0013-7227/90/1266-3271$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 6 Printed in U.S.A.
IDENTIFICATION OF A NOVEL FAMILY OF GROWTH HORMONE-RELATED PROTEINS SECRETED BY RAT PLACENTA S. OGILVIE, W.C. BUHI, J.A. OLSON AND K.T. SHIVERICK Departments of Pharmacology and Therapeutics (S.O., J.A.O, K.T.S.) and Obstetrics and Gynecology (W.C.B.), University of Florida, Gainesville, FL 32610 ABSTRACT: Mid-to-late gestation rat placenta synthesizes a number of proteins related to prolactin, including rat placental lactogen II (rPL-II), rat prolactin-like protein A (rPLP-A) and rat prolactin-like protein B (rPLP-B). This study identifies a new family of proteins synthesized and secreted by gestation day 15 placental explants which exhibit amino acid homology to growth hormone precursors from several species. Placental explant medium was fractionated by ammonium sulfate precipitation and analyzed by two-dimensional SDSpolyacrylamide gel electrophoresis to isolate four distinct proteins with Mr values of 28,000, 23,000, 25,000 and 30,000 and pi values of 5.7, 5.7, 5.4 and 5.3, respectively. These proteins represent a major fraction of secretory proteins with Mr in the 20,000 to 30,000 range. Immunoblot analysis showed that none of the four proteins crossreacted with antipeptide antisera against rPL-II, rPLP-A, or rPLP-B. These proteins were electrophoretically transferred from two-dimensional SDS-polyacrylamide gels onto an Immobilon PVDF membrane and N-terminal amino acid microsequencing carried out with a gas phase sequencer. N-terminal sequences of 45, 37, 37 and 32 amino acid residues were identified for proteins 1, 2, 3 and 4, respectively. The four proteins exhibit 76% to 97% homology. Computer analysis further revealed a 28% identity in a 32 amino acid overlap which begins at residue 14 of the 28,000 Mr protein (protein 1) and at residue 31 of growth hormone precursors of rat, mouse and human. The 32 amino acid overlap is 78% homologous if conservative amino acid replacements are included. The developing hemochorial placenta expresses several proteins in the prolactin (PRL)-growth hormone (GH) gene family, believed to have evolved from a common ancestral gene which diverged into two separate lineages, one PRL-related and one GH-related (1, 2). In the rat placenta, members of this gene family include rat placental lactogen II (rPL-II), rat prolactin-like protein A (rPLP-A) and rat prolactin-like protein B (rPLP-B) which exhibit 52%, 43% and 44% homology at the amino acid level to rat PRL, respectively (3-5). Evidence that rodent placental lactogens are more closely related to PRL is in contrast to primates in which placental lactogen belongs to the GH lineage, suggesting different patterns of evolution for the two lineages in rat and human (1, 2). Despite significant sequence homology to PRL, each of the PRL-related proteins are unique and appear to be synthesized in a specific temporal pattern and by different cell types in the mid-tolate gestation rat placenta (3-8). In this paper, we describe the identification and partial characterization of a group of proteins also secreted by basal zone during mid-to-late gestation which, in contrast to previously identified rat placental proteins, show greater homology to GH than PRL. MATERIALS AND METHODS Placental explant culture: Placental tissue, gestation day 15, was aseptically removed from anesthetized rats and dissected. The nomenclature of Davies and Glasser (9) was used to describe the respective zones of the rat placenta. The term basal zone refers to the zone between the labyrinth and decidua basalis which is without fetal mesenchyme and in which only maternal vascular channels are found. Basal zone tissue was incubated under sterile conditions in modified Eagle's Medium (175 mg tissue/5 ml) for 24 hr at 37CC under 47.5% O 2 : 2.5% CO2: 50% N2. The culture media was supplemented with 3 mg/ml glucose, penicillin (100 units/ml), streptomycin (100 Hg/ml), fungizone (0.25 (ig/ml), 1% (v/v) non-essential amino acids and 1.5 ng/ml methionine (1/10 normal concentration). In some experiments, tunicamycin was added from a 10 mg/ml final concentration stock solution in dimethylsulfoxide at the initiation of the cultures. DMSO alone was added to control explants. [35S]methionine, 40 |J.Ci, was added 16 hr after the initiation of the culture (SA, 1045 Ci/mmol, ICN Radiochemicals, Irvine, CA). Following incubation, culture medium was extensively dialyzed (m.w. cut-off, 3500) at 4°C: first against 10 mM TRIS buffer, pH 8.3, containing 0.02% sodium azide and then against deionized water. The dialyzed medium was stored at -20°C.
