Purification and characterization of a calcium-independent acidic phospholipase A2 from rat lung

Lung surfactant is the surface-active agent comprised of phospholipids and proteins that lines pulmonary alveoli. Surfactant stabilizes the alveolar volume by reducing surface tension. Previously, we identified a lysosomal phospholipase A2, termed LPLA2, with specificity toward phosphatidylcholine and phosphatidylethanolamine. The phospholipase is localized to lysosomes, is calcium-independent, has an acidic pH optimum, and transacylates ceramide. Here, we demonstrate that LPLA2 is selectively expressed in alveolar macrophages but not in peritoneal macrophages, peripheral blood monocytes, or other tissues. Other macrophage-associated phospholipase A2s do not show a comparable distribution. LPLA2 is of high specific activity and recognizes disaturated phosphatidylcholine as a substrate. The lysosomal phospholipase A2 activity is six times lower in alveolar macrophages from mice with a targeted deletion of the granulocyte macrophage colonystimulating factor (GM-CSF), a model of impaired surfactant catabolism, compared with those from wild-type mice. However, LPLA2 activity and protein levels are measured in GM-CSF null mice in which GM-CSF is expressed as a transgene under the control of the surfactant protein C promoter. Thus LPLA2 may be a major enzyme of pulmonary surfactant phospholipid degradation by alveolar macrophages and may be deficient in disorders of surfactant metabolism.Previously, in an attempt to identify the enzyme that transacylates ceramide at the 1-hydroxyl position, a novel phospholipase A2 was characterized (1). The phospholipase A2, termed 1-O-acylceramide synthase or lysosomal phospholipase A2 (LPLA2) 1 has the following properties. In the presence of ceramide, the enzyme catalyzes the formation of 1-O-acylceramide by transacylation of fatty acids from the sn-2 position of phosphatidylcholine or phosphatidylethanolamine. In the absence of ceramide or other alcohols as acceptors, the enzyme acts as a traditional phospholipase A2. However, the phospholipase has a pH optimum of 4.5 and is mannose-rich and calcium-independent (2). The phospholipase amino acid sequence is 49% identical to human LCAT (3). The homology with LCAT is highest within the catalytic domain, but the phospholipase does not recognize cholesterol as an acceptor for the fatty acid. The phospholipase colocalizes with other lysosomal proteins in cell fractionates. Upon the initial characterization of this enzyme, the functional role of this phospholipase A2 was not immediately apparent. A role for an acidic phospholipase A2 activity has previously been suggested for the degradation of pulmonary surfactant phospholipids (4). The pulmonary acidic phospholipase A2 activity is also reported to be calcium-independent and inhibited by a transition state analog of arachidonate, MJ33 (5). In rats treated with MJ33 the surfactant phospholipid catabolism was inhibited by ϳ40 -50%, suggesting that the drug-sensitive phospholipase A2 activity contributes significantly to total surfactant degradation (6). In the present paper LPLA2 was stu...

Section: Discussionmentioning

confidence: 98%

Lysosomal Phospholipase A2 Is Selectively Expressed in Alveolar Macrophages

Abe

Hiraoka

Wild

et al. 2004

“…This enzyme displayed some properties similar to the calf brain transacylase, including insensitivity to 2-mercaptoethanol, ATP, AACOCF 3 , and the binding ability to heparin-Sepharose resin. However, the molecular mass of the lung iPLA 2 was 15 kDa (24). A soluble, lysosomal iPLA 2 partially purified from bovine adrenal medulla showed a basic pI, Triton X-100 inhibition, and binding to concanavalin A-agarose resin (22).…”

Section: Discussionmentioning

confidence: 99%

“…Although a soluble acidic pH-optimum iPLA 2 was purified from rat brain by a protocol similar to ours, the enzyme displayed a molecular mass of 58 kDa and was highly phosphatidic acid-selective (25). Recently Wang et al (24) purified an acidic-pH optimum iPLA 2 from rat lung. This enzyme displayed some properties similar to the calf brain transacylase, including insensitivity to 2-mercaptoethanol, ATP, AACOCF 3 , and the binding ability to heparin-Sepharose resin.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of 1-O-Acylceramide Synthase, a Novel Phospholipase A2 with Transacylase Activity

Abe

Shayman

1998

-independent phospholipase A 2 , had no significant effect on the enzyme activity. The enzyme activity was completely abolished in the presence of greater than 773 M Triton X-100. Partial inhibition of the enzyme activity was observed in the presence of 10 -100 g/ml heparin. In the absence of N-acetylsphingosine, the enzyme acted as a phospholipase A 2 . These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a novel phospholipase A 2 .

“…This sPLA 2 belongs to group IIC and is prevalently expressed in testis. Other low molecular mass PLA 2 s have been characterized from various tissues including spermatozoa, brain, and lung, suggesting a larger diversity of PLA 2 s (41)(42)(43)(44). Moreover, a sPLA 2 -related gene has been cloned from human teratocarcinoma cells and found to code for a protein of 689 amino acids containing two domains of high homology with sPLA 2 s (45).…”

mentioning

confidence: 99%

Cloning, Chromosomal Mapping, and Expression of a Novel Human Secretory Phospholipase A2

Cupillard¹,

Koumanov²,

Matteï³

et al. 1997

247

250

Secretory phospholipases A 2 (sPLA 2 s) represent a rapidly expanding family of structurally related enzymes found in mammals as well as in insect and snake venoms. In this report, a cDNA coding for a novel sPLA 2 has been isolated from human fetal lung, and its gene has been mapped to chromosome 16p13.1-p12. The mature sPLA 2 protein has a molecular mass of 13.6 kDa, is acidic (pI 5.3), and made up of 123 amino acids. Key structural features of the sPLA 2 include: (i) a long prepropeptide ending with an arginine doublet, (ii) 16 cysteines located at positions that are characteristic of both group I and group II sPLA 2 s, (iii) a C-terminal extension typical of group II sPLA 2 s, (iv) and the absence of elapid and pancreatic loops that are characteristic of group I sPLA 2 s. Based on these structural properties, this sPLA 2 appears as a first member of a new group of sPLA 2 s, called group X. A 1.5-kilobase transcript coding for the human group X (hGX) sPLA 2 was found in spleen, thymus, and peripheral blood leukocytes, while a less abundant 0.8-kilobase transcript was detected in the pancreas, lung, and colon. When the hGX sPLA 2 cDNA was expressed in COS cells, sPLA 2 activity preferentially accumulated in the culture medium, indicating that hGX sPLA 2 is an actively secreted enzyme. It is maximally active at physiological pH and with 10 mM Ca 2؉ . hGX sPLA 2 prefers phosphatidylethanolamine and phosphatidylcholine liposomes to those of phosphatidylserine.