Lys 43 and Asp 46 in alpha-helix 3 of uteroglobin are essential for its phospholipase A2 inhibitory activity.

Uteroglobin (UG) is an anti-inflammatory, secreted protein with soluble phospholipase A2 (sPLA2)-inhibitory activity. However, the mechanism by which UG inhibits sPLA2 activity is unknown. UG is a homodimer in which each of the 70-amino acid subunits forms four alpha-helices.

Stable, long-term bacterial production of soluble, dimeric, disulfide-bonded protein pharmaceuticals without antibiotic selection.

Numerous biopharmaceuticals and other recombinant biotechnology products are made in prokaryotic hosts. However, bacterial production of native, biologically active eukaryotic proteins is rarely possible for disulfide-bonded and/or multisubunit proteins. We previously described the production of soluble, native disulfide-bonded dimeric proteins in the Escherichia coli cytoplasm (Miele et al.

Cystic fibrosis gene mutation (deltaF508) is associated with an intrinsic abnormality in Ca2+-induced arachidonic acid release by epithelial cells.

The mechanism(s) of chronic airway inflammation in cystic fibrosis (CF) remains poorly understood. We studied Ca2+-induced release of arachidonic acid (AA), a precursor of proinflammatory lipid mediators, in epithelial cell lines with the deltaF508 mutation in CF transmembrane conductance regulator (CFTR) gene and in those lacking this mutation or cells in which this mutation was corrected by a functional CFTR gene transfer. We found that: (i) the mutant cells manifested an abnormally high Ca2+-induced AA release as compared to controls, (ii) AA release appeared to be catalyzed by a phospholipase A2 (PLA2) but not by phospholipase C followed by diacylglycerol lipase, and (iii) either correction of the CFTR-mutation or inhibition of PLA2 activity rectified this AA release abnormality.

Recombinant human uteroglobin suppresses cellular invasiveness via a novel class of high-affinity cell surface binding site.

The mechanism(s) that regulates invasion of trophoblasts through the uterine epithelium during embryo implantation and nidation in hemochorial placental mammals is poorly understood. While limited trophoblast invasion is essential for the establishment of normal pregnancy, dysregulation of this process may contribute to the pathogenesis of choriocarcinoma, a highly invasive and lethal form of cancer arising from the trophoblasts. We have previously demonstrated that rabbit uteroglobin (UG), a cytokine-like, antiinflammatory protein, produced by the endometrial epithelium during pregnancy, has a potent antichemotactic effect on neutrophils and monocytes in vitro.

Nonradiometric ELISA-based quantitation and validation of polymerase chain reaction-amplified DNA, including detection of point mutations, without allele-specific amplification, or ligation.

We describe two simple novel procedures, one direct and the other involving hybridization, for the enzyme-linked immunosorbent assay (ELISA)-based detection, quantitation, and validation of polymerase chain reaction (PCR)-amplified DNA. Both procedures are applicable to any PCR reaction, and do not require specially synthesized or enzyme-tagged oligonucleotides. We obtained accurate quantitation of PCR-amplified human cc10kDa cDNA with a sensitivity of about 0.

The localization of phospholipase A2 in the secretory granule.

A heat-resistant phospholipase A2 has been detected in the secretory granules of the mast cell [Chock, Rhee, Tang and Schmauder-Chock (1991) Eur. J. Biochem.

Osteopontin: its transglutaminase-catalyzed posttranslational modifications and cross-linking to fibronectin.

Osteopontin (OP) is a component of extracellular, bone, and urinary stone matrices, but the mechanism by which it is stably incorporated into such matrices remains unknown. By SDS-PAGE analysis of [125I]OP, treated with a catalytic amount of TG, we first demonstrate both intra- and intermolecular covalent cross-linking of OP. Most importantly, the analysis of the products generated from reactions containing OP, Fn, and TG by SDS-PAGE, autoradiography, and Western blotting using either OP or Fn antibody, and quantitation of TG-catalyzed epsilon-(gamma-glutamyl)lysine isopeptide formation between OP and Fn demonstrate, for the first time, covalent cross-linking between these two proteins.

Tissue-specific expression of the gene coding for human Clara cell 10-kD protein, a phospholipase A2-inhibitory protein.

Clara cell 10-kD protein (cc10kD), a secretory phospholipase A2 inhibitor, is suggested to be the human counterpart of rabbit uteroglobin (UG). Because cc10kD is expressed constitutively at a very high level in the human respiratory epithelium, the 5' region of its gene may be useful in achieving organ-specific expression of recombinant DNA in gene therapy of diseases such as cystic fibrosis. However, it is important to establish the tissue-specific expression of this gene before designing gene transfer experiments.

