Embryo

Atypical Kunitz-Type Serine Proteinase Inhibitors Produced by the Ruminant Placenta

Abstract
Recently an unusual family of genes was identified whose expression was confined to the trophoblast of ruminant ungulate species. The members of this family (the Trophoblast Kunitz Domain Proteins, or TKDPs) are characterized by the presence of one or more similar ~80 residue repeat sequences placed ahead of a Kunitz serine proteinase inhibitor domain. To examine the specificity of the Kunitz moiety, the Kunitz domains of selected TKDPs and a control Kunitz protein, bovine pancreatic trypsin inhibitor (BPTI), were produced as glutathione-S-transferase fusions and their abilities to inhibit six serine proteinases examined. Circular dichroism spectroscopy confirmed that the Kunitz fold was intact. Three of the TKDPs had unusual residues at their P1 "warhead" (ovine TKDP-1, Asn; bovine TKDP-3, Thr; bovine TKDP-5, Ile) and exhibited no measurable inhibitory activity towards any of the proteinases. Three (ovine TKDP-3, bovine TKDP-3, and bovine TKDP-4) lacked the conserved cysteines at residues 14 and 38 that form one of the highly conserved disulfide bonds that are structurally important in all known mammalian Kunitz proteins. Ovine TKDP-3 and bovine TKDP-4 had P1 lysines and inhibited trypsin and plasmin with Ki values only about 10-fold higher than BPTI. Bovine TKDP-2 had a P1 lysine and the three conserved disulfides, but possessed an unusual residue (Asp) at P2. It exhibited no inhibitory activity. These data suggest that the function of the TKDP, like certain Kunitz proteins found in snake venoms, may not be in proteinase inhibition.

Key words: Implantation • Placenta • Trophoblast • Uterus

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