Characterization of placentation-specific binucleate cell glycoproteins possessing a novel carbohydrate. Evidence for a new family of pregnancy-associated molecules.

The ovine binucleate cell-specific glycoproteins recognized by the monoclonal antibody SBU-3 first appear at the initiation of placentation, and their expression continues throughout gestation. These placenta-specific proteins have not been detected in any other adult or fetal sheep tissues and are specific to the materno-fetal interface. The SBU-3 monoclonal antibody recognizes the carbohydrate epitope common to a group of proteins ranging in molecular mass from 30 to 200 kDa whose function during pregnancy remains undefined. The biochemical properties of these uniquely expressed glycoproteins were investigated by analyzing both the carbohydrate and protein portion of the molecules. Analysis of phytohemagglutinin and concanavalin A binding to electrophoretically separated SBU-3 proteins revealed that the major proteins between 40 and 70 kDa bind phytohemagglutinin. In contrast, concanavalin A bound only to minor proteins in the SBU-3 glycoprotein preparation. Analysis of the carbohydrate conjugated to the SBU-3 glycoproteins revealed that the major chains are sialylated O-linked and complex partially sialylated multiple antennary N-linked chains. The presence of N-glycolylneuraminic acid in an N-linked structure indicates the unique nature of this carbohydrate epitope. The differential binding to phytohemagglutinin and concanavalin A provided a method for further purification and characterization of the major protein components with monoclonal antibody immunoaffinity-purified SBU-3 proteins being further separated by concanavalin A-Sepharose chromatography. Microsequence analysis of the major non-concanavalin A-binding proteins (69, 62, and 57 kDa) revealed partial homology to ovine and bovine pregnancy-associated glycoprotein and rabbit pepsinogen F. Immunoblot analysis of the SBU-3 proteins showed cross-reactivity with polyclonal antisera directed against ovine placental-associated glycoprotein and pregnancy-specific glycoprotein B. These results suggest that together these glycoproteins represent members of a binucleate cell-derived family of pregnancy-associated molecules in the ruminant placenta.

The ovine binucleate cell-specific glycoproteins recognized by the monoclonal antibody SBU-3 first appear at the initiation of placentation, and their expression continues throughout gestation. These placenta-specific proteins have not been detected in any other adult or fetal sheep tissues and are specific to the materno-fetal interface. The SBU-3 monoclonal antibody recognizes the carbohydrate epitope common to a group of proteins ranging in molecular mass from 30 to 200 kDa whose function during pregnancy remains undefined. The biochemical properties of these uniquely expressed glycoproteins were investigated by analyzing both the carbohydrate and protein portion of the molecules. Analysis of phytohemagglutinin and concanavalin A binding to electrophoretically separated SBU-3 proteins revealed that the major proteins between 40 and 70 kDa bind phytohemagglutinin. In contrast, concanavalin A bound only to minor proteins in the SBU-3 glycoprotein preparation. Analysis of the carbohydrate conjugated to the SBU-3 glycoproteins revealed that the major chains are sialylated O-linked and complex partially sialylated multiple antennary N-linked chains. The presence of Nglycolylneuraminic acid in an N-linked structure indicates the unique nature of this carbohydrate epitope. The differential binding to phytohemagglutinin and concanavalin A provided a method for further purification and characterization of the major protein components with monoclonal antibody immunoaffinity-purified SBU-3 proteins being further separated by concanavalin A-Sepharose chromatography. Microsequence analysis of the major non-concanavalin A-binding proteins (69, 62, and 57 kDa) revealed partial homology to ovine and bovine pregnancy-associated glycoprotein and rabbit pepsinogen F. Immunoblot analysis of the SBU-3 proteins showed cross-reactivity with polyclonal antisera directed against ovine placental-associated glycoprotein and pregnancy-specific glycoprotein B. These results suggest that together these glycoproteins represent members of a binucleate cellderived family of pregnancy-associated molecules in the ruminant placenta.
