Proc. Natl. Acad. Sci. USA Vol. 93, pp. 13653–13658, November 1996 Biochemistry

Pepsin-inhibitory activity of the uterine (uterine secretory activity͞aspartic proteinase inhibitor͞progesterone-induced uterine protein͞endometrium–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 , , and during are glyco- both species should produce large quantities of structurally with amino sequences that place them in the similar -inducible products during pregnancy. ( proteinase inhibitor) superfamily of proteins. Hence the studies on uterine serpins have been extended. The inferred sequences for bovine 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 (UABP) and aligns with the first 4 amino 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 A and can weakly inhibit the proteolytic activities of MATERIALS AND METHODS and C (but not D and E). OvUS-1 is the first specific inhibitor of aspartic proteinases to be identified Materials. Porcine gastric pepsin A, bovine spleen in vertebrates and provides another example of a serpin with B, N␣-CBZ-L- p-nitrophenyl ester hydrochloride (CBZ is ‘‘crossover’’ activity. The pregnancy-associated carbobenzoxy), bovine hemoglobin, and CNBr-activated (PAGs), which are secreted by the trophoblast layer of the Sepharose-4B were purchased from Sigma. Recombinant hu- of species and are inactive members of the man and porcine were donated by G. Conner, aspartic proteinase family, can also bind ovUS-1 and may be University of Miami. Recombinant 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 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 ϭ 50,000, 48,000, and 42,000, respectively) are in the serpin cDNA was merged with the 5Ј 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 ϩ two basic glycoproteins, the so-called uterine milk proteins, metrial poly(A) RNA in ␭ZAP 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 ; 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 Tris⅐HCl, pH 9.5͞100 unilaterally pregnant ewes (12). The basic protein fraction of mM NaCl͞5 mM MgCl2) (22). the uterine secretions, which is predominantly ovUS-1, was Microtiter Plate Binding Assay. Proteins (1 ␮g 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 Tris⅐HCl (pH at 25ЊC. 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 ␮g 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 ␮g per well column (1 ϫ 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 ml͞min. Samples (50 ␮l) from antiserum described above (1:10,000 dilution) followed by a each fraction were assayed for pepsin A inhibitory activity. second ( anti- immunoglobulin G conju- Samples (10 ␮l) from the peak protein fractions were analyzed gated to alkaline phosphatase). Bound was assayed by by gel electrophoresis in 12.5% polyacrylamide gels in pres- using p-nitrophenyl phosphate as (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 (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 ␮g), bovine serum albumin, or were prein- passed over the ovUS-1-Sepharose column. Equilibration, cubated with 0.5 ␮g of pepsin A or 5.0 ␮g 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 ␮lat37ЊC 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 SDS͞PAGE 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 ␮l of 1% bovine serum albumin and 0.23 ml bovine pregnancy-associated protein (PAG) 2 (29). The bands of 10% trichloroacetic acid. After centrifugation (10,000 ϫ g; were visualized as described for Western blot analysis. 10 min), 0.2 ml of the supernatant solutions were removed and Determination of Amino-Terminal Sequences. The protein their content of 14C measured. Controls included reactions samples were applied to Prospin cartridges (Applied Biosys- containing no enzyme (to provide background cpm) and ones tems) and sequenced on an Applied Biosystems model 470 without substrate. Inhibition assays on recombinant cathepsin protein sequencer with on-line analysis for phenylthiohydan- toin derivatives. D and recombinant cathepsin E were carried out by the same procedure in 0.2 M sodium acetate (pH 4.5) (24, 25). Inhibition