On the Binding Preference of Human Groups IIA and X Phospholipases A2 for Membranes with Anionic Phospholipids

Abstract

Mammals contain 9 -10 secreted phospholipases A 2 (sPLA 2 s) that display widely different affinities for membranes, depending on the phospholipid composition. The much higher enzymatic activity of human group X sPLA 2 (hGX) compared with human group IIA sPLA 2 (hGIIA) on phosphatidylcholine (PC)-rich vesicles is due in large part to the higher affinity of the former enzyme for such vesicles; this result also holds when vesicles contain cholesterol and sphingomyelin. The inclusion of anionic phosphatidylserine in PC vesicles dramatically enhances interfacial binding and catalysis of hGIIA but not of hGX. This is the result of the large number of lysine and arginine residues scattered over the entire surface of hGIIA, which cause the enzyme to form a supramolecular aggregate with multiple vesicles. Thus, high affinity binding of hGIIA to anionic vesicles is a complex process and cannot be attributed to a few basic residues on its interfacial binding surface, as is also evident from mutagenesis studies. The main reason hGIIA binds poorly to PC-rich vesicles is that it lacks a tryptophan residue on its interfacial binding surface, a residue that contributes to the high affinity binding of hGX to PC-rich vesicles. Results show that the lag in the onset of hydrolysis of PC vesicles by hGIIA is due in part to the poor affinity of this enzyme for these vesicles. Binding affinity of hGIIA, hGX, and their mutants to PC-rich vesicles is well correlated to the ability of these enzymes to act on the PC-rich outer plasma membrane of mammalian cells.

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

“…It may also be noted that the addition of recombinant hGIIA in the low g/ml range to some mammalian cells and tissues such as guinea pig bronchoalveolar lavage cells and lung pleural strips led to fatty acid release that was completely blocked by submicromolar to low micromolar concentration of LY315920 (40), a compound very similar in structure to Me-Indoxam. We have shown previously that the addition of a tryptophan to the interfacial binding surface of hGIIA dramatically promotes the binding of this enzyme to phosphatidylcholine rich vesicles in vitro and also allows the enzyme to more efficiently act on the plasma membrane of mammalian cells when added exogenously (23). As shown in Fig.…”

“…With hGX-transfected HEK293 cells, Me-Indoxam has no effect on [ 3 H]arachidonate release, arguing that the extracellular concentration of this sPLA 2 never builds to a high enough level to allow plasma membrane phospholipid hydrolysis. In the case of hGIIA, which binds orders of magnitude more weakly than hGX to the phosphatidylcholine-rich outer plasma membrane (8,23), [ 3 H]arachidonate release occurs in CHO-K1 and HEK293 cells only prior to enzyme secretion. This model nicely explains why exogenously added hGIIA is orders of magnitude less efficient at phospholipid hydrolysis than hGIIA produced by transcription/translation within the cell; the concentration of membrane that hGIIA "sees" is orders of magnitude higher when enzyme is in the secretory compartment compared with the extracellular medium.…”

“…In marked contrast to hGIIA, hGX binds with high affinity to phosphatidylcholine-rich membranes in vitro (23), and ng/ml amounts of exogenously added hGX are sufficient to liberate arachidonic acid from HEK293 cells and other mammalian cells (8,9,25). Given these observations and the fact that hGX does not bind to heparan sulfate, it has been suggested that hGX acts on the extracellular face of the plasma membrane once secreted from hGX-transfected HEK293 cells (18).…”

“…This is because the extracellular face of the mammalian cell plasma membrane is rich in the zwitterionic phospholipids phosphatidylcholine and sphingomyelin, and it is well established that hGIIA displays extremely low enzymatic activity toward phosphatidylcholine-rich vesicles in vitro because this enzyme cannot bind to the zwitterionic interface (22,23). Indeed, relatively large amounts (typically Ͼ10 g/ml) of hGIIA need to be added exogenously to mammalian cells, including HEK293 cells, to cause detectable arachidonate release (23).…”

Purification and biochemical characterization of a secreted group IIA chicken intestinal phospholipase A2

“…Some bacteria specifically invade the intestinal mucosa from the lamina propria, as it has been postulated to occur in Whipple's disease [35]. The human PLA 2 -IIA enzyme shows low affinity for zwitterionic interfaces, and in the absence of interfacial binding mammalian membrane hydrolysis is not possible [36]. …”