Ruminant trophoblast Kunitz domain proteins

The overall aim of this research is to improve the understanding of maternal-fetal interactions during pregnancy in domestic ruminants. In particular, to characterize the expression and possible actions of a novel sub-family of Kunitz-type proteinase inhibitors produced by the conceptus. Each member of the family contains a classical Kunitz domain at its carboxyl terminus, preceded by one or more slightly hydrophobic domains that resemble no previously known protein motifs. The expression of the first ovine clone was detected only in trophectoderm and was thus termed, Ovine Trophoblast Kunitz: Domain Protein (ovTKDP-1). Subsequently, the bovine homologue of ovTKDP-1 has been identified, as have an additional four distinct cDNA encoding bovine TKDP. Preliminary characterization of boTKDP expression indicates that each has a distinct, though sometimes overlapping, expression, relative to the others. The fact that each demonstrates peak expression at different periods of placental development, along with subtle structural differences between proteins, suggests that each TKDP may play a unique role in maternal-fetal communication.;The present experiments describe the cloning of ovTKDP-4 and ovTKDP-5. The nucleotide and amino acid sequences of five members from both ovine and bovine TKDP families are compared by phylogenetic analysis in order to provide insights into the duplication and subsequent evolution of the TKDP genes. The expression patterns of the ovine TKDP were investigated by ribonuclease protection assay. The majority of these TKDP were detected only in early pregnancy (days 15--17), corresponding to the timing of blastocyst expansion and attachment to the uterine lining.;This work also describes the production of ovTKDP-(1 and 3) and boTKDP-(2,3,4, and 5) Kunitz domain recombinant proteins as fusions to glutathione-S-transferase. The inhibitory specificity of these TKDP was tested against a battery of serine proteinases. Two of these proteins, ovTKDP-3 and boTKDP-4 are shown to be active inhibitors of the serine proteinases plasmin and trypsin. The TKDP contain a variety of P1 or "warhead" residues of unusual distribution that might function against an array of proteinases. A mechanism by which the TKDP protect the placenta from proteinases produced by cytotoxic T cells of the maternal immune system is presented.