Cloning and Characterization of Novel Mouse and Human Secretory Phospholipase A2s

Abstract

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.

Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2

“…The heterogeneous glycosylation pattern of hGIID appears to be similar to those observed for bee venom sPLA 2 or other proteins endogenously or heterogeneously expressed in insect cells (46 -48). Treatment of glycosylated hGIID with N-glycopeptidase (Roche Molecular Biochemicals) followed by mass spectrometry analysis confirmed that hGIID produced in S2 cells is N-glycosylated (not shown), in good agreement with the presence of an N-glycosylation site in its sequence (49). On the other hand, treatment with O-glycosidase and neuraminidase (Roche Molecular Biochemicals) does not affect the molecular mass of the glycosylated hGIID protein, suggesting the absence of sialic acids and Oglycosylation (not shown).…”

“…Although we did not study mouse group XII, this sPLA 2 is also reported to have low phospholipase activity after refolding of inclusion body protein expressed in E. coli (64). Also, the enzymatic activity of group IID, IIE, and XII sPLA 2 s in the culture media of mammalian cells transfected with the genes coding for these enzymes is barely detectable (38,41,43,49,64,65). It may be noted that porcine and bovine pancreatic group IB sPLA 2 s display true interfacial allosteric activation (k cat * and K m * type) upon binding to the vesicle interface (3), although this activation is not yet fully understood at the molecular level.…”

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

“…Briefly, 5 l of the DNA-free RNA preparations from Ficoll-enriched or FACS-sorted CD16 ϩ human neutrophils or 2 g of DNA-free RNA extracted from myocardial tissue (Ambion, Austin, TX) was reverse-transcribed using 40 units of Moloney murine leukemia virus-reverse transcriptase in the presence of 0.5 g of oligo(dT 18 ) primers, 50 mmol/liter Tris-HCl, pH 8.3, 50 mmol/liter KCl, 4 mmol/liter MgCl 2 , 10 mmol/liter dithiothreitol, deoxynucleotide (dNTP) mix (1 mmol/liter each), and 20 units of RNase inhibitor. The reaction mixture was incubated for 60 min at 39°C (transcription) and 10 min at 70°C (inactivation of RT).…”

“…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

“…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).…”