Ammonium sulfate precipitation of proteins: Proteins in the basal zone explant medium were separated by precipitating sequentially with 45%, 60% and 80% saturated ammonium sulfate and centrifuging after each cut to obtain precipitates. Precipitates were resuspended in distilled water and extensively dialyzed against deionized water. The precipitates from each saturation and the final supernatant were analyzed by twodimensional SDS-polyacylamide gel electrophoresis (SDSPAGE). Polyacrylamide gel electrophoresis and immunoblotting: Both two-dimensional SDS-PAGE and fluorography was performed according to methods of Roberts et al. (10) as described in Ogilvie et al. (11). Equal amounts of [35S]methionine-labeled protein (100,000 cpm) were loaded onto each gel. Electrophoretic transfer of gels to nitrocellulose and subsequent immunoblot analysis was performed as described in Ogilvie et al. (6, 11). Rabbit polyclonal antiserum against rPLP-B was generated against an oligopeptide representing amino acids 186-200 in the deduced amino acid sequence (including the signal peptide) of rPLP-B (5), with an additional cysteine at the amino terminus. An oligopeptide representing amino acids 160-175 of the deduced amino acid sequence (including signal peptide) of rPLP-A (4) with an additional cysteine at the amino terminus was used to generate rabbit polyclonal antiserum against rPLP-A. A rabbit polyclonal antiserum against rPL-II was generated against an oligopeptide representing amino acids 56-70 of rPL-II and this was a generous gift from M.J. Soares. N-terminal amino acid microsequencing of secreted proteins: Proteins were electrophoretically transferred after twodimensional SDS-PAGE onto Immobilon PVDF membranes (Millipore, Bedford, MA) in the Towbin buffer system (12). Following transfer, membranes were extensively rinsed in deionized water, stained with 0.01% Coomassie blue in 50% methanol, destained in 50% methanol, rinsed in deionized water, dried and stored at -20°C until sequencing. N-terminal amino acid microsequencing was performed at the University of Florida Protein Chemistry Core Facility using an Applied Biosystems Model 470A Gas Phase Protein Sequencer with an on-line analytical HPLC system. Sequence data were analyzed and compared to the National Biomedical Research Foundation (NBRF, Washington, DC) protein database and the Genbank Genetic Sequence database (Intelligenetics, Mountain View, CA) using FASTA and TFASTA computer programs (13). These programs are included in the sequence analysis software package of the Genetics Computer Group (GCG, University of Wisconsin, Madison, WI).
Received in Iowa City March 6, 1990 3271
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TABLE 1 N-TERMINAL AMINO ACID SEQUENCES OF RAT PLACENTAL PROTEINS 1-4
RESULTS The [35S]methionine-labeled proteins secreted by explants of rat basal zone (day 15 gestation) were characterized using twodimesional SDS-PAGE and a representative fluorograph is shown in Fig. 1A. Athough several proteins with M r values in the 20,000 to 30,000 range have been recently identified (6,7), those indicated by arrows 1-4 were previously unidentified. Visual inspection of Fig. 1A shows that these proteins represent a substantial fraction of the secretory products within this M r range. Initial purification was achieved by precipitation with 45% saturated ammonium sulfate as shown in Fig. IB and ID. The four proteins are distinct and have been separated from the majority of the other secretory products. Proteins 1-4 have M r values of 28,000, 23,000, 25,000 and 30,000 with pi values of 5.7, 5.7, 5.4 and 5.3, respectively. The inclusion of tunicamycin, an inhibitor of N-linked glcosylation, during the explant incubation period, markedly inhibited the secretion of a number of proteins synthesized de novo by the basal zone explants (Fig. 1C). Of the four proteins under investigation, only the secretion of protein 4
E n d o • 1990 Vol 126 • No 6
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#1 IPACLAEEGG CWNPJ.VETFN SAIHKAETLY DLANQIWVEL* #2 IPACMVEDGG CWDPLREAFN SATQRAETLR NLSDQLY #3 IPACMVEEGD CWDPLQETFN SAIQRAETLC NLADQLY #4 IPACMVEDGG CWDPLGEAFN SATQRAETLR NL
Residues identical in all four proteins have been underlined. * Residues 41-45 of protein 1 are YQNKF. acid replacements are considered the four proteins exhibit even greater homology ranging from 76% to 97% (Table 2). The Nterminal amino acid sequences of proteins 2 and 4 are identical with the exception of a single amino acid at position 16. Based on the N-terminal amino acid sequences, proteins 2 and 4 are more closely related to protein 3 than to protein 1. TABLE 2 AMINO ACID HOMOLOGIES OF RAT PLACENTAL PROTEINS 1-4
335 92 76 66
45Z (NH4)2S04 Pellet
Total
.
. . .
45 •
30
*
.•»
1
1
Protein
1
2
3
4
1
100
76
84
78
2
76
100
84
97
3
84
84
100
84
4
78
97
84
100
*
A.
