Characterization and expression analysis of SOLD1, a novel member of the retrotransposon-derived Ly-6 superfamily, in bovine placental villi.

Abstract

Ly-6 superfamily members have a conserved Ly-6 domain that is defined by a distinct disulfide bonding pattern between eight or ten cysteine residues. These members are divided into membrane-type and secretory-type proteins. In the present study, we report the identification of a novel Ly-6 domain protein, secreted protein of Ly-6 domain 1 (SOLD1), from bovine placenta.
SOLD1 mRNA was expressed in trophoblast mononucleate cells and the protein was secreted into and localized in the extracellular matrix of the mesenchyme in cotyledonary villi. SOLD1 bound mainly with type I collagen telopeptide. We confirmed secretion of SOLD1 from the basolateral surface of a bovine trophoblast cell line (BT-1). It may be related to the organization of the extra-cellular matrix in the mesenchyme of fetal villi. Since trophoblast mononucleate cells are epithelial cells, their polar organization is expected to have a crucial role in the SOLD1 secretion system. We established that SOLD1 is an intronless bovine gene containing the Alu retrotransposon, which was integrated via cytoplasmic reverse transcription.
We identified a novel retrotransposon-like Ly-6 domain protein in bovine placenta. SOLD1 is a crucial secreted protein that is involved in the organization of the mesenchyme of the cotyledonary villi. Furthermore, the gene encoding SOLD1 has an interesting genomic structure.

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Characterization and Expression Analysis of SOLD1, a
Novel Member of the Retrotransposon-Derived Ly-6
Superfamily, in Bovine Placental Villi
Koichi Ushizawa1, Toru Takahashi1*, Misa Hosoe1, Keiichiro Kizaki2, Kazuyoshi Hashizume2
1Reproductive Biology Research Unit, Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan, 2Department of Veterinary
Medicine, Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan
Abstract
Background: Ly-6 superfamily members have a conserved Ly-6 domain that is defined by a distinct disulfide bonding
pattern between eight or ten cysteine residues. These members are divided into membrane-type and secretory-type
proteins. In the present study, we report the identification of a novel Ly-6 domain protein, secreted protein of Ly-6 domain 1
(SOLD1), from bovine placenta.
Principal Findings: SOLD1 mRNA was expressed in trophoblast mononucleate cells and the protein was secreted into and
localized in the extracellular matrix of the mesenchyme in cotyledonary villi. SOLD1 bound mainly with type I collagen
telopeptide. We confirmed secretion of SOLD1 from the basolateral surface of a bovine trophoblast cell line (BT-1). It may be
related to the organization of the extra-cellular matrix in the mesenchyme of fetal villi. Since trophoblast mononucleate cells
are epithelial cells, their polar organization is expected to have a crucial role in the SOLD1 secretion system. We established
that SOLD1 is an intronless bovine gene containing the Alu retrotransposon, which was integrated via cytoplasmic reverse
transcription.
Conclusion: We identified a novel retrotransposon-like Ly-6 domain protein in bovine placenta. SOLD1 is a crucial secreted
protein that is involved in the organization of the mesenchyme of the cotyledonary villi. Furthermore, the gene encoding
SOLD1 has an interesting genomic structure.
