Avian oviducts synthesize a tissue specific proteinase inhibitor, ovostatin. The quaternary structure and mechanism for proteinase inhibition are similar to those of a plasma protein, Alpha2-macroglobulin. The long term objectives of the proposed research are: 1) to understand the evolution of structural genes for ovostatin and Alpha2-macroglobulin and 2) to elucidate the possible biological functions of ovostatin during the development of the embryo.
Specific aims for structural and evolutionary studies are to re-examine """"""""trap"""""""" hypothesis as a mechanism for ovostatin to inhibit metalloproteinases, and to deduce the primary structure of chicken ovostatin from cDNA sequencing. The """"""""trap"""""""" hypothesis will be examined by using the differential kinetic labeling technique in order to postulate a geographical arrangement of the enzyme molecule in the enzyme-inhibitor complex. The deduction of the primary structure of chicken ovostatin involves isolation of ovostatin mRNA, construction, cloning and sequencing of DNA complementary to ovostarin mRNA. Evolutionary aspects of a thiolester bond will be investigated. Although chicken ovostatin does not contain a thiolester bond, duck and goose ovostatins do, as does Alpha2-macroglobulin. Such a bond is also found in sea urchin protein with Mr 125,000. The primary structures near the thiolester bond of these proteins will be sequenced and compared with the corresponding sequences of chicken ovostatin, human Alpha2-macroglobulin and complement proteins C3 and C4 in order to understand the evolutionary relationship of these proteins. Biological functions of ovostatin are not known. The possible involvement of ovostatin in connective tissue remodeling during embryogenesis will be examined. Chick fibroblasts and embryonic cells will be examined for the presence of ovostatin receptors with binding assays and receptors will be localized in embryos at various stages with autoradiography and the immunofluorescence localization. Metalloproteinase(s) secreted from the chick fibroblasts in response to stimulation with ovostatin-proteinase complexes will be characterized by their specificities against connective tissue substrata such as fibronectin, laminin, proteoglycans and various collagen types. Such enzymes may play a key role in tissue remodeling that accompanies development of the embryo.