20
c
1? • Tunicamycin (10 ug/ml)
FIG. 1. REPRESENTATIVE FLUOROGRAPHS OF TWODIMENSIONAL SDS-POLYARCYLAMIDE GELS OF PROTEINS SECRETED BY BASAL ZONE EXPLANTS. (A)
Fluorograph of [35S]methionine-labeled proteins in culture medium. (B) Fluorograph of [35S]methionine-labeled proteins shown in (A) precipitated by 45% saturated ammonium sulfate. The proteins of interest are labeled 1-4. (C) Flourograph of [35S]methionine-labeled proteins secreted in the presence of tunicamycin. (D) An enlargement of the area of the polyacrylamide gel outlined by the square in (B) in which the proteins are stained with Coomassie blue. was inhibited by tunicamycin, suggesting it is the only protein in the group that is N-glycosylated. In a previous study, rPL-II, rPLP-A and rPLP-B proteins were identified on Western blots of the total profile of proteins secreted by basal zone explants with antipeptide antisera against rPL-II, rPLP-A and rPLP-B, respectively (6). In data not shown, none of the the antipeptide antisera crossreacted with the four proteins shown in Fig. ID. To further characterize the four proteins, N-terminal amino acid microsequencing was performed following two-dimensional SDS-PAGE and electrophoretic transfer onto an Immobilon PVDF membrane. N-terminal sequences of 45, 37, 37 and 32 amino acid residues were identified for proteins 1, 2, 3 and 4, respectively and are shown in Table 1. The sequence homology for identical amino acids between the four proteins ranges from 60% to 97%. If conservative amino
Numbers represent percent related amino acids between secreted proteins, which are considered to be L=I=V=M=A=G, S=T, D=E, K=R, Q=N and Y=F=W. A computer search of the NBRF protein and Genbank nucleic acid databases using both the FASTA and TFASTA programs of the GCG software package revealed a 28% identity in a 32 amino acid overlap between protein 1 and GH precursors of rat (Fig. 2) as well as mouse, human and human placental variant. The overlap begins at residue 14 of protein 1 and at residue 31 of the GH precursors. Residues 1-26 of GH precursors have been reported to correspond to the signal sequence, which is lacking in secreted GH (14-17). If conservative amino acid replacements are included, the 32 amino acid overlap between protein 1 and GH precursors is 78% homologous. Computer analysis further revealed a 42% identity in a 24 amino acid overlap between protein 3 and GH precursors of rat (Fig. 2), mouse and human. The 24 amino acid overlap begins at residue 14 of protein 3 and residue 31 of the GH precursors. The 24 amino acid overlap is 75% homologous if related amino acids are included. DISCUSSION Three PRL-related proteins, rPL-II, rPLP-A and rPLP-B are expressed in mid-to-late term rat placenta (3-8). This report identifies four proteins synthesized and secreted by gestation day 15 rat placenta which exhibit amino acid sequence homology to GH rather than PRL. Despite different Mr and pi values for all four proteins, the N-terminal amino acid sequences indicate the proteins are highly related to each other and to GH precursors from
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protein 1 IPACUVEEGGCWNPLVETFNSAIHKAETLYDLANQIWVELYQNKF X: . : . . : . . : . : . . : X : ... . K3Hp LLWPQEAGAFPAMPLSSLFANAVLRAQHLHQLAADTYKE-FERAY 20 X: . : . . : . : : . : .:X.. : 60
protein 3 IPACMVEEGDCWDPLQETFNSAIQRAETLCNLADQLY 10
20
30
FIG. 2. AMINO ACID COMPARISON OF NEWLY IDENTIFIED RAT PLACENTAL PROTEINS WITH RAT GROWTH HORMONE PRECURSOR. N-terminal amino acid sequence of
protein 1 and 3 compared to partial sequence of rat GH precursor (rGHp). The amino acid sequence for rGHp is from Page et al. (17). The computer program utilized for the comparison is from Pearson and Lipman (13). X denotes the ends of the initial region of identity found by the program; a colon between the two sequences denotes identity and a dot indicates a conservative replacement. several species. Furthermore, the degree of amino acid homology between the N-terminal sequences of proteins 1 and 3 and GH precursors is similar to that observed between other members of the PRL-GH family (3-5). In addition to amino acid sequence homology to GH, the four newly identified proteins exhibit two cysteine residues, one at position 4 and one at position 11. The presence of two cysteines in the N-terminal region of the sequence is characteristic of PRLs and mouse proliferin (2, 18). The physiologic function of the newly identified placental GH-related peptides is unknown, but expression of GH-related peptides in the placenta is not without precedent. The human growth hormone variant gene (hGH-V) is expressed in term placenta, specifically in the syncytiotrophoblast of the villi (19, 20). Frankenne and coworkers have proposed that placental GH may replace pituitary GH in governing maternal metabolism during the second half of pregnancy (20). Interestingly, the region of the rat placenta which secretes the newly identified GH-related peptides, the basal zone, has only maternal vascular channels. Previous studies have shown that both