Molecular characterization and expression analysis of three membrane-bound complement regulatory protein isoforms in the ginbuna crucian carp Carassius auratus langsdorfii

Fish & Shellfish Immunology 35 (2013) 1333e1337

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Short sequence report

Molecular characterization and expression analysis of three membrane-bound complement regulatory protein isoforms in the ginbuna crucian carp Carassius auratus langsdorfii Indriyani Nur, Hikari Harada, Masakazu Tsujikura, Tomonori Somamoto*, Miki Nakao Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 May 2013 Received in revised form 1 August 2013 Accepted 2 August 2013 Available online 15 August 2013

Regulators of complement activation (RCA) play a role in protecting cells from excessive complement activation in humans. cDNA corresponding to three isoforms of teleost membrane-bound RCA protein (gTecrem) have been identified in the ginbuna crucian carp. gTecrem-1 consists of seven short consensus repeats (SCRs), whereas gTecrem-2 and gTecrem-3 have four SCRs. While gTecrem-1 possesses a tyrosine phosphorylation site in its cytoplasmic region, gTecrem-2 and gTecrem-3 lack the site. Tissue distribution analysis showed that gTecrem-1 and gTecrem-2 mRNAs were expressed in almost all tissues examined, whereas gTecrem-2 expression was not significantly detected in gill, liver, or intestine. Furthermore, analysis showed that gTecrem-1 was expressed in both peripheral blood leukocytes (PBLs) and erythrocytes and was also expressed in T cell subsets such as CD4þ, CD8þ T cells, and IgMþ B cells. gTecrem-2 expression was not detected in either PBLs or erythrocytes, whereas gTecrem-3 was expressed only in erythrocytes. These results suggested that gTecrem isoforms may serve different functional roles; gTecrem-1, which is expressed in T cells and possesses a tyrosine phosphorylation site, may act as a complement regulator and a cellular receptor in adaptive immunity. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Membrane-bound complement regulatory protein Short consensus repeats Ginbuna crucian carp Teleost Isoform

1. Introduction The complement system plays a noteworthy role in biologically important processes, including inflammation, host defense, and maintenance of homeostasis, and it additionally serves as a bridge between the innate and adaptive immune systems [1,2]. Complement activation has been viewed as a “double-edged sword.” Benefits of complement activation include host protection from pathogenic aggression, whereas the downfalls include inappropriate activation, leading to autologous tissue destruction. To minimize this type of damage, the host expresses several kinds of soluble and membrane-bound proteins that inhibit complement activation [3,4]. These proteins, most of which belong to a family of regulators of complement activation (RCA) proteins, inhibit or accelerate complement activation at any of the two main steps convertase and membrane attack complex (MAC) formation, causing transmembrane channel formation and cell lysis [5e7]. RCA contains tandemly arranged short consensus repeats (SCRs)

* Corresponding author. Tel.: þ81 92 642 2895; fax: þ81 92 642 2894. E-mail address: [email protected] (T. Somamoto). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.08.002

that are typically composed of 60e70 amino acids, including four highly conserved cysteines with genes clustered at 1q32; this location is called the RCA gene locus in humans [8]. In humans, RCAs have been studied more extensively and classified into the following types: soluble, such as C1 inhibitor (C1INH), factor H, and C4 binding protein (C4bp) and membranebound proteins, such as complement receptor type 1 (CR1), complement receptor type 2 (CR2), decay accelerating factor (DAF), and membrane cofactor protein (MCP) [9e12]. RCAs have also been characterized in non-mammalians, such as chickens [13], amphibians [14], lampreys [15], and bony fish [16e 20]. Similar to many other complement regulatory proteins, these organisms have a single gene that encodes the SCR protein in the locus corresponding to the human RCA. In bony fish, more soluble RCA proteins have been found than membrane-bound RCA proteins, examples of which include the cofactor protein (SBP1) and its homolog, the cofactor-related protein 1 (SBCRP-1) in barred sand bass [16,17], HCRF in Japanese flounder [18], and ZRC2 in zebrafish [19,20]. The barred sand bass molecule SBP1 has been shown to play a role similar to that of mammalian factor H and C4bp [21]. However, research focused on characterization of membrane-bound RCA protein has been

