Reviews of Reproduction (1998) 3, 62–69

Pepsin-related molecules secreted by trophoblast

Jonathan A. Green, Sancai Xie and R. Michael Roberts

Departments of Animal Sciences, Biochemistry, and Veterinary Pathobiology, University of Missouri–Columbia, Columbia, MO 65211–0001, USA

The pregnancy-associated glycoproteins (PAGs) were first described as placental antigens of cattle that were also present in the blood serum of the mother after implantation. Molecular cloning studies have shown that they are members of the aspartic proteinase gene family and closely related to the pepsinogens. An enzymatic role seems unlikely, as at least some of them have mutations likely to render them enzymatically inactive. Nevertheless, these molecules have retained the substrate-binding cleft of the pepsins and are expressed abundantly in trophectoderm, particularly in the invasive binucleate cell component. There may be as many as 100 PAG genes in cattle and sheep, many of which are transcribed. PAGs are also products of the placenta of the pig, a species whose progenitors diverged from the ruminants at least 55 million years ago. There is even evidence for PAG-like molecules outside the Artiodactyla. Although their function remains elusive, it seems unlikely that these placentally expressed mol- ecules are simply oddities in view of their long-term evolutionary survival and conspicuous presence at the fetal–maternal interface.

The placentation that occurs in ruminant ungulate species, such Identification of placental-specific antigens in ruminants as cattle, sheep and goats, is superficial, relatively noninvasive, and can be referred to as synepitheliochorial cotyledonary In 1982, Sasser and coworkers (Butler et al., 1982) injected ex- (Wooding, 1992). ‘Synepitheliochorial’ describes the fetal– tracts of homogenized bovine cotyledons into rabbits with the maternal syncytium formed by the fusion of trophoblast binu- aim of raising antisera with placental specificity. Antibodies cleate cells and uterine epithelial cells, whereas ‘cotyledonary’ that were not directed against placenta were adsorbed out with describes the gross structure of the placenta and specifically the tissue extracts from nonpregnant animals. The cleared anti- tufts of villous trophoblast (cotyledons) that insinuate them- serum was then used to monitor the partial purification of selves into the crypts of the maternal caruncles. These regions two pregnancy-specific antigens, pregnancy-specific protein A of fused fetal and maternal tissue are referred to as placentomes (PSP-A) and pregnancy-specific protein B (PSP-B). The former and are the main sites for nutrient and gas exchange in the was α-fetoprotein; however, PSP-B represented a novel antigen. placenta. Subsequent studies indicated that PSP-B probably represented The trophectodermal (chorionic) epithelium of the ruminant a mixture of related proteins since multiple molecular weight placenta contains two distinct cell populations: binucleate cells, forms (47 000–90 000) and isoelectric variants (pI 3.7–4.4) were which represent 15–20% of the total cell population within the reported (Butler et al., 1982; Sasser et al., 1989). The placental ex- mature placenta, and mononucleate cells, which comprise pression of PSP-B was restricted to the binucleate cells described the rest (Wooding, 1983). Binucleate cells have two unique above (Eckblad et al., 1985). The antigen was also detectable in characteristics. First, they migrate from the trophectoderm and maternal blood (Sasser et al., 1986), probably as a result of dis- fuse with uterine epithelial cells to form a syncytium. In sheep, charge from secretory granules subsequent to trinucleated cell the syncytium is rather extensive and persists throughout preg- formation. The radioimmunoassay with the anti-PSP-B anti- nancy. However, in cattle, the syncytium that is formed during serum developed by Sasser and his colleagues provided an ac- implantation is subsequently replaced by an intact epithelium curate pregnancy test for cattle as early as 30–35 days after by day 40. After this, the migration and fusion of binucleate insemination (Humblot et al., 1988). In some animals, PSP-B cells is limited to the generation of short-lived trinucleated cells could even be detected in maternal serum before day 20 of preg- (Fig. 1; Wooding, 1992). This fusion event is the extent of in- nancy. Antigens related immunologically to PSP-B were also vasive implantation in these species, but it allows the conceptus found in the blood of pregnant sheep (Ruder et al.,1988), mule to establish a more direct endocrinological, and presumably deer, white tail deer (Wood et al., 1986), and muskoxen (Rowell immunological, dialogue with the mother than is observed in et al., 1989) and appear to be characteristic of all ruminants. species such as the pig, where no erosion of the uterine epi- Zoli et al. (1991) reported the purification of a pregnancy- thelium occurs. Indeed, the second unique characteristic of the specific antigen they named pregnancy-associated glycoprotein binucleate cells is their ability to deliver their secretory products, or PAG. Again, antisera were raised against antigens specific to which are concentrated in dense granules, directly to the ma- fetal cotyledons and used to monitor the purification steps of ternal system. Within these dense secretory granules are the PAG. PAG is an acidic glycoprotein (pI 4.4–5.4) with a mol- placental lactogens (Duello et al., 1986) as well as many of the ecular weight of 67 000. Like PSP-B, it was first detectable in the pregnancy-associated glycoproteins, the subject of this review. maternal circulation of some cows at around the time that © 1998 Journals of Reproduction and Fertility 1359-6004/98 $12.50 Downloaded from Bioscientifica.com at 10/02/2021 09:03:44AM via free access Pepsin-related molecules secreted by trophoblast 63