mouse and rat placenta are a source of GH-releasing hormone (GHRH) and the site of synthesis of GHRH in the rat placenta has been identified as cytotrophoblast cells (21-24). Further, it has been suggested that placental GHRH may be a paracrine factor regulating placental protein expression and secretion (21-24). Evidence from the present study suggests that basal zone cells which synthesize GHrelated proteins may be targets for placental GHRH. The findings of this study, as well as previous investigations indicate the complexity of the placental hormonal milieu and its regulation during pregnancy. Additional studies characterizing the newly identified GH-related proteins produced in the rat placenta may increase our understanding of events governing a successful pregnancy. REFERENCES 1. Miller WL, Eberhardt NL 1983. Structure and evolution of the growth hormone gene family. Endocrine Rev 4:97 2. Nicoll CS, Gregg LM, Russell SM 1986 Structural features of prolactins and growth hormones that can be related to their biological function. Endocrine Rev 7:169 3. Duckworth ML, Kirk KL, Friesen HG 1986 Isolation and identification of a cDNA clone of rat placental lactogen n. J BiolChem 261:10871 4. Duckworth ML, Peden LM, Friesen HG 1986 Isolation of a novel prolactin-like cDNA clone from developing rat placenta. I BiolChem 261:10879 5. Duckworth ML, Peden LM, Friesen, HG 1988 A third prolactin-like protein expressed by the developing rat placenta: complementary deoxyribonucleic acid sequence and partial structure of the gene. Mol Endocrinol 2:912
6. Ogilvie S, Duckworth ML, Larkin LH, Buhi WC, Shiverick KT 1990 De novo synthesis and secretion of prolactin-like protein B by rat placental explants. Endocrinology In Press 7. Campbell WJ, Deb S, Kwok SCM, Joslin JA, Soares MJ 1989 Differential expression of placental lactogen-II and prolactin-like protein-A in the rat chorioallantoic placenta. Endocrinology 125:1565 8. Duckworth ML, Schroedter I, Friesen HG 1990 Cellular localization of rat placental lactogen II and rat prolactin-like proteins A and B by in situ hybridization. Placenta In Press 9. Davies J, Glasser SR 1968 Histological and fine structural observations on the placenta of the rat. Acta Anat (Basel) 69:542 10. Roberts RM, Baumbach GA, Buhi WC, Denny JB, Fitzgerald LA, Babelyn SF, Horst, MN 1984 Analysis of membrane polypeptides by two-dimensional polyacrylamide gel electrophoresis. In: Venter JC, Harrison LC (eds) Receptor Biochemistry and Methodology: Molecular and Chemical Characterization of Membrane Receptors. Alan R. Liss Inc., New York, Vol 111:61 11. Ogilvie S, Kvello-Stenstrom AG, Hammond G, Buhi WC, Larkin LH, Shiverick KT 1989 Identification of proteins immunochemically related to pregnancy specific Bi glycoprotein in the rat placenta. Endocrinology 125:287 12. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Nad Acad Sci USA 76:4350 13. Pearson WR, Lipman DJ 1988 Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444 14. Linzer DIH, Talamantes F 1986 Nucleotide sequence of mouse prolactin and growth hormone mRNAs and expression of these mRNAs during pregnancy. J Biol Chem 260:9574 15. Martial JA, Hallewall RA, Baxter JD, Goodman HM 1979 Human growth hormone: Complementary DNA cloning and expression in bacteria. Science 205:602 16. Seeburg PH, Shine J, Martial JA, Baxter JD, Goodman HM 1977 Nucleotide sequence and amplification in bacteria of the structural gene for rat growth hormone. Nature 270:486 17. Page GS, Smith S, Goodman HM 1981 DNA sequence of the rat growth hormone gene. Location of the 5' terminus of the growth hormone messenger-RNA and identification of an internal transposon-like element. Nucl Acids Res 9:2087 18. Linzer DIH, Nathans D 1985 A new member of the prolactin-growth hormone gene family expressed in mouse placenta. EMBOJ 4:1419-1423 19. Liebhaber SA, Urbanek M, Jharna R, Tuan RS Cooke, NE 1989 Characterization and histologic localization of human growth hormone-variant gene expression in the placenta. J Clin Invest 83:1985 20. Frankenne F, Closset J, Gomez F, Scippo, ML, Smal J, Hennen G 1988 The physiology of growth hormones (GHs) in pregnant women and partial characterization of the placental GH variant. J Clin Endocrinol Metab 66:1171 21. Meigan G, Sasaki A, Yoshinaga K 1988 Immunoreactive growth hormone-releasing hormone in rat placenta. Endocrinology 123:1098 22. Baird A, Wehrenberg WB, Bohlen P, Ling N 1985 Immnoreactive and biologically active growth hormonereleasing factor in the rat placenta. Endocrinology 117:1598 23. Margioris AN, Brockmann G, Bohler HCL, Grino M, Vamvakopoulos N, Chrousos GP 1990 Expression and localization of growth hormone-releasing hormone messenger ribonucleic acid in rat placenta: In vitro secretion and regulation of its peptide product. Endocrinology 126:151 24. Suhr ST, Rahal JO, Mayo KE 1989 Mouse growth hormone-releasing hormone: Precursor structure and expression in brain and placenta. Mol Endocrinol 3:1693
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3273
Vol. 126, No. 6 Printed in U.S.A.
