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Molecular cloning and expression analysis of pig CD138
Research in Veterinary Science 95 (2013) 1021–1025
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Molecular cloning and expression analysis of pig CD138 Joonbeom Bae a, Seonah Jeong a, Ju Yeon Lee a, Hyun-Jeong Lee b, Bong-Hwan Choi b, Ji-Eun Kim c, Inho Choi c,⇑, Taehoon Chun a,⇑ a
Division of Biotechnology, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea Division of Animal Genomics and Bioinformatics, National Institute of Animal Science, Rural Development Administration, Suwon 143-13, Republic of Korea c School of Biotechnology, Yeungnam University, Gyeongsan City 712-749, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 6 August 2013 Accepted 20 September 2013
Keywords: cDNA cloning CD138 Expression Pig Syndecan
a b s t r a c t CD138 (syndecan-1) interacts with various components of the extracellular matrix and associates with the actin cytoskeleton. In this study, we cloned pig CD138 cDNA and determined its complete cDNA sequence. Pig CD138 cDNA contained an open reading frame (930 bp) encoding 309 amino acids with five well conserved putative glycosaminoglycan attachment sites, a putative cleavage site for matrix metalloproteinases, and conserved motifs involved in signal transduction among mammalian species. Pig CD138 mRNA was detected in various tissues, including lymphoid and non-lymphoid organs, indicating the multicellular functions of CD138 in pigs. Western blot and flow cytometry analyses detected an approximate 35 kDa pig CD138 protein expressed on the cell surface. Further immunohistochemistry analysis revealed that CD138 expression was mainly observed in submucosa and lamina propria of the pig small intestine. Further study will be necessary to define the functional importance of CD138 during specific infectious diseases in pigs. Ó 2013 Elsevier Ltd. All rights reserved.
Syndecans are type I transmembrane proteins consisting of a core polypeptide attached with heparin sulfate proteoglycans as an extracellular domain that interacts with various components of the extracellular matrix and associates with the actin cytoskeleton (Couchman, 2010; Teng et al., 2012). Additionally, syndecans interact with many heparin or heparin sulfate binding molecules (Bernfield et al., 1999; Fears and Woods, 2006). Therefore, syndecans are biological significant for their connecting the extracellular matrix to the intracellular cytoskeleton and by regulating many biological activities through specific ligand–receptor interactions. Four isoforms of syndecan proteins have been identified in mammals (Teng et al., 2012). Each protein is encoded by a distinct gene and has a highly conserved transmembrane and cytoplasmic tail with at least three attachment sites for heparin sulfate (Teng et al., 2012). CD138 (syndecan-1) is the first identified protein among mammalian syndecans (Saunders et al., 1989) and is expressed mainly on epithelial cells (Teng et al., 2012). However, CD138 is highly expressed in precursor B cells and plasma cell stages during B cell maturation in secondary lymphoid tissues (Sanderson et al., 1989). The functional roles of CD138 have been proposed to include cell growth, cell migration, and tissue remodeling because CD138 interacts with many cell surface molecules expressed on particular Abbreviation: Gapdh, glyceraldehyde-3-phosphate dehydrogenase.
⇑ Corresponding authors. Tel.: +82 2 3290 3069; fax: +82 2 3290 3499. E-mail addresses: [email protected] (I. Choi), [email protected] (T. Chun). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.09.010
cells (Teng et al., 2012). Indeed, CD138 negatively regulates leukocyte infiltration by inhibiting the interactions between integrins and their binding partners such as intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 during inflammation (Kharabi Masouleh et al., 2009). Some studies have also shown that CD138 inhibits chemokine secretion by activated leukocytes (Li et al., 2002; Xu et al., 2005; Hayashida et al., 2009). Therefore, CD138 has anti-inflammatory activities during the development of several inflammatory diseases such as allergic lung disease, endotoxic shock, and vascular injury (Xu et al., 2005; Fukai et al., 2009; Hayashida et al., 2009). During early infection by several pathogens, CD138 serves as a receptor that facilitates initial attachment and subsequent entry of pathogens into host cells (Bhanot and Nussenzweig, 2002; Kalia et al., 2009; Bacsa et al., 2011) and subverts host defense mechanisms by inhibiting activation of innate immune cells (Park et al., 2001; Hayashida et al., 2011). Therefore, CD138-deficient mice are more resistant to several viral and bacterial pathogens, compared with those of normal mice (Teng et al., 2012). Many viral and bacterial pathogens share the same niche when reproducing in mammalian species. Therefore, identifying other examples of mammalian CD138 may be important to elucidate the functional role of CD138 during infection by specific pathogens and analyze the structural relationship between the receptor–ligand interaction for host cell entry. In this study, we cloned and determined the full-length cDNA sequence of pig CD138 and analyzed pig CD138 mRNAs in various pig organs, according to the detailed ‘‘Materials and methods’’ in
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(A)
95%
100%
90%
80%
85%
Pig CD138
75%
81%