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limited. ZRC1 has been recently identified in zebrafish [20], and teleost complement regulatory membrane protein, termed as Tecrem in our laboratory, has been found in carp and zebrafish (GenBank Accession Number: AB723858, AB723859). Tecrem displays 15e20% homology with the human membrane RCA protein (CR1, CR2, and DAF), and has a threonine/proline-rich region, which is similar to the serine/proline/threonine-rich region (STP-region) in human MCP (CD46). Clonal triploid ginbuna crucian carp is naturally occurring gynogenetic fish, and several clonal strains have been available for laboratory study [22]. The teleost serves as a model for studying innate and adaptive immune functions. In the present study, three sequences of Tecrem have been identified in the ginbuna crucian carp, and their expression in various tissues and blood cells was determined. 2. Materials and methods 2.1. Cloning of Tecrem cDNA from ginbuna crucian carp Total RNA was extracted from ginbuna carp (S3n strain) liver by following the protocol previously described [23]. A gene-specific primer (50 -CAAGATGGGCTGTAAGGTGC-30 ), corresponding to sequences containing the predicted 50 -untranslated region (UTR), was designed on the basis of the Tecrem sequence from common carp. First-strand cDNA from liver RNA was prepared using the SMART RACE cDNA amplification kit (Clontech Laboratories, USA), according to the manufacturer’s protocol. 30 -RACE PCR was performed under the following conditions: 40 cycles at 98  C for 10 s, 61  C for 10 s, and at 72  C for 4 min. The bands were gel-purified and subcloned into pGEM-T vectors (Promega, Madison, USA). Nucleotide sequences were determined using the dideoxy chain termination method [24] using a CEQ8800 DNA Analysis system and dye terminator cycle sequencing kit (Beckman coulter). Multiple sequence alignment of the amino acid sequences was performed using ClustalW (ver. 2) program (http://www.ebi.ac.uk/ Tools/msa/clustalw2/) [25] within Bioedit Ver 7.0.9.0 [26]. Domain organization of gTecrem was predicted using SMART (http://smart. embl-heidelberg.de/) [27]. 2.2. Gene expression analysis of gTecrem isoforms Three pairs of primers specific for gTecrem-1, gTecrem-2, and gTecrem-3 (Table 1) were designed using Primer3 software (http:// frodo.wi.mit.edu/primer3/) [28] on the basis of identified sequences. 40S ribosomal protein S11 gene was amplified and was used as an internal control. To detect Tecrem gene tissue expression patterns in ginbuna crucian carp, RT-PCR was performed. Total RNA was extracted from 13 tissues (brain, gill, heart, thymus, liver, head kidney, body kidney, spleen, ovary, intestine, muscle, skin, and air

Table 1 Primers used in this study. Upper and lower sequences in each primer pair are the forward and reverse primer, respectively. Name

Product Size

Primer sequences (50 -30 )

gTecrem-1

177

gTecrem-2

117

gTecrem-3

421

40S ribosomal protein

159

TCG GGG AAT GTA AAC CGC CTT CTT TTG TCC ACT TGA TCC CCT CAC AGG GTG GGT GTC CAG CAC CTA CAG TTA A GCT CCT TTC ATT TCA TAG CCA TCG TCA ACA TAT CCG AGC GCA ATG CAC CTC CAT TTT CTA TAG CTA GAG GTG G AGA CGG GAC TAC TTG CAT TA ATT GAA CCT CAC TGT CTT GC