of of Bovine Serum Albumin. Pepsin RESULTS A (0.5 ␮g) in 50 ␮l of saline was incubated for 15 min in the Identification and Cloning of a Second cDNA for Porcine presence or absence of purified ovUS (50 ␮g). Bovine serum Uterine Serpin (poUS-2). When the porcine cDNA library was albumin (0.45 ml of a 1% solution in 0.1 M HCl) was then screened with a poUS-1 cDNA probe (9), several clones added and the incubation continued at 37 C (26). Samples (75 Њ containing a related but clearly distinct cDNA (poUS-2) were ␮ l) were removed at 0, 10, 20, and 30 min, proteolysis was identified (sequence not shown, GenBank accession no. stopped by addition of 10 ␮l of 6 M NaOH, and the X62845). The poUS-2 cDNA was 1400 bp in length and longer products were analyzed by electrophoresis in 15% polyacryl- than poUS-1 by 9 bases due to the presence of three additional amide gels (27). codons that resulted in the insertion of a Leu, a Phe, and a Lys Affinity Chromatography of Sheep Uterine Secretions. Af- in the mature protein at positions, 111, 118, and 124, respec- finity matrices (bovine serum albumin, ovalbumin, and porcine tively. The 5Ј and 3Ј untranslated regions of poUS-1 and pepsin) were prepared by coupling 35 mg of each protein to poUS-2 cDNA were identical in length (63 and 77 bases, 1.5 g of CNBr-activated Sepharose 4B (Pharmacia). Coupling respectively) and in sequence. Similarly, the primary structures efficiency was between 95 and 97%. The immobilized pepsin of the two signal sequences and the predicted sites (30) for retained about 10% of its original activity. Glycine (0.2 M) was signal sequence cleavage (CysϪ26–Gluϩ1) were the same. The used for preparing the glycine-Sepharose column. The col- predicted molecular weights of poUS-1 and -2 were 45,123 and umns were equilibrated with phosphate-buffered saline (PBS; 46,009, respectively, values that were close to that (Mr 45,000) 10 mM, pH 7.4, and 0.15 M NaCl). Ovine uterine secretions of the major in vitro translation product of the poUS mRNA that had been dialyzed against PBS at room temperature (Ϸ1 (8). Overall the two cDNA exhibited 97% identity in nucleo- mg of protein) were applied to the column, which was then tide sequence and 89% identity in inferred amino acid se- washed with PBS until absorption at 280 nm was zero. Bound quence. While poUS-1 possessed four sites for potential N- protein was eluted with sodium phosphate (50 mM, pH 7.4) glycosylation (positions 107, 197, 243, and 315), poUS-2 lacked containing 1 M NaCl. Eluted protein fractions (1 ml) were the one at Asn107. pooled, desalted, freeze-dried, and analyzed by electrophore- Identification and Cloning of a cDNA for Bovine Uterine sis. Serpin. The longest cDNA isolated was 1463 bases in length Western Blot Analysis. The proteins from SDS͞PAGE gels [excluding the poly(A) tail] and had short 5Ј and 3Ј untrans- were transferred to nitrocellulose filters and incubated with lated regions (18 and 68 bases, respectively) flanking an open 5% nonfat dry milk in Tris-buffered saline (TBS, 10 mM, pH reading frame of 1377 bases (data not shown; GenBank 8.0) containing 0.15 M NaCl and 0.05% Tween 20 for2hat accession no. L11627). As has been observed with all the other 25ЊC. The blot was washed three times with TBS and incubated uterine serpins (9, 14), the cDNA had two in-frame initiation with rabbit antiserum to ovUS-1 (13, 14) at a dilution of 1:5000 codons (one beginning at base 19 and the second at base 34). inTBSfor4hat25ЊC. The blot was washed again with TBS The boUS cDNA encoded a polypeptide of 459 amino acids and incubated with anti-rabbit immunoglobulin G conjugated and would include a 25-residue signal sequence. If the first to alkaline phosphatase (Promega) in TBS for2hat25ЊC. in-frame were used for initiation, the molecular After incubation with the second antibody, the blot was briefly weight of the mature protein would be 52,371. Unlike the other washed with TBS and placed in a buffer suitable for display of uterine serpins, there was only a single site for potential Downloaded by guest on September 24, 2021 Biochemistry: Mathialagan and Hansen Proc. Natl. Acad. Sci. USA 93 (1996) 13655

