Developmental and Comparative Immunology 35 (2011) 1173–1181 Contents lists available at ScienceDirect Developmental and Comparative Immunology jo ur n al homepage: www.elsevier.com/locate/dci Short communication Transporters associated with antigen processing (TAP) in sea bass (Dicentrarchus labrax, L.): Molecular cloning and characterization of TAP1 and TAP2 Rute D. Pinto a,c , Pedro J.B. Pereira b , Nuno M.S. dos Santos a,∗ a Fish Immunology and Vaccinology Group, IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal b Protein Crystallography Group, IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal c ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Largo Prof. Abel Salazar 2, 4099-003 Porto, Portugal a r t i c l e i n f o Article history: Received 2 February 2011 Received in revised form 15 March 2011 Accepted 17 March 2011 Available online 19 April 2011 Keywords: Dicentrarchus labrax TAP Gene cloning Comparative sequence analysis Protein structure prediction Homology modelling a b s t r a c t The transporters associated with antigen processing (TAP), play an important role in the MHC class I antigen presentation pathway. In this work, sea bass (Dicentrarchus labrax) TAP1 and TAP2 genes and transcripts were isolated and characterized. Only the TAP2 gene is structurally similar to its human orthologue. As other TAP molecules, sea bass TAP1 and TAP2 are formed by one N-terminal accessory domain, one core membrane-spanning domain and one canonical C-terminal nucleotide-binding domain. Homology modelling of the sea bass TAP dimer predicts that its quaternary structure is in accordance with that of other ABC transporters. Phylogenetic analysis segregates sea bass TAP1 and TAP2 into each subfamily cluster of transporters, placing them in the fish class and suggesting that the basic structure of these transport-associated proteins is evolutionarily conserved. Furthermore, the present data provides information that will enable more studies on the class I antigen presentation pathway in this important fish species. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction The transporter associated with antigen processing (TAP) is a key component in the MHC class I-dependent antigen presen- tation pathway [reviewed by Peaper and Cresswell, 2008]. TAP translocates peptides mainly derived from proteasomal cleavage of ubiquitinated proteins in the cytosol to the ER lumen, where these peptides are loaded onto MHC class I molecules (Abele and Tampé, 2009; Michalek et al., 1993). Stable MHC-peptide com- plexes migrate to the cell surface for presentation to CD8 + cytotoxic T lymphocytes and this recognition event triggers elimination of the presenting cell. TAP is a member of the ABC (ATP-binding cas- sette) family of transporters, which translocate a wide range of substrates across membranes in an ATP-dependent manner. ABC transporters have a conserved architecture of two transmembrane domains (TMDs) each with six canonical transmembrane helices that together form the translocation pathway and two cytosolic nucleotide-binding domains (NBDs) together bearing two ATP- binding sites. ATP binding induces the NBDs dimerization, with the two ATP molecules sandwiched at the interface and bridg- ing contacts between the two NBDs (Procko et al., 2006). The TAP ∗ Corresponding author. Tel.: +351 226 074 900; fax: +351 226 099 157. E-mail addresses: rsp@ibmc.up.pt (R.D. Pinto), ppereira@ibmc.up.pt (P.J.B. Pereira), nsantos@ibmc.up.pt (N.M.S. dos Santos). translocation complex is composed of two half-transporters, TAP1 (ABCB2) and TAP2 (ABCB3), each with one TMD and one NBD (Kelly et al., 1992; Powis et al., 1991; Spies and DeMars, 1991). Additionally, TAP molecules have an N-terminal accessory domain (Procko et al., 2005), also with three to four membrane-spanning regions, that is not required for peptide binding and transport, but is essential for tapasin-binding and assembly of the peptide loading complex (Koch et al., 2004). TAP binds 8–16 amino acid long peptides [reviewed in Abele and Tampé, 2009] but its peptide specificity is restricted to the three N-terminal residues and the C- terminal residue (Uebel et al., 1997). Both TAP subunits were found to be essential and sufficient for ATP-dependent peptide translo- cation into the ER lumen (Androlewicz et al., 1993; Meyer et al., 1994; Shepherd et al., 1993). TAP genes have been identified and mapped to the MHC in several non-mammalian species, including cartilaginous and bony fish (Grimholt, 1997; Hansen et al., 1999; Ohta et al., 1999a), chicken (Walker et al., 2005) and the amphibian Xenopus (Ohta et al., 1999b; Ohta et al., 2003). Here, sea bass TAP1 and TAP2 cDNA and genes have been for the first time identified and characterized at the molecular, structural and phylogenetic levels. 2. Materials and methods 2.1. Fish Sea bass, Dicentrarchus labrax, were kept in a recirculating, ozone-treated salt-water (20–25‰) system at 22 ± 1 ◦ C and fed 0145-305X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2011.03.024