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Endocrinology Vol. 141, No. 9 3343-3352
Copyright © 2000 by The Endocrine Society

ARTICLES

Identification and Characterization of a New Member of the Placental Prolactin-Like Protein-C (PLP-C) Subfamily, PLP-Cß1

In-Taek Hwang2, Yong-Hun Lee2, Boung-Cheon Moon, Kyu-Youn Ahn, See-Woo Lee and Jong-Yoon Chun

Kumho Life and Environmental Science Laboratory (Y.H.L, I.T.H., J.Y.C.); Chonnam National University Research Institute of Medical Science and Department of Anatomy, Chonnam National University Medical School (B.C.M., K.Y.A.), Kwangju 506-712, Korea; and Clon Biotech Laboratory (S.W.L.), Seoul 143-721, Korea

Address all correspondence and requests for reprints to: Dr. Jong-Yoon Chun, Kumho Life and Environmental Science Laboratory, 572 Sangam-Dong, Kwangsan-Gu, Kwangju 506–712, Korea. E-mail: jychun{at}ksc.kumho.co.kr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have isolated a complementary DNA (cDNA) clone that encodes a new member of the PRL-like protein-C (PLP-C) subfamily of the PRL gene family. The clone was amplified from a 13.5-day-old mouse conceptus cDNA library by PCR using primers based on conserved regions of PLP-C sequences. The full-length cDNA encodes a predicted protein of 241 residues, which contains a putative signal sequence and 2 putative N-linked glycosylation sites. The predicted protein shares 55–66% amino acid identity with mouse PLP-C{alpha} and rat PLP-D, PLP-H, PLP-Cv, and PLP-C and also contains 6 homologously positioned cysteine residues. Thus, we named this protein PLP-Cß for consistency. We have also isolated rat PLP-Cß from rat placenta cDNA library. Surprisingly, two messenger RNA (mRNA) isoforms of rat PLP-Cß were isolated: one mRNA (rPLP-Cß) encodes a 241-amino acid product, but another mRNA (rPLP-Cß{Delta}39) lacks 39 bases that encode for a region rich in aromatic amino acids. The 39-bp region corresponds to exon 3 of other PLP-C subfamily members, such as PLP-C{alpha}, PLP-Cv, and d/tPRP. It suggests that the two isoforms are probably generated by an alternative splicing from a single gene. RT-PCR analysis revealed that the rPLP-Cß form was dominantly expressed in placenta, although both isoforms are coexpressed during placentation. The mouse PLP-Cß mRNA expression, which was specific to the placenta, was first detected by Northern analysis on embryonic day 11.5 (E 11.5) and persisted until birth. However, in situ hybridization analysis revealed mPLP-Cß expression on E 10.5 in specific trophoblast subsets, such as giant cells and spongiotrophoblast cells. mPLP-Cß mRNA was detected in the labyrinthine zone on E 18.5, suggesting that spongiotrophoblast cells had penetrated the labyrinthotrophoblast zone. Consistent with the observed expression in trophoblast giant cells, PLP-Cß expression was also detected in in vitro differentiated Rcho-1 cells, which express the trophoblast giant cell phenotype. In summary, overall high amino acid identity (79%), the locations of cysteine residues, and consensus sites for N-linked glycosylation between mouse and rat PLP-Cß clearly indicate that PLP-Cß is a bona fide member of the PLP-C subfamily. The conservation between mouse and rat, the presence of alternative isoforms, and the pattern of expression during gestation suggest the biological significance of PLP-Cß during pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PRL FAMILY includes a number of hormones that are involved in the regulation of maternal adaptations to pregnancy. To date, at least 19 distinct members of the PRL family have been isolated from either mouse or rat (1, 2, 3, 4, 5, 6). PRL family proteins are divided into 2 groups, classical and nonclassical, on the basis of their biological activities: classical members (e.g. PRL, PL-I, PL-Iv, and PL-II) use the PRL receptor signaling pathway, whereas nonclassical members use different pathways (4). The family is further divided into subfamilies [e.g. PL-I and PRL-like protein C (PLP-C) subfamilies] on the basis of structural considerations, including gene structure, primary structure, and posttranslational modification. Members of the PLP subfamily are nonclassical PRL family proteins and include PLP-A, PLP-B (7), PLP-C (8), PLP-Cv (9), PLP-C{alpha} (1), PLP-D (10), PLP-F (11), PLP-G (3), PLP-H (2), and d/tPRP (7, 12, 13, 14). Recently, 4 additional members of this family have been identified in the rat and named PLP-I, PLP-J, PLP-K, and PLP-L (5, 6). Of these, the PLP-C subfamily consists of at least 7 different proteins, 5 of which (PLP-C, PLP-Cv, PLP-D, PLP-H, and d/tPRP) have been identified in the rat and 2 (PLP-C{alpha} and d/tPRP) in the mouse. The PLP-C subfamily is characterized by a high degree of amino acid sequence identity, a 6-exon/5-intron gene structure, and the presence of 6 highly conserved cysteine residues (Refs. 1, 4, 9, 13 and this study). However, their biological functions are not yet known. Only the gene structure and expression of the PLP-C subfamily members have been reported (1, 2, 8, 9, 10, 12, 13, 14).