Cow CD138 Human CD138
97% 76%
94%
Chimpanzee CD138 Rhesus CD138
80% Mouse CD138
92% 90%
Rat CD138 Hamster CD138
(B) Pig CD138 Cow CD138 Human CD138 Chimpanzee CD138 Rhesus CD138 Mouse CD138 Rat CD138 Hamster CD138
signal peptide 1 MRRAAFWLWL CALALRLQPA MRRAALWLWL CALALRLQPA MRRAALWLWL CALALSLQPA MRRAALWLWL CALALSLQPA MRRAALWLWL CALALSLQPA MRRAALWLWL CALALRLQPA MRRAALWLWL CALALRLQPA MRRAALWLWL CALALRLQPV
LLETVATNVP LLHSVAVNMP LPQIVATNLP LPQIVATNLP MPQIVATNLP LPQIVAVNVP LPQIVTANVP LPQIMAVNVP
Pig CD138 Cow CD138 Human CD138 Chimpanzee CD138 Rhesus CD138 Mouse CD138 Rat CD138 Hamster CD138
61 TPSTWKDTGL TPSTWKDLGP TPSTWKDTQL .......... TPSTWKDTWL TPSTWKDVWL TPSTWKDVWL TSSTLKDVWL
LTTMPTAPEP VTTTATAPEP LTAIPTSPEP .......... VRATPMSPEP LTATPTAPEP LTATPTAPEP LTATPTAPEP
ECD TSPDTVATST SVLPAGERPG TSPDAIAAST TILPTGEQPE TGLEATAAST STLPAGEGPK .......... .......... TGLEATAAST STIQAGEGPK TSSNTETAFT SVLPAGEKPE TSRDTEATLT SILPAGEKPE TSRDTEATFT SILPAGEKPG
RGRAVLL.DL GGRAVLLAEV EGEAVVLPEV ...EVVMG.. EGEAVVLLEV EGEPVLHVEA EGEPVAHVEA EGEPVLIAEV
120 DPGLTAQ..E EPGLTAQ... EPGLTAR..E .......... EPDLTAR..E EPGFTARDKE EPDFTARDKE DTSSTTWDKE
Pig CD138 Cow CD138 Human CD138 Chimpanzee CD138 Rhesus CD138 Mouse CD138 Rat CD138 Hamster CD138
121 KEATHSPSET KEATHPPSET QEATPRPRET ...APRPK.. QEATPQPTET KEVTTRPRET KEATTRPRET LEVTTRPRET
TQHPTTHRAS TLHPTTHSVS TQLPTTHQAS T.LPTTHQAS TQLPTTHQAP VQLPITQRAS TQLPVTQQAS TQLLVTHRVS
ECD T.AGATTAQV PATSHPHRDV T.ARATMAPG PATSHPHRDV T.TTATTAQE PATSHPHRDM T.TTATTAQE PATSHPHRDM T.ARATTAQE PATSHPHRDM T.VRVTTAQA AVTSHPHGGM TAARATTAQA SVTSHPHGDV T.ARATTAQA PVTSHPHRDV
PPDHPETSAP QPDHHETSAP QPGHHETSTP QPGHHETSTP QPGHHETSAP QPGLHETSAP QPGLHETLAP QPGLHETLAP
180 AGHGQLDPHT TGRGRMEPHR AGPSQADLHT AGPSQADLHT AGPGQADLHT TAPGQPDHQP TAPGQPDHQP TAPGQPDHQP
Pig CD138 Cow CD138 Human CD138 Chimpanzee CD138 Rhesus CD138 Mouse CD138 Rat CD138 Hamster CD138
181 PGVGDGGPAT PHVEEGGPPA PHTEDGGPSA PHTEDGGPSA PRTEDGGPSA PRVEGGGTSV PSVEDGGTSV P...SGGTSV
TEKAAEGEAS TEKAAEEDPS TERAAEDGAS TERAAEDGAS TERAAEDGAS IKEVVEDGTA IKEVVEDETT IKEVAEDGAT
ECD TQLPVGEGSG EPDFTFDVSG TQIPVGEGSG EQDFTFDLSG SQLPAAEGSG EQDFTFETSG SQLPAAEGSG EQDFTFETSG SQLPAAEGSG EQDFTFETSG NQLPAGEGSG EQDFTFETSG NQLPAGEGSG EQDFTFETSG NQLPTGEGSG EQDFTFETSG
ENTAGDALDP ENAAGAAGEP ENTAVVAVEP ENTAVVAVEP ENTAIVAVEP ENTAVAAVEP ENTAVAGVEP ENTAVAAIEP
240 DQRN...EPP GSRNGAPEDP DRR N...QSP DHR N...QSP DHR N...QSP GLRN...QPP DLR N...QSP DQRN...QPP
Pig CD138 Cow CD138 Human CD138 Chimpanzee CD138 Rhesus CD138 Mouse CD138 Rat CD138 Hamster CD138
ECD 241 VDQGTTGASQ SLLDRKEVLG EATGATGASQ GLLDRKEVLG VDQGATGASQ GLLDRKEVLG VDQGATGASQ GLLDRKEVLG VDPGATGASQ GLLDRKEVLG VDEGATGASQ SLLDRKEVLG VDEGATGASQ GLLDRKEVLG VDEGATGASQ GLLDRKEVLG
TM GVIAGGLVGL IFAVCLVGFM GVIAGGLVGL IFAVCLVGFM GVIAGGLVGL IFAVCLVGFM GVIAGGLVGL IFAVCLVGFM GIIAGGLVGL IFAVCLVGFM GVIAGGLVGL IFAVCLVAFM GVIAGGLVGL IFAVCLVAFM GVIAGGLVGL IFAVCLVGFM
CY 300 LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA
Pig CD138 Cow CD138 Human CD138 Chimpanzee CD138 Rhesus CD138 Mouse CD138 Rat CD138 Hamster CD138
301 CY NGGAYQKPSR NGGAYQKPTK NGGAYQKPTK NGGAYQKPTK NGGAYQKPTK NGGAYQKPTK NGGAYQKPTK NGGAYQKPTK
316 QEEFYA QEEFYA QEEFYA QEEFYA QEEFYA QEEFYA QEEFYA QEEFYA
(C)
MLN
K
ECD PEDQDGSGDD SD NFSGSGAG PEDQDGSGDD SD NFSGSGAG PEDQDGSGDD SD NFSGSGAG PEDQDGSGDD SD NFSGSGAG PEDQDGSGDD SD NFSGSGAG PEDQDGSGDD SD NFSGSGTG PEDQDGSGDD SD NFSGSGTG PEDQDGSGDD SD NFSGSGTG
60 ALPDVTLSQQ ALPDIT.SSH ALQDITLSQQ .......... ALQDITLSQQ ALPD.TLSRQ ALPDMTLSRQ ALPDITLSRQ
* * ** Mw
M
H
500 bp 400 bp 300 bp 200 bp
Lv
Lu
Sp
T
Si
Li Cd138 Gapdh
Fig. 1. cDNA cloning and mRNA expression analyses of pig Cd138. (A) Phylogenetic analysis of pig CD138 with other mammalian species. The phylogenetic tree was constructed with the DNAMAN software package using each CD138 deduced amino acid sequence with 1000 trials of bootstrap analyses. Very high homology was generally observed among mammalian species. (B) Alignment of putative amino acid sequences of pig CD138 with other mammalian species. Alignment of putative CD138 amino acid sequences among mammalian species. Amino acid sequences for pig CD138 (EMBL accession HF677512), human CD138 (GenBank accession NM_001006946), chimpanzee CD138 (GenBank accession XM_001140545), rhesus monkey CD138 (GenBank accession XM_001095194), mouse CD138 (GenBank accession NM_011519), rat CD138 (GenBank accession NM_013026), cow CD138 (GenBank accession NM_001075924), and hamster CD138 (GenBank accession L38991) are shown below. The putative glycosaminoglycan attachment sites within the ECD are indicated as black circles and the putative dimerization site (GGLVG265-269 sequence) within TM is indicated as a white circle. The putative cleavage site for matrix metalloproteinases within the ECD (Arg255 and Lys256) is indicated by an arrow and putative N-glycosylation sites are indicated in bold. Three tyrosine residues possibly phosphorylated within CY are underlined, and the binding motif of the PDZ domain within CY is indicated with an asterisk. ECD, extracellular domain; TM, transmembrane; CY, cytoplasmic tail. (C) Expression analyses of pig CD138 mRNA transcripts from various tissues. Total RNA was isolated from each tissue and used as template for the reverse transcription-polymerase chain reaction. Gapdh was used as the internal control. Mw, molecular weight; MLN, mesenteric lymph node; M, muscle; K, kidney; H, heart; Lv, liver; Lu, lung; Sp, spleen; T, thymus; Si, small intestine; Li, large intestine.
