Molecular cloning and sequencing of feline CD56 (N-CAM)

We isolated feline CD56 cDNA to investigate the primary structure of CD56 of feline leukocytes. Sequencing analysis revealed that an open reading frame (2538 bp) encoded a homologue of a 140-kDa isoform of CD56. The predicted amino acid sequence showed 97.3% homology with that of humans. Natural killer (NK) cells exhibit MHC-unrestricted cytotoxicity and play important roles in the immune system, where the NK cells recognize and kill certain tumour cells and virus-infected cells. CD56 (neural cell adhesion molecule: N-CAM), a cell surface antigen, has been used as a marker of NK cells in humans. Several isoforms of CD56 have been identified: the 140- and 180-kDa forms are expressed as transmembrane proteins, whereas the 120-kDa form is a phosphatidylinositol-glycan anchored protein. Differences amongst these polypeptides are found mainly within the transmembrane and cytoplasmic domains and arise from alternative mRNA splicing from a single gene. In humans, the expression of the 140-kDa transmembrane-anchored isoform is recognized on NK cells and ≃ 5% of peripheral blood T lymphocytes. In the cat, no cDNA of CD56 has been cloned and little is known about the function of feline CD56 and relationships between the molecule and NK cells. Here, we report the primary structure of CD56 derived from feline leukocytes. Total RNA was extracted by ISOGEN from feline thymus, and then double-strand (ds) cDNA was synthesized using the Marathon® cDNA Amplification Kit with oligo dT as a primer. Subsequently we tried to amplify part of the open reading frame (ORF) of feline CD56 cDNA by polymerase chain reaction (PCR) using LA Taq polymerase. Primers fCD56F1 and fCD56R1 were designed to amplify about 2.1 kbp of the partial ORF based on the highly conserved sequence among the cDNA of CD56 mRNA of the human and rat. The PCR conditions were as follows: 94°C for 2 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 3 min and then 72°C for 10 min. The PCR product was subcloned into the pMosBlueT vector by the TA-cloning procedure and designated pMBfCD56P. The sequencing reaction was carried out using the dye primer cycle sequencing FS ready reaction kit (- 21M13 and M13 rev.). Sequencing analysis was performed using the GeneScan® Model 373A auto-sequencer. Based on sequence information from pMBfCD56P, we attempted to clone upstream and downstream of the cDNA clone of the partial CD56 ORF. The prepared ds cDNA of mRNA of feline thymocytes was circulized using a DNA ligation kit (Takara) and used as the templates for nested 5'-and 3'-rapid amplification of cDNA ends (RACE) PCR. The first PCR was performed uisng a primer pair of fCD56/5'-1 and fCD56/3'-1. Then the nested PCR was performed using a primer pair of fCD56/5'-2 and fCD56/3'-2. The nested 5'- and 3'-RACE PCR also successfully amplified a specific fragment of about 1 kbp. From sequencing analysis of the PCR product, we obtained sequence data around the 3'-end of the ORF. To amplify the cDNA encompassing the entire ORF, a primer pair of fCD56F1 and fCD56R2 was designed based on the sequence data obtained. The PCR was performed as described above. A specific cDNA fragment of about 2.5 kbp was cloned into pMosBlueT and designated pMBfCD56F140. The pMBfCD56F140 was subjected to sequence analysis and the nucleotide sequences obtained and the amino acid sequences deduced were analysed using genetic information processing software, GENETYX-MAC ver. 9. The sequencing analysis revealed that the cDNA clone encoded a 140-kDa form of the CD56 homologue. The ORF was 2538 bp and encoded 846 amino acid residues with a predicted molecular mass of 93.2 kDa. Considering the putative signal sequence, the size of mature feline CD56 is predicted to be 91.0 kDa, consisting of 835 amino acid residues. Another sequenced clone, pMBfCD56P, lacked 42 bp of the sequence (nucleotide positions from 555 to 596) when compared with pMBfCD56F140. The feline CD56 protein could be divided into four primary regions; the signal peptide and extracellular, transmembrane, and cytoplasmic domains, comparable to chicken CD56. The extracellular domain of feline CD56 was composed of five domains of about 100 amino acid residues, homologous to each other and to Ig domains as in the chicken. In addition, each of the five domains contained a pair of cysteine residues that were separated by about 50 amino acid residues, like the Ig domain. These cysteine residues were perfectly conserved among the human, rat and chicken. The nucleotide sequence of CD56 of the cat showed 93.1 and 87.7% identities with those of human and rat, respectively. The predicted amino acid sequence of feline CD56 showed 97.3, 94.4 and 87.8% identities with those of human, rat and chicken, respectively. Figure 3 shows a comparison of the predicted amino acid sequences between the cat and human. These structural characteristics and the high homology over the whole molecule may imply conservation of the functions of CD56 among species.