Citation: Ushizawa K, Takahashi T, Hosoe M, Kizaki K, Hashizume K (2009) Characterization and Expression Analysis of SOLD1, a Novel Member of the
Retrotransposon-Derived Ly-6 Superfamily, in Bovine Placental Villi. PLoS ONE 4(6): e5814. doi:10.1371/journal.pone.0005814
Editor: Immo A. Hansen, New Mexico State University, United States of America
Received March 21, 2009; Accepted May 11, 2009; Published June 5, 2009
Copyright: ? 2009 Ushizawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by a grant from the Research Project for Utilizing Advanced Technologies (05-1770) from the Ministry of Agriculture,
Forestry and Fisheries of Japan; grants (Houga-kenkyu 19658101, Kiban-kenkyu B 20380159) from the Ministry of Education, Culture, Sport, Science and
Technology of Japan; and a grant from the Animal Remodeling Project (05-201) at the National Institute of Agrobiological Sciences. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: tatoru@affrc.go.jp
Introduction
Ruminants form the cotyledonary placenta at the feto-maternal
interface. Two specific types of trophoblast cells, trophoblast giant
binucleate cells (BNCs) and trophoblast mononucleate cells
(TMCs), play a crucial role in ruminant placentation [1,2]. The
properties of BNC-specific genes, such as anti-apoptotic BCL2-
related protein A1 (BCL2A1), which is involved in cell
maintenance [3], placental lactogen (CSH1) [1,4,5,6], prolactin-
related proteins (PRPs) [7], and pregnancy-associated glycopro-
teins (PAGs) [8], have been investigated, and TMC-expressed
interferon-tau (IFNT) is the molecule for maternal recognition of
pregnancy. BNC and TMC individually produce numerous
proteins of unknown function. It is important to identify the
genes that are specifically expressed in each cell type in order to
systematically decipher the function of the trophoblast cells.
In a recent gene expression profiling analysis using a bovine
placental-specific microarray, we detected the specific expression of
a novel gene during the peri-implantation period [9]. This bovine
gene is composed of only one Ly-6 (lymphocyte antigen-6, Ly-6/
urokinase-type plasminogen activator receptor, uPAR) domain and
a signal peptide. We named this gene ‘‘secreted protein of Ly-6
domain 1’’, and assigned it a gene symbol of ‘‘SOLD1’’. Normally,
the Ly-6 domain consists of 70–100 amino acids and is
characterized by a conserved pattern of 8 or 10 cysteine residues
that have a defined pattern of disulfide bonding in several types of
proteins [10,11,12]. The Ly-6 superfamily is divided into two
groups, membrane-type GPI-anchored proteins and secretory
proteins. Most members of this family are membrane-type proteins,
such as the complement regulatory protein CD59 [13,14,15],
plasminogen activator, urokinase receptor (PLAUR, uPAR)
[10,12,16], lymphocyte antigen 6 complex, and locus D (LY6D,
E48) [17,18,19]. The secreted members of this family, such as
secreted LY6/PLAUR domain containing 1 (SLURP1) [20,21],
Ly6/neurotoxin 1 (LYNX1, SLURP2) [22,23,24], acrosomal
vesicle protein 1 (ACRV1, SP10) [25,26,27], expressed in prostate
and testis (PATE) [28,29,30], and secreted seminal vesicle Ly6
protein (Sslp-1) [31], lack the GPI-anchor. To date, no evidence has
been reported that the members of the Ly-6 superfamily have a
common function. For example, CD59 is an inhibitor of the
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complement cascade and regulates immunosuppression [14,15].
PLAUR has a crucial role in proteolysis of extracellular matrix
proteins [32,33]. SLURP1 and LYNX1 control the pro- and anti-
apoptosis, respectively, of the keratinocyte through the nicotinic
cholinergic receptor (nAChR) [23,34]. ACRV1, PATE, and Sslp-1
are sperm-associated proteins with a possible role in mammalian
sperm maturation. Each Ly-6 protein is reported to have a different
function. Preliminary evidence suggests that SOLD1 also has a
specific function in the placenta [25,30,31].
In the present study, we identified another specific feature of
SOLD1. Secreted SOLD1 protein was detected under the basement
membrane, but only trophoblasts expressed the SOLD1 gene. There
is some evidence that trophoblast cells have bilateral secretion ability
[35,36,37].Sometrophoblastcellshavethesamepolarityasepithelial
cells, and are able to release some enzymes and cytokines at both the
apical and/or the basolateral surface. For example, the bilateral
secretion of interferon-gamma has been confirmed in a porcine
trophoblast cell line [35]. In contrast, the basolateral secretion of
matrix metalloproteinase-2 and -9 (MMP2 and MMP9) has been
confirmed in human syncytiotrophoblasts [36]. The secretion of
leptinwas confirmed at both the apical and basolateral surfaces of the
human trophoblast cell line BeWo [37]. The cotyledonary villi are
composed of the trophoblast and mesenchyme. We explored the
possibilitythatSOLD1hassomefunctioninthemesenchymewhenit
is secreted in the direction of the basement membrane. The
mesenchyme is the connective tissue that contains much extra-
cellular matrix (ECM). The binding properties of ECM and SOLD1
were important clues in our search for the function SOLD1.
