Studies on the structure, function and expression of phospholipase A2

Abstract

Phospholipase A2 (PLA2) enzymes are widely distributed and the enzymes possess different functions such as a role in digestion and inflammation. PLA2 catalyzes the hydrolysis of the sn-2 acylester bond of phospholipids, yielding a free fatty acid and a lysophospholipid. Arachidonic acid is a precursor molecule for important lipid mediators such as leukotrienes, prostaglandins and thromboxanes, collectively called eicosanoids. The level of free arachidonic acid in the cell is usually low since the majority of arachidonic acid is esterified at the sn-2 position of phospholipids. Mammalian cells contain several different PLA2 enzymes, each capable of releasing arachidonic acid from phospholipids. Furthermore, the produced lysophospholipids can be used for synthesis of platelet-activating factor (PAF). Both eicosanoids and PAF are involved in inflammatory reactions.

Atherosclerosis is a disease of large and medium-sized arteries. The athersosclerotic plaque exhibits some features similar to those of chronic inflammation. Furthermore, PLA2 enzymes have been implicated in the pathogenesis of atherosclerosis. Therefore, PLA2 activities and expression of secretory PLA2 (sPLA2), group IIA, and cPLA2 were investigated in normal and atherosclerotic arterial wall by immunohistochemistry. Both healthy and diseased airteries possessed substantial PLA2 activity and expressed sPLA2 group IIA protein. cPLA2 was only detected in atherosclerotic arteries, in regions consisting of inflammatory infiltrate and smooth muscle cells. The expression of cPLA2 in atherosclerotic plaques indicate that this enzyme is associated with the inflammatory component of atherosclerosis. PLA2 has been reported to play a pivotal role in leukotriene synthesis. Leukotrienes are produced by human neutrophils after calcium ionophore A23187 stimulation. However, equine neutrophils, but not eosinophils, require exogenous arachidonic acid for leukotriene synthesis. Studies were undertaken to investigate if equine neutrophils did express an active calcium-dependent PLA2 enzyme. The group IV calcium-dependent cytosolic PLA2 (cPLA2) from equine neutrophils was purified >6500-fold and found to be catalytically active. A possible difference in the degree of phosphorylation of the cPLA2 protein between equine eosinophils and neutrophils was observed. This might explain the inability of equine neutrophils to produce leukotrienes in the absence of exogenous arachidonic acid.

In contrast to human myeloid cells, B-lymphocytes are dependent on exogenous arachidonic acid for leukotriene synthesis. However, it has been demonstrated that homogenates of B-lymphocytes possess PLA2 activity. Therefore, we investigated the expression and regulation of PLA2 enzymes in B-lymphocytes. The results demonstrated that a calcium-independent PLA2 (iPLA2) was abundantly expressed in these cells. A full-length cDNA clone encoding the human iPLA2 was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA clone encoded a 806-Alternativelyamino acid protein. This human iPLA2 protein had an additional 54 amino acid insertion not found in rodent forms of the iPLA2 protein. Alternatively spliced variants of the human iPLA2 transcript were observed in cells of both myeloid and lymphoid origin and in muscle cells. The expression of one splice variant was found to be different in immature and mature myeloid cells. The encoded protein from one of these splice variants was found to function as a negative regulator of iPLA2 activity in transfection experiments. Results obtained with human granulocytes suggest that granulocytes possess calcium- independent PLA2 activity. Furthermore, inhibitor studies indicate that iPLA2 is involved in events leading to the release of arachidonic acid destined for leukotriene synthesis in granulocytes.

The human iPLA2 gene was identified by computational identification and it was localized to chromosome 22qI3. 1. The gene consists of at least 17 exons and spans more than 69 kb. Based upon the organization of the gene, the observed splice variants of iPLA2 can be explained. Subcellular fractionation studies were performed to investigate the subcellular localization of the native human iPLA2 protein. This form of iPLA2 contains 54 additional amino acids as compared to the soluble iPLA2 enzyme found in several rodents. After subcellular fractionation, human calcium-independent PLA2 activity and iPLA2 protein were found in the membrane fraction. It is suggested that the addition of the 54 amino acids leads to a membrane associated protein. In conclusion, iPLA2 and its splice variants are expressed in various cells such as B-lymphocytes, myeloid cells and muscle cells. Furthermore, multiple enzymes with distinct function and localization are derived from the iPLA2 transcript by alternative splicing.