During efforts to molecularly clone additional member genes of the PLP-C subfamily, we have isolated a complementary DNA (cDNA) clone encoding a novel member of the PLP-C subfamily from mouse conceptus by homology-based PCR and also isolated its rat homolog cDNA from rat placenta. Surprisingly, we also isolated an alternative messenger RNA (mRNA) isoform of the rat homolog, which probably generated by an alternative splicing from a single gene.

As the nonclassical PRL family members, little is known about alternative isoforms. Only a few of the hGH and classical PRL family genes have been reported to produce alternative isoforms (15, 16, 17, 18). Here, we describe the structure and expression of this novel mouse molecule mRNA and the alternative isoforms of its rat homologs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
ICR and FVB mice (Dae Han Laboratory Animal Research Center, Korea) were used throughout the study. The day on which a copulation plug first appeared was designated embryonic day 0.5 (E 0.5), assuming that mating had taken place at midnight.

Cloning of a new member of mouse PLP-C subfamily gene, mPLP-Cß
PLP-C subfamily genes were amplified from a 13.5-day-old mouse conceptus cDNA library (19) by PCR using the degenerate oligonucleotide primers 5'-TGT GGG AA/GA AAG CTT/G CAG/T CA-3' (sense) and 5'-G/TTG AGT ATC CAC C/T TA AGG TC-3' (antisense; Fig. 1Go). These oligonucleotides were designed to match the highly conserved regions identified by a comparative analysis of all available sequences of PLP-C subfamily genes. All PCRs were performed in a final reaction volume of 50 µl containing 2.5 U Taq DNA polymerase (Promega Corp., Madison, WI), 2.5 mM MgCl2, 0.2 mM each dNTP, 1 µM each primer (Bioneer, Korea), and 3 µl of cDNA library suspension containing about 108 plaque-forming units of bacteriophage. Samples were denatured for 5 min at 94 C and subjected to 35 cycles of 1 min at 94 C, 1 min at 55 C, and 1 min at 72 C, followed by a 7-min incubation at 72 C. Amplified cDNA fragments of the expecting size (460-bp) were obtained and cloned into the pGEM-T Easy vector (Promega Corp.). The inserts of the resulting clones were sequenced and those containing PLP-C subfamily member sequences were identified by sequence comparisons using the BLAST family of programs (20). The mouse conceptus cDNA library was screened with the radio-labeled PCR product as previously described (19).



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  		Figure 1. Nucleotide and predicted amino acid sequences of the mouse cDNA clone encoding mPLP-Cß. A vertical arrow indicates the putative signal-peptide cleavage site. Putative glycosylation sites are boxed, and cysteine residues are indicated by the shaded box. An asterisk represents the termination codon, and the putative polyadenylation signal is underlined.

 
Construction of rat placenta cDNA library and the isolation of rat PLP-Cß
Poly(A)+RNA was purified using an Oligotex mRNA Kit (QIAGEN, Germany) from total RNA extracted from the 16.5-day-old rat placenta according to the manufacture’s instructions. The poly(A)+ RNA was converted into double stranded cDNA using a ZAP-cDNA Synthesis Kit (Stratagene, La Jolla, CA). Then, cDNA was ligated with EcoRI- and XhoI-digested {lambda} ZAP II vector (Stratagene) and packaged with Gigapack II Gold (Stratagene). The cDNA clones for rat PLP-Cß homolog were isolated by screening the placenta cDNA library using the mouse PLP-Cß cDNA as a probe.