(b) Dividing cell

Tight junctions 1 Trophectoderm 2 Microvillar junction 3 4 Uterine Fetal villi cryptal epithelium (a)

Maternal capillary Sheep, goat Exocytosis

(c)

Cow 1 5 Maternal crypt Trophectoderm 2 Trinucleate 4 3 cells Uterine cryptal epithelium

Exocytosis

Maternal capillary

Fig. 1. The binucleate cell, its contribution to the definitive ruminant placenta and its endocrine characteristics. (a) Part of a mature placentome showing the cotyledonary fetal villi penetrating the crypts of the maternal caruncle. The region is heavily vascularized on both sides of the junction. (b) In sheep and goats, a fetomaternal syncytium is formed as the result of binucleate cell migration (stages 1 and 2) and fusion (stages 3 and 4); secretory granules release their contents basolaterally. After day 40 of pregnancy, bovine binucleate cell migration (c) pro- duces short-lived trinucleate cells (stage 3) which, after exocytosis, are resorbed by the trophectoderm (stages 4 and 5). The granules of binu- cleate cells contain placental lactogens and PAGs that can gain access to maternal capillaries. Presumably, PAGs expressed by mononucleate cells are secreted at the maternal–fetal interface. (Diagram adapted from Wooding, 1992.) the trophoblast formed definitive attachment to the uterine wall. active site that would likely interfere with the catalytic mechan- Concentrations increased gradually thereafter, and reached peak ism, bovine and ovine PAG-1 are probably not capable of acting values just before parturition at about 1 µg ml–1 (Zoli et al., 1992). as enzymes. This feature is discussed in greater detail below. Soon after, PAG and PSP-B were determined to be similar in The expression of PAG-1 is restricted to binucleate cells amino acid sequence (Lynch et al., 1992). within the outer epithelial layer of the placenta (Xie et al., 1991), Xie et al. (1991) cloned PAG (now known as PAG-1) from where it colocalizes with a placental lactogen, and also with late bovine and ovine placenta by screening cDNA libraries another antigen recognized by the monoclonal antibody, SBU-3 with an anti-PAG antiserum. The bovine and ovine cDNAs en- (Fig. 2). The latter recognizes a carbohydrate epitope restricted coding PAG-1 shared 86% nucleotide sequence identity with to binucleate cells (Lee et al., 1986). one another and encoded proteins of 380 and 382 amino acids, respectively, including a 15 amino acid signal sequence. Aspartic proteinases However, protein sequence data already showed that the first amino acid of bovine PAG-1 was an arginine that corresponded The aspartic proteinases, to which the PAGs are related in to one at position 39, downstream of the site of signal sequence structure, are an ancient family of enzymes. Those from higher cleavage, indicating that PAG-1 undergoes post-translational organisms, such as the digestive enzyme, pepsin, or the more processing from a pro-form. specialized hormone-processing proteinase, renin, consist of a The most surprising feature of bovine and ovine PAG-1 re- single polypeptide chain folded to form a bilobed structure vealed by the cloning experiments was that they belonged to a (Davies, 1990). In contrast, the retroviral forms are dimers of family of proteolytic enzymes known as aspartic proteinases. In two identical subunits. Consequently, the eukaryotic enzymes particular, they had greatest sequence identity with pepsino- probably arose by a duplication of a progenitor gene (Tang and gens (Table 1). However, owing to mutations in and around the Wong, 1987). In these enzymes, a pair of essential aspartic acid

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Table 1. Comparison of amino acid sequence identities between representative pregnancy-associated glycoproteins (PAGs) and aspartic proteinases

pPEP A rhPEP boPAG-1 ovPAG-1 boPAG-2 ovPAG-2 pPAG-1 pPAG-2 ePAG rPEP F hCath D hCathE pPEP A 100 rhPEP 86 100 boPAG-1 48 49 100 ovPAG-1 48 50 73 100 boPAG-2 50 51 58 59 100 ovPAG-2 49 49 59 60 65 100 pPAG-1 49 49 49 48 50 53 100 pPAG-2 51 52 54 53 57 57 64 100 ePAG 56 56 52 53 54 52 54 57 100 rPEP F 56 57 51 50 53 58 56 58 69 100 hCath D 49 46 40 40 40 45 36 40 42 44 100 hCath E 54 54 42 42 44 44 43 46 46 46 52 100 pPEP A: pig pepsinogen A; rhPEP: rhesus monkey pepsinogen A; boPAG-1: bovine PAG-1; ovPAG-1: ovine PAG-1; boPAG-2: bovine PAG-2; ovPAG-2: ovine PAG-2; pPAG-1: pig PAG-1; pPAG-2: pig PAG-2; ePAG: equine PAG; rPEP F: rabbit pepsinogen F; hCath D: human cathepsin D; hCath E: human cathepsin E.