Fetal binucleate cells are unique to the ruminant placenta (Wooding, 1992). In the sheep, they differentiate from the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mononucleate cells of the trophectoderm as it establishes close contact with the uterine epithelium on days 14-15 of gestation (Wooding, 1992). At least two subpopulations of binucleate cells are present in the ovine placenta, as defined by the monoclonal antibody (mAb)' SBU-3 which recognizes a proportion of binucleate cells present in the areas where the placenta develops (placentomes), and not those present in the interplacentomal regions (Lee et al., 1986). This mAb, which was raised against trophoblast microvilli isolated from the fetal portion of midgestation ovine placentomes, has been shown to recognize a carbohydrate moiety present on a group of proteins with molecular weights ranging between 30,000 and 200,000 (Gogolin-Ewens et al., 1986). Extensive studies using this antibody failed to detect the presence of the SBU-3 carbohydrate antigen in a wide range of ovine tissues other than in restricted regions of the placenta. Cell surface carbohydrates attached to glycoproteins have been implicated in a wide range of biological processes. The concept of tissue-specific recognition of carbohydrates was first introduced with the discovery of the asialoglycoprotein receptor on hepatocytes (Kawasaki and Ashwell, 1976). With tissuespecific carbohydrate recognition came the concept of tissuespecific carbohydrate expression. The involvement of carbohydrates in cell-cell interaction provides a n excellent illustration of this phenomenon. For example, it was observed that the SSEA-1 antigen expressed on the preimplantation mouse embryo is actually a Gal/3l4(Fuccul-3)GlcNAc/31- structure (Gooi et al., 1981). Exogenous addition of oligosaccharides inhibited morulae compaction and decompacted morulae (Fenderson et al., 1984). Another reproductive process in which carbohydrates are involved includes the adhesion of sperm to eggs during fertilization (Wassarman, 1987).
The unique and pregnancy-specific expression of the SBU-3 carbohydrate epitope which is common to a group of placental glycoproteins, raises the possibility of placental specific glycosylation. We sought to gain an insight into this question by biochemically characterizing some of these glycoproteins. In addition, the expression of a placentation-specific carbohydrate epitope warrants further investigation with a view to defining the molecular mediators of placentation.

EXPERIMENTAL PROCEDURES
Monoclonal Antibody Zmmunoafinity Purification of SBU-3 Conjugated Proteins-The SBU-3 proteins were mAb immunoaffinity-purified as previously described (Gogolin-Ewens et al., 1986). Briefly, SBU-3 mAb was purified from ascites fluid and coupled to M-Gel 10 (Bio-Rad) at 10 mg of protedml beads. Preparations of fetal trophoblast microvilli isolated from midgestation ovine placentomes were centrifuged to yield a supernatant and a membrane fraction. The membrane pellet was diluted and applied to the affinity column. Specifically bound proteins were eluted with 4 M sodium thiocyanate (&x Chemicals, Australia), and column fractions were immediately desalted into distilled water on Sephadex G-25 (Pharmacia, Sweden). Column fractions containing SBU-3 protein were identified on Western blots stained with anti-SBU-3 mAb (Gogolin-Ewens et al., 1986), pooled, and concentrated.
Lectin and Zmmunoblotting of SBU-3 Proteins-SBU-3 proteins were separated by SDS-PAGE on 10% gels (8 x 7 x 1.5 cm) according to the method of Laemmli (1970) and transferred onto 0.2-pm or 0.45-pm nitrocellulose membranes (Schleicher & Schuell) in electrophoretic transfer buffer containing 25 m ?tis base (Sigma), 192 m glycine (Sigma), 0.01% w/v SDS (Sigma), and 10% (v/v) methanol (Towbin et al., 1979). Proteins were transferred at 70 V for 2 h. For lectin and immunoblotting, unbound membrane sites were blocked with phosphate-buffered saline (PBS) containing 0.5% Tween 20 (Sigma) for 1 h a t room temperature. For immunoblotting, membranes were washed three times in PBS + 0.05% Tween 20 (PBSPT) and incubated for 1 h in SBU-3 hybridoma supernatant fluid, anti-ovine placental-associated glycoprotein (oPAG) polyclonal rabbit sera (a generous gift from Prof. J. F. Beckers, Departement d'Endocrinologie et de Reproduction Animales, Universite de l'Etat a Liege), or anti-pregnancy-specific glycoprotein B (PSPB) polyclonal rabbit sera (a generous gift from Dr. R. Garth Sasser, Dept. ofAnimal and Veterinary Science, University of Idaho). Following three washes in PBW, the membrane was incubated in horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins (1500 in PBSPT, Dako), or horseradish peroxidase-conjugated goat anti-rabbit ( M O O in PBW, Sigma) for 1 h at mom temperature. Following two washes in P B W and two washes in PBS, the blot was developed with the chromogen 3,3'-diaminobenzidine (Dako, 0.6 m g h l + 0.0075% H202) at room temperature. For lectin blots, a similar procedure was used, except the first incubation was with 6.25 pg/ml biotinylated C o d (Sigma) or biotinylated PHA (Sigma) in PBSm. The second incubation was with horseradish peroxidase-streptavidin conjugate (Amersham), diluted 1:lOOO with PBSPT. Lectin blots were developed as described above for immunoblots. Dot blots were performed by dotting various concentrations of immunoaffinity-purified SBU-3 proteins onto nitrocellulose. Blots were probed with biotinylated PHA, and binding was detected as described above for lectin blots.
