Veterinary Immunology and Immunopathology 133 (2010) 243–249

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Veterinary Immunology and Immunopathology

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Short communication Cloning and characterization of ovine immunoglobulin G Fc receptor II (FcgRII)§ Yunchao Liu a,b, Aiping Wang b, Songlin Qiao a, Gaiping Zhang a,*, Jun Xi a, Leiming You a, Xiaohui Tian a, Qiaomu Li a, Lina Zhang a, Junqing Guo a a Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China b College of Bioengineering, Zhengzhou University, Zhengzhou 450001, China

ARTICLE INFO ABSTRACT

Article history: Immunoglobulin G (IgG) Fc receptors (FcgRs) bind to immune complexes through Received 31 December 2008 interactions with the Fc region of IgG to initiate or inhibit the defense mechanism of the Received in revised form 1 July 2009 leukocytes on which they are expressed. In this study, we describe the cloning, sequencing Accepted 9 July 2009 and characterization of ovine FcgRII. By screening a translated expression sequence tag (EST) database with the protein sequence of bovine IgG Fc receptor II, we identified a Keywords: putative ovine homologue. Using rapid amplification of cDNA ends (RACE), we isolated the Sheep cDNA encoding ovine FcgRII from peripheral blood leucocyte RNA. The ovine FcgRII cDNA FcgRII contains an 894 bp open-reading frame, encoding a 297 amino acid transmembrane Inhibitory receptor Expression glycoprotein composed of two immunoglobulin-like extracellular domains, a transmem- brane region and a cytoplasmic tail with an immunoreceptor tyrosine-based inhibitory motif (ITIM). The glycoprotein encoded by the cloned cDNA was then expressed on the surface of COS-7 cells and immunoglobulin-binding assays show that it binds ovine IgG1, but not IgG2. Identification of the ovine FcgRII will aid in the understanding of the molecular basis of IgG–FcgR interaction. ß 2009 Elsevier B.V. All rights reserved.

1. Introduction Several structurally related but functionally distinct classes of FcgRs have been extensively characterized in Receptors for the Fc domain of IgG (FcgRs) are central the human and mouse. FcgRII (CD64) is a highly affinity Fc mediators of triggered effector functions. By receptor with three extracellular Ig-like domains, while binding to the antibody Fc-portion, they provide a link FcgRII (CD32) and FcgRII (CD16) have lower affinities for between the specificity of the adaptive immune system their ligands, effectively binding IgG immune complexes, and the powerful effector functions triggered by innate and have two extracellular domains (Ravetch and Bolland, immune effector cells (Nimmerjahn and Ravetch, 2007a). 2001). FcgRIV is a novel FcgR which was found only in mouse with intermediate affinity and restricted subclass specificity (Nimmerjahn et al., 2005).

§ The GenBank accession no. of the nucleotide sequence reported here According to their functions, FcgRs can be divided into is EU589389. two general classes: the activation receptors, character- Abbreviations: FcgRs, IgG Fc receptors; RACE, rapid amplification of ized by the presence of a cytoplasmic immunoreceptor cDNA ends; ITIM, immunoreceptor tyrosine-based inhibitory motif; tyrosine-based activation motif (ITAM) as in the case of DMEM, Dulbecco’s modified Eagle’s minimal essential medium; PAM, human FcgRIIA (a receptor not found in the mouse) or pulmonary alveolar macrophages; EST, expression sequence tag. * Corresponding author. Tel.: +86 0371 65711364; more commonly, as part of an associated subunit, the g or fax: +86 0371 65729031. z chain, as in FcgRI, FcgRIII and FcgRIV and the inhibitory E-mail address: [email protected] (G. Zhang). FcgR, characterized by a ligand-binding extracellular

