Molecular characterization of the feline T-cell receptor γ alternate reading frame protein (TARP) ortholog

Abstract

T-cell receptor γ alternate reading frame protein (TARP) is expressed by human prostate epithelial, prostate cancer, and mammary cancer cells, but is not found in normal mammary tissue. To date, this protein has only been described in humans. Additionally, no animal model has been established to investigate the potential merits of TARP as tumor marker or a target for adoptive tumor immunotherapy. In this study conducted to characterize feline T-cell receptor γ sequences, constructs very similar to human TARP transcripts were obtained by RACE from the spleen and prostate gland of cats. Transcription of TARP in normal, hyperplastic, and neoplastic feline mammary tissues was evaluated by conventional RT-PCR. In felines similarly to the situation reported in humans, a C-region encoding two open reading frames is spliced to a J-region gene. In contrast to humans, the feline J-region gene was found to be a pseudogene containing a deletion within its recombination signal sequence. Our findings demonstrated that the feline TARP ortholog is transcribed in the prostate gland and mammary tumors but not normal mammary tissues as is the case with human TARP.

Fig. 1

Portion of the feline T-cell receptor γ (fTRG) locus containing the hypothetical feline T-cell receptor γ alternate reading frame protein (fTARP) and TARP mRNA (not to scale). Alternatively, two loci may exist: one containing fTRGC4 and the carnivore-specific short interspersed nuclear elements (CAN-SINE), and a second one containing fTARP and fTRGJP. Arrows and numbers indicate primer position and orientation. 1: FeTcRGr2, 2: FeTcRGr3/fTARPr2, 3: FTGR4, 4: FTGVf1/fTARPf1, 5: fTARPf2, 6: FTGJr1, 7: FTGCr1/fTARPr1, fTARPr3, 8: FTGPIr1, C.-S.: CAN-SINE, 5'UTR: 5'untranslated region, 3'UTR: 3'untranslated region, AAAAA: poly-A-tail.

Fig. 2

(A) Alignment of the deduced amino acid sequences of human and putative feline TARP. In human TARP (hTARP), five leucine residues in heptad repeats can be found. In fTARP, only four leucine residues were observed. The basic region downstream of the last leucine displayed high homology. (B) Alignment of clone pFTGII.164, the genomic sequence of fTRGJP (gnl|ti|755161287), and fTRGJ2.3 (gnl|ti|915242736). In fTRGJP, the heptamer of the 12RSS was deleted. The spacer of the fTRGJ2.3 12RSS was either 10- or 11-bp long, or the nonamer ended on 'T'.

Fig. 3

PCR amplification of feline TARP with different primer sets from genomic DNA and cDNA. (A) TARP amplification of genomic DNA from five different cats using FTGVf1/fTARPf1 as the forward primer and FTGJr1 (lanes 1, 11, 21, 31, and 41 are specific for fTRGJP), FTGCr1/fTARPr1 (lanes 2, 12, 22, 32, and 42 are specific for spliced TARP) and FTGPIr1 (lanes 3, 13, 23, 33, and 43 specific for the intron downstream of fTRGJP) as the reverse primers. Lane 0: control (no template), lane M: DNA size marker. (B) Amplification of DNA from cat No. 4 using FTGVf1/fTARPf1 and FTGCr1/fTARPr1 (lanes 1~4 are specific for spliced TARP) or FTGPIr1 (lanes 11~14 are specific for the intron downstream of fTRGJP). DNA was untreated (lanes 1 and 11), treated with RNase (lanes 2, 3, 12 and 13), and treated with DNase (lanes 4 and 14). Lane 0: control (no template), lane M: DNA size marker. (C) Amplification of TARP cDNA/mRNA from feline prostate glands (primers: fTARPf1 and fTARPr2). Lane 1: cat No. 5, lane 2: cat No. 6, lane 3: cat No. 7, lane 4: cat No. 8, lane 5: cat No. 9, lane 0: control (no template). (D) Amplification of TARP cDNA/mRNA with fTARPf2 and fTARPr3 primers from normal mammary gland (lanes 1, 2, and 5), mammary glandular hyperplasia (lane 3), mammary adenomas (lanes 14 and 15), mammary adenocarcinomas (lanes 4, 6, 7, 9, 10, 11, and 12), normal lymph node (lane 13), and lymph node metastases of mammary adenocarcinoma (lane 8). Lane M: DNA size marker, lane 0: control (no template), lane +: positive control.