Fig. 2. Immunoperoxidase localization of bovine pregnancy-associated glycoprotein 1, bovine placental lactogen, and SBU-3 antigen in serial sections of a day 200 bovine placentome. (a) Cross-section of fetal cotyledonary villi showing binucleate cells immunostained with a monoclonal antibody for SBU-3 antigen. CV, fetal cotyledonary villi; END, uterine endometrium. (b) Bovine PAG-1-secreting binucleate cells of fetal cotyledonary villi. (c) Bovine placental lactogen-positive binucleate cells. Only background staining was observed in control sections in which the primary antibodies were omitted or substituted with normal rabbit serum (data not shown). In these sections, the cotyledonary villi have pulled away from the maternal caruncular tissues. Most of the immunopositive cells remain associated with the fetal villi. Antisera were generously donated by J-F. Beckers, University of Liège, Belgium (bovine PAG-1); M. R. Brandon, University of Melbourne, Australia (SBU-3); and R. V. Anthony, University of Missouri (bovine placental lactogen). Scale bar represents 50 µm. residues, one from each lobe, face each other across the sub- into the active site cleft, inhibiting the access of substrate. It is strate-binding cleft. The cleft is approximately 4 nm long and removed either autocatalytically, after a fall in pH, or by the can accommodate a segment of substrate seven or eight amino action of another proteolytic enzyme (Marciniszyn et al., 1976b; acids long. The catalytic mechanism, which leads to peptide James and Sielecki, 1986; Koelsch et al., 1994). bond cleavage in the middle of the bound peptide, is initiated through nucleophilic attack by an hydroxyl ion supplied by a Relationship between bovine and ovine PAG-1 and the aspartic water molecule strategically positioned between the two as- proteinases partic acids (James et al., 1985; Szecsi, 1992). The side chains of the amino acids that line the cleft govern binding specificity PAG-1 from cattle and sheep most resembles pepsinogen A towards the substrate. As a result, enzymes such as pepsin or (approximately 50% at the amino acid level; Table 1). However, cathepsin D have broad peptide bond preferences, while others neither bovine nor ovine PAG-1 is likely to function as a pro- such as renin have narrowly defined activities. Naturally oc- teinase because of critical amino acid changes within the highly curring peptide inhibitors, including pepstatin, and inhibitors conserved residues that form the active site of these molecules engineered by the pharmaceutical industry are designed to fit (Xie et al., 1991; Guruprasad et al., 1996). In ovine PAG-1, the snugly into the substrate binding cleft without being cleaved catalytically essential aspartic acid within the C-terminal lobe (Marciniszyn et al., 1976a; Kay and Dunn, 1992). of the molecule (D215, pepsin numbering) has been mutated to Another feature of eukaryotic aspartic proteinases is that a glycine (Fig. 3). A similar change in the analogous aspartic they are synthesized as inactive zymogens. An amino terminal acid within the amino terminal lobe of pepsin eliminates all propeptide, generally 40–50 amino acids long, becomes folded activity (Lin et al., 1989).

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NH2 COOH Terminus Terminus

Pepsin (human) FDTGSS VDTGTS

Cathepsin E(human) FDTGSS VDTGTS

Cathepsin D(human) FDTGSS VDTGTS

PAG-1 (bovine) FDTASS VDTGTS

PAG-1 (ovine) FDTGSS VGTGTS

Fig. 3. The amino acid residues adjacent to the catalytic aspartic acids from selected mammalian aspartic proteinases. Aspartic pro- teinases exist as bilobed structures. Contained within each lobe is an aspartic acid that is directly involved in the catalytic mechanism. In these enzymes, each catalytic aspartic acid is closely apposed to the corresponding aspartic acid in the other lobe. Highly conserved residues are adjacent to the catalytic aspartic acids. The boxed residues in the pregnancy-associated glycoprotein-1 (PAG-1) se- quences indicate mutations of highly conserved amino acids that are likely to interfere with the catalytic mechanism. In ovine pregnancy-associated glycoprotein 1, the aspartic acid to glycine mutation at position 215 (pepsin numbering) eliminates one of the essential catalytic residues. In bovine pregnancy-associated glyco- protein 1, alanine replaces a highly conserved glycine at position 34 (pepsin numbering). Presumably, the addition of an alanine at this position displaces a water molecule that normally resides between the two catalytic aspartic acids and is directly involved in the catalytic mechanism (see Fig. 4a).

