sPLA2-dependent Catabolism of HDL
Introduction Secretory phospholipase A2 (sPLA2) is a pro-inflammatory enzyme that promotes atherogenesis via several mechanisms. One of these mechanisms probably involves high-density lipoproteins (HDL). Previous in vitro studies of HDL modified by sPLA2 showed increased exposure of certain domains of the major HDL protein, apoA-I, without protein fragmentation. Over-expression of sPLA2 in transgenic mice reduced plasma levels of HDL cholesterol due to rapid HDL catabolism. The origin of this enhanced HDL catabolism is unknown.
Hypothesis In addition to hydrolysis of HDL phospholipids, sPLA2 can also hydrolyze apoA-I, leading to enhanced catabolism of sPLA2 modified HDL.
Methods Human plasma HDL and lipid-free apoA-I were incubated with sPLA2 at 37 oC. The HDL lipids were removed, and the protein integrity was analyzed by gel electrophoresis and mass spectrometry.
Results Consistent with previous studies, HDL hydrolysis by sPLA2 slightly reduced the particle size without release of protein. SDS PAGE of hydrolyzed HDL showed no significant changes in the protein composition. Interestingly, delipidation of sPLA2-hydrolyzed HDL showed that nearly 10% of apoA-I formed well-defined fragments of 6-15 kDa. Incubation of sPLA2 with lipid-free apoA-I produces very similar fragments, indicating direct action of sPLA2 on apoA-I. Similar incubation of sPLA2 with other HDL proteins, apoA-II and apoC-I, did not lead to proteolysis, indicating enzymatic specificity of sPLA2 for apoA-I. ApoA-I hydrolysis by sPLA2 was Ca-independent and hence, occurred via a different mechanism than the phospholipid hydrolysis by sPLA2 that is Ca-dependent. Mass spectrometry analysis of isolated proteolytic fragments indicated several N-and C-terminal fragments of apoA-I, suggesting at least two cleavage sites in the central domain of apoA-I.
Conclusion We have identified a previously unknown proteolytic activity of sPLA2 which is specific to apoA-I and is independent of the apoA-I lipidation. This novel enzymatic activity of sPLA2 helps explain enhanced catabolism of HDL in vivo, and is expected to contribute to the pro-atherogenic action of this pro-inflammatory enzyme.
- © 2013 by American Heart Association, Inc.