DNA sequencing and sequence analysis
Subcloned PCR products and cDNA clones were sequenced either manually with the Sequenase II kit (Amersham Pharmacia Biotech/U.S. Biochemical Corp.) or with the ABI PRISM 310 Genetic Analyzer (Perkin-Elmer Corp., Norwalk, CT) using Taq or T7 DNA polymerase and BigDye Terminator cycle sequencing kit (Perkin-Elmer Corp.). Computer-assisted sequence analysis was carried out using the DNASIS program (Hitachi, San Bruno, CA).

Genomic southern analysis
Genomic DNA was prepared from the placenta of ICR mice using the QIAamp Tissue Kit (QIAGEN, Chatsworth, CA), digested with BamHI, HindIII, XbaI, or EcoRV and resolved on 1% agarose gels. The DNA was then transferred onto Hybond-N+ membranes (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. The 460-bp PLP-Cß PCR product was radiolabeled by a random primer labeling kit (, Mannheim, Germany) and used as a probe. The membrane was incubated in hybridization solution containing the labeled probe as previously described (19).

Northern blot analysis
Aliquots (10–20 µg) of RNA from embryo and extraembryonic tissues, adult tissues, and cultured cells were resolved on denaturing 1% agarose gels containing formaldehyde, transferred onto nylon membranes (Hybond-N, Amersham Pharmacia Biotech, Arlington Heights, IL), and hybridized with a 32P-labeled PLP-Cß cDNA probe in QuikHyb solution (Stratagene) overnight at 58°C as previously described (19). Blots were washed at 65°C twice for 20 min in 2 x SSC (standard saline citrate) and 0.1% SDS, twice for 20 min in 1 x SSC and 0.1% SDS, and once for 20 min in 0.1 x SSC and 0.1% SDS. The membranes were exposed to Kodak X-Omat XK-1 film (Eastman Kodak, Rochester, NY) with a Fuji Photo Film Co., Ltd. intensifying screen at -80°C.

In situ hybridization histochemistry
Mouse tissues were fixed with 4% paraformaldehyde and embedded in Paraplast as previously described (21, 22). Antisense and sense digoxigenin-labeled mPLP-Cß riboprobes (Roche Molecular Biochemicals) were synthesized and hybridized to 6 µm Paraplast sections mounted on gelatin-coated slides. To remove nonspecifically bound probe, the slides were washed in 2 X SSC at room temperature, treated with ribonuclease A (50 µg/ml) at 37 C for 30 min, and then washed with 0.1 x SSC at 50 C as previously described (21, 22). Hybridization signals were visualized with an alkaline phosphatase-conjugated anti-digoxigenin antibody using procedures recommended by Roche.

Culture of Rcho-1 cells
The Rcho-1 cell line was a gift from Dr. M. J. Soares (University of Kansas Medical Center). Rcho-1 trophoblast cells were routinely maintained in subconfluent conditions in RPMI 1640 medium (Life Technologies, Inc., USA) containing 20% heat-inactivated FBS (Life Technologies, Inc.) and induced to differentiate as described elsewhere (22, 23, 24). The cells were grown in a humidified 5% CO2 atmosphere at 37°C.

RT-PCR and Southern blot analysis of rat PLP-Cß isoforms
Total RNA (3 µg) obtained from rat placenta on E 16.5 and E 21.5 was used as a template in a RT-PCR. First strand cDNA was prepared with RT using standard procedures. DNA fragments were amplified by PCR using the following set of primers. The primer set 5'-TGCTGCTGTGGGAGAAAGTT-3' (ß5') and 5'-TCCTAATCAGTTTTGAGTTAAG-3' (ß3'; see Fig. 8AGo) was used to generate the 204- and 165-bp fragments of the rPLP-Cß and rPLP-Cß{Delta}39, respectively, and the primer set ß5' and ß3'{Delta}39 (5'-GTTTTGAGTTAAGCTC-3'; see Fig. 8AGo) was used to generate the rPLP-Cß{Delta}39-specific 156-bp fragment. Thirty-five cycles of PCR were performed under the following conditions: denaturation at 95 C for 1 min, primer annealing at 55 C for 1 min, and primer extension at 72 C for 1 min. The resulting products were analyzed by agarose gel electrophoresis and subjected to Southern blot analysis with the rPLP-Cß cDNA as a probe.