bladder) using ISOGEN reagent (Nippon Gene, Tokyo, Japan), according to the manufacturer’s instruction. First-strand cDNA was synthesized from 1.0 mg of total RNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, Life Technologies, Carlsbad, CA, USA) with oligo (dT) primer, according to the manufacturer’s instructions. To analyze expression of gTecrem isoforms in blood leukocytes, PBLs and erythrocytes were purified using a Histopaque (d ¼ 1.083) gradient (SigmaeAldrich). Cell suspensions were applied to Histopaque and centrifuged for 30 min at 380  g to separate erythrocytes and leukocytes. Leukocytes on the Histopaque were collected, and erythrocytes below the Histopaque were harvested. Separated cells were washed twice with RPMI 1640 (Nissui Pharmaceutical) and adjusted to 1  107 cells/ml. RNA extraction and cDNA synthesis were performed as described above. To determine differential expression of gTecrem isoforms in lymphocyte subsets, T cell subsets and B cells were separated by magnetic activated cell sorting (MACS; MiniMACS; Miltenyi Biotec), according to the method previously described [29]. CD8þ T cells, CD4þ T cells, and B cells were then counted and adjusted to 8  105 cells/ml. Total RNA was extracted using NucleoSpinÒ RNA kits (MachereyeNagel), according to the manufacturer’s instruction. cDNA synthesis was performed as described above. Amplifications from all the cDNA samples were performed with rTaq DNA polymerase (Toyobo, Japan) using first-strand cDNA as a template on a PC808 thermal cycler (Astec, Japan) under the following conditions: (i) for gTecrem-1; denaturation at 95  C for 1 min, 35e42 cycles of 95  C for 30 s, 55  C for 30 s, and 72  C for 1 min, followed by elongation at 72  C for 2 min. (ii) For gTecrem-2; denaturation at 94  C for 2 min, 39e43 cycles of 94  C for 30 s, 56  C for 30 s, and 72  C for 2 min, followed by elongation at 72  C for 4 min. (iii) For gTecrem-3; denaturation at 95  C for 1 min, 40e42 cycles of 95  C for 30 s, 55  C for 30 s, and 72  C for 1 min, followed by elongation at 72  C for 2 min. (iv) For 40S ribosomal protein S11; 94  C for 2 min, 28e31 cycles of 94  C for 30 s, 56  C for 30 s, 72  C for 4 min, and finally 72  C for 4 min. Amplified products were run on 2% agarose gels and stained with ethidium bromide. 3. Results and discussion 3.1. cDNA cloning and sequence analysis of the gTecrem genes Complement regulatory membrane genes have already been identified in zebrafish [20] and common carp (GenBank Accession Number: AB723858). In the present study, we characterized the homologs of complement regulatory membrane protein (gTecrem) in ginbuna crucian carp. RACE PCR yielded three different gTecrem sequences (gTecrem-1, gTecrem-2, and gTecrem-3) from ginbuna crucian carp. Interestingly, gTecrem-1 consists of seven SCRs, whereas gTecrem-2 and gTecrem-3 have four SCRs. The three gTecrem nucleotide sequences contain open reading frames of 1557, 969, and 1005 bp encoding 519, 323, and 335 amino acids, respectively (Fig.1). Based on pairwise alignment, the predicted amino acid sequences of gTecrem isoforms (gTecrem-1, gTecrem-2, and gTecrem-3) exhibited 68, 64, and 65% identity, respectively, with carp Tecrem and 50, 50, and 47%, respectively, with zebrafish Tecrem. These gTecrem isoforms are not the products of alternative splicing because their SCR domains have distinct sequences. SignalP and SMART analysis indicated that gTecrem isoforms have signal peptide sequences and all isoforms have putative transmembrane regions and STP-like domains. These data indicated that gTecrem structure principally conserved all the features of Xenopus, chicken, and human membrane-bound RCA. The cytoplasmic region of gTecrem-1 possesses a putative tyrosine phosphorylation site, whereas gTecrem-2 and gTecrem-3 have no such signature site.

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Fig. 1. Alignment of gTecrem sequences generated using the CLUSTALW2 program. Shaded letters indicate putative signal sequences (SP), short consensus repeats (SCRs), transmembrane protein (TM), and phosphorylation site (P-site). Each SCR consists of 60e70 amino acids, including four highly conserved cysteines. Gaps used to maximize the alignment are indicated by dashes (–). Conserved or similar amino acids are shown with asterisks (*) and dots (: or .) under the sequences, respectively (GenBank Accession Number: gTecrem-1, AB822933; gTecrem-2, AB822934; and gTecrem-3, AB822935).

