Molecular cloning and sequencing of the cDNA encoding the feline FcγRIIIA (CD16) homologue

Veterinary Immunology and Immunopathology 73 (2000) 353±359

Short communication

Molecular cloning and sequencing of the cDNA encoding the feline FcgRIIIA (CD16) homologue$ Yorihiro Nishimuraa, Takayuki Miyazawaa, Yasuhiro Ikedaa, Yoshihiro Izumiyaa, Kazuya Nakamuraa, Eiji Satoa, Takeshi Mikamib, Eiji Takahashia,* a

Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan b The Research Center for Protozoan Molecular Immunology, Obihiro University, Nishi 2 Inada-cho, Obihiro 080-8555, Japan

Received 9 August 1999; received in revised form 21 December 1999; accepted 21 December 1999

Abstract We ampli®ed the cDNA encoding the feline FcgRIIIA (CD16) homologue from peripheral blood mononuclear cells by polymerase chain reaction and cloned two forms of FCGR3A cDNA. Sequencing analysis revealed that the open reading frame of feline FCGR3A cDNA consists of 750 or 747 base pairs encoding 250 or 249 amino acid residues, respectively. Comparison of the predicted amino acid sequence of feline FCGR3A cDNA with those of other mammalians' homologues revealed that the extracellular domain has a relatively low homology. However, the cytoplasmic domain contained an 8-amino acid motif, Leu-Phe-Val-Val-Asp-Thr-Gly-Leu, which was considered to interact with an accessory molecule such as the g chain of Fc receptors for IgE to form heterodimeric complexes. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Cat; CD16; cDNA; Cloning; FcgRIII

1. Introduction Binding of Fc domain of immunoglobulin (Ig) or immune complex to Fc receptors (FcRs) on hematopoietic cells play critical roles for immune reactions mediating a link $

The nucleotide sequence data reported in this paper have been submitted to the DDBJ nucleotide sequence data base and have been assigned the accession numbers AB025314 and AB025315. * Corresponding author. Tel.: ‡81-3-5841-5396; fax: ‡81-3-5841-8184. E-mail address: [email protected] (E. Takahashi). 0165-2427/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 0 ) 0 0 1 5 6 - 2

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between humoral and cellular effector functions. Triggering the FcR initiates a variety of immune responses, including effector functions such as phagocytosis of immune complex, antibody-dependent cellular cytotoxicity (ADCC), and release of in¯ammatory cytokines (van de Winkel and Capel, 1993). In the humans, FcRs for IgG (FcgR) have been divided into three classes. FcgRI (CD64) is a high af®nity class, whereas FcgRII (CD32) and FcgRIII (CD16) are low af®nity classes (Ravetch and Kinet, 1991). The human FcgRIII is a 50±80 kDa glycoprotein expressed on the cell surface (Huizinga et al., 1988) and is encoded by the highly homologous two genes, FCGR3A and FGGR3B (Ravetch and Perussia, 1989). The FcgRIIIA is a transmembrane protein expressed on NK cells, a subset of T-cells, and activated monocytes/macrophages (Ravetch and Perussia, 1989). On the other hand, the FcgRIIIB is a glycosylphosphatidylinositol (GPI)anchored protein and expressed on polymorphonuclear leukocytes (Ravetch and Perussia, 1989). The FcgRIIIA constitutes a multimeric transmembrane complex (Kurosaki and Ravetch, 1989), and mediates ADCC (Ravetch and Kinet, 1991). Tompkins et al. (1983) showed that cytotoxic cells of the cats possess FcgRs. However, there were no speci®c monoclonal antibodies against feline FcRs to clarify the class of the FcgRs. As part of a study to de®ne feline FcRs, we have isolated and sequenced the cDNA encoding the feline FcgRIIIA homologue. 2. Materials and methods 2.1. Preparation of cDNA of feline peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMC) were separated from heparinized whole blood from a speci®c pathogen-free cat (Japanese mongrel) by Ficoll-Paque1 (Amersham Pharmacia, Uppsala, Sweden) gradient centrifugation. The separated cells were collected and washed with phosphate-buffered saline and centrifuged at 150g to remove platelets. The PBMC were cultured in RPMI1640 growth medium (Sigma, St. Louis, MO, USA) supplemented with 10 mg/ml concanavalin A, 100 units/ml recombinant human interleukin 2, antibiotics, and 10% heat-inactivated fetal calf serum for 3 days at 378C in 5% CO2. Then poly A‡ mRNA was extracted from the cultured PBMC by using the Quick PrepTM micro mRNA puri®cation kit (Amersham Pharmacia), and then double stranded (ds) cDNA was synthesized by MarathonTM cDNA Ampli®cation Kit (Clontech, Palo Alto, CA, USA) using oligo dT as a primer with or without adaptor DNA ligation. The ds cDNA without adaptor DNA was circularized by T4 ligase. 2.2. Cloning and sequencing of the feline FCGR3A cDNA We cloned partial feline FCGR3A cDNA by semi-nested 50 - and 30 -rapid ampli®cation of cDNA ends (RACE) polymerase chain reaction (PCR) using Ampli Taq Gold polymerase (Perkin Elmer, Branchburg, NJ, USA) from the circularized ds cDNA as a template as described below. For the ®rst RACE PCR, primers hbrCD16/50 -1 (50 CACYKGTAYTCKCCACTGTC-30 ) and hbrCD16/30 -1 (50 -TCCTKTTTGCAGTGGA-