N-glycosylation (Asn243). This site was conserved in poUS-1, loop is represented by the sequence RPF, which is part of a poUS-2, and ovUS-1. conserved motif, RPF(F͞L)LFV that has been implicated in Comparison of the Inferred Amino Acid Sequences of the binding of serpin–proteinase complexes to clearance receptors Uterine Serpins. As expected, bo- and ovUS-1 exhibit consid- (35). erable amino acid sequence similarity (72%), presumably Inhibitory Activities of Uterine Secretions. Uterine secre- reflecting the relatively recent evolutionary divergence of tory proteins flushed from the uteri of cattle, sheep, and pigs cattle and sheep (18, 19). The differences between them and at times when the uterus was under long-term progesterone poUS-1 and -2 are more considerable. All the uterine serpins maintenance were tested for their ability to inhibit porcine have closely similar signal sequences and predicted signal pepsin. Crude uterine flushings of cattle and sheep were able sequence cleavage sites. Several amino acid residues that are to inhibit pepsin activity in a dose-dependent manner (data not critical for maintaining serpin backbone structure of ␣1- shown). Inhibitory activity was only noted in occasional por- antitrypsin (31) are also present in uterine serpins in identical cine samples, was unstable, and was lost upon standing at 4ЊC locations (data not shown). The basic architecture of serpins is for more than a day. highly conserved across evolutionarily diverse organisms (31). Freshly purified ovUS-1 was able to inhibit pepsin A and Therefore, it is possible to recognize the reactive center loop, (pepsin C) activity at both pH 2.0 and 4.5 in the the region that potentially interacts with proteinase, even in standard assay employing 14C-labeled hemoglobin as substrate serpins that appear to lack inhibitory activities. In Fig. 1A, the (Fig. 2). Neither bovine serum albumin nor ovalbumin had any putative reactive center loops of the uterine serpins have been inhibitory activity. A 50% inhibition of 0.5 ␮g of pepsin A aligned with those of six other serpins, three of which are activity at pH 2.0 required about 20 ␮g, an approximately 35-fold molar excess of ovUS-1, whereas a 50% inhibition of known to be inhibitory and whose P1–PЈ1 bond has been defined. The uterine serpins possess the small conserved Thr pepsin C was achieved with about an 8-fold molar excess of ovUS-1. The ability of ovUS-1 to inhibit pepsin is also illus- and Ala residues found at positions P14 and P10 (relative to the trated in Fig. 2C, where preincubation of pepsin (0.5 ␮g) with P1–PЈ1 bond) found in the majority of inhibitory serpins. However, the loop regions of the uterine serpins are clearly 50 ␮g of ovUS-1 effectively prevented proteolysis of albumin. unusual. All have an invariant KVP sequence, which is found The activity of purified ovUS-1 was unstable. Either dialysis in the propeptide of several aspartic proteinases, including or storage of the samples in 0.9% NaCl or PBS at 4ЊC for 48 h pepsinogens A and C (33) (Fig. 1B). Similarly there is a led to complete loss of antipepsin activity. Freeze–thawing was conserved KEVPVVVK sequence downstream of the KVP also deleterious. A visible precipitate of protein became motif, found in poUS-1 and -2 and in ovUS-1 (Fig. 1A). The evident in each stored sample. Therefore, dialysis and purifi- VVVK portion of this motif aligns almost perfectly with the cation were carried out at room temperature, and freshly first 4 amino acids of pepstatin (isovaleryl-Val-Val-statine- purified protein was used in all experiments. No inhibitory activity of uterine secretions was observed toward recombi- Ala-statine; ref. 34). The same valine-rich motif is present in nant human cathepsin D, porcine cathepsin D, or recombinant boUS-1 but is preceded by a 39-residue insertion, which cathepsin E. Activity of the cysteine proteinase cathepsin B incorporates three imperfect KEVPVVVK repeats. In addi- was also unaffected (data not