the Supplementary file. Then, a phylogenetic tree was constructed using the deduced amino acid sequences of the CD138 molecules thus far identified among mammalian species. The amino acid
sequence identity of pig CD138 deduced with that of cow was 81%, which shared the highest degree of homology (Fig. 1A). The identity of the pig CD138 deduced amino acid sequence with those
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(A)
Jurkat Mock pCD138
PK15
3D4/31
HeLa
500 bp
Cd138
400 bp 300 bp
Gapdh
200 bp
Jurkat
(B)
Mock pCD138 PK15
3D4/31
HeLa CD138
β-actin
(C)
pCD138 (Jurkat)
HeLa
cell number
Mock (Jurkat)
CD138
(D) Submucosa
Lamina propria
Submucosa
Lamina propria
Lumen
Lumen
100 βm
100 βm
Fig. 2. Recognition of pig CD138 by anti-human CD138 antibody. cDNA encoding pig CD138 was transfected into Jurkat cells (human T cells) using a retroviral system and analyzed by RT-PCR, western blot, and flow cytometry (A–C). (A) RT-PCR analyses with total RNAs from mock transfectants (mock), pig CD138 transfectants (pCD138), PK15 cells (pig epithelial cells), 3D4/31 cells (pig macrophages), and HeLa cells (human epithelial cells). (B) Western blot analyses with various cell lysates using anti-human CD138 antibody. (C) Flow cytometry analyses with mock transfectants (mock), pig CD138 transfectants (pCD138), and HeLa cells. Dotted line indicates staining with an isotype control and the thin line indicates staining with the anti-human CD138 antibody. (D) Immunohistochemistry analyses of pig small intestine with anti-human CD138 antibody. Upper left and lower left panels show hematoxylin and eosin stained images. Upper right and lower right panels show staining with anti-human CD138 antibody. Inset box (upper panels) is displayed at a higher resolution (lower panels).
of other mammalian species was 76% (Fig. 1A). The full-length cDNA encoding pig CD138 consists of a 930 nucleotide base pair open reading frame, and the putative amino acid sequence is composed of 309 amino acids (data not shown; EMBL accession HF677512). The results of aligning other mammalian CD138 proteins with the deduced pig CD138 amino acid sequence are shown
in Fig. 1B. Chimpanzee CD138 is unique among mammalian species because the extracellular domain of chimpanzee CD138 has a large deletion (between the Ala51 residue and Ala123 residue in pig CD138) compared with those of other mammalian species (Fig. 1B). The mammalian CD138 protein consists of a characteristic large extracellular domain with putative glycosaminoglycan attachment
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sites (Saunders et al., 1989). As shown in Fig. 1B, five putative glycosaminoglycan attachment sites (black circles: Ser47, Ser55, Ser57, Ser199 and Ser209 residues) were well conserved among mammalian CD138 proteins. The C-terminus of the CD138 extracellular domain has a putative cleavage site for matrix metalloproteinases (Saunders et al., 1989; Mali et al., 1990). Cleavage of the CD138 extracellular domain is critical for the shedding effect of CD138, which leads to several biological functions, including establishing the chemokine gradient, stimulating cell growth, increasing bone resorption, and facilitating angiogenesis (Teng et al., 2012). The putative cleavage site for matrix metalloproteinases (arrow in Fig. 1B; Arg255 and Lys256 residues) was also well conserved among mammalian CD138 proteins. The number of putative N-glycosylation sites within the CD138 protein varies depending on the mammalian species. Two putative N-glycosylation sites (bold; Asn43 and Asn234 residues) within the CD138 protein were found in humans, chimpanzees, Rhesus monkeys, and rats (Fig. 1B). Only one putative N-glycosylation site (bold; Asn43 residue) within the CD138 protein was found in pigs, mice, hamsters, and cows (Fig. 1B). Each syndecan molecule has a conserved sequence (GXXXG sequence; G is glycine and X is any amino acid residue) that mediates homodimerization with its own syndecan molecule or heterodimerizes with another syndecan molecule within the transmembrane region (Dews and MacKenzie, 2007). The GXXXG sequence (GGLVG265–269 sequence) is well conserved within the transmembrane region of mammalian CD138 (white circle in Fig. 1B). Based on the amino acid sequence alignment, the cytoplasmic tail of CD138 consists of three distinct regions: the first conserved region (Arg283–Tyr292), the variable region (Ser293–Glu312), and the second conserved region (Glu313–Ala316) (Couchman, 2010). Each distinct region within the cytoplasmic tail of CD138 mediates specific signal transduction activities involved in several cellular functions (Couchman, 2010). In particular, the second conserved region (Glu313–Ala316) within the cytoplasmic tail of CD138 interacts with PDZ domain-containing proteins such as CASK and syntenin-1 involved in remodeling of cytoskeletal architecture (Hsueh and Sheng, 1999; Sulka et al., 2009). Among mammalian CD138 molecules, three tyrosine residues, possibly phosphorylated, were well conserved (underline in Fig. 1B; Tyr292, Tyr305 and Tyr315 residues). Additionally, the binding motif of the PDZ domain in the C-terminus of the cytoplasmic tail (the second conserved region within the CD138 cytoplasmic tail) was well conserved among mammalian CD138 molecules (asterisk in Fig. 1B; EFYA313–316 motif). RT-PCR analyses were performed to determine the expression of pig Cd138 mRNA in various tissues. Total RNAs were isolated from each tissue sample, and RT-PCR was conducted. Equal amounts of RT-PCR products for Gapdh mRNA (internal control) indicated the equivalent template used in this experiment (Fig. 1C). The pig Cd138 mRNA transcripts were detected in almost all tissues examined (Fig. 1C). A relatively higher level of pig Cd138 mRNA expression was observed in kidney, liver, lung, large intestine, and small intestine. Relatively lower levels of pig