Pepsin-Inhibitory Activity of the Uterine Serpins
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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 13653–13658, November 1996 Biochemistry Pepsin-inhibitory activity of the uterine serpins (uterine secretory activityyaspartic proteinase inhibitoryprogesterone-induced uterine proteinyendometrium–trophoblast interaction) NAGAPPAN MATHIALAGAN*† AND THOMAS R. HANSEN‡ *Department of Animal Sciences, University of Missouri, Columbia, MO 65211; and ‡Department of Animal Sciences, University of Wyoming, Laramie, WY 82071 Communicated by Michael Roberts, University of Missouri, Columbia, MO, September 19, 1996 (received for review June 20, 1996) ABSTRACT Among the major products secreted by the distinct (20). Therefore, it was of considerable interest that uteri of cattle, sheep, and pigs during pregnancy are glyco- both species should produce large quantities of structurally proteins with amino acid sequences that place them in the similar progesterone-inducible products during pregnancy. serpin (serine proteinase inhibitor) superfamily of proteins. Hence the studies on uterine serpins have been extended. The inferred amino acid sequences for bovine uterine serpin Herein we demonstrate that these uterine serpins interact with (boUS-1) and ovine uterine serpin (ovUS-1) exhibit about 72% members of the aspartic proteinase family rather than with sequence identity to each other but only about 50% and 56% serine proteinases. They provide another example of serpins identity, respectively, to two distinct porcine uterine serpins with crossover function. (poUS-1 and poUS-2). Despite these differences in primary Because various acronyms were used for these uterine structure, the uterine serpins possess well-conserved reactive serpins before their general relatedness was revealed by mo- center loop regions that contain several motifs present in the lecular cloning studies, it is proposed that the previous desig- propeptide regions of pepsinogens. One such motif, VVVK, nations [e.g., uteroferrin-associated basic protein (UABP) and aligns with the first 4 amino acids of the aspartic proteinase uterine milk protein (UTMP)] be abandoned and instead they inhibitor pepstatin. Although no inhibitory activity toward should be named uterine serpins (or US) preceded by the any serine proteinase has been found, at least one of the species name, e.g., bovine (bo), ovine (ov), and porcine (po). uterine serpins, ovUS-1, can bind specifically to immobilized pepsin A and can weakly inhibit the proteolytic activities of MATERIALS AND METHODS pepsin A and C (but not cathepsins D and E). OvUS-1 is the first specific inhibitor of aspartic proteinases to be identified Materials. Porcine gastric pepsin A, bovine spleen cathepsin in vertebrates and provides another example of a serpin with B, Na-CBZ-L-lysine p-nitrophenyl ester hydrochloride (CBZ is ‘‘crossover’’ activity. The pregnancy-associated glycoproteins carbobenzoxy), bovine hemoglobin, and CNBr-activated (PAGs), which are secreted by the trophoblast layer of the Sepharose-4B were purchased from Sigma. Recombinant hu- placentas of ungulate species and are inactive members of the man and porcine cathepsin D were donated by G. Conner, aspartic proteinase family, can also bind ovUS-1 and may be University of Miami. Recombinant cathepsin E was a gift from the natural target partners for the uterine serpins. B. Dunn, University of Florida, Gainesville, FL. Pepsin C was provided by J. Tang, University of Oklahoma, Oklahoma City, 14 The porcine uterus produces large quantities of several pro- OK. [ C]Formaldehyde was obtained from American Radio- 14 teins in response to progesterone, the hormone of pregnancy labeled Chemicals, St. Louis. [ C]Hemoglobin was prepared (1, 2). These proteins are secreted into the uterine lumen and, by the procedure described by Means and Feeney (21). during pregnancy, contribute to the so-called histotrophe or Screening of Porcine Endometrial cDNA Library. About uterine milk that bathes the conceptuses. Among them are 40,000 recombinant phages from a porcine endometrial cDNA uteroferrin (3) and a retinol-binding protein (4, 5) (both of library were screened with a random-primed poUS-1 cDNA which probably have nutritional roles), growth factors, growth probe (9, 22). factor binding proteins (6), a group of low molecular weight Phage DNA was isolated from 10 positive plaques. The sizes proteinase inhibitors belonging to the Kunitz family (7), and of cDNA inserts ranged from 400 bp to 1250 bp. Clone 12.1 three related basic glycoproteins known collectively as the cDNA (1250 bp) was subcloned and sequenced (23). To obtain a full-length cDNA (1400 bases), the sequence of clone 12.1 uteroferrin-associated basic proteins (8, 9). The latter (Mr 5 50,000, 48,000, and 42,000, respectively) are in the serpin cDNA was merged with the 59 sequence of a previously superfamily and arise by proteolytic processing and differential reported poUS clone (2.1) (9). This sequence was confirmed glycosylation of a larger precursor molecule (9). from the genomic sequence of poUS-2 (data not shown). The uterus of the ewe also synthesizes abundant amounts of Screening of Bovine Endometrial cDNA Library. The bo- vine library was constructed from day 17 pregnant cow endo- progesterone-induced secretory protein composed largely of 1 two basic glycoproteins, the so-called uterine milk proteins, metrial poly(A) RNA in lZAP vector (Stratagene). The library was amplified in XL1-blue cells, and about 106 plaques which have a Mr of 57,000 and 55,000, respectively (10–13), and 32 which are also in the serpin superfamily (14). These ovine were screened with a P-labeled full-length ovUS-1 cDNA. uterine serpins (now called ovUS) have been reported to be About 20 positives were plaque-purified, and ones with the immunosuppressive (15, 16) and may prolong the ability of skin largest inserts were identified by PCR with M13r and M13f grafts to survive within the uterus (17). They have not been primers. Clone 1.38 (1.4 kb) was selected for further charac- shown to possess any antiproteinase activity (14). terization. The plasmid was excised from the phage by in vivo Although pigs and sheep are both ungulate species, their excision and sequenced in both directions. ancestors diverged at least 55 million years ago (18, 19). Moreover, the types of placentation they exhibit are quite Abbreviations: bo, bovine; ov, ovine; PAG, pregnancy-associated glycoprotein; po, porcine; US, uterine serpin. Data deposition: The sequences reported in this paper have been The publication costs of this article were defrayed in part by page charge deposited in the GenBank data base (accession nos. X62845 and payment. This article must therefore be hereby marked ‘‘advertisement’’ in L11627). accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed. 13653 Downloaded by guest on September 24, 2021 13654 Biochemistry: Mathialagan and Hansen Proc. Natl. Acad. Sci. USA 93 (1996) Purification of ovUS. Sheep uterine milk was collected from alkaline phosphatase activity (100 mM TriszHCl, pH 9.5y100 unilaterally pregnant ewes (12). The basic protein fraction of mM NaCly5 mM MgCl2) (22). the uterine secretions, which is predominantly ovUS-1, was Microtiter Plate Binding Assay. Proteins (1 mg in 0.2 ml of obtained by chromatography on CM-cellulose at pH 8.2 (12). PBS) were allowed to bind to microtiter well surfaces for 12 h Protein was eluted with 0.5 M NaCl in 10 mM TriszHCl (pH at 258C. Remaining sites were blocked with 2% nonfat dry milk 8.2) and dialyzed for 6 h against three changes of 0.9% NaCl for 1 h. OvUS-1 (1 mg in 0.2 ml of PBS) was then allowed to at room temperature. About 1.0 mg of the eluted protein from bind to the adsorbed proteins in presence or absence of control CM-cellulose was further chromatographed on a Superose-12 proteins (bovine serum albumin or uteroferrin; 5 mg per well column (1 3 30 cm, Pharmacia), equilibrated with 0.9% NaCl for 1 h). Bound ovUS-1 was detected by using the anti-ovUS-1 and eluted at a flow rate of 0.5 mlymin. Samples (50 ml) from antiserum described above (1:10,000 dilution) followed by a each fraction were assayed for pepsin A inhibitory activity. second antibody (goat anti-rabbit immunoglobulin G conju- Samples (10 ml) from the peak protein fractions were analyzed gated to alkaline phosphatase). Bound enzyme was assayed by by gel electrophoresis in 12.5% polyacrylamide gels in pres- using p-nitrophenyl phosphate as substrate (Sigma). ence of SDS (12). OvUS Affinity Chromatography of Ovine Placental Secre- Enzyme Inhibitory Activity Measurements. Inhibitory ac- tions. Secretory proteins were collected by in vitro incubation tivity of ovUS-1 toward pepsin A and pepsin C (gastriscin) was of explants prepared from day 100 sheep placenta (28). determined by using [14C]methyl-hemoglobin as substrate Purified ovUS-1 (25 mg) was coupled to CNBr-activated (24). Increasing protein concentrations of purified ovUS-1 Sepharose 4B. About 1 mg of placental secretory proteins were (1–250 mg), bovine serum albumin, or ovalbumin were prein- passed over the ovUS-1-Sepharose column. Equilibration, cubated with 0.5 mg of pepsin A or 5.0 mg of pepsin C in 0.9% washing, and elution of the affinity column were carried out as NaCl in water in a total volume of 50 mlat378C for 15 min. described earlier for the pepsin affinity column. Bovine serum After the preincubation, 0.1 ml of hemoglobin [0.25% labeled albumin- and ovalbumin-Sepharose matrices were used as hemoglobin in 0.2 M sodium citrate (pH 2.0) or in 0.2 M controls. Polypeptides in the flow-through and eluted fractions sodium acetate (pH 4.5)] was added, and the incubation were analyzed by SDSyPAGE and Western blot analysis with continued for 30 min. At 30 min, the reactions were terminated a rabbit antiserum (diluted 1:1000) raised against recombinant by addition of 20 ml of 1% bovine serum albumin and 0.23 ml bovine pregnancy-associated protein (PAG) 2 (29).