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Fig. 1. Schematic structure of the feline FCGR3A cDNA and cloning strategies by PCR. The location and direction of PCR primers are indicated by arrow heads. The ampli®ed cDNA fragments are shown by solid lines. Nested 50 - and 30 -RACE PCR from circularized ds cDNA (A, B). Nested 50 -RACE PCR from adaptor-ligated ds cDNA (C). Ampli®cation of the cDNA encompassing the entire ORF of feline FCGR3A (D).

CAC-30 ) were designed based on the highly conserved sequence of the FCGR3A cDNA of humans (Ravetch and Perussia, 1989), cattle (Collins et al., 1997), and rats (Farber and Sears, 1991) (Fig. 1A). The PCR condition was as follows; 948C for 8 min, followed by 35 cycles of 948C for 1 min, 558C for 1 min and 728C for 2 min and then 728C for 10 min. Then the semi-nested RACE PCR was performed using a primer pair of hbrCD16/50 -2 (50 -TCCCTGGCACKTCAGMGTCAC-30 ) and hbrCD16/30 -1. Consequently, a speci®c fragment 0.5 kilobase pairs (kbp) was ampli®ed (Fig. 1A), and cloned into pCR2.1 vector (Invitrogen, Carlsbad, CA, USA) by the TA-cloning procedure and designated as pCRfCD16P1. Sequencing analysis was performed by GeneScanTM ABI PRIZMTM 377XL auto sequencer (Perkin Elmer). The cDNA encompassing the entire open reading frame (ORF) was ampli®ed with a primer pair of fCD16F0 (50 GACTTGTTCATTCTACCGTG-30 ) and fCD16R0 (50 -ATGGTCGTGTTGCTGTGC-30 ), which was designed based on the sequence data obtained (Fig. 1D). The ampli®ed fragments from three independent PCRs were cloned into pCR2.1 and designated as pCRfCD16FR1, pCRfCD16FR2, and pCRfCD16FR3, respectively. The obtained nucleotide sequences and the deduced amino acid sequences were analyzed using a genetic information processing software, GENETYX-MAC ver. 9 (Software development, Tokyo, Japan). 3. Results and discussion The pCRfCD16P1 did not contain the start codon and the poly A signal sequence. To amplify the upstream and downstream of the cDNA of pCRfCD16P1, we performed another nested 50 - and 30 -RACE PCR using a primer pair of fCD16/50 -1 (50 CAGGTTCTAGGACCACCATG-30 ) and fCD16/30 -1 (50 -CAAAGTCACGTGGAGCCACG-30 ), followed by fCD16/50 -2 (50 -GCCTTCGAGAGGTCAGCTCG-30 ) and fCD16/30 -2 (50 -TTCAGGTGCCCACAGTGACG-30 ) designed from the obtained sequence (pCRfCD16P1) (Fig. 1B). The PCR product 1 kbp was cloned into pCR2.1 and named pCRfCD16P2. Unfortunately, the pCRfCD16P2 also did not contain the start codon, and we carried out the nested 50 -RACE PCR from the adaptor-ligated ds cDNA template using the adaptor speci®c primer and fCD16/50 -1, followed by the adaptor

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Fig. 2. The nucleotide sequence and deduced amino acid sequence of feline FCGR3A cDNA (pCRfCD16FR1) (accession No. AB025314). Italic letters are the nucleotide sequence of 50 -region (from 1 to 120) of pCRfCD16FR2 and pCRfCD16FR3 (accession No. AB025315) and corresponding amino acid sequence. The absence of nucleotides and an amino acid residue in pCRfCD16FR1 are indicated by bars (ÿ). The locations of PCR primers are indicated with solid lines above the corresponding nucleotide sequences and arrows indicate the directions of extension. The polyadenylation signal sequence is underlined with a solid line.