0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.07.020 244 Y. Liu et al. / Veterinary Immunology and Immunopathology 133 (2010) 243–249 domain highly homologous to its activation counterparts, protein-A sepharose (Pharmacia) using the method but containing the distinctive inhibitory or immunor- described previously (Goudswaard et al., 1978; Schmerr eceptor tyrosine-based inhibition motif (ITIM) sequence and Goodwin, 1991). in its cytoplasmic domain (Ravetch and Bolland, 2001). The role of activating FcgRs function in providing a 2.3. Isolation of ovine immunocytes and preparation critical link between ligands and effector cells in type II of their cDNA and type III inflammation is well established (Dijstel- bloem et al., 2001). The inhibitory FcgRs function in the Ovine peripheral blood mononuclear cells (PBMC) and maintenance of peripheral tolerance, in regulating the polymorphonuclear granulocytes (PMN) were purified threshold of activation responses, and ultimately in from the peripheral blood of sheep by density centrifuga- terminating IgG mediated effector stimulation (Katz, tion on lymphoprep (Nycomed). After PBMCs had been 2002; Ravetch and Bolland, 2001). One of the most harvested, monocytes were recovered by plastic adher- important features of FcgR system is the co-expression of ence, and peripheral blood lymphocytes (PBL) were activating and inhibitory receptors on the same cell, recovered from media. PMN cells were recovered from there by setting thresholds for cell activation (Nimmer- the red cell pellet by isotonic lysis. Pulmonary alveolar jahn and Ravetch, 2007b). Additionally, it has recently macrophages (PAMs) were collected by lung lavage using been appreciated that the IgG Fc receptors display ice-cold phosphate-buffered saline (PBS). Cell preparations substantial differences in their affinity for individual that were less than 90% viable prior to experimentation antibody isotypes, rendering certain isotypes more were not used. Ovine mRNA was isolated using Micro strictly regulated than others (Fukuyama et al., 2005). mRNA Purification Kit (TAKARA) according to the manu- For this reason, a better understanding of ovine FcgRs on facturer’s instructions, and cDNA was subsequently the immune effector cells should conduce to comprehend synthesized using an olig-dT-3sites adaptor primer the pathway of immunoregulation. (TAKARA). In contrast to the detailed information available on the structure and function of human and mouse FcgRs, little is 2.4. Amplification, cloning, and sequencing of ovine FcgRII known about equivalent structure in ruminant animals (Kacskovics, 2004). Previously, we identified four distinct Blood samples were collected from a 6-month-old, classes of FcgRs in cattle, three of them are homologous to crossbred sheep from a commercial farm. Ovine peripheral those identified in humans and mice (Zhang et al., 1994, blood leucocytes were isolated by centrifugation of 1995; Yan et al., 2000a,b). The novel type of FcgR is the peripheral blood anticoagulated with ethylenediamine bovine IgG2 Fc receptor (boFcg2R), which is genetically tetraacetic acid (EDTA) and hypotonic lysis of erythrocytes more closely related to a novel family of human proteins as suggested (Carlson and Kaneko, 1973). Ovine PBL mRNA that includes FcaR, killer cell Ig-like receptor. Although it was extracted from approximately 107 cells using Micro shows a high level of amino acid identity with FcaR within mRNA Purification Kit (TAKARA) according to the manu- the EC domains, boFcg2R binds only bovine IgG2 but not facturer’s instruction and cDNA was subsequently synthe- IgG1 nor IgA (Zhang et al., 1995). Regarding livestock sized using an oligo-dt-3sites adaptor primer (TAKARA). animals, the FcgRII has also been cloned and characterized The resultant cDNA was then subjected to 30-RACE using in pig (Qiao et al., 2006). In this report, we describe the the adapter primer and a gene-specific primer SH0 (All cloning, sequencing and characterization of ovine FcgRII. primers used in this paper were listed in Table 1.) designed The comparative analysis of this FcgR has allowed us to from an EST (GenBank accession no. EE755662) identified begin an exploration of some immunological characteristic as being highly homologous to bovine FcgRII. The resulting of ruminants. PCR product was isolated, cloned and sequenced. The 50- full RACE system (TAKARA) were then used to obtain the putative full-length ovine cDNA with the 50RACE primers 2. Materials and methods and a gene-specific primer SH1. The obtained fragment 2.1. Cell culture was subsequently cloned and sequenced. According to the newly obtained sequence for the full-length cDNA, a pair of COS-7 cells were maintained in Dulbecco’s modified primers SHE-F and SHE-R was designed to amplify the Eagle’s minimal essential medium (DMEM) (Invitrogen) sequence covering the ORF (open-reading fragment) supplemented with 10% fetal bovine serum.

