Abstract
Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.
Highlights
Among the many mammalian secreted phospholipase A2 enzymes, PLA2G3 is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s
Tail biopsies were taken on day 28, and the genomic DNAs isolated were subjected to PCR genotyping with a CAG forward primer and a reverse primer specific for PLA2G3
PLA2G3 Tg mice did not display noticeable phenotypic changes in the skin, which is profoundly affected in PLA2G2A mice [24], or lung, whose architecture is disrupted in PLA2G5 Tg mice [22] and macrophage-specific PLA2G10 Tg mice [23], suggesting that phospholipids in mouse skin and lung surfactant may not be main targets for PLA2G3 in vivo
Summary
Animals—All mice were housed in climate-controlled (21 °C) specific pathogen-free facilities with a 12-h light-dark cycle, with free access to standard laboratory food (Picolab mouse diet 20; Laboratory Diet, Brentwood) and water. The specificity of the antibodies obtained was evaluated by immunoblotting (Fig. S1A) using Sf9 cells (cell homogenates and culture supernatants) infected with baculovirus expressing III-S of human PLA2G3. Mouse monoclonal antibodies for human PLA2G3 were prepared by a standard protocol using recombinant human III-S protein as an antigen (see above). After washing the column with 10 times the column volume of 20 mM phosphate buffer, the anti-PLA2G3 antibody was eluted with 100 mM citric acid buffer (pH 2.7) and dialyzed against 10 mM Tris-HCl (pH 7.4) containing 150 mM NaCl (TBS). Histochemistry—Immunohistochemistry of mouse and human tissue sections was performed as described previously [19]. University of Washington, Seattle), or PLA2G3 (see above) (0.2 and 1 g/ml) was incubated with 1 mg/ml human LDL or HDL (both from Sigma) for 4 h in 100 mM Tris-HCl, pH 7.4, containing 10 mM CaCl2. The particle size was estimated using ferritin (12.2 nm), thyroglobulin (17 nm), and latex beads (30 nm) as standards [47]
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