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  		Figure 8. RT-PCR analysis of rPLP-Cß and rPLP-Cß{Delta}39 mRNA isoform expression during rat placenta development. A, RT-PCR strategy for the expression of rPLP-Cß and rPLP-Cß{Delta}39 isoforms. Specific primers for the amplification of rPLP-Cß{Delta}39 (ß5' and ß3'{Delta}39) or both isoforms (ß5' and ß3') were designed based on sequences around the 39-bp region of the rPLP-Cß cDNA. The primer sets used in the RT-PCR reactions are indicated by arrows, and the expected PCR products for each set of primers are shown by thick bars. The lack of 39-bp region, which is boxed in rPLP-Cß or shown by a triangle in rPLP-Cß{Delta}39, is indicated by dashes in the ß3'{Delta}39 primer. B, The primer set ß5' and ß3' generated an amplified product of 204 bp from rPLP-Cß cDNA and 165 bp from rPLP-Cß{Delta}39 cDNA. However, the ß3'{Delta}39 produces a product with an expected size of 156 bp from rPLP-Cß{Delta}39 cDNA, but not from rPLP-Cß cDNA. RT-PCR was performed with both primer sets using total RNA isolated from E 16.5 and E 21.5 rat placenta. Unlike our expectation that the primers ß5' and ß3' amplify two PCR products of 204 and 165 bp, only the 204-bp products were amplified. However, the expression of the rPLP-Cß{Delta}39 isoform was detected in E 16.5 and E 21.5 placenta by RT-PCR using the specific primer set ß5' and ß3'{Delta}39. PCR products were separated on 2% agarose gels and stained with ethidium bromide. They were transferred to a nylon membrane and hybridized with the 32P-labeled rPLP-Cß cDNA probe.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of an additional member of the PLP-C subfamily, PLP-Cß
To isolate additional members of mouse PLP-C subfamily, a mouse conceptus cDNA library was screened by PCR using degenerate oligonucleotides matching the most conserved region among seven different PLP-C subfamily cDNAs. An amplicon of the expected size (460 bp) was obtained, and sequencing of randomly chosen subclones identified a partial cDNA fragment with a significant sequence homology to known PLP-C subfamily members. The full-length cDNA was isolated by screening the mouse conceptus cDNA library (19) with the radiolabeled 460-bp PCR-derived fragment. The complete cDNA and deduced amino acid sequences of the cDNA thus obtained are shown in Fig. 1Go. The cDNA includes a termination codon (TAA) at nucleotides 767–769 and a putative polyadenylation signal sequence (AATAAA) 19-bp upstream of the polyadenylation site and encodes a predicted protein of 241 amino acids.

Database searching using the full-length sequence of the predicted protein revealed significant homology with all known members of the PLP-C subfamily, including rPLP-H (65%), rPLP-D (64%), rPLP-Cv (63%), rPLP-C (56%), and rd/tPRP (55%). The closest match was to mouse PLP-C{alpha} (66%), so we have named the novel protein PLP-Cß for consistency. In addition to the overall sequence homology, PLP-Cß contains six cysteine residues, the positions of which are consistent with the positions of cysteine residues in other PLP-C subfamily members. Cysteine residues at positions 4, 11, and 71 are absolutely conserved in all members of the PLP-C subfamily, including PLP-Cß. The first 30 amino acids of PLP-Cß appear to comprise a secretion signal sequence, given the level of homology shared by this region with signal sequences of known PLP-C subfamily proteins. PLP-Cß contains two potential N-glycosylation sites, Asn-Leu-Ser and Asn-Glu-Thr, at amino acid positions 183–185 and 190–192, respectively.