Y. Nishimura et al. / Veterinary Immunology and Immunopathology 73 (2000) 353±359

Fig. 3. Comparison of the predicted amino acid sequences of FCGR3A among the cats, cattle (accession No. X99695), humans (accession No. X52645), and mice (accession No. M14215). The predicted amino acid sequences of feline FCGR3A are shown as cat-1 (pCRfCD16FR1 (accession No. AB025314)) and cat-2 (pCRfCD16FR2 and pCRfCD16FR3 (accession No. AB025315)). Identical amino acid residues are indicated by dots (
) and gaps are indicated by bars (ÿ). The predicted transmembrane region is boxed with a dotted line. The N-glycosylation sites within the extracellular domain are underlined. The highly conserved cysteine residues are indicated by closed triangles. The amino acid sequence considered to interact with an accessory molecule (Leu-Phe-Ala/Val-Val-Asp-Thr-Gly-Leu) is boxed with a solid line.

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speci®c nested primer and fCD16/50 -2 (Fig. 1C). The PCR product contained the 50 untranslated region (UTR) and start codon of the feline FCGR3A cDNA. The lack of the 50 - and 30 -region of the cDNA of the pCRfCD16P1 and pCRfCD16P2 might be arti®cial deletions. Fig. 2 shows the nucleotide sequence of the obtained cDNAs and predicted amino acid sequence of feline FCGR3A. The pCRfCD16FR1 contained an ORF consisting of 747 nucleotides encoding 249 amino acids, whereas the ORFs of pCRfCD16FR2 and pCRfCD16FR3 were identical (750 nucleotides) and had an additional codon `GCA' encoding Ala between nucleotide acid positions 85 and 86 from the start codon of pCRfCD16FR1. It was unexpected result that there were two forms of FCGR3A cDNA in cats. It is unclear whether this insertion was produced by an alternative splicing or these mRNAs were transcribed from separate genes. The nucleotide sequence from 1908 to 1913 was a typical polyadenylation signal sequence, indicating that the pCRfCD16P2 contained a full length of 30 -UTR. The alignment of the deduced amino acid sequences of FCGR3A cDNA among the species indicates that our cloned cDNAs encoded feline homologue of FcgRIIIA (Fig. 3). It is not known yet whether a homologue of FcgRIIIB exists in cats. The predicted amino acid sequence of feline FCGR3A (pCRfCD16FR1) showed 63.6, 61.4 and 45.2% homologies with those of the humans (Ravetch and Perussia, 1989), cattle (Collins et al., 1997), and mice (Ravetch et al., 1986). An 8-amino acid motif, Leu-Phe-Ala-Val-AspThr-Gly-Leu, is considered to interact with an accessory molecule such as the g chain of FcRs for IgE to form heterodimeric complexes in the mice (Ra et al., 1989, Zeger et al., 1990). This motif was highly conserved in the cytoplasmic domain in the cats, humans, cattle, and mice, however, a substitution of Ala to Val was present in cats. This substitution may re¯ect some structural characteristics of accessory molecules that interact with feline FcgRIIIA. In summary, we cloned feline FCGR3A cDNA, and determined the nucleotide sequence including the entire ORF. The availability of full-length feline FCGR3A ORF will allow the synthesis of feline FcgRIIIA recombinant protein to raise monoclonal antibodies, which will be useful as tools for further understanding of feline immunology. Acknowledgements The authors thank Dr. M. Hattori (Kyoto University, Kyoto, Japan) for providing recombinant human interleukin-2-producing Ltk-IL-2.23 cells. This study was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan. Y. Nishimura, Y. Ikeda, Y. Izumiya, and E. Sato are supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. References Collins, R.A., Gelder, K.I., Howard, C.J., 1997. Nucleotide sequence of cattle FcGRIII: its identi®cation in g d T cells. Immunogenetics. 45, 440±443.

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