Lectin Purification of the Major SBU-3 Proteins-One hundred micrograms of immunoaffinity-purified SBU-3 proteins in 200 pl were applied to a 200-1.11 column of Cod-Sepharose (Pharmacia, Sweden) in 10 m "is-HC1, pH 10.0, under gravity flow. The column was washed with 3 ml of 20 m Tris-HCI, 0.5 M NaCI, pH 7.4. Fractions containing non-Cod-binding SBU-3 proteins were pooled and desalted into 10 m Tris-HC1, pH 10.0, 0.1% CHAPS (Sigma) using Sephadex G-24 SF (Pharmacia) and analyzed on 12% polyacrylamide gels. Proteins were visualized by silver staining, according to the method of Chaudhuri and Green (1987). The major non-Cod-binding proteins were further separated by lengthened electrophoresis times on 12% gels.
Monosaccharide Analysis of the SBU-3 Carbohydrate Epitope-The immunoaffinity-purified SBU-3 glycoprotein sample was evaporated in a screw top septum vial with 10 pg of perseitol internal standard and dried over PzOS. Two hundred pl of 1 M methanolic HCI was added under a stream of nitrogen, and the sample was heated in the capped vial for 18 h at 80 "C. On cooling, silver carbonate was added to neutrality, and the sample was re-N-acetylated by the addition of 50 pl of acetic anhydride. After 4 h, the supernatant was removed, and the residual silver carbonate was washed three times with methanol. The combined supernatants were evaporated under nitrogen and dried over P20s before addition of 20 1.11 trimethylsilylating reagent (Sylon H T P , Supelco, Saffron Walden, UK). The sample was heated at 60 "C for 5 mi n, evaporated under nitrogen, taken up in 20 1.11 of dry toluene, and injected onto a capillary GC column (25M x 0.22-mm inside diameter BPIO, SGE Milton Keynes, UK), programmed from 130-230 "C at 2 Wmin.
For oligosaccharide profiling, the N-linked oligosaccharide chains were released from glycoprotein using peptide N-glycanase F (Boehringer Mannheim, Lewes, UK) in 40 m potassium dihydrogen orthophosphate, 10 m EDTA adjusted to pH 6.2 with 1 M NaOH (37 "C, 72 h). The protein was precipitated by addition of a 2-fold excess of ice cold ethanol and centrifugation at 15,000 x g for 20 min. The pellet was washed three times with ice-cold ethanol, and the combined supernatants were evaporated and analyzed by HPAE-PAD as described above in a gradient of eluent A (0.1 M NaOH) and eluent B (0.1 M NaOH, 0.5 M sodium acetate) as follows: 0-15 min 95% eluent A, 5% eluent B; 15-70 min to 40% eluent A, 60% eluent B; 70-75 min 40% eluent A, 60% eluent B; 75-78 min to 100% eluent A, 0% eluent B.