Owing to the replacement of a highly conserved glycine at position 34 with an alanine, bovine PAG-1 also appears to be inactive. The presence of the relatively bulky methyl group of alanine in this position probably displaces the water molecule residing between the two catalytic aspartates, thus rendering bovine PAG-1 incapable of functioning as a proteinase (Xie et al., 1991; Figs 3 and 4a). In addition, there are other changes in both Fig. 4. (a) Modelling the pig pepsin (orange) and bovine pregnancy- ovine and bovine PAG-1 that are likely to interfere with the associated glycoprotein-1 (purple) active site. Bovine pregnancy- access of peptide substrate to the catalytic centre (Guruprasad associated glycoprotein 1 is predicted to be unable to act as a et al., 1996). Finally, denatured [14C]haemoglobin is not degraded proteinase owing to the mutation of a highly conserved glycine with when used as a substrate in a proteinase assay with purified an alanine. The predicted position of the water molecule that resides bovine PAG-1 (Xie et al., 1991). At present, it appears that both between the two catalytic aspartic acids (blue) of pepsin (H355 HOH) and bovine pregnancy-associated glycoprotein 1 (H355 WAT) (black) PAG-1 molecules are enzymatically inactive. is shown. The presence of the relatively bulky methyl group of the Because of their close resemblance to pepsin A and other as- alanine 34 β-carbon (CB) substituted for the normally invariant gly- partic proteinases whose crystal co-ordinates are available, it cine at this position of bovine pregnancy-associated glycoprotein 1 has been possible to derive accurate atomic models of ovine is predicted to displace the catalytic water molecule from between and bovine PAG-1 (Fig. 4b). It is also known that some PAGs the carboxyl groups on the side chains of Asp32 and Asp215. This can bind pepstatin since they can be selectively removed from displacement is expected to interfere with the catalytic mechanism. placental-conditioned media by affinity chromatography on The figure has been modified from one published by Xie et al. (1991). pepstatin–agarose columns (J. A. Green, R. J. Nagel and R. M. The modelling was performed by Jordan Tang, University of Roberts, unpublished). Consequently, at least some of these Oklahoma. (b) Pepstatin superimposed on the binding cleft of ovine proteins have retained the ability to bind peptide ligands. The pregnancy-associated glycoprotein 1. The molecular surface of the space-filling model of ovine pregnancy-associated glycoprotein 1 generally acidic nature of the binding cleft of ovine and bovine shows the charge distribution, with negative charges as red and PAG-1 suggests that they would exhibit a preference for positive charges as blue. The binding cleft appears electronegatively basic, somewhat hydrophobic peptides (Guruprasad et al., charged and is likely to have a preference for basic peptides. The 1996; Fig. 4b). P4 (isovaline) and P3′ (statine) residues of pepstatin are labelled. The conservation between PAG-1 and the aspartic pro- Modelling was performed by Kunchur Guruprasad and Tom teinases is also evident at the genomic level. Three PAG genes Blundell, University of London.

Downloaded from Bioscientifica.com at 10/02/2021 09:03:44AM via free access 66 J. A. Green et al. have been identified from bovine and pig genomic libraries (Xie et al., 1995; J.A. Green, B. Szafranska and R.M. Roberts, un- published) and each exhibit the nine exon–eight intron struc- ture typical of mammalian aspartic proteinase genes. However, the promoter regions of these genes (bovine PAG-1, bovine PAG-2, and pig PAG-2; see below) showed no obvious se- quence similarity to the promoters of other aspartic proteinase genes. This apparent evolutionary shift in the genetic elements likely to be involved in transcriptional control may provide an explanation for the placental-specific expression of the PAGs.

