Uteroglobin: structure, molecular biology, and new perspectives on its function as a phospholipase A2 inhibitor.

UG or blastokinin is a low molecular weight protein which is secreted by the endometrium of the rabbit during early pregnancy. Its synthesis and secretion by the endometrium are regulated by ovarian steroids, especially P. However, the protein is also produced by tracheo-bronchial, gastrointestinal, prostatic, and seminal vesicular epithelium. In the respiratory tract, UG synthesis is under glucocorticoid control. The hormonal regulation of UG synthesis in organs other than the endometrium and tracheobronchial epithelium is poorly understood. The structure of this protein and its gene has been extensively investigated while its physiological function is still unclear. Since UG, after reduction of its two disulfide bonds, has the ability to bind P and related steroids, it has been suggested that this protein is a P carrier or a P scavenger. However, the protein does not bind glucocorticoids, estrogens, or androgens and its presence in organs other than the uterus cannot be explained on the basis of its P binding. Recent data indicate that UG has other interesting biological properties. These include antichemotactic/antiphagocytic effects on macrophages, monocytes, and neutrophils, tolerogenic effect on both blastomeres and spermatozoa against recognition by maternal lymphocytes, and its ability to inhibit thrombin-induced platelet aggregation. Moreover, UG has been shown to be a potent phospholipase A2 inhibitor. The latter property could suggest a possible mechanism of some of the observed biological effects of this protein. The structural similarities of UG with phospholipase A2 and with other PLA2 inhibitory proteins like lipocortins may suggest that the physiological function of this protein may be primarily immunomodulatory through its function as a PLA2 inhibitor. The possible occurrence of similar proteins in other species, including humans, may confirm this hypothesis. Additionally, our hypothesis may lend support to the suggestion that P may be nature's immunosuppressant during pregnancy.

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. Since the UG gene in the rabbit is expressed in many other organs besides the lung and the endometrium, we investigated the organ and tissue specificity of human cc10kD gene expression using polymerase chain reaction, nucleotide sequence analysis, immunofluorescence, and Northern blotting. Our results indicate that, in addition to the lung, cc10kD is expressed in several nonrespiratory organs, with a distribution pattern very similar, if not identical, to that of UG in the rabbit. These results underscore the necessity for more detailed analyses of the 5' region of the human cc10kD gene before its usefulness in gene therapy could be fully assessed. These data also suggest that cc10kD and UG may have similar physiological function(s).

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. In this study, the anti-inflammatory effects of antiflammins were compared with those of corticosteroids on endotoxin-induced uveitis as measured by phospholipase A2 enzyme activity, inflammatory cell counts in the aqueous humor, and histopathologic features. Antiflammins are as effective as corticosteroids in their ability to suppress endotoxin-induced uveitis.

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. Chem. 265, 17180-17188]. Here, we report the effect of transglutaminase substrates such as mono-, di-, and polyamines on this transglutaminase-catalyzed post-translational modification of phospholipase A2. Incorporation of radioactively labeled polyamines into phospholipase A2 was demonstrated by using porcine pancreatic phospholipase A2 as a substrate in a conventional transglutaminase assay. These results were further confirmed by SDS-polyacrylamide gel electrophoresis followed by fluorography. Additionally, gamma-glutamyl-polyamine was detected and unequivocally identified in proteolytic digests of polyaminated phospholipase A2. When phospholipase A2 was incubated with transglutaminase in the presence of putrescine, spermine, spermidine, dansylcadaverine, or methylamine, a 2-3-fold increase in phospholipase A2 activity was observed. The increase of phospholipase A2 activity was found to be dependent upon the concentration of phospholipase A2, preincubation time, and the duration of the reaction. Increase in phospholipase A2 activity after transglutaminase treatment in the presence of polyamines was demonstrated using two different assay systems. Kinetic studies on phospholipase A2 pretreated with spermidine and transglutaminase demonstrated a significant increase of the apparent Vmax but no significant change in the apparent Km. Unlike phospholipase A2 pretreated with transglutaminase alone, polyaminated phospholipase A2 does not undergo non-covalent dimerization in solution. Polyaminated phospholipase A2 was further purified by chromatofocusing and was found to contain N-mono(gamma-glutamyl)-spermidine in a molar ratio of about 1:1 to phospholipase A2. Freshly purified, polyaminated phospholipase A2 had a specific activity approximately 3-fold higher than that of control phospholipase A2 treated in an identical way except for the absence of transglutaminase. To our knowledge, this is the first demonstration that transglutaminase catalyzes the incorporation of amines into a phospholipase, and that this post-translational modification increases phospholipase A2 activity.