RESULTS
Differential Binding of SBU-3 Proteins to PHA and ConA -Preliminary experiments showed that immunoaffinity-purified SBU-3 proteins inhibited lymphocyte proliferation in response to PHA, yet had no effect on other IL-2-mediated proliferative responses (Murray e t al., 1978;Segerson, 1988). Dotblot experiments confirmed that the SBU-3 proteins bound to PHA in sufficient quantities to inhibit the mitogenic response. Since dot-blot analysis revealed that the SBU-3 proteins bind PHA and, to a lesser degree, ConA, we wished to define more specifically which of the spectmm of SBU-3 proteins binds to these two lectins. Therefore, SBU-3 proteins were electrophoretically separated and transferred to nitrocellulose and immu-Biochemical Characterization of the SBU-3 Proteins Western blot analysis of the lectin binding pattern of SBU-3 glycoproteins. Immunoaffinity-purified SRU-3 proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose. Blots were probed with either anti-SBU-3 antibody or biotin-labeled PHA or ConA. The reaction was detected by horseradish peroxidase-conjugated second antibody and developed with diaminobenzidine.  nostained with SBU-3 mAb or stained with biotin-labeled PHA or ConA (Fig. 1). The major proteins between 40 and 70 kDa which stain with SBU-3 antibody also bind PHA. In contrast, ConA bound only to minor proteins in the SBU-3 preparation. Notably, ConA did not bind to the major SBU-3-reactive proteins localized between 40 and 70 kDa. These binding patterns are consistent with the differential intensities of binding seen in the dot blots. Lower molecular mass proteins (<40 kDa) were lost during this transfer due to the use of 0.45-pm nitrocellulose and a longer transfer time which was employed to enable us to focus on the higher molecular mass proteins.
Carbohydrate Analysis of the SBU-3 Proteins-Gas chromatographic (GC) analysis of the immunoafinity-purified SBU-3 proteins revealed that they contained 18% carbohydrate with the molar ratios shown in Table I. The large amount of N-acetylgalactosamine suggests that 0-linked chains are present, having the core linkage GalNAcal-Ser/Thr. The ratio of the remaining monosaccharides with respect to mannose at 3.0 suggests a generalized N-linked chain having three to four Gal-GlcNAc antennae, with additional GlcNAc (bisecting), terminal sialylation of branches, and the possibility of a core or terminal fucose (Fig. 2). Binding of the SBU-3 glycoproteins to the lectin PHA suggests that not all N-linked antennae are sialylated, but that some of the sialic acid is on 0-linked Gal-NAc. Analysis of the sialic acid component of the carbohydrate indicated that this residue is N-glycolylneuraminic acid (NeuGc ; Fig. 3). These results are consistent with the oligosac- charide profiles obtained by both HPAE and PGC chromatography. In the former technique, N-glycolated oligosaccharides elute later than their N-acetylated counterparts. Comparison of the HPAE elution profile of the oligosaccharides released from SBU-3 (Fig. 4A) and a standard glycoprotein, fetuin (Fig.  4B), shows that except for a peak at 26 min in the region of disialylated oligosaccharides containing NeuAc (25-30 min), the SBU-3 oligosaccharides elute later than trisialylated oligosaccharides containing NeuAc (36-40 min). The peaks for SBU-3 a t around 46, 51, and 56 min (Fig. 4A) are therefore consistent with multiple sialylated complex oligosaccharides containing NeuGc. The various peaks represent different numbers of sialic acid residues, different linkage isomers, and a different number of antennae as shown for peaks around 29, 38, and 50 min in the standard (Fig. 4B) for oligosaccharides having N-acetylneuraminic acids. On PGC chromatography, sialylated oligosaccharides differing only in their content of NeuAc or NeuGc co-elute: monosialylated oligosaccharides chromatograph from 22-25 min, disialylated from 29 to 32 min, and trisialylated from 30 min.2 The oligosaccharide profile of SBU-3 (Fig. 5) is therefore consistent with the presence of mainly monoor disialylated oligosaccharides with either NeuAc or NeuGc. Enrichment

FIG. 5. Porous graphitic carbon
gest of the carbohydrate conjugated to the SBU-3 glycoproteins. The SBU-3 oligosaccharides were injected onto a Hypercarb S column, eluted in a gradient of 0.05% aqueous trifluoroacetic acid and acetonitrile, 0.05% trifluoroacetic acid, and detected at 206 nm. The elution profile revealed the presence of mainly monoor disialylated oligosaccharides with either NeuAc or NeuGc. Proteins-The differential binding of SBU-3 glycoproteins to PHA and ConA provided a method for further purification of the major protein components. The SBU-3 proteins were applied to a Cod-Sepharose column, and the flow-through was collected, desalted, and analyzed by SDS-PAGE. A proportion of the SBU-3 glycoproteins, ranging in molecular mass from 97 kDa to 37 kDa, did not bind to the ConA-Sepharose column (Fig. 6). Major bands of 69,62, and 57 kDa were easily detected, whereas the 97-kDa (not seen in this photograph), 84-kDa, 64-kDa, 51-kDa, and 37-kDa bands represented minor components of the non-ConA-binding SBU-3 proteins. The major 69-kDa, 62-kDa, and 57-kDa proteins were separated further on SDS-PAGE for subsequent amino-terminal microsequence analysis.