Cd138 mRNA expression were observed in mesenteric lymph nodes, heart, spleen, and thymus. The lowest level of pig Cd138 mRNA expression was detected in muscle (Fig. 1C). These results demonstrate that broad and constitutive expression of CD138 in most tissues may be associated with various cellular functions of CD138 in pigs. Next, we expressed pig CD138 in Jurkat cells using a retroviral system and determined whether an anti-human CD138 antibody (EPR6454 clone) would recognize the pig CD138 molecule, according to the detailed ‘‘Materials and methods’’ in the Supplementary file. After obtaining stable Jurkat cells expressing pig CD138, we confirmed expression of pig CD138 using RT-PCR and Western blot analyses. Mock (empty vector) transfectants were used as a negative control and HeLa cells were used as a positive control. The RT-PCR results confirmed that CD138 was overexpressed in
the pig CD138 transfectants (pCD138) compared with that of the mock transfectants (Fig. 2A). Pig epithelial cells (PK15 cells) and macrophages (3D4/31 cells) were also detected by the pig Cd138 mRNA transcripts (Fig. 2A). Equal amounts of Gapdh mRNA were detected after RT-PCR analyses of various cells (Fig. 2A). A single protein was detected after western blot analyses using either pig or human cell lysates (Fig. 2B), and the size of the protein was approximately 35 kDa (Fig. 2B). As shown in Fig. 2B, enhanced expression of the CD138 protein in pig CD138 transfectants (pCD138) was observed compared with that in the mock transfectants. Pig epithelial cells (PK15 cells) and macrophages (3D4/31 cells) also constitutively expressed CD138 (Fig. 2B). Equal amounts of protein loading from cell lysates were confirmed by b-actin expression (Fig. 2B). Flow cytometry analyses were performed on the mock transfectants (negative control), pig CD138 transfectants (pCD138), and HeLa cells (positive control), according to the detailed ‘‘Materials and methods’’ in the Supplementary file. As shown in Fig. 2C, the anti-human CD138 antibody recognized pig CD138 expression on the cell surface. As pig Cd138 mRNA transcripts were detected at a relatively higher level in the small intestine, we examined CD138 expression in pig small intestine by immunohistochemistry using the anti-human CD138 antibody. CD138 is predominantly expressed by epithelial cells and B cells in adult human and mice tissues (Bernfield et al., 1999; Teng et al., 2012). As shown Fig. 2D, pig CD138 localized to both the submucosal layer and lamina propria. These results suggest that pig CD138 is also expressed on the epithelial cells of basal and suprabasal cell layers and on B cells of the lamina propria. In conclusion, we cloned the cDNA encoding pig CD138, and examined its expression patterns. The extracellular region, transmembrane region, and cytoplasmic tail of pig CD138 showed significant amino acid homology with those of other mammalian species. Results from western blot, flow cytometry, and immunohistochemistry analyses indicated that pig CD138 was recognized by an anti-human CD138 antibody, and that pig CD138 was expressed on macrophages, epithelial cells, and some B cells. Future studies will focus on the functional role of CD138 during the course of infection by pig-specific pathogens. Conflict of interest statement The authors have no conflict of interest. Acknowledgments This study was supported by a grant from the Research Cooperating Program for Agricultural Science & Technology Development (project No. PJ006406) and Next-Generation BioGreen 21 program (No. PJ008089), Rural Development Administration, Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.rvsc.2013.09.010. References Bacsa, S., Karasneh, G., Dosa, S., Liu, J., Valyi-Nagy, T., Shukla, D., 2011. Syndecan-1 and syndecan-2 play key roles in herpes simplex virus type-1 infection. Journal of General Virology 92, 733–743. Bernfield, M., Götte, M., Park, P.W., Reizes, O., Fitzgerald, M.L., Lincecum, J., Zako, M., 1999. Functions of cell surface heparin sulfate proteoglycans. Annual Review of Biochemistry 68, 729–777. Bhanot, P., Nussenzweig, V., 2002. Plasmodium yoelii sporozoites infect syndecan-1 deficient mice. Molecular and Biochemical Parasitology 123, 143–144.
J. Bae et al. / Research in Veterinary Science 95 (2013) 1021–1025 Couchman, J.R., 2010. Transmembrane signaling proteoglycans. Annual Review of Cell and Developmental Biology 26, 89–114. Dews, I.C., MacKenzie, K.R., 2007. Transmembrane domains of the syndecan family of growth factor coreceptors display a hierarchy of homotypic and heterotypic interactions. Proceedings of the National Academy of Sciences of the United States of America 104, 20782–20787. Fears, C.Y., Woods, A., 2006. The role of syndecans in disease and wound healing. Matrix Biology 25, 443–456. Fukai, N., Kenagy, R.D., Chen, L., Gao, L., Daum, G., Clowes, A.W., 2009. Syndecan-1: an inhibitor of arterial smooth muscle cell growth and intimal hyperplasia. Arteriosclerosis, Thrombosis, and Vascular Biology 29, 1356–1362. Hayashida, K., Parks, W.C., Park, P.W., 2009. Syndecan-1 shedding facilitates the resolution of neutrophilic inflammation by removing sequestered CXC chemokines. Blood 114, 3033–3043. Hayashida, A., Amano, S., Park, P.W., 2011. Syndecan-1 promotes Staphylococcus aureus corneal infection by counteracting neutrophil-mediated host defense. Journal of Biological Chemistry 285, 3288–3297. Hsueh, Y.P., Sheng, M., 1999. Regulated expression and subcellular localization of syndecan heparin sulfate proteoglycans and the syndecan-binding protein CASK/LIN-2 during rat brain development. Journal of Neuroscience 19, 7415– 7425. Kalia, M., Chandra, V., Rahman, S.A., Sehgal, D., Jameel, S., 2009. Heparan sulfate proteoglycans are required for cellular binding of the hepatitis E virus ORF2 capsid protein and for viral infection. Journal of Virology 83, 12714–12724.
