Molecular cloning, sequencing, and characterization of bovine transporter associated with antigen processing 2 ( BoTAP2)

• Published in: Immunogenetics (New York) 2002-04, Vol.54 (1), p.30-38
• Main Authors: Ambagala, Aruna P N, Feng, Zhengyu, Barletta, Raul G, Srikumaran, Subramaniam
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; ATP-Binding Cassette Transporters - analysis; ATP-Binding Cassette Transporters - chemistry; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette, Sub-Family B, Member 3; Cattle - immunology; Cloning, Molecular; DNA, Complementary - chemistry; Green Fluorescent Proteins; Luminescent Proteins - analysis; Microscopy, Immunoelectron; Molecular Sequence Data; RNA, Messenger - analysis
• ISSN: 0093-7711; 1432-1211
• DOI: 10.1007/s00251-001-0416-6

The transporter associated with antigen processing (TAP) 1/2 heterodimer is essential for the transport of antigenic peptides from the cytosol into the lumen of the endoplasmic reticulum. Bovine herpesvirus 1 (BHV-1) inhibits bovine TAP (BoTAP) activity, as a means of down-regulating MHC class I expression on the cell surface, and hence evasion of the cytotoxic T-lymphocyte response of the host. Identification of BHV-1 protein(s) responsible for TAP inhibition, and elucidation of the mechanism of TAP inhibition necessitate cloning and high-level expression of BoTAP1 and 2. In this study, we cloned and sequenced BoTAP2. Cytoplasmic RNA isolated from bovine peripheral blood mononuclear cells was used for cDNA synthesis. Rapid amplification of cDNA ends was used to amplify the 5' and the 3' ends of BoTAP2 cDNA. Based on the 5' and 3' sequences, primers were designed and the full-length BoTAP2 cDNA was PCR-amplified and sequenced. The full-length cDNA encodes a 719-amino acid polypeptide with a predicted molecular weight of M(r) 79,000. BoTAP2 has approximately 80% homology, at the amino acid level, to its mammalian counterparts. Similar to human TAP2, BoTAP2 consists of seven putative transmembrane segments followed by an ATP-binding cassette. As expected, the level of BoTAP2 mRNA expression was up-regulated by treatment with recombinant bovine interferon-gamma. In Northern blot analysis, BoTAP2 transcripts were detected in several bovine tissues with the highest level observed in jejunum. BoTAP2, when expressed as a green fluorescent fusion protein, exhibited a typical endoplasmic reticulum localization pattern.