Microsequence analyses of the 69-kDa, 62-kDa, and 57-kDa non-ConA-binding SBU-3 proteins determined 27, 25, and 23 amino acids, respectively (Fig. 7). These sequences are homologous to each other to varying degrees. SBU-369 is 84% homologous (identical and conserved amino acids) to SBU-362 and 57% homologous to    6. SDS-PAGE analysis of non-Cod-binding immunoaffinity-purified SBU-3 proteins. One hundred pg of SBU-3 mAb immunoaffinity-purified glycoprotein was applied to Cod-Sepharose under gravity flow and washed with 20 m Tris-HC1, 0.5 M NaCl, pH 7.4. Fractions containing SBU-3 proteins that did not bind to C o d were pooled and desalted, then resolved on a 12% polyacrylamide gel, and visualized following silver staining.

78%
Pro-Leu-Arg-Asn-I l e -Lys-Asp-Leu-Arg-Gly-Ser-Asn-Leu-Thr-Thr-His-5 5 5 6 5 7 5 8 5 9 6 0 6 1 6 2 6 3 6 4 6 5 Gln-sp-Pro-Asp-Val-Ser-Phe-Glu-Pro-Leu-Arg-Asn-Tyr-Leu-Asp-Leu-A l a -T y r -I l e -G l y -Ile-I l e -S e r -Ile-Gly-Thr-pro  with the immunoaffinity-purified SBU-3 proteins. Proteins were separated by 10% SDS-PAGE and then transferred to nitrocellulose. m e r incubation with either anti-SBU-3, anti-oPAG, or anti-PSPB, binding was detected with a horseradish peroxidase-conjugated second antibody and developed using diaminobenzidine. 21 in the 69-kDa protein and 2 and 19 in the 62-kDa protein yielded no identifiable amino acid derivative, but are followed by the consensus sequence for a n N-glycosylation site (N-X- [ST]-X, X cannot be a P). The SBU-369 protein contains a consensus sequence for a n N-myristoylation site (GSXVTI), and all three protein sequences contain the consensus sequence for sulfation of carbohydrate (PLR).
A search of the protein and DNA sequence databases reveals a 61-78% identity between the three SBU-3 proteins and the binucleate cell-specific protein, ovine (and bovine) pregnancyassociated glycoprotein (oPAG, Fig. 8). In addition, the SBU-369, SBU-362, and SBU-357 sequences are 6 6 4 8 % homologous to rabbit pepsinogen F. The amino-terminal sequence of SBU-369 starts a t Arg-39 in the oPAG sequence, which is the proposed cleavage site for the aspartic protease.
To determine the extent of homology between the SBU-3 proteins, oPAG, and PSPB, immunoblotting of the SBU-3 protein was performed. Immunoaffinity-purified SBU-3 proteins were separated by SDS-PAGE and transferred onto 0.2-pm nitrocellulose. The blots were then probed with SBU-3 mAb or polyclonal antisera directed against oPAG or PSPB as shown in Fig. 9. Both the oPAG and the PSPB antibodies recognized the three major SBU-3 proteins characterized in this paper, indicating that a high degree of homology exists between these proteins. The oPAG antibody also recognizes all of the minor SBU-3 proteins (both Cod-binding and non-Cod-binding), whereas the PSPB antibody recognizes only five such bands.

