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...

Proliferation of vascular smooth muscle cells (VSMC) is triggered by two types of growth factors. One activates tyrosine kinase-type receptors and the other activates G-protein-coupled receptors. We found that a conditioned medium of rat VSMC contained a growth-potentiating activity for the latter type of growth factor, and we purified a 70-kDa growth-potentiating factor (GPF) from the conditioned medium. Analyses of GPF and its cDNA revealed GPF to be a gamma-carboxyglutamic acid-containing protein encoded by a growth arrest-specific gene, gas6, which related to protein S. GPF specifically potentiated cell proliferation mediated by Ca(2+)-mobilizing receptors. The presence of a specific binding site suggests that the effect of GPF is mediated by a receptor. Thus, GPF may be a new type of extracellular factor regulating VSMC proliferation.

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.

Gas6 (encoded by growth-arrest-specific gene 6) is a gamma-carboxyglutamic acid (Gla)-containing protein which is released from growth-arrested vascular smooth muscle cells (VSMCs) and potentiates VSMC proliferation induced by Ca2+-mobilizing growth factors, but not that induced by receptor tyrosine kinases. In this study we examined the importance of Gla residues for the biological activities of Gas6 and tried to assess the importance of endogenous Gas6 in VSMC proliferation. We demonstrated that Gla-deficient Gas6 lacked receptor-binding and growth-potentiating activities. Therefore the vitamin K-dependent modification of Gas6 appeared to be essential for its biological activities. Next we used warfarin, an inhibitor of vitamin K-dependent gamma-carboxylation, to estimate the contribution of endogenous Gas6 to VSMC proliferation. Warfarin markedly inhibited the thrombin-induced proliferation of VSMC without affecting the mRNA or protein expression of Gas6. Therefore the inhibition seems to be due to prevention of the vitamin K-dependent modification of Gas6. However, warfarin did not affect epidermal growth factor-induced proliferation. A neutralizing antibody against Gas6 gave a similar result, i.e. it inhibited thrombin-induced VSMC proliferation but not that induced by epidermal growth factor. These results indicate that endogenously produced Gas6 is very important for VSMC proliferation induced by Ca2+-mobilizing growth factors.