Structural analysis showed that the extracellular domain of gTecrem proteins is composed of SCRs, each of which spans 60e70 amino acids containing four highly conserved cysteine (C) residues and one tryptophan (W) or phenylananine (F) residue (Fig. 1). The

most significant difference among gTecrem isoforms is the number of SCRs. gTecrem-1 has seven SCR domains, whereas gTecrem-2 and gTecrem-3 have four SCRs (Fig. 2), and the identities among the SCRs range from 54% to 89% (data not shown). Theoretical molecular

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Fig. 2. Domain organization of gTecrem isoforms. SP, signal peptide; SCR, short consensus repeat; STP, serine/proline/threonine-rich region; TM, transmembrane region, and P, phosphorylation site. Numbering of amino acids and molecular weights is shown.

masses of the predicted gTecrem-1, gTecrem-2, and gTecrem-3 polypeptide chains are 56.7, 35.1, and 36.5 kDa, respectively. However, the membrane-bound protein ZCR1in zebrafish has five SCRs and molecular weight of 42.8 kDa [20]. Human DAF and CD46 contain four SCRs, CR1 contains 30 SCRs, and mouse Crry contains five SCRs [9,10]. In lower vertebrates, Xenopus ARC2 and ARC3 have four and eight SCRs, respectively, and carp and zebrafish Tecrem contain four and five SCRs, respectively [14]. These findings indicated that the number of SCRs in regulatory membrane-bound protein is different across isoforms and animal species. Because the functional significance of the number of SCRs in complement regulatory proteins is still unclear, it would be interesting to understand the functional diversification among gTecrem isoforms. 3.2. Expression analysis of gTecrem isoforms Expression of gTecrem isoforms in various tissues was analyzed by RT-PCR. As shown in Fig. 3, gTecrem-1 and gTecrem-3 mRNA

were detected in most organs, but gTecrem 2 expression was not significantly detected in gill, liver, and intestine. Similarly, ZRC1, which encodes a putative membrane protein, was constitutively expressed in all the examined zebrafish tissues [20]. Human CD46 is ubiquitously expressed, except on erythrocytes [10,30], whereas mouse CD46 expression is limited to the testis [31]. It has been reported that four isoforms of human CD46 (BC1, BC2, C1, and C2) arise by alternative splicing and show distinct tissue-specific expression patterns [10,32e35]. It is yet to be analyzed if the gTecrem isoforms can be further diversified by alternative splicing. Even if not, the three gTecrem isoforms seem to be functional divergent, possibly as diverse as the splicing variants of human CD46. gTecrem expression in blood cells is shown in Fig. 4. gTecrem1 expression was detected in both PBLs and erythrocytes. gTecrem-2 was not expressed in either PBLs or erythrocytes, whereas gTecrem-3 was expressed only in erythrocytes. gTecrem-1 was also expressed on CD8þ and CD4þ T cells and IgMþ B cells, suggesting the involvement of gTecrem-1 in activation of adaptive immunity. In humans, CR1 and CD46 protect host cells from complement-mediated cell damage [30]. Therefore, gTecrem-1 may play an important role in preventing PBLs from spontaneous complement attack. In addition, it is known that CD46 is expressed on T cells and promotes T cell activation and differentiation toward regulatory T cells [36,37]. Tyrosine phosphorylation site in the cytoplasmic tail of CD46 is an important motif for activating or inhibiting T cell activity [38,39]. The present study showed that only gTecrem-1 had the tyrosine phosphorylation site and was expressed in T cell subsets. These findings suggested that gTecrem-1 had functional similarities to mammalian CD46.

Fig. 3. Tissue distribution of gTecrem isoform genes in ginbuna crucian carp. 40S ribosomal protein S11 was used as an internal control. Data are representative of two fish analyzed.

