MOLECULAR REPRODUCTION AND DEVELOPMENT 70:262–270 (2005) Identification and Cloning of Caprine Uterine Serpin 1,2 1 3 1 S¸ABAN TEKIN, MARIA B. PADUA, GARY R. NEWTON, AND PETER J. HANSEN * 1Department of Animal Sciences, University of Florida, Gainesville, Florida 2Department of Biology, University of Gaziosmanpas¸a, Tokat, Turkey 3Cooperative Agricultural Research Center, Prairie View A&M University, Prairie View, Texas ABSTRACT The uterine serpins have been des- istics suggesting it is not an inhibitory serpin. Mol. cribed in sheep, cattle, and pigs as a highly diverged Reprod. Dev. 70: 262–270, 2005. group of the large superfamily of serpin proteins that ß 2005 Wiley-Liss, Inc. typically function as serine proteinase inhibitors. Here, the range of species that possess and express a uterine Key Words: caprine; ovine; serpin; uterus; endome- serpin gene is extended to the goat. Sequencing of trium; pregnancy 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 INTRODUCTION serpin (CaUS). The predicted amino acid sequence of The uterine serpins found in sheep (Ing and Roberts, the caprine precursor showed 96%, 82%, 55%, and 1989), cattle (Mathialagan and Hansen, 1996), and pigs 56% identity to OvUS, BoUS, PoUS1, and PoUS2, (Malathy et al., 1990) are part of a large superfamily of respectively. The signal peptide extends from amino proteins of about 500 known members that, prototypi- acids 1 to 25, resulting in a secreted protein of 404 cally, fold into a conserved structure and inhibit serine amino acids and 46,227 Mr (excluding carbohydrate). proteinases through a unique suicide-like mechanism Both the goat and sheep uterine serpins have a nine (Irving et al., 2000; Silverman et al., 2001). Like several amino acid insert in the Helix I region that is not found other members of the serpin family, the uterine serpins in bovine or porcine uterine serpins. A total of 13 amino have functions distinct from proteinase inhibition. The acids in CaUS are different than those for the nearest best studied of these, ovine uterine serpin can inhibit homologue, ovine uterine serpin. One of these is in proliferation of ab T lymphocytes (Segerson et al., 1984; the site of cleavage of the signal sequence, where a Skopets and Hansen, 1993; Skopets et al., 1995; Peltier single nucleotide substitution (G ! C) changed the et al., 2000a) and has been proposed to protect the cysteine for the sheep, bovine, and porcine genes to a conceptus from destruction by the maternal immune serine. In addition, the amino acid at the putative P1– system (Hansen, 1998). The porcine uterine serpins P10 site (the scissile bond for antiproteinase activity) is form complexes with the iron-containing protein, uter- a valine for CaUS, BoUS, PoUS1, and PoUS2 versus oferrin (Baumbach et al., 1986), and may be involved in an alanine for OvUS. The hinge region of all five of metabolism of that protein. the uterine serpins (P17–P9) is distinct from the In the present report, we provide data that extend the consensus pattern for inhibitory sequences and it is range of species in which uterine serpins exist to the goat unlikely, therefore, that the uterine serpins possess (Caprus hircus). Moreover, the coding regions for ovine prototypical proteinase inhibitory activity. The goat and caprine uterine serpin (CaUS) exhibit very similar uterine serpin was immunolocalized to the glandular sequence identity and the caprine protein shows the epithelium of the endometrium from a pregnant nanny same tissue specificity in the uterus as other uterine at day 25 of pregnancy. There was also immunoreac- serpins. tive product in scattered luminal epithelial cells. No immunoreaction product was detected in endometrium MATERIALS AND METHODS from a nanny at day 5 of the estrous cycle. Western RNA Isolation blotting of uterine fluid collected from the pregnant Intercaruncular endometrium was collected from the uterine horn of a unilaterally-pregnant goat revealed uterus of a nanny at day 25 of pregnancy and snap frozen the presence of a protein band at Mr 56,000 that reacted with monoclonal antibody to OvUS. In conclu- sion, the range of species in which uterine serpins are Grant sponsor: USDA NRICGP; Grant number: 2001-35204-10797. present and expressed in the uterus includes the goat in *Correspondence to: Dr. Peter J. Hansen, Department of Animal addition to the previously described sheep, cow, and Sciences, University of Florida, PO Box 110910, Gainesville, FL 32611-0910. E-mail: [email protected]fl.edu pig. In all of these species, the uterine serpin is derived Received 6 July 2004; Accepted 27 August 2004 primarily from glandular epithelium, is secreted into Published online in Wiley InterScience (www.interscience.wiley.com). the uterine lumen, and contains sequence character- DOI 10.1002/mrd.20206 ß 2005 WILEY-LISS, INC. CAPRINE UTERINE SERPIN 263 in liquid nitrogen. Total RNA was isolated from endo- The concentration of the