The expanding PAG family Although initially it was believed that there was only a single PAG, it has become increasingly clear that there are possibly >100 PAG genes in ruminants and that many of these genes are expressed. Genomic Southern blot analysis performed with exon-specific probes under stringent conditions provided the initial hint that the PAG gene family was large and complex (Xie et al., 1995; Fig. 5). The frequency of PAG-related genes in genomic libraries also far exceeded what would be expected if only a single gene was present. These predictions were borne out as additional cDNAs encoding PAGs were cloned from cattle and sheep placental libraries. For example, Xie et al. (1994) and Nagel et al. (1993) described the identification of bovine PAG-2 and ovine PAG-2. Not only did these PAGs fail to crossreact with the original anti-bovine PAG-1 antiserum, but their transcripts were also much more abundant than those of bovine PAG-1. Although these molecules do not con- tain any obvious inactivating mutations within the active site, modelling studies suggest that they too may not be proteinases (Guruprasad et al., 1996). Screening cDNA libraries at different stages of pregnancy with mixtures of cDNA probes and the application of reverse transcription–PCR techniques have so far allowed our group to identify nine unique, full-length ovine cDNAs encoding PAGs and at least 12 bovine clones. Partially sequenced cDNA hint at the presence of many more. While the expression of many of these PAGs is restricted to binucleate cells, others are localized to the entire trophectoderm (Xie et al., 1994; S. Xie and R. M. Roberts, unpublished). Not only has a rich array of PAG transcripts been identified, but several different PAGs have also been purified as well. Our laboratory has recently purified at least ten distinct ovine Fig. 5. Genomic Southern blot of bovine DNA. Bovine genomic µ PAG-1-like proteins from placental conditioned media by using DNA (5 g) was digested with the indicated restriction endonucle- a series of chromatography procedures (Xie et al, in press). ases. The digestion products were resolved on a 0.8% (w/v) agarose Amino-terminal sequencing of four of the purified products gel by electrophoresis and transferred to nylon membrane. An approximately 800 bp DNA probe representing a portion of the revealed that three of them were novel and that each of them bovine pregnancy-associated glycoprotein 1 gene (part of intron 6, had been processed by removal of the propeptide within what exon 7, intron 7, exon 8, and part of intron 8) was labelled by PCR appeared to be a consensus sequence between the pro-peptide and hybridized to the blot under stringent conditions. The many and the mature molecule, ISF↓RD/GSN. The arrow indicates bands produced by hybridization of the probe are indicative of the the site of cleavage between the pro-peptide and the mature large number of pregnancy-associated glycoprotein-1-related genes protein. None of these purified molecules demonstrated any in the bovine genome. The hybridization conditions were such that enzymatic activity in a standard proteinase assay with de- only those genes closely related to bovine pregnancy-associated natured haemoglobin. glycoprotein 1 would hybridize to the probe. Another subset of PAGs was discovered by chance by Atkinson et al. (1993) who used a monoclonal antibody, carbohydrate epitope; second, when used immunohistochemi- SBU3, to immunopurify placental antigens from extracts of cally, it stains trophoblast binucleate cells specifically (Gogolin- sheep placenta. The SBU3 antibody has two interesting Ewens et al., 1986; Atkinson et al., 1993; Fig. 2). The three features. First, as mentioned earlier, it is directed towards a proteins affinity-purified with the SBU-3 antibody were distinct