shown). tion, the VVKVP sequence repeated three times in the insert Association of Antipepsin Activity with Purified ovUS. on boUS-1 is present in the propeptide sequences of several Sheep uterine flushings were passed through CM-cellulose at pepsinogens (Fig. 1B). The distal end of the reactive center pH 8.2 to bind ovUS-1 selectively (11–13). The ovUS-1 was eluted with 0.5 M NaCl (12) and subjected to gel filtration on a Superose-12 column. Individual fractions were tested for their ability to inhibit pepsin (Fig. 3A). Antipepsin activity was associated with the small excluded volume peak, which was probably aggregated ovUS-1 (Fig. 3B, lane 2), and with the leading shoulder of the main peak (Fig. 3B, lane 3). The latter, containing the majority of inhibitory activity, provided a single band of protein of Mr 55,000 upon electrophoresis, while fractions within the center and trailing edge of the peak contained polypeptides of smaller size (Fig. 3B, lane 4). Amino acid sequencing of the total protein in each of the three peaks gave the identical sequence EKQQHS, which corresponded to the amino terminus of ovUS-1. No significant secondary signals were observed. Moreover, all protein bands within peak 3 that were detectable by silver staining also reacted with ovUS-1 antiserum on Western blots (data not shown). Thus, the pepsin inhibitory activity of ovine uterine secretion is associated with a specific subfraction of ovUS-1. The material FIG.1. (A) Reactive center loop regions of the uterine serpins and of various inhibitory and noninhibitory serpins. The 39-residue insert in the trailing edge of the peak was presumed to be ovUS-1 that sequence present in the reactive center loop of boUS-1 is shown was partially denatured (11, 12), already cleaved (36–39), or separately to compare all serpin sequences. Conserved residues of present in a stable but latent form, as has been observed for uterine serpins are indicated by boldface type. The following three plasminogen activator inhibitor 1 (40). (human plasminogen activator inhibitor-2, PAI-2; human ␣1- Inhibitory serpins form 1:1 complexes with their partner antitrypsin, hu␣1-AT; and human , AT III) are sequences proteinases, which are often so stable that they can be resolved of inhibitory serpins. Ovalbumin, human angiotensinogen (huAng); electrophoretically in presence of SDS (41). OvUS-1 com- and corticosteroid binding globulin (rabbit CBG) are noninhibitory plexed with pepsin could not be detected in this manner (data serpins. The arrowhead indicates the P1-PЈ1 bond of the not shown). B inhibitory serpins. ( ) Alignment of propeptide amino acid sequences Pepsin Affinity Column Chromatography. of aspartic proteinases from various species (32). The conserved KVP Sheep uterine residues and VVKVP motifs are indicated by boldface type. These flushings were passed over a pepsin affinity column. Electro- motifs (underlined sequences in A) are present in the reactive center phoretic analysis of the protein that bound revealed a single loop region of uterine serpins. The sequences were obtained from band (Mr Ϸ 55,000) that was recognized by ovUS-1 antiserum Swiss-Prot data bank. (Fig. 3C, lane 7). The control affinity matrices did not retain Downloaded by guest on September 24, 2021 13656 Biochemistry: Mathialagan and Hansen Proc. Natl. Acad. Sci. USA 93 (1996)

FIG. 2. Concentration-dependent inhibition of pepsin A and pepsin C activities by purified ovUS-1. (A) pepsin (0.5 ␮g) activity in presence of increasing concentrations of ovUS-1 at pH 2.0 (E)orpH4.5(●). Control protein (ovalbumin, Ⅺ) gave no inhibition. (B) Same as A, except pepsin C(5␮g) was used as proteinase. (C) Electrophoretic analysis of the silver-stained products formed during proteolysis of bovine serum albumin by pepsin A in presence (Right) or absence (Left) of purified ovUS. Bovine serum albumin (4.5 mg) was incubated with pepsin A (0.5 ␮g) that had been preincubated for 15 min at 37ЊC in presence or absence of 50 ␮g of ovUS. Samples (20 ␮g of protein) were removed at specified time points and analyzed by SDS͞PAGE.