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Kharabi Masouleh, B., Ten Dam, G.B., Wild, M.K., Seelige, R., Van Der Vlag, J., Rops, A.L., Echtermeyer, F.G., Vestweber, D., Van Kuppevelt, T.H., Kiesel, L., Götte, M., 2009. Role of the heparan sulfate proteoglycan syndecan-1 (CD138) in delayedtype hypersensitivity. Journal of Immunology 182, 4985–4993. Li, Q., Park, P.W., Wilson, C.L., Parks, W.C., 2002. Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury. Cell 111, 635–646. Mali, M., Jaakkola, P., Arvilommi, A.M., Jalkanen, M., 1990. Sequence of human syndecan indicates a novel gene family of integral membrane proteoglycans. Journal of Biological Chemistry 265, 6884–6889. Park, P.W., Pier, G.B., Hinkes, M.T., Bernfield, M., 2001. Exploitation of syndecan-1 shedding by Pseudomonas aeruginosa enhances virulence. Nature 411, 98–102. Sanderson, R.D., Lalor, P., Bernfield, M., 1989. B lymphocytes express and lose syndecan at specific stages of differentiation. Cell Regulation 1, 27–35. Saunders, S., Jalkanen, M., O’Farrell, S., Bernfield, M., 1989. Molecular cloning of syndecan, an integral membrane proteoglycan. Journal of Cell Biology 108, 1547–1556. Sulka, B., Lortat-Jacob, H., Terreux, R., Letourneur, F., Rousselle, P., 2009. Tyrosine dephosphorylation of the syndecan-1 PDZ binding domain regulates syntenin-1 recruitment. Journal of Biological Chemistry 284, 10659–10671. Teng, Y.H., Aquino, R.S., Park, P.W., 2012. Molecular functions of syndecan-1 in disease. Matrix Biology 31, 3–16. Xu, J., Park, P.W., Kheradmand, F., Corry, D.B., 2005. Endogenous attenuation of allergic lung inflammation by syndecan-1. Journal of Immunology 174, 5758– 5765.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Molecular cloning and expression analysis of pig CD138 Joonbeom Bae a, Seonah Jeong a, Ju Yeon Lee a, Hyun-Jeong Lee b, Bong-Hwan Choi b, Ji-Eun Kim c, Inho Choi c,⇑, Taehoon Chun a,⇑ a
Division of Biotechnology, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea Division of Animal Genomics and Bioinformatics, National Institute of Animal Science, Rural Development Administration, Suwon 143-13, Republic of Korea c School of Biotechnology, Yeungnam University, Gyeongsan City 712-749, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 6 August 2013 Accepted 20 September 2013
Keywords: cDNA cloning CD138 Expression Pig Syndecan
a b s t r a c t CD138 (syndecan-1) interacts with various components of the extracellular matrix and associates with the actin cytoskeleton. In this study, we cloned pig CD138 cDNA and determined its complete cDNA sequence. Pig CD138 cDNA contained an open reading frame (930 bp) encoding 309 amino acids with five well conserved putative glycosaminoglycan attachment sites, a putative cleavage site for matrix metalloproteinases, and conserved motifs involved in signal transduction among mammalian species. Pig CD138 mRNA was detected in various tissues, including lymphoid and non-lymphoid organs, indicating the multicellular functions of CD138 in pigs. Western blot and flow cytometry analyses detected an approximate 35 kDa pig CD138 protein expressed on the cell surface. Further immunohistochemistry analysis revealed that CD138 expression was mainly observed in submucosa and lamina propria of the pig small intestine. Further study will be necessary to define the functional importance of CD138 during specific infectious diseases in pigs. Ó 2013 Elsevier Ltd. All rights reserved.
Syndecans are type I transmembrane proteins consisting of a core polypeptide attached with heparin sulfate proteoglycans as an extracellular domain that interacts with various components of the extracellular matrix and associates with the actin cytoskeleton (Couchman, 2010; Teng et al., 2012). Additionally, syndecans interact with many heparin or heparin sulfate binding molecules (Bernfield et al., 1999; Fears and Woods, 2006). Therefore, syndecans are biological significant for their connecting the extracellular matrix to the intracellular cytoskeleton and by regulating many biological activities through specific ligand–receptor interactions. Four isoforms of syndecan proteins have been identified in mammals (Teng et al., 2012). Each protein is encoded by a distinct gene and has a highly conserved transmembrane and cytoplasmic tail with at least three attachment sites for heparin sulfate (Teng et al., 2012). CD138 (syndecan-1) is the first identified protein among mammalian syndecans (Saunders et al., 1989) and is expressed mainly on epithelial cells (Teng et al., 2012). However, CD138 is highly expressed in precursor B cells and plasma cell stages during B cell maturation in secondary lymphoid tissues (Sanderson et al., 1989). The functional roles of CD138 have been proposed to include cell growth, cell migration, and tissue remodeling because CD138 interacts with many cell surface molecules expressed on particular Abbreviation: Gapdh, glyceraldehyde-3-phosphate dehydrogenase.
⇑ Corresponding authors. Tel.: +82 2 3290 3069; fax: +82 2 3290 3499. E-mail addresses: [email protected] (I. Choi), [email protected] (T. Chun). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.09.010
cells (Teng et al., 2012). Indeed, CD138 negatively regulates leukocyte infiltration by inhibiting the interactions between integrins and their binding partners such as intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 during inflammation (Kharabi Masouleh et al., 2009). Some studies have also shown that CD138 inhibits chemokine secretion by activated leukocytes (Li et al., 2002; Xu et al., 2005; Hayashida et al., 2009). Therefore, CD138 has anti-inflammatory activities during the development of several inflammatory diseases such as allergic lung disease, endotoxic shock, and vascular injury (Xu et al., 2005; Fukai et al., 2009; Hayashida et al., 2009). During early infection by several pathogens, CD138 serves as a receptor that facilitates initial attachment and subsequent entry of pathogens into host cells (Bhanot and Nussenzweig, 2002; Kalia et al., 2009; Bacsa et al., 2011) and subverts host defense mechanisms by inhibiting activation of innate immune cells (Park et al., 2001; Hayashida et al., 2011). Therefore, CD138-deficient mice are more resistant to several viral and bacterial pathogens, compared with those of normal mice (Teng et al., 2012). Many viral and bacterial pathogens share the same niche when reproducing in mammalian species. Therefore, identifying other examples of mammalian CD138 may be important to elucidate the functional role of CD138 during infection by specific pathogens and analyze the structural relationship between the receptor–ligand interaction for host cell entry. In this study, we cloned and determined the full-length cDNA sequence of pig CD138 and analyzed pig CD138 mRNAs in various pig organs, according to the detailed ‘‘Materials and methods’’ in
