We have synthesized oligopeptides corresponding to a region of high amino acid sequence similarity between two phospholipase A2 (PLA2) inhibitory proteins: Uteroglobin(UG) and lipocortin-1(lip-1). These novel peptides (antiflammins) are potent PLA2 inhibitors in vitro and very potent antiiflammatory agents in vivo suggesting possible therapeutic applications. Additionally, these peptides inhibit collagen and thrombin induced platelet aggregation at micromolar concentrations. The mechanism of action of these oligopeptides seems to be by interaction with the active site of the PLA enzyme. In order to obtain UG at a preparative scale for further studies we reconstructed the plasmid cloning vector pLE- 101 and pLE-102 (described last year) so that we can obtain higher level of expression than was possible by using the above plasmids. This has been achieved and the new plasmids pLE 103-1, when introduced into a bacterial strain BL21 (DE-3) and induced with IPTG produced 20-30 mg of UG per ml of bacterial culture. The recombinant UG seems to be a dimer as normally secreted by the rabbit endometrial and tracheobronchial epithelium. To our knowledge, this is the first report of a dimeric mammalian protein that has been expressed in a bacterial host in its natural dimeric form. The full characterization of this recombinant protein is now under way. The successful development of this expression system paves the way for future site-directed mutagenesis studies to delineate the structural-functional relationships of this protein. We have also discovered that major N-terminal fragments of Xenopus antimicrobial peptides are potent PLA2 inhibitors in vitro and antiiflammatory agents in vivo suggestings that these antimicrobial peptides degrade to produce antiinflammatory agents. The presence of UG-like protein is now confirmed in three human organs eg. tracheobronchus, uterus, and the prostate. The prostatic and tracheobronchial protein have been partially purified and preliminary results suggest that these proteins are also PLA2 inhibitors as rabbit UG. Using cDNA probes for PLA2, UG and lip- 1 the regulation of expression of these genes is being investigated in established cell lines from several organs. In a clinical study an inverse relationship between the level of UG-like protein and leukotriene C4 has been found. During viral functional of the upper and lower respiratory tracts this inverse relationship was even more pronounced. Studies, currently in progress, may delineate a cause and effect relationship between UG-like protein and proinflammatory leukotriene C4 in the human tracheobronchial mucosa.
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