concentrated DNA was deter- metrial tissue samples with the TriPure reagent (Roche mined spectrophotometrically and purity was asses- Biochemicals, Indianapolis, IN). The quantity of RNA sed by electrophoresis as described in the previous was assessed spectrophotometrically and integrity of section. RNA was examined by electrophoresis. Sheep inter- Sequencing of the DNA samples was performed at the caruncular endometrium was collected at 140 days of University of Florida DNA Sequencing Core Laboratory gestation from the pregnant uterine horn of a Rambouil- using ABI Prism BigDye Terminator cycle sequencing let crossbred ewe made unilaterally-pregnant as des- protocols developed by Applied Biosystems (Perkin- cribed earlier (Bazer et al., 1979). The uterus was Elmer Corp., Foster City, CA). The fluorescently-labeled removed after captive bolt stunning and exsanguina- extension products were analyzed on an Applied Bio- tion, and tissue was snap frozen in liquid nitrogen. Total systems Model 373 Stretch DNA Sequencer or 377 DNA RNA was isolated from endometrial tissue samples Sequencer or on a 3100 Genetic Analyzer (Perkin-Elmer). using TRI reagent (Sigma-Aldrich, St. Louis, MO) ac- Double strand sequencing of CaUS cDNA amplified cording to the manufacturer’s protocol. RNA concentra- using primer set 1 was made by using primer set 1 and tion was determined spectrophotometrically. primer set 3 (forward primer, TTT TCA GCC CAA TCT CAC C; reverse primer, GGC ATC TTA ACC ATC GTA) Cloning of Caprine Uterine Serpin synthesized by Integrated DNA Technologies Inc. CaUS cDNA was amplified from total endometrial (Coralville, IA). Partial sequencing (single strand) of 50 RNA by reverse transcription-polymerase chain reac- and 30 ends of CaUS cDNA amplified using primer set 2 tion (RT-PCR) using primer sets 1 (forward primer, CAC was performed using primer set 2. Nucleotide sequences CAT GTC CCA CAG GAG AAT G; reverse primer, CTC were aligned and assembled using programs in the AAC TTG GGG GTT GAG GAC T) and 2 (forward Sequencher 3.0 software package (Gene Codes Corp., primer, TCA GTA GAT AAC AGC GGG CTC C; reverse Ann Arbor, MI). primer, GAA TTG TAC TCT TTT TAT TCA TGG), The consensus sequence of CaUS cDNA (submitted which, based on the sequence for OvUS (Ing and to GenBank Third Party Annotation database and Roberts, 1989; GenBank accession number M21027), assigned accession number TPA: BK005554) was were estimated to produce products of 1,290 and aligned with other uterine serpin sequences (OvUS, 1,400 bp, respectively. The RT-PCR was performed GenBank accession number M21027; BoUS, GenBank using SuperScript One Step RT-PCR kit with Platinium accession number L22095; PoUS1, GenBank accession Taq (Invitrogen, Carlsbad, CA). To verify that PCR pro- number M30315; PoUS2, GenBank accession number ducts were amplified from RNA only, the SuperScript NM213845) using multiple sequence alignment analy- reverse transcriptase was omitted from control reac- sis with the ClustalW program (Higgins et al., 1994) at tions. cDNA synthesis and pre-denaturation reactions the European Bioinformatics Institute (http://www.ebi. were 1 cycle of 508C for 30 min and 948C for 2 min, ac.uk/clustalw/) and with the Genedoc program at http:// respectively. PCR amplification consisted of 35 cycles of www.psc.edu/biomed/genedoc/. The inferred amino acid 948C for 30 sec, 468C for 30 sec, and 728C for 2 min and sequence of CaUS was determined using the TRANS- 1 cycle of 728C for 5 min for final extension. A 2Â reaction LATE program of EXPASY (http://us.expasy.org/) and buffer (containing 0.4 mM of each dNTP and 2.4 mM aligned with inferred amino acid sequences of other MgSO4) supplied in the kit was used in all PCR runs. uterine serpins using ClustalW and Genedoc. The puta- PCR products were separated by electrophoresis using a tive signal sequence was determined by use of SignalP 0.8% (w/v) agarose (Fisher Scientific, Fair Lawn, NJ) gel 3.0 from Center for Biological Sequence Analysis, Tech- in Tris-acetate buffer (40 mM Tris-acetate, 2 mM EDTA) nical University of Denmark (Bendtsen et al., 2004), pH 8.5 containing 0.5 mg/ml ethidium bromide (Sigma- available at http://www.cbs.dtu.dk/services/SignalP/. Aldrich), visualized on an ultraviolet UV translumina- Identification of start codons was achieved using the tor and photographed using either a Polaroid camera TRANSLATE program at EXPASY. The N-glycosyla- and film (Fisher Scientific, Pittsburgh, PA) or a Sony tion sites were found using the NetNGlyc1.0 from Mavica CD400 digital camera (Sony, Japan). Center for Biological Sequence Analysis, Technical Uni- versity of Denmark at http://www.cbs.dtu.dk/services/ Sequencing of Caprine Uterine Serpin NetNGlyc/as. Identification of the putative reactive site Amplicons were gel-purified using the S.N.A.P. gel scissile bond of CaUS (i.e., the P1–P10 site) was based on purification kit from Invitrogen before sequencing.