any proteins under these conditions. The amino-terminal ovUS-1, whereas ovalbumin and bovine serum albumin were sequence of the eluted protein was that of ovUS-1 (EKQQHS). not. Electrophoretic analysis of the products from explant A second signal (TDNLLKV) of about two-thirds the intensity cultures that were selectively adsorbed by an ovUS-1 affinity of the first was also detected and corresponded to a region column revealed several protein bands in the range of 70–50 within the reactive center loop (Fig. 1A). The cleaved C- kDa, typical of PAGs (28, 29, 43, 44), two of which cross- terminal fragment appears to be stably associated with ovUS reacted with the anti-bovine PAG-2 antiserum (Fig. 4B). since there was no shift in the electrophoretic mobility of the eluted ovUS-1. However, it remains unclear whether the DISCUSSION second peptide resulted from specific cleavage of the P1–PЈ1 bond (in which case P1 would be a residue) or from The serpin family is so named because, among its members, it less specific proteolysis occurring after partial dissociation of contains a range of serine proteinase inhibitors with a similar the inhibitory complex. The reactive center loop of serpins is structural organization. However, earlier failures by this lab- notoriously susceptible to nonspecific proteolytic cleavage oratory to demonstrate inhibitory activity of uterine serpins (41). toward the commoner serine proteinases such as , Binding of ovUS-1 to PAG. PAGs are among the major , and elastase were not unexpected because the secretory products of the placenta of ungulate species (28, 29, reactive center loop region sequences of uterine serpins were 42–44) and, despite lacking evident proteolytic activity, belong atypical. The decision to examine whether uterine serpins to the aspartic proteinase family. To determine whether could inhibit aspartic proteinases was prompted by three PAGs were possible partners for ovUS-1, their binding to the related observations. The first was the KVP motif, present in serpin was examined. Fig. 4A illustrates that both pepsin and the inhibitory propeptide region (residues 4, 5, and 6) of proteins secreted by ovine placental explants were able to bind pepsinogens (ref. 32; Fig. 1B). The second was a sequence

FIG.3. (A) Superose-12 gel filtration chromatography of ovUS-1 purified from ovine uterine secretions and the distribution of pepsin A inhibitory activity. The column was equilibrated and eluted with 0.15 M NaCl. Approximately 1.0 mg of protein was analyzed, and absorption at 280 nm in the eluant was followed. Arrows (I–IV) represent the elution positions of blue dextran, bovine serum albumin, ovalbumin, and cytochrome c.(B) Silver-stained SDS͞PAGE analysis of peak protein fractions of Superose-12 chromatography. Lanes: 1, molecular weight markers; 2–4, 50-␮l aliquots of peak protein fractions 1–3; 5, 10 ␮g of ovUS-1 before Superose-12 chromatography. (C) Silver-stained SDS͞PAGE analysis of proteins purified from ovine uterine secretions by affinity chromatography on a pepsin A-Sepharose affinity column. Lanes: 1, molecular weight markers; 2, 2 ␮g of unfractionated uterine secretions; 3–6, protein fractions eluted from bovine serum albumin, ovalbumin, glycine, and pepsin affinity columns, respectively. Western blot analysis of protein eluted from pepsin affinity column developed with ovUS-1 antiserum is shown in lane 7. Downloaded by guest on September 24, 2021 Biochemistry: Mathialagan and Hansen Proc. Natl. Acad. Sci. USA 93 (1996) 13657

FIG.4. (A) Binding of ovUS-1 to proteins immobilized on surface of microtiter wells. Proteins used for coating on the surface of microtiter plate are printed vertically. Proteins used for binding are shown on the horizontal axis. (B) OvUS-1 specifically binds polypeptides of the aspartic proteinase family released by ovine placental explant cultures. Silver-stained SDS͞PAGE analysis of proteins purified from ovine placental secretions by ovUS-1 affinity column. Lanes: 1, molecular weight markers; 2, 20 ␮g of placental secretory proteins; 3–5, eluates from bovine serum albumin, ovalbumin, and ovUS-1 affinity columns, respectively. Lanes 6 and 7 are Western blots of protein samples in lanes 2 and 5 developed with antiserum to recombinant bovine PAG-2 (44). Arrowheads indicate the protein bands that are cross-reacting with PAG antiserum.