To determine whether the PLP-Cß is a mouse counterpart for a known member of the rat PLP-C subfamily or is an additional novel member of PLP-C subfamily, we next screened the rat placenta cDNA library using the mouse PLP-Cß cDNA as a probe and isolated the rat homolog cDNA clones. Surprisingly, two mRNA isoforms were isolated: one mRNA encodes a 241-amino acid product (designated rPLP-Cß; Fig. 2Go), but another mRNA lacks 39 bases encoding for a region rich in aromatic amino acids (designated rPLP-Cß{Delta}39; Fig. 2Go). The 39-bp region corresponds to exon 3 of other PLP-C subfamily members such as PLP-C{alpha}, PLP-Cv, and d/tPRP (1, 9, 13). It suggests that the two isoforms are probably generated by an alternative splicing from a single gene. In addition to these two isoforms, an aberrant mRNA was also frequently isolated from the screening of rat placenta cDNA library. These clones lack three bases, CAG, in the putative signal sequence, which caused the replacement of serine11 and glycine12 by cysteine11 (Fig. 2Go).



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  		Figure 2. Nucleotide and predicted amino acid sequences of the rat cDNA clones encoding rPLP-Cß isoforms. The lack of 39 bp in the alternative isoform (rPLP-Cß{Delta}39) is indicated by a long black box covering 13 amino acids, and the lack of three bases (CAG) in the aberrant mRNA is indicated by a short black box covering two amino acids. A vertical arrow indicates the putative signal-peptide cleavage site. Putative glycosylation sites are boxed, and cysteine residues are indicated by the shaded box. An asterisk represents the termination codon, and the putative polyadenylation signal is underlined.

 
Alignment of the mature rat and mouse PLP-Cß proteins showed 79% overall identity (Fig. 3Go). In addition, the mouse and rat PLP-Cß proteins have the same locations of the two putative N-linked glycosylation sites (Asn183 and Asn190) and the same locations of cysteine residues (Fig. 3Go). Thus, the identification of the rat PLP-Cß homolog clearly indicates that this PLP-Cß is a bona fide member of the PLP-C subfamily. A comparison of the rPLP-Cß to other members of the PLP-C subfamily revealed significant amino acid identities with rPLP-D (71%), rPLP-Cv (68%), rPLP-H (66%), rPLP-C (63%), d/tPRP, and mPLP-C{alpha} (66%).



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  		Figure 3. Comparison of amino acid sequences of mouse and rat PLP-Cß. Vertical arrows indicate putative signal-peptide cleavage sites. Shaded areas denote amino acid sequences identical between mouse and rat proteins. Conserved cysteine residues are black-boxed, and putative glycosylation sites are indicated by the shaded box.

 
Genomic Southern blot analysis, using radiolabeled mPLP-Cß cDNA as a probe, suggested the existence of a single mouse PLP-Cß gene (Fig. 4Go).



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  		Figure 4. Southern blot analysis of mouse PLP-Cß-related sequences in mouse genomic DNA. Mouse genomic DNA digested with the indicated restriction endonucleases hybridized to a mouse PLP-Cß cDNA probe.

 
Expression of mPLP-Cß mRNA in mouse tissue
The expression patterns of mPLP-Cß mRNA during embryonic development and in embryonic and adult tissues were analyzed by Northern blot hybridization using the 32P-labeled mPLP-Cß cDNA as a probe. To determine the stage at which mPLP-Cß is first expressed, total RNA was prepared from conceptuses at various stages from E 4.5–18.5. A transcript of about 0.9 kb was first detected on E 11.5 and was expressed continually throughout development (Fig. 5AGo). The expression of mPLP-Cß was also examined in embryonic and adult tissues. mPLP-Cß mRNA was detected only in placenta (Fig. 5BGo), consistent with the expression patterns of other PLP family members. mPLP-Cß mRNA was not detected in other tissues, including heart, kidney, liver, uterus, testis, ovary, fetus, and yolk sac (Fig. 5BGo). To examine the expression pattern of mPLP-Cß mRNA during placenta development, placenta RNA was isolated at embryonic stages from E 10.5 to E 18.5 and further examined by Northern blotting analysis. mPLP-Cß mRNA was first detected in the placenta on E 11.5 and persisted until birth (Fig. 5CGo), although its expression level fluctuated during gestation.