Fig. 4. Expression analysis of gTecrem isoform genes in PBL and erythrocyte (Eryt) (A) and T cells and B cells (B). Head kidney (HK) was used as a positive control template. 40S ribosomal protein S11 was used as an internal control. Data are representative of four fish analyzed.

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3.3. Conclusion We have identified three complement regulatory membrane genes (gTecrem) in ginbuna crucian carp and have demonstrated differential expression among the three isoforms. Unlike the other gTecrem isoforms, gTecrem-1 has a tyrosine phosphorylation site in its cytoplasmic tail and is expressed in T cell subsets, suggesting that gTecrem-1 has functional similarity to CD46. In contrast, gTecrem-2 is not expressed in all blood cells, and gTecrem-3 is expressed in erythrocytes but not leukocytes. This finding suggested that gTecrem isoforms play different roles in complement regulation or activation of adaptive immunity. Acknowledgments This work was supported by the Grant-in-Aid for Scientific Research (KAKENHI) to NM (22380111). IN is supported by a scholarship from the Indonesian Directorate General of Higher Education (DIKTI). The authors would like to thank Enago (www. enago.jp) for the English language review. References [1] Mastellos D, Morikis D, Isaacs SN, Holland MC, Strey CW, Lambris JD. Complement: structure, functions, evolution, and viral molecular mimicry. Immunol Res 2003;27:367e86. [2] Morgan BP, Marchbank KJ, Longhi MP, Harris CL, Gallimore AM. Complement: central to innate immunity and bridging to adaptive responses. Immunol Lett 2005;97:171e9. [3] Brennan FH, Anderson AJ, Taylor SM, Woodruff TM, Ruitenberg MJ. Complement activation in the injured central nervous system: another dual-edged sword? J Neuroinflammation 2012;9:137. [4] Zipfel PF, Skerka C. Complement regulators and inhibitory proteins. Nat Rev Immunol 2009;9:729e40. [5] Weiler JM, Daha MR, Austen KF, Fearon DT. Control of the amplification convertase of complement by the plasma protein beta1H. Proc Natl Acad Sci U S A 1976;73:3268e72. [6] Fearon DT. Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane. Proc Natl Acad Sci USA 1979;76:5867e71. [7] Ricklin D, Hajishengallis G, Yang K, Lambris JD. Complement: a key system for immune surveillance and homeostasis. Nat Immunol 2010;11:785e97. [8] Reid KB, Day AJ. Structureefunction relationships of the complement components. Immunol Today 1989;10:177e80. [9] Kim DD, Song WC. Membrane complement regulatory proteins. Clin Immunol 2006;118:127e36. [10] Liszewski MK, Post TW, Atkinson JP. Membrane cofactor protein (MCP or CD46): newest member of the regulators of complement activation gene cluster. Annu Rev Immunol 1991;9:431e55. [11] Jiang H, Wagner E, Zhang H, Frank MM. Complement 1 inhibitor is a regulator of the alternative complement pathway. J Exp Med 2001;194:1609e16. [12] Post TW, Liszewski MK, Adams EM, Tedja I, Miller EA, Atkinson JP. Membrane cofactor protein of the complement system: alternative splicing of serine/ threonine/proline-rich exons and cytoplasmic tails produces multiple isoforms that correlate with protein phenotype. J Exp Med 1991;174:93e102. [13] Inoue N, Fukui A, Nomura M, Matsumoto M, Nishizawa Y, Toyoshima K, et al. A novel chicken membrane-associated complement regulatory protein: molecular cloning and functional characterization. J Immunol 2001;166:424e31. [14] Oshiumi H, Suzuki Y, Matsumoto Y, Seya T. Regulator of complement activation (RCA) gene cluster in Xenopus tropicalis. Immunogenetics 2009;61: 371e84. [15] Kimura Y, Inoue N, Fukui A, Oshiumi H, Matsumoto M, Nonaka M, et al. A short consensus repeat-containing complement regulatory protein of lamprey that participates in cleavage of lamprey complement 3. J Immunol 2004;173:1118e28.

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