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. Similar reactions in the presence of polyamine substrates of TG show OP-Fn intermolecular cross-linking via N,N-bis-(gamma-glutamyl)polyamine formation. Finally, immunoprecipitation of 125I-labeled NRK cell surface proteins with anti-OP and anti-Fn antibodies, SDS-PAGE analysis, and autoradiography provides critical evidence for nonreducible OP-Fn cross-linking in vivo. These results clearly suggest that TG-mediated cross-linking between OP and Fn represents one of the most likely mechanisms by which OP becomes covalently linked to bone matrix, urinary stone matrix, and to ECM.

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

Phospholipases A2 (PLA2s; E.C.3.1.1.4) are a family of esterases that are involved in myriads of physiological and pathological processes. The involvement of these enzymes, especially the extracellular PLA2s, in the generation of proinflammatory lipid mediators makes them a very important target for investigation. These PLA2s have been suggested to be involved in the pathogenesis of several human inflammatory diseases. Thus, delineating the mechanism(s) of regulation of the activity of these enzymes may provide a better understanding of the pathophysiology of these diseases and allow the rational design and development of novel therapeutic agents. In this article, we provide a brief description of PLA2s in general with a special emphasis on extracellular enzymes, their mechanism(s) of action, and possible role in the pathogenesis of inflammatory diseases. Additionally, we describe: (i) a novel mechanism of activation of extracellular PLA2s by transglutaminases and (ii) the development of one class of antiinflammatory agents, antiflammins, derived from the active site structure of endogenous PLA2-inhibitory proteins.

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 0.6 fmoles. This cDNA was also used to detect single-base insertions, deletions, and substitutions specifically. Additionally, we could readily distinguish zinc-finger Y chromosome-specific genomic sequences in mixtures of male and female cells and normal and mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene sequences in crude fibroblast lysates. To our knowledge, this is the first report of point mutation detection in solution by ELISA without allele-specific amplification or ligation. These novel procedures have vast potential for basic and clinical applications, including gene expression studies, rapid screening of genetic diseases, detection of oncogene and anti-oncogene mutations, and identification of pathogens (e.g., HIV-1) in clinical specimens.

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. Taken together, our results suggest that CFTR mutation is associated with an intrinsic abnormality in AA release by epithelial cells carrying the deltaF508 mutation and suggest that the mechanism of chronic airway inflammation in CF, at least in part, involves this abnormality. These results also partly explain the effectiveness of high-dose ibuprofen therapy in arresting the progression of destructive lung disease in CF. Furthermore, they raise the possibility that correction of abnormal AA release by inhibiting PLA2 activity may improve the therapeutic benefits of ibuprofen.

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. In addition, we identified a sequence similarity between the antiflammin peptides, the putative active region of uteroglobin and a region in pancreatic phospholipase A2. This region of phospholipase A2 has been previously identified as being involved in the regulation of dimerization of this enzyme, and is conserved in the pancreatic-type enzymes. Taken together, these observations suggest that uteroglobin and antiflammins interact with porcine pancreatic phospholipase A2 and this may, at least in part, explain the enzyme inhibitory effect of these molecules observed in vitro. One possible mechanism of this effect may be an interference with the dimerization process of phospholipase A2 which is associated with interfacial activation.

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., 1990; Mantile et al., 1993). Native, biologically active proteins with up to six disulfide bonds have been produced with our expression system (Garces et al., 1997). However, plasmid instability during induction limited its usefulness. We now report the stable, high-level expression of soluble, disulfide-bonded human uteroglobin without antibiotic selection. We designed a new vector containing a multifunctional stabilization region that confers complete plasmid stability and increased protein yields without copy number increases. Recombinant expression remains fully inducible after long-term continuous culture in nonselective liquid medium (at least 260 generations). This system may significantly expand the applications of bacterial expression to recombinant production of soluble, bioactive proteins for biochemical studies and biopharmaceutical/industrial purposes. As a result of the very broad activity spectrum of the stabilization region we selected, its use could be extended to bacterial hosts other than enterobacteria.

