Exogenously Added Human Group X Secreted Phospholipase A2 but Not the Group IB, IIA, and V Enzymes Efficiently Release Arachidonic Acid from Adherent Mammalian Cells

Abstract

Mammalian secreted phospholipases A(2) (sPLA2s) comprise a group of at least eight enzymes, including the recently identified group X sPLA2. A bacterial expression system was developed to produce human group X sPLA2 (hGX). Inhibition studies show that the sPLA2 inhibitor LY311727 binds modestly more tightly to human group IIA sPLA2 than to hGX and that a pyrazole-based inhibitor of group IIA sPLA2 is much less active against hGX. The phospholipid head group preference of vesicle-bound hGX was determined. hGX binds tightly to phosphatidylcholine vesicles, which is thought to be required to act efficiently on cells. Tryptophan 67 hGX makes a significant contribution to interfacial binding to zwitterionic vesicles. As little as 10 ng/ml hGX releases arachidonic acid for cyclooxygenase-2- dependent prostaglandin E(2) generation when added exogenously to adherent mammalian cells. In contrast, human group IIA, rat group V, and mouse group IB sPLA2s are virtually inactive at releasing arachidonate when added exogenously to adherent cells. Dislodging cells from the growth surface enhances the ability of all the sPLA2s to release fatty acids. Studies with CHO-K1 cell mutants show that binding of sPLA2s to glycosaminoglycans is not the basis for poor plasma membrane hydrolysis by group IB, IIA, and V sPLA2s.

Transcellular Secretion of Group V Phospholipase A2 from Epithelium Induces β2-Integrin-Mediated Adhesion and Synthesis of Leukotriene C4 in Eosinophils

“…By contrast, gIIaPLA 2 is measurable during cell activation because the preferential substrates for this secretory PLA 2 are phosphatidylserine and phosphatidylethanolamine, which are located at the inner leaflet of cell membrane (2)(3)(4). In this study, we also demonstrated that Naja naja naja PLA 2 and hXPLA 2 (37,38), both of which have greater PChydrolyzing activity than hVPLA 2 (36, 37), did not induce eosinophil adhesion. This is likely the result of the low affinity of each secretory PLA 2 for cell surface HSPG that mediate the internalization of secretory PLA 2 .…”

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

“…In the present study no hydrolysis of surfactant PC was observed in sPLA 2 -IIA-treated mice. This is not surprising, since sPLA 2 -IIA, which bears basic residues resulting in positive charges of its interfacial binding surface, cannot bind well to PC vesicles, as described for PG (33,41,42).…”

Arachidonic Acid Release from Mammalian Cells Transfected with Human Groups IIA and X Secreted Phospholipase A2 Occurs Predominantly during the Secretory Process and with the Involvement of Cytosolic Phospholipase A2-α

“…For example, group V sPLA 2 is present in the macrophage-like cell line p388D1 and contributes a portion of the arachidonate released in response to lipopolysaccharide (6). Exogenous addition of groups V and X sPLA 2 s to a variety of mammalian cells leads to arachidonate release (7)(8)(9)(10). There is some evidence to suggest that the action of cPLA 2 -␣ is a prerequisite for sPLA 2 function in cells (11,12) and even for the vice versa scenario (13)(14)(15)), but such cross-talk between PLA 2 s remains poorly understood.…”