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(A)
95%
100%
90%
80%
85%
Pig CD138
75%
81%
Cow CD138 Human CD138
97% 76%
94%
Chimpanzee CD138 Rhesus CD138
80% Mouse CD138
92% 90%
Rat CD138 Hamster CD138
(B) Pig CD138 Cow CD138 Human CD138 Chimpanzee CD138 Rhesus CD138 Mouse CD138 Rat CD138 Hamster CD138
signal peptide 1 MRRAAFWLWL CALALRLQPA MRRAALWLWL CALALRLQPA MRRAALWLWL CALALSLQPA MRRAALWLWL CALALSLQPA MRRAALWLWL CALALSLQPA MRRAALWLWL CALALRLQPA MRRAALWLWL CALALRLQPA MRRAALWLWL CALALRLQPV
LLETVATNVP LLHSVAVNMP LPQIVATNLP LPQIVATNLP MPQIVATNLP LPQIVAVNVP LPQIVTANVP LPQIMAVNVP
Pig CD138 Cow CD138 Human CD138 Chimpanzee CD138 Rhesus CD138 Mouse CD138 Rat CD138 Hamster CD138
61 TPSTWKDTGL TPSTWKDLGP TPSTWKDTQL .......... TPSTWKDTWL TPSTWKDVWL TPSTWKDVWL TSSTLKDVWL
LTTMPTAPEP VTTTATAPEP LTAIPTSPEP .......... VRATPMSPEP LTATPTAPEP LTATPTAPEP LTATPTAPEP
ECD TSPDTVATST SVLPAGERPG TSPDAIAAST TILPTGEQPE TGLEATAAST STLPAGEGPK .......... .......... TGLEATAAST STIQAGEGPK TSSNTETAFT SVLPAGEKPE TSRDTEATLT SILPAGEKPE TSRDTEATFT SILPAGEKPG
RGRAVLL.DL GGRAVLLAEV EGEAVVLPEV ...EVVMG.. EGEAVVLLEV EGEPVLHVEA EGEPVAHVEA EGEPVLIAEV
120 DPGLTAQ..E EPGLTAQ... EPGLTAR..E .......... EPDLTAR..E EPGFTARDKE EPDFTARDKE DTSSTTWDKE
Pig CD138 Cow CD138 Human CD138 Chimpanzee CD138 Rhesus CD138 Mouse CD138 Rat CD138 Hamster CD138
121 KEATHSPSET KEATHPPSET QEATPRPRET ...APRPK.. QEATPQPTET KEVTTRPRET KEATTRPRET LEVTTRPRET
TQHPTTHRAS TLHPTTHSVS TQLPTTHQAS T.LPTTHQAS TQLPTTHQAP VQLPITQRAS TQLPVTQQAS TQLLVTHRVS
ECD T.AGATTAQV PATSHPHRDV T.ARATMAPG PATSHPHRDV T.TTATTAQE PATSHPHRDM T.TTATTAQE PATSHPHRDM T.ARATTAQE PATSHPHRDM T.VRVTTAQA AVTSHPHGGM TAARATTAQA SVTSHPHGDV T.ARATTAQA PVTSHPHRDV
PPDHPETSAP QPDHHETSAP QPGHHETSTP QPGHHETSTP QPGHHETSAP QPGLHETSAP QPGLHETLAP QPGLHETLAP
180 AGHGQLDPHT TGRGRMEPHR AGPSQADLHT AGPSQADLHT AGPGQADLHT TAPGQPDHQP TAPGQPDHQP TAPGQPDHQP
Pig CD138 Cow CD138 Human CD138 Chimpanzee CD138 Rhesus CD138 Mouse CD138 Rat CD138 Hamster CD138
181 PGVGDGGPAT PHVEEGGPPA PHTEDGGPSA PHTEDGGPSA PRTEDGGPSA PRVEGGGTSV PSVEDGGTSV P...SGGTSV
TEKAAEGEAS TEKAAEEDPS TERAAEDGAS TERAAEDGAS TERAAEDGAS IKEVVEDGTA IKEVVEDETT IKEVAEDGAT
ECD TQLPVGEGSG EPDFTFDVSG TQIPVGEGSG EQDFTFDLSG SQLPAAEGSG EQDFTFETSG SQLPAAEGSG EQDFTFETSG SQLPAAEGSG EQDFTFETSG NQLPAGEGSG EQDFTFETSG NQLPAGEGSG EQDFTFETSG NQLPTGEGSG EQDFTFETSG
ENTAGDALDP ENAAGAAGEP ENTAVVAVEP ENTAVVAVEP ENTAIVAVEP ENTAVAAVEP ENTAVAGVEP ENTAVAAIEP
240 DQRN...EPP GSRNGAPEDP DRR N...QSP DHR N...QSP DHR N...QSP GLRN...QPP DLR N...QSP DQRN...QPP
Pig CD138 Cow CD138 Human CD138 Chimpanzee CD138 Rhesus CD138 Mouse CD138 Rat CD138 Hamster CD138
ECD 241 VDQGTTGASQ SLLDRKEVLG EATGATGASQ GLLDRKEVLG VDQGATGASQ GLLDRKEVLG VDQGATGASQ GLLDRKEVLG VDPGATGASQ GLLDRKEVLG VDEGATGASQ SLLDRKEVLG VDEGATGASQ GLLDRKEVLG VDEGATGASQ GLLDRKEVLG
TM GVIAGGLVGL IFAVCLVGFM GVIAGGLVGL IFAVCLVGFM GVIAGGLVGL IFAVCLVGFM GVIAGGLVGL IFAVCLVGFM GIIAGGLVGL IFAVCLVGFM GVIAGGLVGL IFAVCLVAFM GVIAGGLVGL IFAVCLVAFM GVIAGGLVGL IFAVCLVGFM
CY 300 LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA LYRMKKKDEG S YSLEEPKQA
Pig CD138 Cow CD138 Human CD138 Chimpanzee CD138 Rhesus CD138 Mouse CD138 Rat CD138 Hamster CD138
301 CY NGGAYQKPSR NGGAYQKPTK NGGAYQKPTK NGGAYQKPTK NGGAYQKPTK NGGAYQKPTK NGGAYQKPTK NGGAYQKPTK
316 QEEFYA QEEFYA QEEFYA QEEFYA QEEFYA QEEFYA QEEFYA QEEFYA
(C)
MLN
K
ECD PEDQDGSGDD SD NFSGSGAG PEDQDGSGDD SD NFSGSGAG PEDQDGSGDD SD NFSGSGAG PEDQDGSGDD SD NFSGSGAG PEDQDGSGDD SD NFSGSGAG PEDQDGSGDD SD NFSGSGTG PEDQDGSGDD SD NFSGSGTG PEDQDGSGDD SD NFSGSGTG
60 ALPDVTLSQQ ALPDIT.SSH ALQDITLSQQ .......... ALQDITLSQQ ALPD.TLSRQ ALPDMTLSRQ ALPDITLSRQ
* * ** Mw
M
H
500 bp 400 bp 300 bp 200 bp
Lv
Lu
Sp
T
Si
Li Cd138 Gapdh
Fig. 1. cDNA cloning and mRNA expression analyses of pig Cd138. (A) Phylogenetic analysis of pig CD138 with other mammalian species. The phylogenetic tree was constructed with the DNAMAN software package using each CD138 deduced amino acid sequence with 1000 trials of bootstrap analyses. Very high homology was generally observed among mammalian species. (B) Alignment of putative amino acid sequences of pig CD138 with other mammalian species. Alignment of putative CD138 amino acid sequences among mammalian species. Amino acid sequences for pig CD138 (EMBL accession HF677512), human CD138 (GenBank accession NM_001006946), chimpanzee CD138 (GenBank accession XM_001140545), rhesus monkey CD138 (GenBank accession XM_001095194), mouse CD138 (GenBank accession NM_011519), rat CD138 (GenBank accession NM_013026), cow CD138 (GenBank accession NM_001075924), and hamster CD138 (GenBank accession L38991) are shown below. The putative glycosaminoglycan attachment sites within the ECD are indicated as black circles and the putative dimerization site (GGLVG265-269 sequence) within TM is indicated as a white circle. The putative cleavage site for matrix metalloproteinases within the ECD (Arg255 and Lys256) is indicated by an arrow and putative N-glycosylation sites are indicated in bold. Three tyrosine residues possibly phosphorylated within CY are underlined, and the binding motif of the PDZ domain within CY is indicated with an asterisk. ECD, extracellular domain; TM, transmembrane; CY, cytoplasmic tail. (C) Expression analyses of pig CD138 mRNA transcripts from various tissues. Total RNA was isolated from each tissue and used as template for the reverse transcription-polymerase chain reaction. Gapdh was used as the internal control. Mw, molecular weight; MLN, mesenteric lymph node; M, muscle; K, kidney; H, heart; Lv, liver; Lu, lung; Sp, spleen; T, thymus; Si, small intestine; Li, large intestine.