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. Analysis of aqueous fluid demonstrated a similar decrease in protein concentration and phospholipase A2 (PLA-2) activity in the treated animals. The ability of CPZ to inhibit the development of EIU may be related to its properties as a calcium channel blocker and inhibitor of the enzyme phospholipase A2.

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. The inhibition is virtually complete and noncompetitive with respect to phosphatidylcholine substrate. The formation of precipitating immunocomplexes is not involved in the inhibition. The region of phospholipase A2 (residues 21-40) recognized by this antibody includes a highly conserved sequence which contains several functionally important residues of both group I and group II phospholipases A2. These data suggest that amino acid residues in this region of porcine pancreatic phospholipase A2 are accessible for interaction with inhibitors such as neutralizing antibodies and that agents specifically interacting with this region may have potent phospholipase A2 inhibitory activity. Thus, this conserved region of low molecular weight, extracellular phospholipases A2 is a potential target for structure-based design of specific noncompetitive inhibitors of these enzymes. Since these extracellular phospholipases A2 are suggested to play a pathogenic role in several important human diseases, the development of such pharmacologic inhibitors is of potential clinical importance.

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. To our knowledge, correct intracellular formation of multimeric structures containing more than one interchain disulfide bridge has not been reported so far. We have constructed three plasmids which are able to direct expression of recombinant rabbit uteroglobin, a homodimeric protein with two interchain disulfide bridges, in Escherichia coli. Among these, the plasmid pLE103-1, in which the expression of recombinant uteroglobin is controlled by a bacteriophage T7 late promoter, is by far the most efficient. With pLE103-1, recombinant uteroglobin production reached about 10% of total bacterial soluble proteins. This protein accumulated in bacterial cells in dimeric form, as it is naturally found in the rabbit uterus. Recombinant uteroglobin was purified to near-homogeneity and its NH2-terminal amino acid sequence was confirmed to be identical to that of its natural counterpart, except for 2 Ala residues the codons for which were added during the plasmid construction. This protein was found to be as active a phospholipase A2 inhibitor as natural uteroglobin on a molar basis. To our knowledge, this is the first report of high level bacterial expression of a full length eukaryotic homodimeric protein with two interchain disulfide bridges in its natural, biologically active form. The plasmid pLE103-1 may be useful to explore structure-function relationships of rabbit uteroglobin. In addition, this plasmid may be useful in obtaining high level bacterial expression of other eukaryotic proteins with quaternary structure, as well as for other general applications requiring efficient bacterial expression of cDNAs.

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. This enzyme has been suggested to be pathophysiologically related to the initiation and propagation of several inflammatory diseases including juvenile rheumatoid and rheumatoid arthritis. Here, we describe a novel TG-catalyzed post-translational modification of PLA2 which dramatically increases the activity of this enzyme. This increase was dependent upon the time of preincubation, the concentration of TG and the presence of Ca2+. Size exclusion chromatography of TG-treated PLA2 yielded two peaks of PLA2 activity, with apparent molecular masses of 26 and 13 kDa, respectively. The 26-kDa species, a putative PLA2 dimer, contained epsilon-(gamma-glutamyl)-lysine isopeptide in about 1:1 molar ratio to PLA2, suggesting an intramolecular rather than intermolecular cross-linking. This hypothesis was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 26- and 13-kDa species under denaturing conditions. The specific activity of the dimeric peak was 10-fold higher with respect to that of the monomeric enzyme. These data suggest that TG-catalyzed covalent cross-linking of PLA2 is intramolecular and that this may promote a noncovalent dimerization and subsequent activation of this enzyme via a conformational change. To our knowledge, this is the first demonstration that TG-mediated post-translational modification of an enzyme (e.g. PLA2) causes a striking increase in the catalytic activity of the enzyme.