Cloning and Recombinant Expression of a Novel Mouse-secreted Phospholipase A2

Abstract

Secreted phospholipases A2 (sPLA2s) form a class of structurally related enzymes that are involved in a variety of physiological and pathological effects including inflammation and associated diseases, cell proliferation, cell adhesion, and cancer, and are now known to bind to specific membrane receptors. Here, we report the cloning and expression of a novel sPLA2 isolated from mouse thymus. Based on its structural features, this sPLA2 is most similar to the previously cloned mouse group IIA sPLA2 (mGIIA sPLA2). As for mGIIA sPLA2, the novel sPLA2 is made up of 125 amino acids with 14 cysteines, is basic (pI = 8.71) and its gene has been mapped to mouse chromosome 4. However, the novel sPLA2 has only 48% identity with mGIIA and displays similar levels of identity with the other mouse group IIC and V sPLA2s, indicating that the novel sPLA2 is not an isoform of mGIIA sPLA2. This novel sPLA2 has thus been called mouse group IID (mGIID) sPLA2. In further contrast with mGIIA, which is found mainly in intestine, transcripts coding for mGIID sPLA2 are found in several tissues including pancreas, spleen, thymus, skin, lung, and ovary, suggesting distinct functions for the two enzymes. Recombinant expression of mGIID sPLA2 in Escherichia coli indicates that the cloned sPLA2 is an active enzyme that has much lower specific activity than mGIIA and displays a distinct specificity for binding to various phospholipid vesicles. Finally, recombinant mGIID sPLA2 did not bind to the mouse M-type sPLA2 receptor, while mGIIA was previously found to bind to this receptor with high affinity.

Inhibitory Effects of Surfactant Protein A on Surfactant Phospholipid Hydrolysis by Secreted Phospholipases A2

“…This finding is consistent with findings that sPLA 2 -IIA is Ͼ100-fold more active on anionic, rather than zwitterionic, phospholipids (9,30). Remarkably, hydrolysis of PG occurs as early as 1 h after sPLA 2 -IIA administration, indicating that this enzyme could account for surfactant abnormalities observed at early phases in ARDS (17).…”

“…The sPLA 2 s form a growing family of enzymes that catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids, leading to the production of free fatty acids and lysophospholipids that serve as precursors for a variety of lipid-derived mediators involved in numerous biological activities (5,6). To date, 10 different sPLA 2 s, referred to as groups IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII, have been characterized in mammals (7)(8)(9)(10)(11)(12). Of particular interest, levels of sPLA 2 activity in bronchoalveolar lavage fluids (BALF) of patients with ARDS often correlate positively with the severity of the disease (13).…”

“…Recombinant guinea pig sPLA 2 -IIA (r-GP sPLA 2 -IIA) was produced in our laboratory (22). The recombinant mouse sPLA 2 s were expressed and purified as described previously (9,10,23). The rabbit polyclonal anti-rat SP-A Ab was a gift from F. X. McCormack (University of Cincinnati, Cincinnati, OH).…”

Cloning and Recombinant Expression of a Structurally Novel Human Secreted Phospholipase A2

“…1) and the reverse primer given above. The purified PCR product was digested with BamHI and HindIII and was subcloned in frame with the truncated glutathione S-transferase (ϳ10 kDa) encoded by the modified pGEX-2T vector (pAB 3 ), which had been previously used to express several sPLA 2 s in Escherichia coli (17). Protein production in E. coli BL21, purification of inclusion bodies, and refolding and cleavage of the fusion protein with factor Xa were carried out as described previously (17).…”

“…In the last few years, six mouse and five human sPLA 2 s structurally related to GIB and GIIA sPLA 2 s (mGIIC, hGIID, mGIID, hGIIE, mGIIE, mGIIF, hGIIF, hGV, mGV, hGX, and mGX) 2 have been identified (15)(16)(17)(18)(19)(20). 3 All of these group I/II/V/X sPLA 2 s have similar primary structures, including identical catalytic site residues and partially overlapping sets of disulfides (21).…”