IDENTIFICATION OF A NOVEL FAMILY OF GROWTH HORMONE-RELATED PROTEINS SECRETED BY RAT PLACENTA S. OGILVIE, W.C. BUHI, J.A. OLSON AND K.T. SHIVERICK Departments of Pharmacology and Therapeutics (S.O., J.A.O, K.T.S.) and Obstetrics and Gynecology (W.C.B.), University of Florida, Gainesville, FL 32610 ABSTRACT: Mid-to-late gestation rat placenta synthesizes a number of proteins related to prolactin, including rat placental lactogen II (rPL-II), rat prolactin-like protein A (rPLP-A) and rat prolactin-like protein B (rPLP-B). This study identifies a new family of proteins synthesized and secreted by gestation day 15 placental explants which exhibit amino acid homology to growth hormone precursors from several species. Placental explant medium was fractionated by ammonium sulfate precipitation and analyzed by two-dimensional SDSpolyacrylamide gel electrophoresis to isolate four distinct proteins with Mr values of 28,000, 23,000, 25,000 and 30,000 and pi values of 5.7, 5.7, 5.4 and 5.3, respectively. These proteins represent a major fraction of secretory proteins with Mr in the 20,000 to 30,000 range. Immunoblot analysis showed that none of the four proteins crossreacted with antipeptide antisera against rPL-II, rPLP-A, or rPLP-B. These proteins were electrophoretically transferred from two-dimensional SDS-polyacrylamide gels onto an Immobilon PVDF membrane and N-terminal amino acid microsequencing carried out with a gas phase sequencer. N-terminal sequences of 45, 37, 37 and 32 amino acid residues were identified for proteins 1, 2, 3 and 4, respectively. The four proteins exhibit 76% to 97% homology. Computer analysis further revealed a 28% identity in a 32 amino acid overlap which begins at residue 14 of the 28,000 Mr protein (protein 1) and at residue 31 of growth hormone precursors of rat, mouse and human. The 32 amino acid overlap is 78% homologous if conservative amino acid replacements are included. The developing hemochorial placenta expresses several proteins in the prolactin (PRL)-growth hormone (GH) gene family, believed to have evolved from a common ancestral gene which diverged into two separate lineages, one PRL-related and one GH-related (1, 2). In the rat placenta, members of this gene family include rat placental lactogen II (rPL-II), rat prolactin-like protein A (rPLP-A) and rat prolactin-like protein B (rPLP-B) which exhibit 52%, 43% and 44% homology at the amino acid level to rat PRL, respectively (3-5). Evidence that rodent placental lactogens are more closely related to PRL is in contrast to primates in which placental lactogen belongs to the GH lineage, suggesting different patterns of evolution for the two lineages in rat and human (1, 2). Despite significant sequence homology to PRL, each of the PRL-related proteins are unique and appear to be synthesized in a specific temporal pattern and by different cell types in the mid-tolate gestation rat placenta (3-8). In this paper, we describe the identification and partial characterization of a group of proteins also secreted by basal zone during mid-to-late gestation which, in contrast to previously identified rat placental proteins, show greater homology to GH than PRL. MATERIALS AND METHODS Placental explant culture: Placental tissue, gestation day 15, was aseptically removed from anesthetized rats and dissected. The nomenclature of Davies and Glasser (9) was used to describe the respective zones of the rat placenta. The term basal zone refers to the zone between the labyrinth and decidua basalis which is without fetal mesenchyme and in which only maternal vascular channels are found. Basal zone tissue was incubated under sterile conditions in modified Eagle's Medium (175 mg tissue/5 ml) for 24 hr at 37CC under 47.5% O 2 : 2.5% CO2: 50% N2. The culture media was supplemented with 3 mg/ml glucose, penicillin (100 units/ml), streptomycin (100 Hg/ml), fungizone (0.25 (ig/ml), 1% (v/v) non-essential amino acids and 1.5 ng/ml methionine (1/10 normal concentration). In some experiments, tunicamycin was added from a 10 mg/ml final concentration stock solution in dimethylsulfoxide at the initiation of the cultures. DMSO alone was added to control explants. [35S]methionine, 40 |J.Ci, was added 16 hr after the initiation of the culture (SA, 1045 Ci/mmol, ICN Radiochemicals, Irvine, CA). Following incubation, culture medium was extensively dialyzed (m.w. cut-off, 3500) at 4°C: first against 10 mM TRIS buffer, pH 8.3, containing 0.02% sodium azide and then against deionized water. The dialyzed medium was stored at -20°C.