Table 1 2.2. Immunoglobulin G preparations The primers used in this study.

Primer Sequence 50–30 Ovine IgGl and IgG2 were purified from hyperimmune serum collected from a sheep that had been inoculated SH0 AGGGATGCATTAGAAGGAGGATTCA with chicken erythrocytes and used to specifically SH1 TGA ATCCTCCTTCTAATGCATCCCT SHE-F CCTAAGCTTTCCAGGAGGTGATGGG sensitize erythrocytes. The total IgG was precipitated with SHE-R AAGACAGAATTCCCTGGCCTCTACGAAT ammonium sulphate ((NH4)2SO4) from the sheep’s hyper- SHQ-F CTCCATCCATACCCAGAACCA immune serum and further repurified by DEAE chromato- SHQ-R AGCCAGTCAGAAATCACATCCA graphy (Temponi et al., 1989). The IgG subclasses were GAPDH-F AGCAGTTGGTGGTGCAGGAG GAPDH-R ATGTTTGTGATGGCCGTGAA separated by fractionating the purified total IgG on Y. Liu et al. / Veterinary Immunology and Immunopathology 133 (2010) 243–249 245 region of ovine FcgRII. The amplified cDNA was also 2.5. Transfection and immunoglobulin-binding assays subcloned, in the correct orientation, into the mammalian expression vector pCDNA3.1(+) (Invitrogen) for transfec- COS-7 cells were transiently transfected with ovine tion studies. FcgRII cDNA constructs using the Lipofectamine 2000

Fig. 1. Nucleotide sequence of ovine FcgRII with translated protein sequence of the long open-reading frame underneath. Putative signal peptide sequence and transmembrane region are underlined. Cysteine residues in the extracellular region that form the disulphide bonds of the two Ig-like domains are underlined twice. Potential N-linked glycosylation sites are marked with a +. ITIM motif is indicated by open box. Stop codon is indicated with a *. Polyadenylation recognition site (AATAAAA) is shown in bold. 246 Y. Liu et al. / Veterinary Immunology and Immunopathology 133 (2010) 243–249

Fig. 2. Comparison of amino acid sequences of FcgRIIB from sheep, cattle, pig, human and mouse. Amino acid sequence determined from the ovine FcgRII cDNA is shown on the top line. Amino acid sequence for cattle FcgRIIB (accession no. NP_776964), swine FcgRIIB (NP_001028185.1), human FcgRIIB2 Y. Liu et al. / Veterinary Immunology and Immunopathology 133 (2010) 243–249 247

Fig. 3. COS-7 cells transfected with the ovine FcgRII cDNA bind ovine IgG1-sensitized chicken erythrocytes, but not IgG2-sensitized erythrocytes. (A) COS-7 cells with ovine FcgRII cDNA incubated with erythrocytes specifically sensitized with ovine IgG1. Extensive binding of erythrocytes to individual cells was evident. (B) COS-7 cells transfected with the ovine FcgRII cDNA incubated with erythrocytes specifically sensitized with ovine IgG2. No binding of erythrocytes was evident. Control COS-7 cells that had not been transfected did not bind ovine IgG-sensitized erythrocytes (data not shown).