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  		Figure 5. Stage- and tissue-specific expression of mouse PLP-Cß. Northern blot analysis was performed using radiolabeled mouse PLP-Cß cDNA as a probe. A, Stage-specific expression of mPLP-Cß. Total RNA (20 µg/lane) was prepared from mouse conceptuses at the gestation times indicated. B, Tissue-specific expression of mPLP-Cß. Total RNA (20 µg/lane) was prepared from the indicated mouse tissues. C, Time course of mPLP-Cß expression in placental tissues. Total RNA (10 µg/lane) was isolated from placental tissues at various stages of gestation. The control panels (the lower part of each panel) show each gel before blotting, stained with ethidium bromide and photographed under UV light, demonstrating similar levels of 18S and 28S ribosomal RNA as a loading control.

 
To determine the cellular localization of mPLP-Cß in mouse placenta, we performed in situ hybridization analysis of tissue sections prepared from mouse conceptus between 8.5 and 18.5 days gestation, using sense and antisense mPLP-Cß riboprobes labeled with digoxigenin (Fig. 6Go). No hybridization signal was observed with the sense probe at any of the stages examined. With the antisense probe, mPLP-Cß transcripts were localized to spongiotrophoblast and trophoblast giant cells of the placenta (Fig. 6Go, D–L). No mPLP-Cß expression was detected on E 8.5 and E 9.5 (Fig. 6Go, B and C). However, mPLP-Cß mRNA was detected on E 10.5 by in situ hybridization, although it was not detected by Northern blot analysis until E 11.5 (compare Fig. 6Go, D and E, and Fig. 5Go). On E 10.5, mPLP-Cß expression could be detected in some, but not all, spongiotrophoblast and giant cells. This restricted expression might account for the absence of a signal on E 10.5 in Northern blot hybridizations. mPLP-Cß transcripts were detected in the labyrinthine zone on E 18.5, suggesting that the spongiotrophoblast cells had penetrated the labyrinthotrophoblast zone. No mPLP-Cß-specific hybridization signal was detected in decidua or any embryonic tissue.



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  		Figure 6. Cellular localization of mPLP-Cß mRNA in mouse by in situ hybridization analysis. Conceptus sections on E 8.5 (B), E 9.5 (C), E 10.5 (D and E), E 11.5 (F), E 12.5 (G), E 13.5 (A and H), E 14.5 (I), E 15.5 (J), and E 18.5 (K and L) were hybridized to mPLP-Cß-specific sense (A) and antisense (B–L) riboprobes. Hybridization signals were detected in trophoblast giant cells and spongiotrophoblast cells. Arrows indicate the position of the secondary giant cell layer. Sp, Spongiotrophoblast; La, labyrinthine zone; De, decidua; Ch, chorionic ectoderm. Magnifications: x20; B, x32; C, x10; D, x27; E, x66; F–L, x27.

 
Expression of PLP-Cß mRNA during Rcho-1 cell differentiation
We next investigated the expression of PLP-Cß in the Rcho-1 trophoblast cell line. This cell line was derived from a rat placental choriocarcinoma (23) and can be induced to differentiate along the trophoblast giant cell pathway (23, 25, 26, 27). RNA was isolated from Rcho-1 cells at various times after the induction of differentiation and was analyzed by Northern blot. PLP-Cß expression was detected on day 8 postinduction (Fig. 7Go).



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  		Figure 7. Expression of PLP-Cß in differentiated Rcho-1 cells. Stem cells were harvested by brief trypsinization after growth in RPMI 1640 medium containing 20% FBS for 3 days. After removal of stem cells, the adherent cells (day 0) were harvested. Differentiation was induced in NCTC 135 medium containing 10% horse serum for up to 8 days, and the cells were harvested on days (D) 1, 2, 4, 6, and 8 of differentiation culture. Each lane contains 10 µg of total RNA. Radiolabeled mPLP-Cß cDNA was used as a probe.