Ammonium sulfate precipitation of proteins: Proteins in the basal zone explant medium were separated by precipitating sequentially with 45%, 60% and 80% saturated ammonium sulfate and centrifuging after each cut to obtain precipitates. Precipitates were resuspended in distilled water and extensively dialyzed against deionized water. The precipitates from each saturation and the final supernatant were analyzed by twodimensional SDS-polyacylamide gel electrophoresis (SDSPAGE). Polyacrylamide gel electrophoresis and immunoblotting: Both two-dimensional SDS-PAGE and fluorography was performed according to methods of Roberts et al. (10) as described in Ogilvie et al. (11). Equal amounts of [35S]methionine-labeled protein (100,000 cpm) were loaded onto each gel. Electrophoretic transfer of gels to nitrocellulose and subsequent immunoblot analysis was performed as described in Ogilvie et al. (6, 11). Rabbit polyclonal antiserum against rPLP-B was generated against an oligopeptide representing amino acids 186-200 in the deduced amino acid sequence (including the signal peptide) of rPLP-B (5), with an additional cysteine at the amino terminus. An oligopeptide representing amino acids 160-175 of the deduced amino acid sequence (including signal peptide) of rPLP-A (4) with an additional cysteine at the amino terminus was used to generate rabbit polyclonal antiserum against rPLP-A. A rabbit polyclonal antiserum against rPL-II was generated against an oligopeptide representing amino acids 56-70 of rPL-II and this was a generous gift from M.J. Soares. N-terminal amino acid microsequencing of secreted proteins: Proteins were electrophoretically transferred after twodimensional SDS-PAGE onto Immobilon PVDF membranes (Millipore, Bedford, MA) in the Towbin buffer system (12). Following transfer, membranes were extensively rinsed in deionized water, stained with 0.01% Coomassie blue in 50% methanol, destained in 50% methanol, rinsed in deionized water, dried and stored at -20°C until sequencing. N-terminal amino acid microsequencing was performed at the University of Florida Protein Chemistry Core Facility using an Applied Biosystems Model 470A Gas Phase Protein Sequencer with an on-line analytical HPLC system. Sequence data were analyzed and compared to the National Biomedical Research Foundation (NBRF, Washington, DC) protein database and the Genbank Genetic Sequence database (Intelligenetics, Mountain View, CA) using FASTA and TFASTA computer programs (13). These programs are included in the sequence analysis software package of the Genetics Computer Group (GCG, University of Wisconsin, Madison, WI).
Received in Iowa City March 6, 1990 3271
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3272
TABLE 1 N-TERMINAL AMINO ACID SEQUENCES OF RAT PLACENTAL PROTEINS 1-4
RESULTS The [35S]methionine-labeled proteins secreted by explants of rat basal zone (day 15 gestation) were characterized using twodimesional SDS-PAGE and a representative fluorograph is shown in Fig. 1A. Athough several proteins with M r values in the 20,000 to 30,000 range have been recently identified (6,7), those indicated by arrows 1-4 were previously unidentified. Visual inspection of Fig. 1A shows that these proteins represent a substantial fraction of the secretory products within this M r range. Initial purification was achieved by precipitation with 45% saturated ammonium sulfate as shown in Fig. IB and ID. The four proteins are distinct and have been separated from the majority of the other secretory products. Proteins 1-4 have M r values of 28,000, 23,000, 25,000 and 30,000 with pi values of 5.7, 5.7, 5.4 and 5.3, respectively. The inclusion of tunicamycin, an inhibitor of N-linked glcosylation, during the explant incubation period, markedly inhibited the secretion of a number of proteins synthesized de novo by the basal zone explants (Fig. 1C). Of the four proteins under investigation, only the secretion of protein 4
E n d o • 1990 Vol 126 • No 6
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#1 IPACLAEEGG CWNPJ.VETFN SAIHKAETLY DLANQIWVEL* #2 IPACMVEDGG CWDPLREAFN SATQRAETLR NLSDQLY #3 IPACMVEEGD CWDPLQETFN SAIQRAETLC NLADQLY #4 IPACMVEDGG CWDPLGEAFN SATQRAETLR NL
Residues identical in all four proteins have been underlined. * Residues 41-45 of protein 1 are YQNKF. acid replacements are considered the four proteins exhibit even greater homology ranging from 76% to 97% (Table 2). The Nterminal amino acid sequences of proteins 2 and 4 are identical with the exception of a single amino acid at position 16. Based on the N-terminal amino acid sequences, proteins 2 and 4 are more closely related to protein 3 than to protein 1. TABLE 2 AMINO ACID HOMOLOGIES OF RAT PLACENTAL PROTEINS 1-4
335 92 76 66
45Z (NH4)2S04 Pellet
Total
.
. . .
45 •
30
*
.•»
1
1
Protein
1
2
3
4
1
100
76
84
78
2
76
100
84
97
3
84
84
100
84
4
78
97
84
100
*
A.