(Invitrogen), according to the manufacturer’s instructions. (Livak and Schmittgen, 2001). Real-time PCR efficiencies Cells were incubated in DMEM medium containing 10% were estimated by amplification of dilution series of RNA fetal calf serum at 37 8C in a humidified CO2 atmosphere according to the equation 10 (1/slope) and were for 36 h prior for test. Transfected COS-7 cells were consistent between target mRNA and GAPDH. Negative subsequently incubated in DMEM medium without serum controls were performed in which water was substituted for 2 h. Chicken erythrocytes were sensitized with ovine for template. IgG1 and IgG2 for 2 h at 4 8C and negative control chicken erythrocytes were treated with PBS. IgG-sensitized chicken 3. Results and discussion erythrocytes were resuspended in serum-free medium and added to the ovine FcgRII expression COS-7 cell mono- We searched the translated EST database (which is layer. After 45 min incubation at room temperature with composed of sequences from species other than humans or occasional gentle agitation, non-adherent erythrocytes mice) at the National Center for Biotechnological Informa- were washed off with PBS. The monolayer cells were fixed tion (NCBI) with the bovine FcgRII protein sequence with methanol for 10 min, and the cells stained with HRP (accession no. NP_776964) using TBLASTN and found one conjugated rabbit anti-sheep IgG antibody (Sigma), EST (accession no. EE755662) which is highly identity to washed and incubated with 3-amino-9-ethylcarbazole bovine FcgRII. Using a RACE approach, the full-length (AEC) (Sigma) before taking photographs. cDNA was obtained. The nucleotide and predicted amino acid sequences of putative ovine FcgRII were shown in 2.6. Detection of ovine FcgRII expression pattern Fig. 1. The complete sequence is 1480 bp long, including a 75 bp 50 untranslated region (UTR) and a 511 bp 30 Real-time PCR was used to detect the expression of untranslated region. The cDNA contains a single ORF, ovine FcgRII in the cDNA from selective cellular subsets which extends 894 bp, encoding a 297 amino acids protein prepared as described previously with the primers SHQ-F (GenBank accession no. EU589389). The first 42 amino and SHQ-R. All real-time PCR reactions were performed on acids were predicted to be an N-terminal secretory signal an Bio-Rad I-Cycler Real Time PCR Instrument (Bio-Rad), peptide. The mature protein is composed of a 182 amino and the fluorochrome was SYBR green I (TAKARA). The acid extracellular region, a 25 amino acid hydrophobic thermal cycling conditions included an initial denaturation transmembrane domain and a 48 amino acid cytoplasmic step at 95 8C for 30 s, 40 cycles at 95 8C for 5 s, and 62 8C for tail. The extracellular region was found to include five 25 s. The level of expression of the transcript was potential N-glycosylation sites (Asn 37, Asn 44, Asn 63, Asn calculated in relation to the expression of the GAPDH 137 and Asn 144) and four conserved cysteines that form gene. Melting curve analysis was used to confirm the the characteristic disulphide bonds of the immunoglobulin specificity of each product, and the size of products was domains. From the conserved immunoglobulin domain verified on ethidium bromide-stained 2% agarose gels. structure it is suggested that Cys 28 is linked to Cys 70 to Data from three independent experiments were used to form domain 1 (EC1), and that Cys 109 is paired with Cys DD calculate the relative expression ratio of mRNA by 2 Ct 153 to form domain 2 (EC2). The overall identity of the