(VVVK) just distal to KVP motif that can probably assume a that only a fraction of the ovUS-1 isolated from uterine configuration similar to that of pepstatin (45). Third, the secretions retains any inhibitory capability. A high proportion VVKVP sequence, which is repeated three times in the insert may already be cleaved (Fig. 3B). Serpins, in general, seem to of boUS-1 reactive loop region, is also found, with only minor be excellent substrates for their companion proteinase, most variations, in most mammalian pepsinogens (44). Exactly how likely because the correct conformation leading to a stable 1:1 these amino acids contribute to pepsin inactivation, if indeed complex is not continually maintained (31, 41). In addition, they do, is not clear, but the uterine serpins now provide, to our conducting the proteinase assays at low pH probably shortened knowledge, the first known examples of inhibitors of aspartic the half-life of the inhibitory complex. In any case, pepsin is proteinases in vertebrates, and it seems likely that the peptide unlikely to be a proteinase encountered by the uterine serpins. regions discussed above occupy the substrate-binding cleft and Based on their apparent preference for aspartic proteinases prevent access of substrate. The inhibitor from the intestinal and their site of synthesis, the most likely natural partners for nematode , which inhibits cathepsins E and D as well as the uterine serpins are the PAGs, a heterogeneous group of pepsin, is not a serpin and the presumed inhibitory region macromolecules secreted in abundance by the placentas of possesses none of the amino acid motifs discussed above (46). sheep, , and probably other ungulate species (28, 29, 42–44). Serpin inhibitory activity is not confined to serine protein- Although related structurally to pepsin and possessing a ases. A serpin coded by virulent pox viruses has been dem- functional peptide binding cleft, the majority of PAGs appear onstrated to be an inhibitor of the cysteine proteinase that to be catalytically inactive (28, 29, 43, 44, 54). Why the mother processes the precursor of interleukin ␤1 (47). Thus, the would find it necessary to complex PAG with a serpin is reactive center loop region, which is quite variable in sequence, unclear, but it may reflect her need to limit the activities of the may have become adapted for a range of very different trophoblast during pregnancy. specificities. The structures of several serpins are now known through We gratefully acknowledge Dr. R. M. Roberts for his advice and x-ray diffraction analysis (36–40, 48, 49), and there are now helpful discussions, thank Laura Paul for technical assistance, Gail sufficient data to allow some serpin structures to be modeled Foristal for editing the manuscript, and Dr. Ben Dunn (University of (50) and the reactive center loops to be identified (see Fig. 1). Florida), Dr. Greg Conner (University of Miami), and Dr. Jordan The P14 threonine, which is conserved in the uterine serpins, Tang (University of Oklahoma) for supplying and for helpful is the critical hinge residue of the reactive center loop of discussion. We are especially indebted to Dr. Tom Quinn (Department inhibitor serpins (40). When poised in the inhibitory confor- of Biochemistry, University of Missouri) for assistance with the mation, the region containing this small amino acid is partially computer modeling of . This work was supported by Grants HD21980 (R.M.R.) from the National Institutes of Health, 91–37203- ␤ inserted into the -sheet superstructure that makes up the bulk 6682 (T.R.H.) and 95–37203-2035 (N.M.) from the U.S. Department of the molecule (51). The mechanism of serine proteinase of Agriculture, and RB94-108 from the University of Missouri Re- inhibition involves the formation of a covalent bond between search Board (N.M.). This paper is contribution 12,036 from the inhibitor and proteinase (52), with hydrolysis of the peptide Missouri Agricultural Experiment Station Journal Series. bond between the P1 and PЈ1 residues occurring only slowly (38). As cleavage occurs, the loop amino acids on the amino- 1. Roberts, R. M. & Bazer, F. W. (1980) in Steroid-Induced Uterine terminal side of the cleavage site undergo a major conforma- Proteins, ed. Beato, M. (Elsevier͞North–Holland, New York), tional change as the segment pivots about the hinge and swings pp. 133–149. downward to become inserted in the body of the molecule (39, 2. Roberts, R. M. & Bazer, F. W. (1988) J. Reprod. Fertil. 82, 40, 53). Between binding and subsequent release from the 875–892. pepsin affinity column, the loop region of ovUS-1 had clearly 3. Roberts, R. M., Raub, T. J. & Bazer, F. W. (1986) Fed. Proc. 45, 2513–2518. become cleaved between His and Thr (Fig. 1), but it is unclear 4. Trout, W. E., Hall, J. A., Stallings-Mann, M. L., Galvin, J. A., whether this bond corresponded to the P1–PЈ1 warhead site and Anthony, R. V. & Roberts, R. M. (1992) Endocrinology 130, whether strand insertion had occurred. 2557–2564. The amount of ovUS-1 needed to inhibit pepsin clearly 5. Stallings-Mann, M. L., Trout, W. E. & Roberts, R. M. (1993) exceeds that required to provide a 1:1 stoichiometry. This may Biol. Reprod. 48, 998-1005. be due to inactivation of the ovUS-1 by pepsin. Fig. 3A suggests 6. Simmen, F. A. & Simmen, R. C. M. (1991) Biol. Reprod. 44, 1–5. Downloaded by guest on September 24, 2021 13658 Biochemistry: Mathialagan and Hansen Proc. Natl. Acad. Sci. USA 93 (1996)

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