20
c
1? • Tunicamycin (10 ug/ml)
FIG. 1. REPRESENTATIVE FLUOROGRAPHS OF TWODIMENSIONAL SDS-POLYARCYLAMIDE GELS OF PROTEINS SECRETED BY BASAL ZONE EXPLANTS. (A)
Fluorograph of [35S]methionine-labeled proteins in culture medium. (B) Fluorograph of [35S]methionine-labeled proteins shown in (A) precipitated by 45% saturated ammonium sulfate. The proteins of interest are labeled 1-4. (C) Flourograph of [35S]methionine-labeled proteins secreted in the presence of tunicamycin. (D) An enlargement of the area of the polyacrylamide gel outlined by the square in (B) in which the proteins are stained with Coomassie blue. was inhibited by tunicamycin, suggesting it is the only protein in the group that is N-glycosylated. In a previous study, rPL-II, rPLP-A and rPLP-B proteins were identified on Western blots of the total profile of proteins secreted by basal zone explants with antipeptide antisera against rPL-II, rPLP-A and rPLP-B, respectively (6). In data not shown, none of the the antipeptide antisera crossreacted with the four proteins shown in Fig. ID. To further characterize the four proteins, N-terminal amino acid microsequencing was performed following two-dimensional SDS-PAGE and electrophoretic transfer onto an Immobilon PVDF membrane. N-terminal sequences of 45, 37, 37 and 32 amino acid residues were identified for proteins 1, 2, 3 and 4, respectively and are shown in Table 1. The sequence homology for identical amino acids between the four proteins ranges from 60% to 97%. If conservative amino
Numbers represent percent related amino acids between secreted proteins, which are considered to be L=I=V=M=A=G, S=T, D=E, K=R, Q=N and Y=F=W. A computer search of the NBRF protein and Genbank nucleic acid databases using both the FASTA and TFASTA programs of the GCG software package revealed a 28% identity in a 32 amino acid overlap between protein 1 and GH precursors of rat (Fig. 2) as well as mouse, human and human placental variant. The overlap begins at residue 14 of protein 1 and at residue 31 of the GH precursors. Residues 1-26 of GH precursors have been reported to correspond to the signal sequence, which is lacking in secreted GH (14-17). If conservative amino acid replacements are included, the 32 amino acid overlap between protein 1 and GH precursors is 78% homologous. Computer analysis further revealed a 42% identity in a 24 amino acid overlap between protein 3 and GH precursors of rat (Fig. 2), mouse and human. The 24 amino acid overlap begins at residue 14 of protein 3 and residue 31 of the GH precursors. The 24 amino acid overlap is 75% homologous if related amino acids are included. DISCUSSION Three PRL-related proteins, rPL-II, rPLP-A and rPLP-B are expressed in mid-to-late term rat placenta (3-8). This report identifies four proteins synthesized and secreted by gestation day 15 rat placenta which exhibit amino acid sequence homology to GH rather than PRL. Despite different Mr and pi values for all four proteins, the N-terminal amino acid sequences indicate the proteins are highly related to each other and to GH precursors from
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protein 1 IPACUVEEGGCWNPLVETFNSAIHKAETLYDLANQIWVELYQNKF X: . : . . : . . : . : . . : X : ... . K3Hp LLWPQEAGAFPAMPLSSLFANAVLRAQHLHQLAADTYKE-FERAY 20 X: . : . . : . : : . : .:X.. : 60
protein 3 IPACMVEEGDCWDPLQETFNSAIQRAETLCNLADQLY 10
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FIG. 2. AMINO ACID COMPARISON OF NEWLY IDENTIFIED RAT PLACENTAL PROTEINS WITH RAT GROWTH HORMONE PRECURSOR. N-terminal amino acid sequence of
protein 1 and 3 compared to partial sequence of rat GH precursor (rGHp). The amino acid sequence for rGHp is from Page et al. (17). The computer program utilized for the comparison is from Pearson and Lipman (13). X denotes the ends of the initial region of identity found by the program; a colon between the two sequences denotes identity and a dot indicates a conservative replacement. several species. Furthermore, the degree of amino acid homology between the N-terminal sequences of proteins 1 and 3 and GH precursors is similar to that observed between other members of the PRL-GH family (3-5). In addition to amino acid sequence homology to GH, the four newly identified proteins exhibit two cysteine residues, one at position 4 and one at position 11. The presence of two cysteines in the N-terminal region of the sequence is characteristic of PRLs and mouse proliferin (2, 18). The physiologic function of the newly identified placental GH-related peptides is unknown, but expression of GH-related peptides in the placenta is not without precedent. The human growth hormone variant gene (hGH-V) is expressed in term placenta, specifically in the syncytiotrophoblast of the villi (19, 20). Frankenne and coworkers have proposed that placental GH may replace pituitary GH in governing maternal metabolism during the second half of pregnancy (20). Interestingly, the region of the rat placenta which secretes the newly identified GH-related peptides, the basal zone, has only maternal vascular