(accession no. NP_001002273) and mouse FcgRIIB2 (accession no. NP_034317) are shown below. Cysteine residues involved in Ig-like domain disulphide bonds are in bold. The putative signal sequence and transmembrane region are underlined. Dashes indicate identity of amino acid to FcgRII. Italics define the potential glycosylation sites. Dots indicate the gaps inserted to maintain alignment. 248 Y. Liu et al. / Veterinary Immunology and Immunopathology 133 (2010) 243–249 ovine FcgRII with its cattle, pig, human and mouse counterparts at the amino acid sequence level was 91%, 60%, 68% and 67%, respectively; however a comparison of the two extracellular domains alone between the four species shows a higher identity of 93%, 69%, 75% and 72%, respectively (Fig. 2). This is in agreement with the generally highly conserved nature of the individual FcR binding domains. In mice different FcgRII isoforms are expressed in macrophages and B lymphocytes. The FcgRIIB2 transcript expressed by macrophages mediates endocytosis and delivery into lysosomes. The FcgRIIB1 transcript expressed by B lymphocytes has an in-frame insertion which increases the cytoplasmic region from 47 to 99 Fig. 4. Expression pattern of ovine FcgRII mRNA in monocytes, amino acids. This isoform is inefficient in mediating lymphocytes, PAMs (pulmonary alveolar macrophages) and PMNs endocytosis (Amigorena et al., 1992a; Miettinen et al., (polymorphonuclear leucocyte). Each sample was individually assayed in triplicate by real-time PCR. All samples were normalized using GAPDH 1989). The ovine FcgRII have a cytoplasmic sequence of 48 expression as an internal control. Relative level of ovine FcgRII mRNA was amino acids and appears most similar to the FcgRIIB2 analyzed by the 2DDCt method. isoform in mice. Therefore we conjecture that the newly cloned ovine receptor may also have a similar endocytic function. ovine homology of human FcgRII (CD32). Future studies In human, mouse, pig and cattle, FcgRIIB is character- will identify the detail expression pattern of this FcgR and ized by containing an inhibitory or ITIM sequence in its its function in ovine immune responses. cytoplasmic region. In ovine FcgRII, there is an IxYxxL sequence in the cytoplasmic domain which is in agreement Acknowledgment with the consensus sequence of an ITIM (V/I/L/SxYxxL/V/I/ S, x denotes any amino acid) suggesting that the receptor This work was supported by the grant from program of may behave as an inhibitory receptor (Hibbs et al., 1988; the National Natural Science Foundation of China (No. Isnardi et al., 2004; Zhang et al., 1994; Qiao et al., 2006). 30730068). This motif has been shown to be both necessary and sufficient to mediate the inhibition of BCR co-ligation and References is required for its inhibitory activity (Amigorena et al., 1992b). Amigorena, S., Bonnerot, C., Drake, J.R., Choquet, D., Hunziker, W., Guillet, J.G., Webster, P., Sautes, C., Mellman, I., Fridman, W.H., 1992a. Cyto- Rosetting analysis was performed to confirm the plasmic domain heterogeneity and functions of IgG Fc receptors in B identity of the ovine cDNA as a putative IgG receptor lymphocytes. Science 256, 1808–1812. and characterize the ligand specificity of this receptor. Amigorena, S., Salamero, J., Davoust, J., Fridman, W.H., Bonnerot, C., COS-7 cells that were transiently transfected with ovine 1992b. Tyrosine-containing motif that transduces cell activation signals also determines internalization and antigen presentation FcgRII were assayed using erythrocytes sensitized with via type III receptors for IgG. Nature 358, 337–341. ovine IgG subclass. As shown in Fig. 3, COS-7 cells Carlson, G.P., Kaneko, J.J., 1973. Isolation of leukocytes from bovine transfected with the putative ovine FcgRII cDNA were peripheral blood. Proc. Soc. Exp. Biol. Med. 142, 853–856. Dijstelbloem, H.M., van de Winkel, J.G., Kallenberg, C.G., 2001. Inflamma- able to efficiently bind erythrocytes sensitized with tion in autoimmunity: receptors for IgG revisited. Trends Immunol. purified ovine IgG1, while the reaction with IgG2 was 22, 510–516. undetectable. Non-sensitized erythrocytes did not attach Fukuyama, H., Nimmerjahn, F., Ravetch, J.V., 2005. The inhibitory Fcgamma receptor modulates autoimmunity by limiting the accu- to the transfected COS-7 cells and sensitized red cells did mulation of immunoglobulin G+ anti-DNA plasma cells. Nat. Immu- not attach to the nontransfected COS-7 cells (data not nol. 6, 99–106. shown). This result further confirmed the identity of the Goudswaard, J., van der Donk, J.A., Noordzij, A., van Dam, R.H., Vaerman, J.P., 1978. Protein A reactivity of various mammalian immunoglobu- ovine cDNA as an IgG receptor. lins. Scand. J. Immunol. 8, 21–28. In order to examine the cell distribution of ovine FcgRII, Hibbs, M.L., Bonadonna, L., Scott, B.M., McKenzie, I.F., Hogarth, P.M., 1988. real-time PCR was performed on mRNA from selective Molecular cloning of a human immunoglobulin G Fc receptor. Proc. Natl. Acad. Sci. U.S.A. 85, 2240–2244. cellular subsets. As shown in Fig. 4, the monocytes had the Isnardi, I., Lesourne, R., Bruhns, P., Fridman, W.H., Cambier, J.C., Daeron, highest ovine FcgRII mRNA abundance. The ovine FcgRII M., 2004. Two distinct tyrosine-based motifs enable the inhibitory mRNA level was significantly higher in the PAMs and receptor FcgammaRIIB to cooperatively recruit the inositol phospha- lymphocytes than in PMNs. In human and mouse, FcgRII tases SHIP1/2 and the adapters Grb2/Grap. J. Biol. Chem. 279, 51931– 51938. are widely expressed by cells of hematopoietic origin. The Kacskovics, I., 2004. Fc receptors in livestock species. Vet. 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