Here, we studied SOLD1, a novel and crucial TMC-secreted
protein, and examined its secretion polarity from TMC, along
with the temporo-spatial expression of SOLD1. We also investi-
gated SOLD1 binding properties to the ECM. Since the SOLD1
gene contains a retrotransposon in the bovine genome, we further
explored the genomic properties of this gene.
Results
mRNA expression of SOLD1
Figure 1A depicts the tissue distribution of SOLD1, as
determined by RT-PCR analysis. No SOLD1 mRNA expression
was detected in the heart, liver, lung, spleen, and kidney tissues. In
contrast, SOLD1 mRNA was found in the placenta (cotyledon).
Quantitative expression of SOLD1 is depicted in Fig. 1B. In
ovoid-shaped conceptus on Day 11, expression of SOLD1 was
stable, but barely detectable. In the extra-embryonic membrane
(EEM) on Day 17 to 34, expression of SOLD1 was consistently
detected, but the expression level was temporarily reduced on Day
21 (Fig. 1B). In the cotyledon (COT: villous trophoblast), the
expression of SOLD1 decreased after Day 60 of gestation. In
contrast, the expression increased after Day 60 of gestation in the
intercotyledon (ICOT: extravillous trophoblast, the areas between
cotyledonary villi) (Fig. 2A, B). We determined the localization of
SOLD1 mRNA by in situ hybridization on Day 60 of bovine
gestation (Fig. 2). SOLD1 was expressed in TMCs in the COT and
the ICOT. Small amounts of SOLD1 mRNA were detected in the
maternal tissues (caruncle and intercaruncle endometrium). No
significant signal was detected with the sense probes.
Localization of SOLD1 protein
First, we examined the specificity of the custom-made anti-
bovine SOLD1 (anti-bSOLD1) antibody. Recombinant SOLD1
proteins were made using either HEK 293 cells or Rapid
translation systems (RTS; Roche Diagnostics, Basel, Switzerland).
The SOLD1 protein produced by HEK 293 cells was approxi-
mately 25 kDa, and included sugar chains, as detected by western
blot analysis (Fig. 3). The SOLD1 protein synthesized by RTS was
approximately 12 kDa, and lacked sugar chains (Fig. 3).
The results of immunohistochemistry using the anti-bSOLD1
antibody are shown in Fig. 4. Intense staining of the SOLD1
protein was detected in the mesenchyme area of stem (primary)
and branch (secondary) villi in the COT. Weak staining was found
in TMC, which expressed SOLD1 mRNA (as seen in Fig. 2). The
protein signal was absent in both the carucular and intercar-
uncular regions of the maternal tissues (Fig. 4).
SOLD1 binding properties to ECM
Specific binding was detected between SOLD1 and the type I
collagen (COL1) coated plate (Fig. 5A). No specific binding was
detected between SOLD1 and any other ECM. SOLD1 bound
specifically to type I collagen (COL1-A) telopeptide, and weakly to
type III collagen (COL3) (Fig. 5B).
Localization of type I and type III collagens
COL1 was detected in the caruncular epithelium, caruncular
stroma, trophoblast, mesenchyme of primary villi, and the interstice
area between COT and the caruncle, and faint staining was
detected in the mesenchyme of secondary villi (Fig. 6A). Similar
expression patterns were found using the COL3 antibody (Fig. 6B).
Apico-basal polarity of SOLD1 secretion from BT-1 cells
From the gene expression data in Fig. 2 and the protein
expression data in Fig. 4, we hypothesized that TMCs secreted
SOLD1 at their basolateral surfaces. We examined the in vitro
secretion of TMCs using the bovine trophoblast cell line, BT-1.