the Supplementary file. Then, a phylogenetic tree was constructed using the deduced amino acid sequences of the CD138 molecules thus far identified among mammalian species. The amino acid
sequence identity of pig CD138 deduced with that of cow was 81%, which shared the highest degree of homology (Fig. 1A). The identity of the pig CD138 deduced amino acid sequence with those
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(A)
Jurkat Mock pCD138
PK15
3D4/31
HeLa
500 bp
Cd138
400 bp 300 bp
Gapdh
200 bp
Jurkat
(B)
Mock pCD138 PK15
3D4/31
HeLa CD138
β-actin
(C)
pCD138 (Jurkat)
HeLa
cell number
Mock (Jurkat)
CD138
(D) Submucosa
Lamina propria
Submucosa
Lamina propria
Lumen
Lumen
100 βm
100 βm
Fig. 2. Recognition of pig CD138 by anti-human CD138 antibody. cDNA encoding pig CD138 was transfected into Jurkat cells (human T cells) using a retroviral system and analyzed by RT-PCR, western blot, and flow cytometry (A–C). (A) RT-PCR analyses with total RNAs from mock transfectants (mock), pig CD138 transfectants (pCD138), PK15 cells (pig epithelial cells), 3D4/31 cells (pig macrophages), and HeLa cells (human epithelial cells). (B) Western blot analyses with various cell lysates using anti-human CD138 antibody. (C) Flow cytometry analyses with mock transfectants (mock), pig CD138 transfectants (pCD138), and HeLa cells. Dotted line indicates staining with an isotype control and the thin line indicates staining with the anti-human CD138 antibody. (D) Immunohistochemistry analyses of pig small intestine with anti-human CD138 antibody. Upper left and lower left panels show hematoxylin and eosin stained images. Upper right and lower right panels show staining with anti-human CD138 antibody. Inset box (upper panels) is displayed at a higher resolution (lower panels).
of other mammalian species was 76% (Fig. 1A). The full-length cDNA encoding pig CD138 consists of a 930 nucleotide base pair open reading frame, and the putative amino acid sequence is composed of 309 amino acids (data not shown; EMBL accession HF677512). The results of aligning other mammalian CD138 proteins with the deduced pig CD138 amino acid sequence are shown
in Fig. 1B. Chimpanzee CD138 is unique among mammalian species because the extracellular domain of chimpanzee CD138 has a large deletion (between the Ala51 residue and Ala123 residue in pig CD138) compared with those of other mammalian species (Fig. 1B). The mammalian CD138 protein consists of a characteristic large extracellular domain with putative glycosaminoglycan attachment
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sites (Saunders et al., 1989). As shown in Fig. 1B, five putative glycosaminoglycan attachment sites (black circles: Ser47, Ser55, Ser57, Ser199 and Ser209 residues) were well conserved among mammalian CD138 proteins. The C-terminus of the CD138 extracellular domain has a putative cleavage site for matrix metalloproteinases (Saunders et al., 1989; Mali et al., 1990). Cleavage of the CD138 extracellular domain is critical for the shedding effect of CD138, which leads to several biological functions, including establishing the chemokine gradient, stimulating cell growth, increasing bone resorption, and facilitating angiogenesis (Teng et al., 2012). The putative cleavage site for matrix metalloproteinases (arrow in Fig. 1B; Arg255 and Lys256 residues) was also well conserved among mammalian CD138 proteins. The number of putative N-glycosylation sites within the CD138 protein varies depending on the mammalian species. Two putative N-glycosylation sites (bold; Asn43 and Asn234 residues) within the CD138 protein were found in humans, chimpanzees, Rhesus monkeys, and rats (Fig. 1B). Only one putative N-glycosylation site (bold; Asn43 residue) within the CD138 protein was found in pigs, mice, hamsters, and cows (Fig. 1B). Each syndecan molecule has a conserved sequence (GXXXG sequence; G is glycine and X is any amino acid residue) that mediates homodimerization with its own syndecan molecule or heterodimerizes with another syndecan molecule within the transmembrane region (Dews and MacKenzie, 2007). The GXXXG sequence (GGLVG265–269 sequence) is well conserved within the transmembrane region of mammalian CD138 (white circle in Fig. 1B). Based on the amino acid sequence alignment, the cytoplasmic tail of CD138 consists of three distinct regions: the first conserved region (Arg283–Tyr292), the variable region (Ser293–Glu312), and the second conserved region (Glu313–Ala316) (Couchman, 2010). Each distinct region within the cytoplasmic tail of CD138 mediates specific signal transduction activities involved in several cellular functions (Couchman, 2010). In particular, the second conserved region (Glu313–Ala316) within the cytoplasmic tail of CD138 interacts with PDZ domain-containing proteins such as CASK and syntenin-1 involved in remodeling of cytoskeletal architecture (Hsueh and Sheng, 1999; Sulka et al., 2009). Among mammalian CD138 molecules, three tyrosine residues, possibly phosphorylated, were well conserved (underline in Fig. 1B; Tyr292, Tyr305 and Tyr315 residues). Additionally, the binding motif of the PDZ domain in the C-terminus of the cytoplasmic tail (the second conserved region within the CD138 cytoplasmic tail) was well conserved among mammalian CD138 molecules (asterisk in Fig. 1B; EFYA313–316 motif). RT-PCR analyses were performed to determine the expression of pig Cd138 mRNA in various tissues. Total RNAs were isolated from each tissue sample, and RT-PCR was conducted. Equal amounts of RT-PCR products for Gapdh mRNA (internal control) indicated the equivalent template used in this experiment (Fig. 1C). The pig Cd138 mRNA transcripts were detected in almost all tissues examined (Fig. 1C). A relatively higher level of pig Cd138 mRNA expression was observed in kidney, liver, lung, large intestine, and small intestine. Relatively lower levels of pig Cd138 mRNA expression were observed in mesenteric lymph nodes, heart, spleen, and thymus. The lowest level of pig Cd138 mRNA expression was detected in muscle (Fig. 1C). These results demonstrate that broad and constitutive expression of CD138 in most tissues may be associated with various cellular functions of CD138 in pigs. Next, we expressed pig CD138 in Jurkat cells using a retroviral system and determined whether an anti-human CD138 antibody (EPR6454 clone) would recognize the pig CD138 molecule, according to the detailed ‘‘Materials and methods’’ in the Supplementary file. After obtaining stable Jurkat cells expressing pig CD138, we confirmed expression of pig CD138 using RT-PCR and Western blot analyses. Mock (empty vector) transfectants were used as a negative control and HeLa cells were used as a positive control. The RT-PCR results confirmed that CD138 was overexpressed in
