Structures, Enzymatic Properties, and Expression of Novel Human and Mouse Secretory Phospholipase A2s

Abstract

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.

Groups IV, V, and X Phospholipases A2s in Human Neutrophils

“…However, we were unable to detect GIIE sPLA 2 mRNA in myocardium. Hanasaki and co-workers (19) reported that GIIE sPLA 2 mRNA was detected only after multiple rounds of PCR amplification, indicating that this protein is expressed at very low levels, and we have not been able to detect GIIE sPLA 2 using a variety of human tissue cDNAs (Invitrogen multiple tissue panel). RT-PCR analysis of neutrophils that had been prepared by centrifugation on a discontinuous Ficoll-Paque density gradient identified GIIA, GIID, GV, and GX sPLA 2 mRNA species (Fig.…”

“…For GIID and GIII sPLA 2 , only RT-PCR was used for detection, with successful execution of positive controls, because antisera for these proteins are not yet available. Although GIIE sPLA 2 mRNA could not be detected by RT-PCR (Hanasaki and co-workers (19) reported that GIIE sPLA 2 cDNA could be detected by PCR only after multiple rounds of extensive amplification), GIIE sPLA 2 protein was not detected by Western blot analysis even though spiking the neutrophil lysate with 1.5 ng of recombinant GIIE sPLA 2 gave a readily detected band. In contrast to our results, a previous study (75) reported that GV sPLA 2 is not present in human neutrophils.…”

“…GIIC sPLA 2 is present in the mouse but appears as a pseudo gene in humans (4). GIID sPLA 2 mRNA was detected in human spleen, thymus, small intestine, colon, lung, pancreas, and placenta (17,18), whereas GIIE sPLA 2 expression in humans is restricted to the brain, heart, lung, and placenta (19). mRNA coding for GIIF sPLA 2 has been identified in many human tissues, with the highest levels detected in the placenta, thymus, prostate, testes, kidney, liver, and thyroid (17).…”

Cloning and Recombinant Expression of a Structurally Novel Human Secreted Phospholipase A2

“…Furthermore, analysis by RT-PCR with commercial human tissue cDNA panels indicates a pattern of hGXII expression that is Cloning of Human Group XII sPLA 2 39825 consistent with the Northern blot data and additionally shows that this sPLA 2 is strongly expressed in pancreas and weakly in ovaries, testis, and prostate (not shown). The pattern of expression of hGXII thus appears distinct from that of other known human sPLA 2 s (16,19,22), 3 suggesting specific function(s) for this novel sPLA 2 .…”

“…In the last few years, six mouse and five human sPLA 2 s structurally related to GIB and GIIA sPLA 2 s (mGIIC, hGIID, mGIID, hGIIE, mGIIE, mGIIF, hGIIF, hGV, mGV, hGX, and mGX) 2 have been identified (15)(16)(17)(18)(19)(20). 3 All of these group I/II/V/X sPLA 2 s have similar primary structures, including identical catalytic site residues and partially overlapping sets of disulfides (21).…”