DISCUSSION
The SBU-3 glycoproteins represent a group of at least 10 proteins linked to a fetally derived pregnancy-specific carbohydrate antigen recognized by the SBU-3 mAb (Gogolin-Ewens et al., 1986). These glycoproteins are initially detectable in the binucleate cells of the fetal trophoblast at the time of placentome formation, and their expression continues throughout gestation (Gogolin-Ewens et al., 1986;Morgan et al., 1987). It remains to be established whether their expression profile varies as placentation progresses. In this paper, we show that within this glycoprotein family, the major PHA-binding proteins are homologous to oPAG and rabbit pepsinogen F. The SBU-3 carbohydrate antigen appears to be a complex trior tetra-antennary chain containing N-glycolylneuraminic acid. These results provide a clue as to the identity of the SBU-3 glycoproteins. The unique structure of the SBU-3 carbohydrate antigen, combined with a restricted distribution in large quantities at the feto-maternal interface, suggests that modification of the glycoprotein may play an important role in the establishment and/or maintenance of pregnancy.
Our preliminary study of the immunosuppressive properties of the SBU-3 glycoproteins revealed a n ability to suppress PHA-induced mitogenesis without affecting basal proliferation. However, these proteins had no effect in a mixed lymphocyte reaction, which represents an IL-2-driven proliferative response, or on IL-2-dependent lymphoblast proliferation, suggesting that the SBU-3 proteins do not influence IL-2-dependent lymphocyte proliferation. Dot-blot analyses revealed that the SBU-3 proteins bind to PHA at concentrations which are inhibitory in the mitogenesis assay. These findings together indicate that the SBU-3 proteins may inhibit PHA-induced mitogenesis by binding and sequestering the PHA rather than by directly influencing lymphocyte function. However, these results do not preclude the possibility that the SBU-3 glycoproteins play a n immunosuppressive role during pregnancy. Analysis of the carbohydrate moiety conjugated to the SBU-3 proteins was performed in order to define the carbohydrate content and structure. The results were consistent with the major chains being sialylated, 0-linked and complex partially sialylated, multiple antennary N-linked chains. The sialic acid assay indicated that this is all present as N-glycolylneuraminic acid. NeuGc is reported to be completely absent from human serum and from serum glycoprotein, such as al-acid glycoprotein (Corfield and Schauer, 1982). Human erythrocytes contain only NeuAc linked to glycoproteins (Corfield and Schauer, 1982). In contrast, high proportions of NeuGc are found in mice, rat, cat, pig, sheep, cow, horse, mule, and donkey erythrocytes (Corfeld and Schauer, 1982). NeuGc on horse erythrocytes is the structure causing serum sickness in humans (Higashi et al., 1977), suggesting the absence of this moiety from all human tissues. The majority of NeuGc-containing molecules of nonhuman origin have been characterized as 0-linked glycoprotein chains (Chai et al., 1992) and gangliosides. Few oligosaccharides of N-linked type containing NeuGc have been isolated and fully characterized, and no systematic studies have been carried out to look at their tissue distribution.
Analysis of the SBU-3 proteins revealed heterogeneity in their lectin binding properties. The major proteins between 40 and 70 kDa bind PHA, but not C o d , whereas minor proteins ranging in molecular mass up to 200 kDa bind only to ConA. These lectin-binding characteristics provide further information about the possible structures of the carbohydrate attached to the SBU-3 proteins. The biotinylated PHA preparation used in this study contains both PHA-L and PHA-E. PHA-L recognizes triand tetra-antennary N-linked chains with outer Gal and a Manu residue disubstituted at C2 and C6 (Cummings and Kornfeld, 1982). PHA-E recognizes bi-or triantennary oligosaccharides containing a bisecting GlCNAc and a Galpl-4GlcNAc sequence linked pl-2 to Manal-6 (Yamashita et al., 1983). ConA does not bind to the major SBU-3 proteins indicating that they are not high mannose, hybrid, or biantennary oligosaccharides with 2-0-unsubstituted trimannosyl core residues. These lectin binding data are consistent with the carbohydrate analysis indicating that the major SBU-3 proteins are complex triand tetraantennary oligosaccharides.
Microsequence analyses of the 69-kDa, 62-kDa, and 57-kDa SBU-3 proteins demonstrated that they show partial identity with one another (Fig. 7). The SBU-369 and SBU-362 exhibit the highest degree of homology, followed by the SBU-369 and SBU-357. The alignment of these sequences with each other suggests that they may be a related family of pregnancy-specific glycoproteins.