 
Expression patterns of rat PLP-Cß mRNA isoforms
We investigated the expression patterns of rat PLP-Cß isoforms during placenta development by RT-PCR. To distinguish between two isoforms, rPLP-Cß and rPLP-Cß{Delta}39, we designed a specific 3'-primer (ß3'{Delta}39) that allows amplification of only the rPLP-Cß{Delta}39 isoform, but not the rPLP-Cß form. Figure 8AGo shows the strategy of the PCR-based assay. When the rPLP-Cß and rPLP-Cß{Delta}39 cDNAs were used as templates, the primer set ß5' and ß3{Delta}39 amplified the 156-bp product from the rPLP-Cß{Delta}39 cDNA, but not from the rPLP-Cß cDNA (Fig. 8BGo). In contrast, the primer set ß5' and ß3' amplified a 204-bp product from the rPLP-Cß cDNA and a 165-bp from the rPLP-Cß{Delta}39 cDNA (Fig. 8BGo). The 156-bp product was confirmed as the rPLP-Cß{Delta}39 isoform by sequencing analysis. These results indicate that our PCR-based assay can distinguish the two rPLP-Cß mRNA isoforms. Thus, this assay was applied to examine the expression patterns of rPLP-Cß mRNA isoforms. As shown in Fig. 8BGo, the rPLP-Cß and rPLP-Cß{Delta}39 mRNA isoforms were detected in E 16.5 and E 21.5 rat placenta, indicating that the isoforms are coexpressed during placentation. We expected that the primer set ß5' and ß3' would produce two products, 204 and 165 bp, for the rPLP-Cß and rPLP-Cß{Delta}39, respectively (Fig. 8AGo). To our surprise, we could detect only the 204-bp product by RT-PCR (Fig. 8BGo), indicating that the rPLP-Cß mRNA is the dominantly expressed isoform in the placenta, and that in PCR production of the 204-bp product competed out production of the 165-bp product. These results were confirmed by Southern blot analysis using the rPLP-Cß cDNA probe (Fig. 8B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although PLP-C{alpha}, a new mouse PLP-C subfamily member, has been recently isolated, further attempts to isolate mouse homologs of rat PLP-C subfamily members have not been successful, except for d/tPRP (1, 14). Thus, it was proposed that mouse PLP-C{alpha} might possess a broad range of functions, encompassing the range of actions carried out by the closely related rat PLP-C group that includes PLP-C, PLP-Cv, PLP-D, and PLP-H. Therefore, we initially tried to isolate cDNA clones encoding additional members of the mouse PLP-C subfamily. We identified a cDNA that encodes a new member of the mouse PLP-C subfamily. The predicted protein encoded by this cDNA showed significant amino acid sequence identity with known members of the PLP-C subfamily, with the best match being mouse PLP-C{alpha} (66%); we therefore termed the novel protein PLP-Cß. In addition to overall sequence homology, PLP-Cß possesses six cysteine residues similarly positioned with other PLP-C subfamily members. Taken together, it is concluded that PLP-Cß is a bona fide member of the mouse PLP-C subfamily. However, we could not rule out the possibility that the mPLP-Cß may be a mouse counterpart for one of the closely related rat PLP-C subfamily members, such as PLP-H, PLP-D, PLP-C, and PLP-Cv. Thus, we screened the rat placenta cDNA library using the mPLP-Cß as a probe and isolated cDNA clones that appear to encode the rat PLP-Cß. Although a functional homology between the corresponding mouse and rat proteins has not been characterized, the rat PLP-Cß was determined based on overall high amino acid identity (79%) to mouse mature PLP-Cß and the same locations of cysteine residues and consensus sites for N-linked glycosylation as the mouse PLP-Cß.

Members of the prototypical PRL family typically possess a five-exon/four-intron gene organization that has been extremely well conserved across species (15, 28, 29, 30, 31, 32). However, the PLP-C subfamily members have a six-exon/five-intron gene arrangement, which is a feature of all PLP-C subfamily members (4). They have an additional small exon located between exons 2 and 3 of the prototypical PRL gene structure. This unique exon 3 contains 39 bp encoding a region rich in aromatic amino acids. Surprisingly, we isolated an alternative mRNA isoform (rPLP-Cß{Delta}39) of the rat PLP-Cß that lacks 39 bases, which clearly corresponds to the unique exon 3 of PLP-C subfamily members such as PLP-C{alpha}, PLP-Cv, and d/tPRP (1, 9, 13), based on the sequence alignment of rPLP-Cß to other member of the PLP-C subfamily. This new isoform is probably generated by alternative splicing from a single gene. Thus, we hypothesize that other PLP-C subfamily members with the unique exon 3, such as PLP-C{alpha}, PLP-Cv, and d/tPRP, produce alternative isoforms similar to that of the rPLP-Cß. The generation of alternative isoforms by the presence or absence of the aromatic domain suggests that this domain is critical in the biological actions of the PLP-C subfamily members. This finding is the first case demonstrating the presence of alternative isoforms in the nonclassical PRL family genes. As the classical PRL family members, the bovine placental lactogen gene has been reported to produce alternative isoforms (15). Although rPLP-Cß and rPLP-Cß{Delta}39 isoforms are coexpressed during placentation, the rPLP-Cß form is dominantly expressed in placenta.

