Recently, the cloning of a novel Ca 2؉ -independent phospholipase A 2 (iPLA 2 ) from Chinese hamster ovary cells as well as from mouse and rat sources containing a C-terminal lipase motif and eight N-terminal ankyrin repeats has been described. In this report we describe the cloning of the human iPLA 2 cDNA and its expression in B-cells and show that the iPLA 2 gene undergoes extensive alternative splicing generating multiple isoforms that contribute to a novel mechanism to control iPLA 2 activity. The full-length cDNA clone encodes a 806-amino acid protein with a calculated molecular mass of 88 kDa. The protein contains a lipase motif, GXSXG, and ankyrin repeats, as described for the hamster and rodent forms of the enzyme but has an additional 54-amino acid proline-rich insertion in the last of the eight ankyrin repeats (residues 395-449). Furthermore, at least three additional isoforms most likely due to alternative splicing were identified. One that is present as a partial cDNA in the expressed sequence tag data base is similar to iPLA 2 but terminates just after the lipase active site, and two other isoforms contain only the iPLA 2 ankyrin repeat sequence (ankyrin-iPLA 2 -1 and -2). Ankyrin repeats are involved in protein-protein interactions and because the purified iPLA 2 enzyme exists as a multimeric complex of 270 -350 kDa, the expression of just the ankyrin-iPLA 2 sequence suggested that these may also interact with the iPLA 2 oligomeric complexes and perhaps modulate PLA 2 activity. Transfection of the human iPLA 2 cDNA into COS cells resulted in a substantial increase in calcium-independent PLA 2 activity in cell lysate. No activity above background was observed following ankyrin-iPLA 2 -1 cDNA transfection. However, co-transfection of the ankyrin-iPLA 2 -1 and the iPLA 2 cDNAs resulted in a 2-fold reduction in activity compared with iPLA 2 alone. A similar co-transfection of ankyrin-iPLA 2 -1 cDNA with the cPLA 2 cDNA had no effect on PLA 2 activity. These results suggest that the ankyrin-iPLA 2 sequence can function as a negative regulator of iPLA 2 activity and that the alternative splicing of the iPLA 2 gene can have a direct effect on the attenuation of enzyme activity.

Oxidized low-density lipoprotein (oxLDL) consists of both lipid components and apoprotein B100. OxLDL has both proinflammatory and cytotoxic properties. The present study was undertaken to investigate the effects of components in the lipid moiety of oxLDL on immune activation as determined by cytokine and immunoglobulin secretion. LPC induced interferon-gamma (IFN-gamma) secretion in peripheral blood mononuclear leucocytes from healthy blood donors. The effect varied between individuals, and there were both responders and non-responders. Furthermore, LPC induced enhanced antibody production, indicating B cell activation. None of eight oxysterols, arachidonic acid (AA), or 15-lipoxygenase products of AA tested had immune stimulatory properties. We recently demonstrated that PAF and oxLDL induce IFN-gamma secretion by a common mechanism. LPC-induced IFN-gamma secretion was inhibited by a specific PAF receptor antagonist, WEB 2170, indicating that the PAF receptor is involved in LPC-induced immune activation. Both oxLDL- and LPC-induced antibody formation was inhibited by WEB 2170. Furthermore LPC also induced tumour necrosis factor-alpha secretion, and this effect was inhibited by WEB 2170. LPC is produced during lipid oxidation (as in oxLDL), but also by enzymes such as phospholipase A2. The findings indicate that LPC may play an important role in inflammatory reactions, including atherosclerosis.

Human eosinophils contain abundant amounts of 15-lipoxygenase (LO)-1. The biological role of 15-LO-1 in humans, however, is unclear. Incubation of eosinophils with arachidonic acid led to formation of a product with a UV absorbance maximum at 282 nm and shorter retention time than leukotriene (LT)C4 in reverse-phase HPLC. Analysis with positive-ion electrospray tandem MS identified this eosinophil metabolite as 14,15-LTC4. This metabolite could be metabolized to 14,15-LTD4 and 14,15-LTE4 in eosinophils. Because eosinophils are such an abundant source of these metabolites and to avoid confusion with 5-LO-derived LTs, we suggest the names eoxin (EX)C4, -D4, and -E4 instead of 14,15-LTC4, -D4, and -E4, respectively. Cord blood-derived mast cells and surgically removed nasal polyps from allergic subjects also produced EXC4. Incubation of eosinophils with arachidonic acid favored the production of EXC4, whereas challenge with calcium ionophore led to exclusive formation of LTC4. Eosinophils produced EXC4 after challenge with the proinflammatory agents LTC4, prostaglandin D2, and IL-5, demonstrating that EXC4 can be synthesized from the endogenous pool of arachidonic acid. EXs induced increased permeability of endothelial cell monolayer in vitro, indicating that EXs can modulate and enhance vascular permeability, a hallmark of inflammation. In this model system, EXs were 100 times more potent than histamine and almost as potent as LTC4 and LTD4. Taken together, this article describes the formation of proinflammatory EXs, in particular in human eosinophils but also in human mast cells and nasal polyps.

The nomenclature of lipoxygenases (LOXs) is partly based on the positional specificity of arachidonic acid oxygenation, but there is no unifying concept explaining the mechanistic basis of this enzyme property. According to the triad model, Phe-353, Ile-418, and Ile-593 of the rabbit 12/15-LOX form the bottom of the substrate-binding pocket, and introduction of less spacefilling residues at either of these positions favors arachidonic acid 12-lipoxygenation. The present study was aimed at exploring the validity of the triad concept for two novel primate 12/15-LOX (Macaca mulatta and Pongo pygmaeus) and for five known members of the mammalian LOX family (human 12/15-LOX, mouse 12/15-LOX, human 15-LOX2, human platelet type 12-LOX, and mouse (12R)-LOX). The enzymes were expressed as N-terminal His tag fusion proteins in E. coli, the potential sequence determinants were mutated, and the specificity of arachidonic acid oxygenation was quantified. Taken together, our data indicate that the triad concept explains the positional specificity of all 12/15-LOXs tested (rabbit, human, M. mulatta, P. pygmaeus, and mouse). For the new enzymes of M. mulatta and P. pygmaeus, the concept had predictive value because the positional specificity predicted on the basis of the amino acid sequence was confirmed experimentally. The specificity of the platelet 12-LOX was partly explained by the triad hypothesis, but the concept was not applicable for 15-LOX2 and (12R)-LOX.