Identification of a specific region of low molecular weight phospholipases A2 (residues 21-40) as a potential target for structure-based design of inhibitors of these enzymes.

We have identified a specific region of porcine pancreatic phospholipase A2 (residues 21-40) which interacts with a neutralizing antibody causing a dramatic inhibition of its enzymatic activity (Ki in the order of 10(-8) M). The binding equilibrium of the antibody-phospholipase A2 complex is reached in < 3 min at 37 degrees C. Fab fragments are equally effective phospholipase A2 inhibitors, as are intact IgG molecules.

Human Clara cell 10-kDa protein is the counterpart of rabbit uteroglobin.

Human Clara cell 10-kDa protein has been suggested to be a counterpart of rabbit uteroglobin, an immunomodulatory and antiinflammatory secretory protein. Since this human protein is not readily available in substantial quantity for detailed characterization of its biochemical, biological, and pharmacological properties, we sought to express it in Escherichia coli in order to study its structure-function relationship. However, bacterial overproduction of homodimeric proteins with interchain disulfide bonds, such as Clara cell 10-kDa protein, was thought to be impossible until we achieved expression of native uteroglobin (Miele, L.

Transglutaminase-catalyzed incorporation of polyamines into phospholipase A2.

We have previously demonstrated that when phospholipase A2 is treated with either tissue transglutaminase or human plasma Factor XIIIa, a striking increase of its catalytic activity is observed, due to the formation of an intramolecular epsilon-(gamma-glutamyl)-lysine crosslink [Cordella-Miele et al. (1990) J. Biol.

The effect of chlorpromazine on endotoxin-induced uveitis in the Lewis rat.

Chlorpromazine (CPZ) has been used extensively in the treatment of psychiatric disorders, and has recently been shown to possess systemic anti-inflammatory properties as well. To investigate the potential effects of CPZ on ocular inflammation, we evaluated its action on endotoxin-induced uveitis (EIU) in Lewis rats. At three different dosage levels, CPZ produced highly significant reductions in the mean aqueous aspirate inflammatory cell counts and histological inflammatory scores as compared to controls treated with vehicle only.

Regulation of extracellular phospholipase A2 activity: implications for inflammatory diseases.

Phospholipases A2 (PLA2s; E.C.3.

Effects of antiflammins on endotoxin-induced uveitis in rats.

Antiflammins are phospholipase A2-inhibitory, anti-inflammatory, synthetic oligopeptides derived from the region of the highest amino-acid sequence similarity between uteroglobin and lipocortin I. Endotoxin-induced uveitis is a model for anterior uveitis of the eye, which has been suggested to be induced through phospholipase A2 activation. In a preliminary report we demonstrated that topical administration of antiflammins could inhibit endotoxin-induced uveitis in rats.

Inhibition of pancreatic phospholipase A2 activity by uteroglobin and antiflammin peptides: possible mechanism of action.

We investigated the possible mechanism of inhibition of porcine pancreatic phospholipase A2 in vitro by rabbit uteroglobin and by the antiflammin peptides. We optimized the conditions of phospholipase A2 assay using a deoxycholate-phosphatidylcholine mixed micellar substrate and established the activity of these inhibitors under optimized conditions. The results of fluorescence studies and crosslinking experiments indicate that the inhibitors interact with the enzyme in solution and affect the increase in intrinsic fluorescence of phospholipase A2 observed upon interaction with a mixed micellar substrate.

A novel transglutaminase-mediated post-translational modification of phospholipase A2 dramatically increases its catalytic activity.

Transglutaminases (TG), which include coagulation Factor XIIIa, are calcium-dependent ubiquitous enzymes. TGs catalyze the formation of an isopeptide bond by cross-linking a specific glutamine and a lysine residue between two proteins or within the same protein molecule. Phospholipase A2 (PLA2) is a key enzyme in the regulation of prostaglandin and leukotriene biosynthetic pathways, which catalyzes the release of free fatty acids from the sn-2 position of membrane glycerophospholipids.

High level bacterial expression of uteroglobin, a dimeric eukaryotic protein with two interchain disulfide bridges, in its natural quaternary structure.

Bacterial expression of eukaryotic proteins is a tool of ever-increasing importance in biochemistry and molecular biology. However, the majority of the recombinant eukaryotic proteins that have been expressed in bacteria are produced as fusion proteins and not in their native conformation. In particular, correct formation of quaternary structures by recombinant proteins in bacterial hosts has been reported very rarely.

Novel anti-inflammatory peptides from the region of highest similarity between uteroglobin and lipocortin I.

Significant future developments in the effective treatment of inflammatory diseases may arise from non-toxic dual inhibitors of both cyclooxygenase and lipoxygenase pathways in the arachidonate cascade. Inhibition of phospholipase A2(PLA2)(EC3.1.