Immunohistological analysis of ovine placentomes demonstrated that SBU-3 glycoproteins are expressed initially at the time of placentome formation, and for the duration of gestation (Morgan et al., 1987). However, it remains to be established whether some or all of the proteins recognized by the SBU-3 antibody are expressed simultaneously andor in the same lo-cation. For example, one form of SBU-3 may be expressed in migrating binucleate cells, another in fusing binucleate cells, and still another only in syncytium. This pattern of expression would give rise to all three proteins being present as major components of a protein preparation from midgestation placentomes. Therefore, the presence of all three may be an indication of variable expression during placentation.
A search of protein and DNA sequence databases revealed a 61-78% identity between the three SBU-3 proteins and the binucleate cell-specific protein, ovine (and bovine) PAG (Fig. 8).
In addition, all three proteins are 6648% homologous to rabbit pepsinogen F. This observation suggests that at least some of the glycoproteins recognized by SBU-3 mAb, as well as oPAG and PSPB, are members of a binucleate cell-derived family of pregnancy-associated molecules in ruminants.
It is known that binucleate cells produce several pregnancyspecific proteins which include the lactogenic and somatotrophic hormone placental lactogen (Lee et al., 1986;Morgan et al., 19871, ovine pregnancy-associated glycoprotein (oPAG), and a pregnancy-specific protein B (PSPB, Reimers et al. (1985)). Lynch et al. (1992) have recently shown that PSPB is identical with the oPAG characterized by Xie et al. (1991). Ovine PAG appears to be an inactive member of the aspartic proteinase family of proteins, but its role in pregnancy remains undefined.
A number of interesting similarities highlight the homology between the SBU-3 proteins and oPAG. First, the oPAG antibody recognizes an array of proteins synthesized by the binucleate cells at the feto-maternal interface (Xie et al., 1991) which are similar in molecular weight to the SBU-3 proteins. Second, the SBU-369 amino-terminal sequence starts at the putative cleavage point for the oPAG protein, i.e. Arg-39. This was also reported by Zoli et al. (1991) for the bovine PAG amino-terminal sequence. Third, the three SBU-3 protein sequences and oPAG all possess the consensus sequence for sulfation of carbohydrate, i.e. Pro-Leu-Arg (Smith et al., 1992). Fourth, residues 4 and 21 in SBU-369 and residues 2 and 19 in SBU-362 were blocked during microsequencing and are followed by the consensus sequence for an N-glycosylation site. These glycosylation sites correspond to the putative N-glycosylation sites described in the oPAG sequence by Xie et al. (1991). Finally, Xie et al. (1991) described oPAG as an inactive aspartic proteinase. The SBU-3 proteins exhibited no proteolytic activity on hemoglobin when assayed in a standard assay for proteinases at pH 2.0 and pH 7.4.3 Immunohistological analyses of SBU-3 and bovine PAG (bPAG) expression in bovine placentomes demonstrated that SBU-3-positive binucleate cells are located only deep within the trophoblast microvilli, whereas bPAG-positive binucleate cells are further away from the feto-maternal interfa~e.~ Such patterns of expression support the notion of differential expression of these proteins within the placentome. It has recently been determined that the PAG proteins and PSPB are the same proteins (Lynch et al., 1992). Although highly homologous, the SBU-3 proteins and oPAG may also be distinct molecules representing a family of structurally related placental proteins such as the human pregnancy-specific PI glycoprotein family (PSG, Tatarinov and Masyukevich, 1970;Bohn, 1971). Like oPAG, these proteins are synthesized in large amounts by the trophoblast (Home et al., 1976;Watanabe and Chou, 1988a), released into the maternal circulation, and increase in concentration as pregnancy progresses (Bohn, 1971). They have recently been characterized as members of the carcinoembryonic Y. H. Atkinson, unpublished observations. B. Low, S. Xie, and R. M. Roberts, personal communication. antigen gene family, which belongs to the immunoglobulin superfamily (Paxton et al., 1987;Watanabe and Chou, 1988b). Although the function of the PSGs is unknown, carcinoembryonic antigen has recently been shown to function as a Ca2+independent homophilic cell adhesion molecule in vitro (Benchimol et al., 1989).
We have shown in the present study that the SBU-3 proteins expressed exclusively at the feto-maternal interface during placentation have sequences that are homologous to oPAG and contain a unique carbohydrate structure. Experiments are in progress to determine whether the SBU-3 epitope is cleaved or modified from oPAG or PSPB. If so, these results may provide an important insight into placenta-specific glycosylation patterns and their role in glycoprotein trafficking.