Downloaded from Bioscientifica.com at 10/02/2021 09:03:44AM via free access Pepsin-related molecules secreted by trophoblast 67 in their amino-termini from other PAGs (61–78% similarity) Vecchio et al. (1990, 1995a,b) have reported that bovine PSP-B and again emphasize the surprising size and diversity of this (PAG-1) can increase the production of prostanoids in general, family of glycoproteins. and the luteotrophic compound PGE2 in particular, in both cul- tured luteal cells and endometrial explants. However, the re- ported action of PSP-B upon progesterone production has been PAG-related molecules within non-ruminant species variable. In some experiments, it showed no ability to increase Molecules related in sequence to bovine and ovine PAGs have progesterone production (Del Vecchio et al., 1995a,b), whereas been identified within the pig, another member of the in another report, PSP-B treatment increased progesterone pro- Artiodactyla order (Szafranska et al., 1995), by homology duction by luteal cells (Del Vecchio et al., 1996). Further work screening of a pig day 13–17 conceptus cDNA library. The two needs to be done before the results of these studies in vitro can cDNAs that were fully characterized (pig PAG-1 and PAG-2) be considered relevant to the situation in vivo. shared 48–57% amino acid sequence identity with ruminant An alternative hypothesis, and one that we prefer, is that the PAGs and, like the latter, were glycoproteins containing mul- PAGs act locally at the placental interface, and that their pres- tiple putative N-linked glycosylation sites. Western blotting ence in the maternal serum is an unintended consequence of of conditioned media from pig conceptuses performed with the invasive nature of the trophoblast binucleate cells and their antisera against bovine PAG-1 and bovine PAG-2 revealed release close to maternal capillaries (Fig. 1). It should be noted an array of immunoreactive proteins ranging in size from 50 to that some PAGs, for example bovine PAG-2 and ovine PAG-2, 70 kDa. Like PAG-1 from cattle and sheep, one of these pro- are expressed predominantly in mononucleated trophoblast teins (pig PAG-1) appeared to be enzymatically inactive, cells which make up at least 80% of the cell population of because of mutations within the conserved amino acids sur- trophectoderm. Furthermore, PAGs are also produced in species rounding the catalytic aspartic acid residues (Guruprasad et al., with an epitheliochorial placentation, such as the pig, where 1996). Conversely, the other protein (pig PAG-2) possessed there is no erosion of the uterine epithelium and a less ready all the hallmarks of an active aspartic proteinase, although access to the maternal circulation. Presumably pig PAGs, as well the recombinant form did not display any activity toward as those ruminant PAGs that are restricted to mononucleate [14C]haemoglobin in a proteinase assay (J. A. Green and R. M. cells of the trophectoderm, are destined to act within the uterine Roberts, unpublished). The expression of these molecules lumen. became apparent at day 15 of pregnancy and was detectable The lack of enzymatic activity of some, and possibly the until at least day 90 of gestation (Szafranska et al., 1995; preg- majority, of the PAGs tends to argue against a role as pro- nancy lasts for approximately 114 days in pigs). teinases. Of course, a lack of activity is difficult to rule out Molecules related to PAGs with a trophectoderm-specific except where there are clearly inactivating mutations within localization have been identified in species outside the Artio- the catalytic centre. In those members where the changes are dactyla, namely in the horse (Perrisodactyla) and cat (Carni- more subtle, such as those preventing close access of substrates vora). The horse cDNA (equine PAG) was identified from a to the active site, the evidence is less certain. Proteinase assays day 25 placental library and had greatest sequence similarity, with recombinant or purified native PAG also tend to be equivo- not to other PAGs (52–57% amino acid identity), but to a re- cal since the appropriate substrate may not have been used or cently characterized aspartic proteinase, rabbit pepsinogen F the proteins may have become denatured during purification. (69% amino acid identity; Table 1). Rabbit pepsinogen F is a Whether or not some of the PAGs are active as enzymes, gastric proteinase expressed for a short time within the new- many can certainly bind peptide ligands such as pepstatin A. born rabbit (Kageyama et al., 1990). Unlike other known PAGs, Conceivably, this ability to interact with peptides or proteins equine PAG appears not to be extensively glycosylated. without hydrolysing them reflects their function. Possibly, they Another feature of equine PAG is that its recombinant form has are carrier molecules or their substrate-binding cleft may target hydrolytic activity towards [14C]haemoglobin in a proteinase them to specific receptors. It is clear from modelling studies assay; it is the only PAG-like molecule so far proven to be a that the peptide-binding specificities of different PAGs may proteinase (J. A. Green and R. M. Roberts, unpublished). vary. Indeed, this variability may be a key to their function. The A transcript similar to that in the horse (73% amino acid multigenic nature of the family may reflect selective pressures identity) has recently been cloned from the placenta of the that have led to the evolution of a group of molecules that can domestic cat by using RT–PCR with oligonucleotides comple- present an extensive array of peptide binding specificities in mentary to highly conserved regions within different cDNAs the ‘war zone’ between the conceptus and the mother (Roberts encoding PAGs (X. Gan and R. M. Roberts, unpublished). The et al., 1996). discovery of PAG-like transcripts in Carnivora, a group that Of course, PAG function may not involve the binding cleft at may have diverged from the ungulates as long as 100 million all. The variability observed may have no significance if there years ago (Novacek, 1992), emphasizes the relatively ancient have been no selective pressures to maintain this region. origin of these placentally expressed molecules. Function may reside in either the more conserved regions of the molecules or in their carbohydrate motifs or pro-peptides. PAG function Conclusions The fact that PAG-related proteins are detectable in the ma- ternal circulation of ruminant species has been taken to imply In conclusion, the PAGs are represented by a large family of that they are targeted to the mother and are placental hor- genes and are expressed abundantly in the placental trophecto- mones, perhaps acting upon the corpus luteum. Indeed, Del derm of species within the Artiodactyla order. In ruminants,