the pig CD138 transfectants (pCD138) compared with that of the mock transfectants (Fig. 2A). Pig epithelial cells (PK15 cells) and macrophages (3D4/31 cells) were also detected by the pig Cd138 mRNA transcripts (Fig. 2A). Equal amounts of Gapdh mRNA were detected after RT-PCR analyses of various cells (Fig. 2A). A single protein was detected after western blot analyses using either pig or human cell lysates (Fig. 2B), and the size of the protein was approximately 35 kDa (Fig. 2B). As shown in Fig. 2B, enhanced expression of the CD138 protein in pig CD138 transfectants (pCD138) was observed compared with that in the mock transfectants. Pig epithelial cells (PK15 cells) and macrophages (3D4/31 cells) also constitutively expressed CD138 (Fig. 2B). Equal amounts of protein loading from cell lysates were confirmed by b-actin expression (Fig. 2B). Flow cytometry analyses were performed on the mock transfectants (negative control), pig CD138 transfectants (pCD138), and HeLa cells (positive control), according to the detailed ‘‘Materials and methods’’ in the Supplementary file. As shown in Fig. 2C, the anti-human CD138 antibody recognized pig CD138 expression on the cell surface. As pig Cd138 mRNA transcripts were detected at a relatively higher level in the small intestine, we examined CD138 expression in pig small intestine by immunohistochemistry using the anti-human CD138 antibody. CD138 is predominantly expressed by epithelial cells and B cells in adult human and mice tissues (Bernfield et al., 1999; Teng et al., 2012). As shown Fig. 2D, pig CD138 localized to both the submucosal layer and lamina propria. These results suggest that pig CD138 is also expressed on the epithelial cells of basal and suprabasal cell layers and on B cells of the lamina propria. In conclusion, we cloned the cDNA encoding pig CD138, and examined its expression patterns. The extracellular region, transmembrane region, and cytoplasmic tail of pig CD138 showed significant amino acid homology with those of other mammalian species. Results from western blot, flow cytometry, and immunohistochemistry analyses indicated that pig CD138 was recognized by an anti-human CD138 antibody, and that pig CD138 was expressed on macrophages, epithelial cells, and some B cells. Future studies will focus on the functional role of CD138 during the course of infection by pig-specific pathogens. Conflict of interest statement The authors have no conflict of interest. Acknowledgments This study was supported by a grant from the Research Cooperating Program for Agricultural Science & Technology Development (project No. PJ006406) and Next-Generation BioGreen 21 program (No. PJ008089), Rural Development Administration, Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.rvsc.2013.09.010. References Bacsa, S., Karasneh, G., Dosa, S., Liu, J., Valyi-Nagy, T., Shukla, D., 2011. Syndecan-1 and syndecan-2 play key roles in herpes simplex virus type-1 infection. Journal of General Virology 92, 733–743. Bernfield, M., Götte, M., Park, P.W., Reizes, O., Fitzgerald, M.L., Lincecum, J., Zako, M., 1999. Functions of cell surface heparin sulfate proteoglycans. Annual Review of Biochemistry 68, 729–777. Bhanot, P., Nussenzweig, V., 2002. Plasmodium yoelii sporozoites infect syndecan-1 deficient mice. Molecular and Biochemical Parasitology 123, 143–144.
J. Bae et al. / Research in Veterinary Science 95 (2013) 1021–1025 Couchman, J.R., 2010. Transmembrane signaling proteoglycans. Annual Review of Cell and Developmental Biology 26, 89–114. Dews, I.C., MacKenzie, K.R., 2007. Transmembrane domains of the syndecan family of growth factor coreceptors display a hierarchy of homotypic and heterotypic interactions. Proceedings of the National Academy of Sciences of the United States of America 104, 20782–20787. Fears, C.Y., Woods, A., 2006. The role of syndecans in disease and wound healing. Matrix Biology 25, 443–456. Fukai, N., Kenagy, R.D., Chen, L., Gao, L., Daum, G., Clowes, A.W., 2009. Syndecan-1: an inhibitor of arterial smooth muscle cell growth and intimal hyperplasia. Arteriosclerosis, Thrombosis, and Vascular Biology 29, 1356–1362. Hayashida, K., Parks, W.C., Park, P.W., 2009. Syndecan-1 shedding facilitates the resolution of neutrophilic inflammation by removing sequestered CXC chemokines. Blood 114, 3033–3043. Hayashida, A., Amano, S., Park, P.W., 2011. Syndecan-1 promotes Staphylococcus aureus corneal infection by counteracting neutrophil-mediated host defense. Journal of Biological Chemistry 285, 3288–3297. Hsueh, Y.P., Sheng, M., 1999. Regulated expression and subcellular localization of syndecan heparin sulfate proteoglycans and the syndecan-binding protein CASK/LIN-2 during rat brain development. Journal of Neuroscience 19, 7415– 7425. Kalia, M., Chandra, V., Rahman, S.A., Sehgal, D., Jameel, S., 2009. Heparan sulfate proteoglycans are required for cellular binding of the hepatitis E virus ORF2 capsid protein and for viral infection. Journal of Virology 83, 12714–12724.
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