Western blot analysis did not detect any SOLD1 protein in BT-1
cell conditioned medium (Fig. 7). In contrast, 28-kDa SOLD1
protein was detected in the BT-1 cell lysate (Fig. 7). The protein
was detected on the collagen-coated plate after the cultured BT-1
cells were removed from the binding assay plate (Fig. 7). We
confirmed that the protein was secreted to the basolateral surface
of the trophoblast (BT-1) cell.
Properties of mRNA and deduced amino acid sequences
We cloned a full-length cDNA of SOLD1 from the bovine
placentome and identified the 303-bp open-reading-frame (ORF)
cDNA. The amino acid sequence deduced from full-length SOLD1
cDNA was 100 amino acids (aa). The N-terminal region (22-aa) of
the SOLD1 protein was rich in hydrophobic amino acid residues,
which are characteristic of a signal peptide (Fig. 8A). There was
31,73% similarity between SOLD1 and rat urinary proteins (Rup-
1, Rup-2, Rup-3), rat spleen protein (Rsp-1), pig protein (PIP-1),
and mouse Sslp-1, and the C-terminal regions of ACRV1 proteins
from various species, when domain retrieval was carried out based
onProDom[http://prodom.prabi.fr/prodom/current/html/
home.php] (Fig. 8A and Table 1). Although identity among the
amino acid sequences was low, the Cysteine (Cys) configuration was
completely conserved in all proteins. This conserved region is called
the Ly-6 domain [22,23]. The mature SOLD1 protein was
predicted to consist of only one Ly-6 domain. SOLD1 had three
consensus sequences for N-glycosylation (Asn-X-Ser/Thr) at amino
acid positions 32 to 34, 60 to 62, and 81 to 83 (Fig. 8A). We
submitted the mRNA sequences to the DNA Data Bank of Japan
(DDBJ). The DDBJ/GenBank accession number is AB297495.
Genome organization of SOLD1
A high level of similarity between bovine SOLD equences was
identified on chromosomes 17 (chr17) and 29 (chr29) using the
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NCBI BLAST search. On chr17, the full-length SOLD1 ORF was
located between nucleotides 29,757,120 and 29,757,423, and
lacked an intron. We established that the SOLD1 ORF on chr17 is
flanked by reversed Alu sequences (Alu - upstream region - ORF -
reversed Alu) (Fig. 8B). This feature was similar to the structure of
the Alu integrated retrotransposon. On chr29, nucleotides 1-64 of
the SOLD1 ORF were located between nucleotides 24,532,076
and 24,532,139, and nucleotides 179–303 were located between
nucleotides 24,537,293 and 29,537,417. However, the sequence of
the portion between nucleotides 65–178 on chr17 was missing on
chr29.
Discussion
In cattle, SOLD1 was mainly expressed in fetal tissues, such as
the conceptus, placentome, and ICOT, throughout gestation
(Fig. 1B). SOLD1 may have the specific role of building up the
fetal placentomal architecture. As shown in Fig. 2 and 4, TMCs
produced SOLD1, and secreted it into the mesenchyme.
COL1 was a component of connective tissue in the placentome,
and supported the mesenchyme of primary villi and the
trophoblast (Fig. 6) [38,39]. We established that SOLD1
specifically bound to the telopeptide in fibrillar type COL1,
because it bound to the tropocollagen (COL1-A) but not to the
atelocollagen (COL1-P) (Fig. 5). Moreover, SOLD1 also bound
slightly to the reticular type COL3 (Fig. 5). Various kinds of
growth factors, cytokines, and hormones, such as platelet-derived
growth factor (PDGF) [40,41], hepatocyte growth factor (HGF)
[42], transforming growth factor beta 1 (TGF-beta1) [43,44],
tumor necrosis factor alpha (TNF-alpha) [45], interleukin-2 (IL2)
[46], interleukin-7 (IL7) [47], oncostation M (OSM) [48], and
prolactin-related protein-I (PRP1) [49], bind ECM, and they may
perform a pivotal role in the modulation of local bioavailability.
