Smelty P et al. (2010), Identification of the pre-T-cell receptor alpha...

XB-ART-42327

Proc Natl Acad Sci U S A 2010 Nov 16;10746:19991-6. doi: 10.1073/pnas.1010166107.

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Identification of the pre-T-cell receptor alpha chain in nonmammalian vertebrates challenges the structure-function of the molecule.

Smelty P , Marchal C , Renard R , Sinzelle L , Pollet N , Dunon D , Jaffredo T , Sire JY , Fellah JS .

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In humans and mice, the early development of αβ T cells is controlled by the pre-T-cell receptor α chain (pTα) that is covalently associated with the T-cell receptor β (TCRβ) chain to form the pre-T-cell receptor (pre-TCR) at the thymocyte surface. Pre-TCR functions in a ligand-independent manner through self-oligomerization mediated by pTα. Using in silico and gene synteny-based approaches, we identified the pTα gene (PTCRA) in four sauropsid (three birds and one reptile) genomes. We also identified 25 mammalian PTCRA sequences now covering all mammalian lineages. Gene synteny around PTCRA is remarkably conserved in mammals but differences upstream of PTCRA in sauropsids suggest chromosomal rearrangements. PTCRA organization is highly similar in sauropsids and mammals. However, comparative analyses of the pTα functional domains indicate that sauropsids, monotremes, marsupials, and lagomorphs display a short pTα cytoplasmic tail and lack most residues shown to be critical for human and murine pre-TCR self-oligomerization. Chicken PTCRA transcripts similar to those in mammals were detected in immature double-negative and double-positive thymocytes. These findings give clues about the evolution of this key molecule in amniotes and suggest that the ancestral function of pTα was exclusively to enable expression of the TCRβ chain at the thymocyte surface and to allow binding of pre-TCR to the CD3 complex. Together, our data provide arguments for revisiting the current model of pTα signaling.

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Species referenced: Xenopus
Genes referenced: acss1 atl2 cd3g cnpy3 eif4a3.1 entpd6 fbln7 fbxo42 gnmt klc4 klhdc3 mapt mea1 mrpl2 npat prph prph2 rpl7l1 slc22a7 tbcc trpc5 ttbk1 ttl ubr2 vsx1 zc3h6

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	Fig. 1. Gene organization around PTCRA from representative osteichthyan genomes. In mammals (human, mouse, opossum, and platypus), gene synteny is conserved with CNPY3 and RPL7L1 located respectively downstream and upstream of PTCRA. In sauropsids (chicken, zebrafinch, and lizard), gene synteny is conserved downstream of PTCRA (CNPY3, GNMT,…) but is different upstream (POLR1B, TTL, …) than in mammals. In the frog, a gene synteny similar to that in sauropsids was found on chromosome 5, but PTCRA (marked “?”) could not be identified. In the zebrafish, genes and/or groups of genes belonging to tetrapod gene syntenies were found distributed on at least three chromosomes. The candidate region (13 kb) located upstream of CNPY3 on chromosome 1 (double arrow) does not house PTCRA. Genes are depicted by oriented pentagons. Official, full names of the genes annotated in the genomic regions and not mentioned in the text are as follows: ACSS1, acyl-CoA synthetase short-chain family member 1; ATL2, atlastin GTPase 2; CUL7, cullin 7; DACHB, dachshund B; DDX48, eukaryotic translation initiation factor; ENTPD6, ectonucleoside triphosphate diphosphohydrolase 6; FBLN7, fibulin 7; FBXO42, F-box protein 42; KIAA0240, no official name; KLC4, kinesin light chain 4; KLHDC3, kelch domain containing 3; MEA1, male-enhanced antigen 1; MRPL2, mitochondrial ribosomal protein L2; PPP2R5D, protein phosphatase 2, regulatory subunit B′, delta isoform; PRPH2, peripherin 2 (retinal degeneration, slow); SLC22A7, solute carrier family 22 (organic anion transporter), member 7; TBCC, tubulin folding cofactor C; TRPC5, transient receptor potential cation channel, subfamily C, member 5; TTBK1, tau tubulin kinase 1; UBR2, ubiquitin protein ligase E3 component n-recognin 2; VSX1, visual system homeobox 1; ZC3H6, zinc finger CCCH-type containing 6.
	Fig. 2. Amino acid comparison of pTα sequences in amniotes. The Homo sapiens pTα sequence is aligned with that of four sauropsids [chicken (Gallus gallus), turkey (Meleagris gallopavo), zebra finch (Taenopygia guttata), and lizard (Anolis carolinensis)] and six representative species of the main mammalian lineages [monotremes (platypus, Ornithorhynchus anatinus), marsupials (opossum, Monodelphis domestica), afrotherians (elephant, Loxodonta africana), leurasiatherians (cow, Bos taurus; and dog, Canis familiaris), and glires (mouse, Mus musculus)]. The signal peptide (shown within a box) is encoded by exon 1a. The highly variable C-terminal region encoded by exon 4 (shown in gray was aligned tentatively to highlight a few conserved residues in human, cow, dog, and elephant (Fig. S1). For convenience of presentation, amino acids (numbers are given within parentheses) were removed from the human, cow, and dog sequences. Residues identified as important in the mouse pTα sequence are indicated by gray columns. ‖, limits of exons; (.), residue identical to the human pTα residue; -, indel; #, unchanged residue; ?, unknown residue; *, Stop codon. Potential N-glycosylation sites are underlined. Amino acids not identified in the text: F, phenylalanine; I, isoleucine, M, methionine; Y, tyrosine. Cp, connecting peptide; Ct, cytoplasmic tail; Tm, transmembrane.
	Fig. 3. Alignment of mammalian and sauropsidian sequences in two pTα regions in which some amino acids (shown in gray) were found to play an important role. Symbols are as in Fig. 2.
	Fig. 4. Expression of PTCRA transcripts. (A) PTCRA expression in hematopoietic tissues of E18, E14, and E10 chicken embryos was analyzed by RT-PCR. Three bands of 600 bp, 500 bp, and 400 bp, respectively, were identified in all of the stages. Bm, bone marrow; RT-, control PCR in which reverse transcriptase was omitted during first-strand synthesis; Sp, spleen; Th, thymus. S17 primers were used to assess the amount of cDNA samples used for PCR experiments. (B) (Left) Flow cytometric isolation of E18 chicken thymocyte subsets analyzed in Right. (Right) RT-PCR analysis of PTCRA expression on thymocyte subsets. PTCRA transcripts were detected in DN and DP thymocytes. S17 primers were used to assess the amount of cDNA samples used for PCR experiments.

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