channels. Previous studies have shown that both mouse and rat placenta are a source of GH-releasing hormone (GHRH) and the site of synthesis of GHRH in the rat placenta has been identified as cytotrophoblast cells (21-24). Further, it has been suggested that placental GHRH may be a paracrine factor regulating placental protein expression and secretion (21-24). Evidence from the present study suggests that basal zone cells which synthesize GHrelated proteins may be targets for placental GHRH. The findings of this study, as well as previous investigations indicate the complexity of the placental hormonal milieu and its regulation during pregnancy. Additional studies characterizing the newly identified GH-related proteins produced in the rat placenta may increase our understanding of events governing a successful pregnancy. REFERENCES 1. Miller WL, Eberhardt NL 1983. Structure and evolution of the growth hormone gene family. Endocrine Rev 4:97 2. Nicoll CS, Gregg LM, Russell SM 1986 Structural features of prolactins and growth hormones that can be related to their biological function. Endocrine Rev 7:169 3. Duckworth ML, Kirk KL, Friesen HG 1986 Isolation and identification of a cDNA clone of rat placental lactogen n. J BiolChem 261:10871 4. Duckworth ML, Peden LM, Friesen HG 1986 Isolation of a novel prolactin-like cDNA clone from developing rat placenta. I BiolChem 261:10879 5. Duckworth ML, Peden LM, Friesen, HG 1988 A third prolactin-like protein expressed by the developing rat placenta: complementary deoxyribonucleic acid sequence and partial structure of the gene. Mol Endocrinol 2:912
6. Ogilvie S, Duckworth ML, Larkin LH, Buhi WC, Shiverick KT 1990 De novo synthesis and secretion of prolactin-like protein B by rat placental explants. Endocrinology In Press 7. Campbell WJ, Deb S, Kwok SCM, Joslin JA, Soares MJ 1989 Differential expression of placental lactogen-II and prolactin-like protein-A in the rat chorioallantoic placenta. Endocrinology 125:1565 8. Duckworth ML, Schroedter I, Friesen HG 1990 Cellular localization of rat placental lactogen II and rat prolactin-like proteins A and B by in situ hybridization. Placenta In Press 9. Davies J, Glasser SR 1968 Histological and fine structural observations on the placenta of the rat. Acta Anat (Basel) 69:542 10. Roberts RM, Baumbach GA, Buhi WC, Denny JB, Fitzgerald LA, Babelyn SF, Horst, MN 1984 Analysis of membrane polypeptides by two-dimensional polyacrylamide gel electrophoresis. In: Venter JC, Harrison LC (eds) Receptor Biochemistry and Methodology: Molecular and Chemical Characterization of Membrane Receptors. Alan R. Liss Inc., New York, Vol 111:61 11. Ogilvie S, Kvello-Stenstrom AG, Hammond G, Buhi WC, Larkin LH, Shiverick KT 1989 Identification of proteins immunochemically related to pregnancy specific Bi glycoprotein in the rat placenta. Endocrinology 125:287 12. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Nad Acad Sci USA 76:4350 13. Pearson WR, Lipman DJ 1988 Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444 14. Linzer DIH, Talamantes F 1986 Nucleotide sequence of mouse prolactin and growth hormone mRNAs and expression of these mRNAs during pregnancy. J Biol Chem 260:9574 15. Martial JA, Hallewall RA, Baxter JD, Goodman HM 1979 Human growth hormone: Complementary DNA cloning and expression in bacteria. Science 205:602 16. Seeburg PH, Shine J, Martial JA, Baxter JD, Goodman HM 1977 Nucleotide sequence and amplification in bacteria of the structural gene for rat growth hormone. Nature 270:486 17. Page GS, Smith S, Goodman HM 1981 DNA sequence of the rat growth hormone gene. Location of the 5' terminus of the growth hormone messenger-RNA and identification of an internal transposon-like element. Nucl Acids Res 9:2087 18. Linzer DIH, Nathans D 1985 A new member of the prolactin-growth hormone gene family expressed in mouse placenta. EMBOJ 4:1419-1423 19. Liebhaber SA, Urbanek M, Jharna R, Tuan RS Cooke, NE 1989 Characterization and histologic localization of human growth hormone-variant gene expression in the placenta. J Clin Invest 83:1985 20. Frankenne F, Closset J, Gomez F, Scippo, ML, Smal J, Hennen G 1988 The physiology of growth hormones (GHs) in pregnant women and partial characterization of the placental GH variant. J Clin Endocrinol Metab 66:1171 21. Meigan G, Sasaki A, Yoshinaga K 1988 Immunoreactive growth hormone-releasing hormone in rat placenta. Endocrinology 123:1098 22. Baird A, Wehrenberg WB, Bohlen P, Ling N 1985 Immnoreactive and biologically active growth hormonereleasing factor in the rat placenta. Endocrinology 117:1598 23. Margioris AN, Brockmann G, Bohler HCL, Grino M, Vamvakopoulos N, Chrousos GP 1990 Expression and localization of growth hormone-releasing hormone messenger ribonucleic acid in rat placenta: In vitro secretion and regulation of its peptide product. Endocrinology 126:151 24. Suhr ST, Rahal JO, Mayo KE 1989 Mouse growth hormone-releasing hormone: Precursor structure and expression in brain and placenta. Mol Endocrinol 3:1693
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