Eleonora Cordella-Miele

Modulation of cellular response to antigens by uteroglobin and transglutaminase.

Inflammation and immunity are intimately related processes and in the vertebrate species most inflammatory responses have an immunological component (1). This concept also applies to transplantation biology where an acute or chronic rejection may be biologically very similar to an inflammatory response, although the basis of rejection of a graft is immunologically mediated. It is now well accepted that in vertebrate species isografts and autografts are usually endured, but allografts and xenografts (heterografts) are uniformly rejected. A few exceptional circumstances exist in nature where an allograft is normally accepted by the host, at least for a prolonged period of time. One of these unusual allograft tolerances in nature occurs routinely during mammalian pregnancy.

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.

Inhibition of Phospholipase A2 by Uteroglobin and Antiflammin Peptides

Blastokinin (1) or uteroglobin (UG; 2) is a secretory protein with a low molecular mass (15.8 kDa). This protein was originally discovered in the rabbit uterine fluid during early pregnancy (1, 2). Its synthesis and secretion in the endometrium are stimulated by progesterone (2–5). Since its discovery in 1967, UG has been one of the most thoroughly studied markers of progesterone action in the rabbit endometrium. In addition to the uterus, UG has been found in several other organs of rabbits, namely, the oviduct, the male genital tract and the tracheobronchial tree of both male and female animals (4–9). More recently, we detected UG in the circulation of the rabbit (10). The source of this protein in circulation seems to be the tracheobronchial epithelium and/or the progesterone-induced uterine endometrium (10).

Lys 43 and Asp 46 in α-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. We previously reported that sPLA2-inhibitory activity of UG may reside in a segment of alpha-helix 3 that is exposed to the solvent. In addition, it has been suggested that UG may inhibit sPLA2 activity by binding and sequestering Ca++, essential for sPLA2 activation. By site-specific mutation, we demonstrate here that Lys 43 Glu, Asp 46 Lys or a combination of the two mutations in the full-length, recombinant human UG (rhUG) abrogates its sPLA2-inhibitory activity. We demonstrate further that recombinant UG does not bind Ca++ although when it is expressed with histidine-tag (H-tag) it is capable of binding Ca++. Taken together our results show that: (i) Lys 43 and Asp 46 in rhUG are critical residues for the sPLA2-inhibitory activity of UG and (ii) Ca++-sequestration by rhUG is not likely to be one of the mechanisms responsible for its sPLA2-inhibitory activity.

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.

Stimulation of phospholipases A2 by transglutaminases.

Among the enzymes catalyzing post-translational modifications of proteins, transglutaminases (TG; EC 2.3.2.13) have been extensively characterized from the enzymological point of view (1–4). Nevertheless, the physiological role(s) of these enzymes, particularly the intracellular TGs, are still poorly understood. TGs are a class of enzymes which catalyze a Ca++-dependent acyl-transfer reaction in which the γ-carboxamide group of a peptide-bound glutamine residue is the acyl-donor (1–4). Primary amino groups of many low-molecular weight amines may act as acyl-acceptors with the formation of mono-substituted γ-carboxamides of peptide-bound glutamic acid. In the absence of small molecular weight amines, TGs catalyze the formation of an e-(γ-glutamyl) - lysine isopeptide bond between endo-γ-glutaminyl and endo-e-lysyl residues in polypeptides (1–4). The latter reaction results in the formation of inter or intramolecular covalent crosslinks. These enzymes have been detected both intra and extracellularly in higher animals including man. The best characterized extracellular TG is variously known as fibrin-stabilizing factor, Laki-Lorand factor or coagulation Factor XIII.

Amino Acid Residues in α‐Helix‐3 of Human Uteroglobin Are Critical for Its Phospholipase A2 Inhibitory Activity

Rabbit blastokinin1 or uteroglobin (UG)2 is a steroid-dependent, multifunctional, secreted protein.3,4 This protein was first discovered in the rabbit uterus during early pregnancy and subsequently found in many other nonreproductive organs. Interestingly, the highest level of UG expression occurs in the rabbit1,2 and human endometrium5 during the progesterone-dominated phase of the ovarian menstrual cycle, when implantation of the embryo in the uterus takes place. This protein is also expressed in many extrauterine tissues, including the thymus, pituitary gland, lungs, gastrointestinal tract, pancreas, mammary gland, prostate, and seminal vesicle.6 UG is also present in the blood7,8 and urine,9 although it is not synthesized in the kidneys. Currently, this protein is known by several names, which are primarily derived from the organ or body fluid in which it is detectable or from the type of xenobiotics with which it interacts. Thus, it is called progesterone-binding protein,10 Clara cell 10-kDa protein,11 urine protein-1,9 polychlorinated biphenyl–binding protein,12 and retinol-binding protein.13 A unified nomenclature is being developed (see Nomenclature Committee Report in this volume). Structurally, UG is a homodimer in which the two identical 70-amino-acid subunits are covalently linked in an antiparallel orientation by two interchain disulfide bonds.14–20 Each monomer consists of four α-helices and one β-turn between α-helix-2 and -3, but there is no β-structure. Recently, we and others have identified a high-affinity binding site (putative receptor) of UG on several cell types.21–23 Through this pathway, UG appears to inhibit cellular motility and invasion of the extracellular matrix,21 suggesting an antichemokinelike property of this protein. Interestingly, human UG (hUG) is encoded by a single copy gene located on chromosome 11q12.3-13.1,24 a region in which a number of candidate disease genes have been mapped by linkage analyses.

Development of Novel, Sensitive, Nonradioactive, Quantitative ELISA‐PCR Methods Potentially Applicable to the Detection of Fetal Cells in Maternal Circulation

The field of DNA diagnostics, including prenatal diagnostics, has been revolutionized by the introduction of rapid DNA amplification methods such as the polymerase chain reaction (PCR) or the ligase chain reaction (LCR). These technologies allow the analysis of very small amounts of genomic DNA for the presence of specific alleles or mutations. Nanogram amounts of RNA from clinical specimens can be analyzed by reverse-transcription PCR (RT-PCR) for the presence of specific transcripts. Additionally, infectious agents including eukaryotes, prokaryotes, and viruses can be easily detected by PCR procedures that amplify specific regions of their genomes. The validation of amplified DNA is generally carried out by hybridization with specific probes, either in solution or after blotting onto membranes. Many of these techniques can be applied to the prenatal diagnosis of genomic defects on fetal cells recovered from maternal blood, and at least one method has been published for the diagnosis of fetal sex by nested PCR on maternal blood samples.’ However, one area in which novel developments are urgently needed is that of nonradiometric techniques for quantitation and validation of amplified DNA. Such techniques are indispensable for the processing of large numbers of samples in clinical laboratories, where the use of 32P-labeled nucleotides and/or membrane blotting can be timeconsuming and expensive and can create occupational and environmental hazards. Several microtiter plate-based nonradiometric techniques for PCR product detection and validation have been described.*-’ However, these techniques use either chemically modified or enzymatically labeled oligonucleotides as primers or probes. Chemically modified oligonucleotides are expensive and cumbersome to prepare, and the use of