SOLD1 may also participate in ECM remodeling.
Figure 1. Expression of SOLD1 mRNA. (A) Expression of SOLD1 mRNA in various bovine tissues, including heart, liver, lung, spleen, and kidney,
was analyzed by RT-PCR. Cotyledonary tissue at Day 150 of gestation was used as a bovine placental sample. GAPDH expression in each tissue is
presented as a positive control.CSH1 expression in each tissue except for the placenta is presented as a negative control (NC). ALB expression in the
other tissue is presented as a negative control (NC).(B)Quantitative expression of SOLD1 in bovine placental tissues during the initial to rate stage of
pregnancy by qRT-PCR analysis. Pre-Im, pre-implantaion; Peri-Im, peri-implantation; and Post-Im, post-implantation. CON, conceptus; EEM, extra-
embryonic membrane; EMB, embryo; COT, cotyledon; and ICOT, intercotyledon. D, day after insemination. Expression of these mRNAs was
normalized to the expression of GAPDH measured in the corresponding RNA preparation. Values are means6SEM. Values with different letters (a, b, c
and d) are significantly different (P,0.05).
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Thebovineplacentomaltrophoblastcellsconsistoftwodistinctcell
types, BNCs and TMCs [50]. BNCs account for approximately 20%
of the total trophoblast cell population [50]. BNCs are directly
involved in modifying the uterine epithelium, beginning at
implantation and continuing until term. [1,50,51] Hence, BNCs
plays a pivotal role in fetomaternal communication in cattle. BNCs
possess two nuclei and large populations of characteristic granules
that produce an array of compounds [2]. BNCs secrete placental-
Figure 2. mRNA localization of SOLD1 in the bovine placentome on Day 60 of gestation. SOLD1 mRNA was detected by in situ
hybridization. (A, D) DIG-labeled anti-sense cRNA probes were used. (B, E) Enlarged images of frames in A and D, respectively. (C, F) DIG-labeled sense
cRNA probes were used. CE, caruncular epithelium; CS, caruncular stroma; T, trophoblast; TMC, trophoblast mononucleate cells; BNC, trophoblast
binucleate cells; MPV, mesenchyme of primary villi; and MSV, mesenchyme of secondary villi. Scale bars=100 mm (A, C, D and F) and 20 mm (B and E).
doi:10.1371/journal.pone.0005814.g002
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specific prolactin-related hormones (placental lactogen, prolactin-
related proteins), matrix protease-related molecules (i.e., heparanase,
MMP14, TIMP2), aspartic protease family (i.e., pregnancy-associat-
ed glycoproteins),steroid
[2,5,6,7,8,52,53]. In the current study, we provide evidence that
TMCs are the primary cells responsible for SOLD1 production.
TMCshavebeenknowntoproduceinterferon-tau(IFNT),whichisa
substance for maternal recognition of pregnancy in ruminants. This
cytokine is specifically produced in TMCs during the pre-
implantation period [54,55,56]. IFNT acts on the uterine epithelium
and maintains corpus luteum function [57]. The actual functions of
SOLD1 are not currently clear; however, it may have a role in ECM
degradation, as do the cathepsins (CTSs) and the trophoblast Kunitz
domain proteins (TKDPs). CTSs are proteases that have biological
roles in degrading ECM, catabolizing intracellular proteins, and
processing pro-hormones. CTSs were expressed in both bovine
BNCs and TMCs [58]. TKDPs are trophoblast-specific serine
protease inhibitors in ruminants [59,60]. The localization of TKDPs
has not been established; however, several TKDPs were expressed
either in BNCs or in TMCs, or in both BNCs and TMCs. SOLD1
was only expressed in TMCs, and not in BNCs. SOLD1 thus has the
potential to be used as a marker gene of TMCs.
hormones, andprostanoids
SOLD1 was secreted at the basolateral surface of TMCs (Fig. 7).
