Group X secretory phospholipase A 2 (sPLA 2 -X) possesses several structural features characteristic of both group IB and IIA sPLA 2 s (sPLA 2 -IB and -IIA) and is postulated to be involved in inflammatory responses owing to its restricted expression in the spleen and thymus. Here, we report the purification of human recombinant COOH-terminal His-tagged sPLA 2 -X, the preparation of its antibody, and the purification of native sPLA 2 -X. The affinity-purified sPLA 2 -X protein migrated as various molecular species of 13-18 kDa on SDS-polyacrylamide gels, and N-glycosidase F treatment caused shifts to the 13-and 14-kDa bands. NH 2 -terminal amino acid sequencing analysis revealed that the 13-kDa form is a putative mature sPLA 2 -X and the 14-kDa protein possesses a propeptide of 11 amino acid residues attached at the NH 2 termini of the mature protein. Separation with reverse-phase high performance liquid chromatography revealed that N-linked carbohydrates are not required for the enzymatic activity and prosPLA 2 -X has a relatively weak potency compared with the mature protein. The mature sPLA 2 -X induced the release of arachidonic acid from phosphatidylcholine more efficiently than other human sPLA 2 groups (IB, IIA, IID, and V) and elicited a prompt and marked release of arachidonic acid from human monocytic THP-1 cells compared with sPLA 2 -IB and -IIA with concomitant production of prostaglandin E 2 . A prominent release of arachidonic acid was also observed in sPLA 2 -X-treated human U937 and HL60 cells. Immunohistochemical analysis of human lung preparations revealed its expression in alveolar epithelial cells. These results indicate that human sPLA 2 -X is a unique N-glycosylated sPLA 2 that releases arachidonic acid from human myeloid leukemia cells more efficiently than sPLA 2 -IB and -IIA.Phospholipase A 2 (PLA 2 ) 1 comprises a diverse family of lipolytic enzymes that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acid and lysophospholipids (1, 2). PLA 2 s participate in pathophysiological processes by releasing arachidonic acid from membrane phospholipids, leading to the production of various types of proinflammatory lipid mediators, such as prostaglandins (PGs) and leukotrienes (LTs) (3). Over the past two decades, a number of PLA 2 s have been identified and characterized. From their biochemical features, these PLA 2 s are classified into several families (4), including secretory PLA 2 (sPLA 2 ) (5-11), arachidonoyl-specific cytosolic PLA 2 (cPLA 2 ) (12, 13), and Ca 2ϩ -independent PLA 2 (14).Low molecular mass sPLA 2 s (13-18 kDa) have several features including a high disulfide bond content, a requirement for millimolar concentrations of Ca 2ϩ for catalysis, and a broad specificity for phospholipids with different polar head groups and fatty acyl chains (15, 16). Mammalian sPLA 2 s are classified into different groups depending on the primary structure characterized by the number and positions of cysteine residues. At present, five types of functional sPL...

Mammalian secretory phospholipase A 2 s (sPLA 2 s) form a family of structurally related enzymes that are involved in a variety of physiological and pathological processes via the release of arachidonic acid from membrane phospholipids or the binding to specific membrane receptors. Here, we report the cloning and characterization of a novel sPLA 2 that is the sixth isoform of the sPLA 2 family found in humans. The novel human mature sPLA 2 consists of 123 amino acids (M r ‫؍‬ 14,000) and is most similar to group IIA sPLA 2 (sPLA 2 -IIA) with respect to the number and positions of cysteine residues as well as overall identity (51%). Therefore, this novel sPLA 2 should be categorized into group II and called group IIE (sPLA 2 -IIE) following the recently identified group IID sPLA 2 (sPLA 2 -IID). The enzymatic properties of recombinant human sPLA 2 -IIE were almost identical to those of sPLA 2 -IIA and IID in terms of Ca 2؉ requirement, optimal pH, substrate specificity, as well as high susceptibility to the sPLA 2 inhibitor indoxam. Along with the biochemical properties of proteins, genetic and evolutional similarities were also observed among these three types of group II sPLA 2 s as to the chromosomal location of the human gene (1p36) and the exon/intron organization. The expression of sPLA 2 -IIE transcripts in humans was restricted to the brain, heart, lung, and placenta in contrast to broad expression profiles for sPLA 2 -IIA and -IID. In sPLA 2 -IIA-deficient mice, the expression of sPLA 2 -IIE was markedly enhanced in the lung and small intestine upon endotoxin challenge, which contrasted with the reduced expression of sPLA 2 -IID mRNA. In situ hybridization analysis revealed elevation of sPLA 2 -IIE mRNA at alveolar macrophage-like cells in the lung of endotoxin-treated mice. These findings suggest a distinct functional role of novel sPLA 2 -IIE in the progression of inflammatory processes.

In this study, we generated PLA 2 R-deficient mice to define its biological roles further. These mice are viable, fertile, and without evident histopathological abnormalities. There was no difference in the clearance of circulating PLA 2 -IB between wild-type and mutant mice. After challenge with bacterial lipopolysaccharide (LPS), PLA 2 R-deficient mice exhibited longer survival than wild-type mice. The mutant mice were also resistant to lethal effects of exogenous PLA 2 -IB after sensitization with sublethal dose of LPS. The plasma levels of tumor necrosis factor-␣ and interleukin-1␤ elevated after LPS treatment were significantly reduced in mutant mice compared with wild-type mice. These findings suggest a potential role of PLA 2 R in the progression of endotoxic shock.

Mammalian secretory phospholipase A 2 s (sPLA 2 s) are classified into several groups according to molecular structure and the localization of intramolecular disulfide bridges. Among them, group IIA sPLA 2 has been thought to be one of the key enzymes in the pathogenesis of inflammatory diseases owing to its augmented expression under various inflammatory conditions. However, in a number of inbred mouse strains, the group IIA sPLA 2 gene is naturally disrupted by a frameshift mutation. Here, we report the cloning of a cDNA encoding a novel sPLA 2 expressed in the spleen of group IIA sPLA 2 -deficient mouse. We also cloned its human homolog and mapped its gene location on chromosome 1p36.12 near the loci of group IIA and V sPLA 2 genes. The human mature sPLA 2 protein consists of 125 amino acids (M r ‫؍‬ 14,500) preceded by a 20-residue prepeptide and is most similar to group IIA sPLA 2 with respect to the number and positions of cysteine residues as well as overall identity (48%). Based on these structural properties, the novel sPLA 2 should be categorized into group II, called group IID to follow the already identified IIA to IIC sPLA 2 s. When the cDNA was expressed in COS-7 cells, PLA 2 activity preferentially accumulated in the culture medium. It is maximally active at neutral to alkaline pH and with 2 mM Ca 2؉ . In assays with individual substrates, L-␣-1-palmitoyl-2-linoleoyl phosphatidylethanolamine was more efficiently hydrolyzed than the other phospholipids examined. An RNA blot hybridized with the cDNA exhibited two transcripts (2.0 and 1.0 kb) in human spleen, thymus, and colon. The expression of a novel sPLA 2 mRNA was elevated in the thymus after treatment with endotoxin in rats as well as in group IIA sPLA 2 -deficient mice, suggesting its functional role in the progression of the inflammatory process.

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