Identification and cloning of caprine uterine serpin

Authors


Abstract

The uterine serpins have been described in sheep, cattle, and pigs as a highly diverged group of the large superfamily of serpin proteins that typically function as serine proteinase inhibitors. Here, the range of species that possess and express a uterine serpin gene is extended to the goat. Sequencing of cDNA amplified from total RNA from a pregnant goat at day 25 of pregnancy resulted in a 1,292 bp full-length consensus cDNA sequence for caprine uterine serpin (CaUS). The predicted amino acid sequence of the caprine precursor showed 96%, 82%, 55%, and 56% identity to OvUS, BoUS, PoUS1, and PoUS2, respectively. The signal peptide extends from amino acids 1 to 25, resulting in a secreted protein of 404 amino acids and 46,227 Mr (excluding carbohydrate). Both the goat and sheep uterine serpins have a nine amino acid insert in the Helix I region that is not found in bovine or porcine uterine serpins. A total of 13 amino acids in CaUS are different than those for the nearest homologue, ovine uterine serpin. One of these is in the site of cleavage of the signal sequence, where a single nucleotide substitution (G → C) changed the cysteine for the sheep, bovine, and porcine genes to a serine. In addition, the amino acid at the putative P1–P1′ site (the scissile bond for antiproteinase activity) is a valine for CaUS, BoUS, PoUS1, and PoUS2 versus an alanine for OvUS. The hinge region of all five of the uterine serpins (P17–P9) is distinct from the consensus pattern for inhibitory sequences and it is unlikely, therefore, that the uterine serpins possess prototypical proteinase inhibitory activity. The goat uterine serpin was immunolocalized to the glandular epithelium of the endometrium from a pregnant nanny at day 25 of pregnancy. There was also immunoreactive product in scattered luminal epithelial cells. No immunoreaction product was detected in endometrium from a nanny at day 5 of the estrous cycle. Western blotting of uterine fluid collected from the pregnant uterine horn of a unilaterally-pregnant goat revealed the presence of a protein band at Mr ∼56,000 that reacted with monoclonal antibody to OvUS. In conclusion, the range of species in which uterine serpins are present and expressed in the uterus includes the goat in addition to the previously described sheep, cow, and pig. In all of these species, the uterine serpin is derived primarily from glandular epithelium, is secreted into the uterine lumen, and contains sequence characteristics suggesting it is not an inhibitory serpin. Mol. Reprod. Dev. 70: 262–270, 2005. © 2005 Wiley-Liss, Inc.

Ancillary