ECM-degrading metalloproteinases, such as MMP2 and MMP9,
are known to be basolateral secreting molecules in the human
syncytiotrophoblast [36]. These results suggest that SOLD1
participates in the remodeling of ECM and the proliferation of
fetal villi.
Sequences similar to SOLD1 were detected in other species by
BLAST analysis (Fig. 8A). Although the percentage of similarity
was not high between these proteins, the Cys configuration was
completely conserved in each encoded protein. All of the proteins
examined had at least one potential N-glycosylation site. We
anticipate that these molecules evolved from a single origin.
However, we do not yet know whether these genes have a
common function, because the gene is detectable in various tissues.
TheSOLD1mRNAsequence,asdeterminedbygenomicBLAST
analysis, was retrieved on chr17 and chr29. The SOLD1 sequence
on chr17 contained a retrotransposon-like structure, flanked by
forward and reverse Alu sequences (Fig. 8B). Two partial mRNA
fragments of the SOLD1 sequence coincided with the sequence on
chr29. The bovine ACRV1 gene is located in the vicinity of the
above SOLD1 partial fragments on chr29 (nucleotide 24,507,600 to
24,514,200). The Cys configuration of the Ly-6 domain in SOLD1
and bovine ACRV1 are completely identical. ACRV1 specifically
appears in the spermatozoon [25,26,27], and it has been suggested
that this protein has a function related to fertility potential [61]. We
predict that SOLD1 and ACRV1 evolved from the same origin on
chr29, and that both have a reproductive function. Therefore,
SOLD1 might have been transferred from chr29 to chr17 in some
cases. We suggest that SOLD1 is a retrotransposon transferred from
the bovine cytoplasmic genome via mRNA. No Ly-6 domain
protein has thus far been reported for the intronless sequences of
primates or rodents. This genome structure would be an interesting
difference between species if it were specific for cattle. Placental
mammals may have evolved by the insertion of retrotransposons
[62], and the insertion of SOLD1 may have given rise to ruminants.
The Ly-6 superfamily has been detected in various tissues.
ACRV1 structurally resembles SOLD1, and is a spermatid-specific
gene in several species [25,26,63]. Mouse Sslp-1 is also a spermatid-
specific gene [31]. Rat Rup-1, Rup-2, and Rup-3 are expressed in
urinary organs, and rat Rsp-1 is expressed in the spleen [10].
SOLD1 was mainly expressed in placental tissues (Figs. 1 and 2).
Recently, the expression of PATE-P and –Q (Pate-P and –Q) was
demonstrated in human and mouse placental tissue [29]. Mouse
Pate-P was reported to modulate the activity of the alpha4beta2
heteromeric nicotinic acetylcholine receptor (nAChR). SOLD1
may have a similar role as PATE-P in the placenta.
In conclusion, we identified the secreted protein SOLD1, which
contains the Ly-6 domain. SOLD1 mRNA appeared in TMCs in the
bovine placenta, and its protein localized in the mesenchyme of
primary and secondary villi. SOLD1 bound to the telopeptide of
fibrillar type I collagen and the reticular type III collagen in
mesenchymevilli.SOLD1 hasbasolateralsecretionpolarityin TMC.
SOLD1 is related to the mesenchyme organization in villi. SOLD1
was found to be an intronless structure in the bovine genome, and we
deduced that the Alu sequence integrated as a retrotransposon of the
cytoplasmic genome derivation through mRNA. We propose that
retrotransposable SOLD1 organizes bovine cotyredonary villi.
Materials and Methods
Animal and tissue collection
All procedures for following animal experiments were carried out
in accordance with the guidelines and ethics approved by the Animal
Ethics Committee of the NationalInstitute ofAgrobiologicalSciences
Figure 3. Western blot analysis of recombinant SOLD1
proteins. Conditioned media from HEK 293 cells transiently transfect-
ed with the bovine SOLD1 gene were collected. The protein was also
produced by RTS. The purified proteins (1 ng) were loaded onto
separate lanes. The proteins were separated by SDS-PAGE and specific
proteins were detected by western blot analysis using a bovine anti-
SOLD1 antibody.
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