Furthermore, we frequently detected aberrant cDNA clones encoding rPLP-Cß or rPLP-Cß{Delta}39 from the screening of rat placenta cDNA library. These clones lack three bases, CAG, in the putative signal sequence, which caused the replacement of serine 11 and glycine 12 by cysteine 11. Although we screened the rat placenta cDNA library, this aberrant mRNA form was also observed in the rPLP-D cDNA. Based on the sequence alignment of the rPLP-Cß to other members of the PLP-C subfamily, this site appears to be the boundary of the exon-intron junction in other PLP-C subfamily members, such as PLP-C{alpha} and PLP-Cv (1, 9). Thus, the lack of three bases may be generated by an alternative use of the splicing site. For example, one of the phosphatidylserine-specific phospholipase A1 mRNA isoforms has four extra bases inserted at the boundary of the exon-intron junction, which was probably produced via an alternative use of the 5'-splicing site (33).

Mouse PLP-Cß expression was first detected by Northern blot analysis on E 11.5 in mouse placenta and persisted until birth, an expression pattern similar to those of the related rat proteins PLP-C (8), PLP-Cv (9), PLP-D (10), and PLP-H (2). The rat PLP-C subfamily members are first expressed in rats on E 14.5, which corresponds to mouse E 11.5. It is noteworthy that in the mouse, E 11.5 corresponds to the transition from PL-I to PL-II expression during gestation (23). The expression profile of mPLP-Cß mRNA is distinct from that of PLP-C{alpha}. The level of mPLP-Cß expression fluctuated during pregnancy, whereas PLP-C{alpha} expression gradually increased as gestation advanced (1). The physiological significance of this expression pattern remains to be determined.

PLP-Cß expression was restricted to specific subsets of trophoblast cell layers, such as trophoblast giant cells and spongiotrophoblast cells, both of which express PLP-C, PLP-Cv, PLP-D, PLP-H, and PLP-C{alpha}. In situ analysis detected mPLP-Cß mRNA as early as E 10.5, 1 day earlier than Northern blot analysis. On E 10.5, mPLP-Cß mRNA was detected in some spongiotrophoblast and giant cells, but not in all of them, which may reflect the different differentiation stages of the trophoblast cells. PLP-Cß expression was detected in Rcho-1 cells on day 8 after the induction of differentiation, at which stage other PLP-C subfamily genes, including PLP-Cv (9), PLP-D (10), and PLP-H (2), are also expressed in Rcho-1 cells. In contrast, PL-I is expressed in Rcho-1 cells 5–6 days after induction of differentiation (22, 25). Thus, expression of PLP-Cß may require complete or at least relatively advanced differentiation.

Although many members of the nonclassical PRL family, including the PLP-C subfamily, have been isolated, their biological and physiological functions are not yet known. Only limited information is available on possible cellular targets for some members of the rat PLP-C subfamily (34, 35) and possible biological actions of d/tPRP directed toward immune targets such as macrophages or natural killer cells (36). Thus, current and future research targets will be to determine the biological role of each PLP family member in the regulation of pregnancy and fetal development. The function of PLP-Cß remains to be elucidated, but the conservation between mouse and rat and the presence of alternative isoforms suggests the biological significance of PLP-Cß during pregnancy.


   Footnotes
 
1 This work was supported by a grant (1999–2-209–011-5) from the Korea Science and Engineering Foundation. The complete sequences for the mouse and rat PLP-Cß cDNAs have been submitted to GenBank and assigned accession no. AF158744 for mouse PLP-Cß, AF239745 for rat PLP-Cß, and AF239748 for rat PLP-Cß{Delta}39. Back

2 I.T.H. and Y.H.L. contributed equally to this work. Back

Received February 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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