Downloaded from Bioscientifica.com at 10/02/2021 09:03:44AM via free access 68 J. A. Green et al. many seem to be restricted to invasive binucleated cells and, as Del Vecchio RP, Sasser RG and Randel RD (1990) Effect of pregnancy- a consequence, some are able to enter the maternal circulation specific protein B on prostaglandin F2α and prostaglandin E2 release by day 16 perifused bovine endometrial tissue Prostaglandins 40 271–282 where their presence has been used as the basis for a pregnancy Del Vecchio RP, Sutherland WD and Sasser RG (1995a) Effect of pregnancy- test in cattle. Molecular cloning revealed that the PAGs are specific protein B on luteal cell progesterone, prostaglandin, and oxytocin members of the aspartic proteinase gene family, but at least production during two stages of the bovine estrous cycle Journal of Animal some are unable to act as enzymes, raising fascinating ques- Science 73 2662–2668 tions concerning their role in pregnancy. Del Vecchio RP, Sutherland WD and Sasser RG (1995b) Prostaglandin F2 alpha, progesterone and oxytocin production by cultured bovine luteal Despite 15 years of study, the function of the PAGs in repro- cells treated with prostaglandin E2 and pregnancy-specific protein B duction is not known. Especially perplexing is the fact that Prostaglandins 50 137–150 there appear to be multiple, related proteins within species of Del Vecchio RP, Sutherland WD and Sasser RG (1996) Bovine luteal cell the Artiodactyla. If PAGs function in fetal–maternal communi- production in vitro of prostaglandin E2, oxytocin and progesterone in re- sponse to pregnancy-specific protein B and prostaglandin F2α Journal of cation, as implied by the work of Del Vecchio et al. (1990, Reproduction and Fertility 107 131–136 1995a,b), then the reason for so many expressed forms becomes Duello TM, Byatt JC and Bremel RD (1986) Immunohistochemical local- particularly puzzling. Do these different molecules have indi- ization of placental lactogen in binucleate cells of bovine placentomes vidual specificities or is there simply a good deal of redun- Endocrinology 119 1351–1355 dancy in a single important function? Eckblad WP, Sasser RG, Ruder CA, Panlasigui P and Kuczynski T (1985) Localization of pregnancy-specific protein B (PSPB) in bovine placental The presence of PAG-related molecules expressed in the cells using a glucose oxidase-anti-glucose oxidase immunohistochemical placentae of species outside the Artiodactyla order emphasizes stain Journal of Animal Science (Supplement) 61 149–150 their long term conservation and hints at their evolutionary Gogolin-Ewens KJ, Lee CS, Mercer WR, Moseby AM and Brandon MR origins. The PAG-like molecule from horse is an active (1986) Characterization of a sheep trophoblast-derived antigen first ap- pearing at implantation Placenta 7 243–255 proteinase and appears not to be heavily glycosylated. Guruprasad K, Blundell TL, Xie S, Green J, Szafranska B, Nagel RJ, Furthermore, cat and horse PAG resemble pepsinogens at least McDowell K, Baker CB and Roberts RM (1996) Comparative modelling as closely as they resemble the PAGs from ruminants and pigs and analysis of amino acid substitutions suggests that the family of (Table 1). The number of PAG-like genes within these species pregnancy-associated glycoproteins includes both active and inactive seems to be small as well; only a single transcript has been aspartic proteinases Protein Engineering 9 849–856 Haig D (1996) Altercation of generations: genetic conflicts of pregnancy identified from both horses and cats (J. A. Green, X. Gan, American Journal of Reproductive Immunology 35 226–232 R. M. Roberts, unpublished). Hearn JP (1986) The embryo–maternal dialogue during early pregnancy in As the PAG genes have evolved from their progenitors, their primates Journal of Reproduction and Fertility 76 809–819 functions may also have changed. The placenta is the hallmark Humblot P, Camous S, Martal J, Charlery J, Jeanguyot N, Thibier M and Sasser RG (1988) Pregnancy-specific protein B, progesterone concen- of the mammal, yet rather than being the most anatomically trations and embryonic mortality during early pregnancy in dairy cows conserved organ, it is arguably the most diverse. Haig (1996) Journal of Reproduction and Fertility 83 215–223 has argued that the rapid pace of placental evolution has been James MNG and Sielecki AR (1986) A molecular structure of an aspartic pro- powered by conflict between maternal genes and those pater- teinase zymogen, porcine pepsinogen, at 1.8 Å resolution Nature 319 33–38 nal genes expressed by the conceptus. A similar reasoning James MNG, Sielecki AR and Hofmann T (1985) X-ray diffraction studies on penicillopepsin and its complexes: the hydrolytic mechanism. In Aspartic might be used to explain the ontogeny of the PAGs. Human Proteinases and their Inhibitors pp 163–177 Ed. V Kostda. Walter de Gruyter, chorionic gonadotrophin in the higher primates (Hearn, 1986) New York and IFN-τ (Roberts et al., 1992) within the ruminant ungulates Kageyama T, Tanabe K and Koiwai O (1990) Structure and development of are examples of placental products that have evolved to fulfil rabbit pepsinogens: stage-specific zymogens, nucleotide sequences of cDNAs, molecular evolution, and expression during development Journal specific physiological roles required by only a restricted group of Biological Chemistry 265 17 031–17 038 of species (see Roberts et al., 1996). That the PAGs lack a role in Kay J and Dunn BM (1992) Substrate specificity and inhibitors of aspartic pregnancy or are incidental oddities seems unlikely in view of proteinases Scandinavian Journal of Clinical Laboratory Investigations 52 the magnitude of their production and their survival over (Supplement 210) 23–30 millions of years of selective pressure. Koelsch G, Mares M, Metcalf P and Fusek M (1994) Multiple functions of pro-parts of aspartic proteinase zymogens FEBS Letters 343 6–10 Lee CS, Wooding RBP and Brandon MR (1986) Immunogold co-localization The work of the authors described in this review was supported by of ovine placental lactogen and the antigen recognized by the SBU-3 NIH grant HD29483 and USDA grant 96-01842 to R. M. Roberts. The monoclonal antibody in sheep placental granules Journal of Reproduction authors are indebted to Robert J. Nagel and Xensheng Gan, whose un- and Fertility 78 653–662 published work is cited in the text, and to Boon G. Low for performing Lin X, Wong RNS and Tang J (1989) Synthesis, purification, and active site the immunoperoxidase localization in Figure 2. mutagenesis of recombinant porcine pepsinogen Journal of Biological Chemistry 264 4482–4489 Lynch RA, Alexander BM and Sasser RG (1992) The cloning and expression of the bovine pregnancy-specific protein B (bPSPB) gene Biology of References Reproduction (Supplement) 46 Abstract 89 Key references are indicated by asterisks. 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Roberts RM, Cross JC and Leaman DW (1992) Interferons as hormones of Wooding FBP (1983) Frequency and localization of binucleate cells in the pregnancy Endocrine Reviews 13 432–452 placentomes of ruminants Placenta 4 527–540 *Roberts RM, Xie S and Mathialagan N (1996) Maternal recognition of preg- Wooding FBP (1992) Current Topic: the synepitheliochorial placenta of nancy Biology of Reproduction 54 294–302 ruminants: binucleate cell fusions and hormone production Placenta 13 Rowell JE, Flood PF and Sasser RG (1989) Pregnancy-specific protein in the 101–113 plasma of captive muskoxen Journal of Wildlife Management 53 899–901 *Xie S, Low BG, Kramer KK, Nagel RJ, Anthony RV, Zoli AP, Beckers J-F Ruder CA, Stellflug JN, Dahmen JJ and Sasser RG (1988) Detection of and Roberts RM (1991) Identification of the major pregnancy-specific pregnancy in sheep by radioimmunoassay of sera for pregnancy-specific antigens of cattle and sheep as inactive members of the aspartic proteinase protein B Theriogenology 29 905–912 family Proceedings of the National Academy of Sciences USA 88 *Sasser RG, Ruder CA, Ivani KA, Butler JE and Hamilton WC (1986) 10 247–10 251 Detection of pregnancy by radioimmunoassay of a novel pregnancy Xie S, Low BG, Nagel RJ, Beckers J-F and Roberts RM (1994) A novel specific protein in serum of cows and a profile of serum concentrations glycoprotein of the aspartic proteinase gene family expressed in bovine during gestation Biology of Reproduction 35 936–942 placental trophectoderm Biology of Reproduction 51 1145–1153 Sasser RG, Crock J and Ruder-Montgomery CA (1989) Characteristics of Xie S, Green J, Beckers J-F and Roberts RM (1995) The gene encoding pregnancy-specific protein B in cattle Journal of Reproduction and Fertility bovine pregnancy-associated glycoprotein1, an inactive member of the Supplement 37 109–113 aspartic proteinase family Gene 159 193–197 Szafranska B, Xie S, Green J and Roberts RM (1995) Porcine pregnancy- Xie S, Green J, Bao B, Beckers J-F, Valdez KE, Hakami L and Roberts RM associated glycoproteins: new members of the aspartic proteinase gene Multiple pregnancy-associated glycoproteins (PAG) are secreted by day family expressed in trophectoderm Biology of Reproduction 53 21–28 100 ovine placental tissue Biology of Reproduction (in press) Szecsi PB (1992) The aspartic proteinases Scandinavian Journal of Clinical *Zoli AP, Beckers J-F, Woutters-Ballman P, Closset J, Falmagne P and Laboratory Investigations 52 (Supplement 210) 5–22 Ectors F (1991) Purification and characterization of a bovine pregnancy- Tang J and Wong RNS (1987) Evolution in the structure and function of as- associated glycoprotein Biology of Reproduction 45 1–10 partic proteinases Journal of Cellular Biochemistry 33 53–63 *Zoli AP, Guilbault LA, Delabaut P, Ortiz WB and Beckers J-F (1992) Wood AK, Short RE, Darling AE, Dusek GL, Sasser RG and Ruder CA Radioimmunoassay of a bovine pregnancy-associated glycoprotein in (1986) Serum assays for detecting pregnancy in mule and white-tailed deer serum: its application in pregnancy diagnosis Biology of Reproduction 46 Journal of Wildlife Management 50 684–687 83–92

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