Expression of tapasin in rainbow trout tissues and cell lines and up regulation in a monocyte/macrophage cell line (RTS11) by a viral mimic and viral infection Lital Sever, Nguyen T.K. Vo, Niels C. Bols, Brian Dixon ⇑ Department of Biology, University of Waterloo, 200 University Ave W., Waterloo, Ontario N2L 3G1, Canada article info Article history: Received 19 October 2013 Revised 26 November 2013 Accepted 27 November 2013 Available online 7 December 2013 Keywords: Antigen presentation Histocompatibility Virus Glycosylation abstract Tapasin is a transmembrane glycoprotein that acts as a bridge between the transporter associated with antigen processing and the MHC class I receptor in mammals. Through the development of antibody against trout tapasin, this report demonstrates the detection of trout tapasin as a N-glycosylated 48 kDa protein. Tissue and cell line distribution revealed that tapasin protein is expressed mainly in immune system organs and in rainbow trout epithelial cell lines from gill (RTgill-W1), liver (RTL-W1), and intestine (RTgutGC). An additional 20 kDa band was observed in tissues and cell lines, and appeared to be most prominent in RTgutGC but was absent in peripheral blood leukocytes. Tapasin 48 kDa protein was most strongly expressed in RTS11 (monocyte/macrophage cell line) and its regulation following dsRNA stimulation was explored. Upon poly I:C treatment and Chum Salmon Reovirus (CSV) infection, tapasin protein expression was upregulated up to 3.5 fold and 3 fold respectively, in parallel with increased expression of the glycosylated MH class I heavy chain, whereas the expression of the 20 kDa form remained unchanged. Overall this work demonstrates the induction of tapasin protein by dsRNA stimulation, which implies its possible conserved regulation during viral infection in teleost cells. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Major histocompatibility complex (MHC) class I receptors are expressed on the cell surface of all nucleated cells and present pep- tides derived from intracellular proteins to CD8 + T lymphocytes. Peptides are generated by the proteasome and delivered into the lumen of the endoplasmic reticulum (ER) by the transporter asso- ciated with antigen processing (TAP), which associates with the MHC class I receptor through a endogenous antigen presentation pathway specific chaperone: tapasin also known as TAP binding protein (TAPBP) (Wearsch and Cresswell, 2008; Pamer and Cresswell, 1998; Rizvi and Raghavan, 2010). Co-immunoprecipitation studies in human cells have identified tapasin as a 48 kDa protein associated with TAP, MHC class I and calreticulin (Sadasivan et al., 1996). Molecular cloning revealed its identity as a transmembrane glycoprotein containing immuno- globulin superfamily motifs and putative ER retention signal, encoded by a gene that is linked to the MHC (Ortmann et al., 1997). Evidence for its possible function came from a study using the human cell line .220, a lymphoblastoid cell line which does not express tapasin, which demonstrated a lack of any association between MHC class I and TAP, and displayed low levels of MHC class I on the cell surface. The normal phenotype was restored upon transfection with a gene producing recombinant tapasin (Ortmann et al., 1997). Later, studies with tapasin deficient mice showed that not only was the MHC class I surface expression impaired but CD8+ responses during viral infection were also compromised (Garbi et al., 2000). Additional insights into the functional role of tapasin during antigen presentation came from studies in mammals which focused on its role in optimizing the selection of peptides loaded into the MHC class I receptor, favour- ing peptides with longer half-life (Williams et al., 2002; Howarth et al., 2004), similar to the role of HLA-DM with MHC class II molecules (Brocke et al., 2002). However the mechanisms by which this role is carried out are still not completely understood. In mammals, the expression levels of tapasin along with other molecules involved in MHC class I assembly such as TAP, b2 micro- globulin and the proteasomal subunits were shown to be induced by the interferons (IFNs) which are cytokines involved in antiviral response, primarily by interferon gamma in APC, but by type I IFN in non-hematopoietic cells. This induction was shown to be coordi- nated by the presence of interferon factor binding elements in the promoter regions of these genes (Wright et al., 1995; Herberg et al., 1998). Tapasin expression in mammals was shown to enhance upon infection with the intracellular bacteria: Listeria monocytoge- nes and also during treatment with IFNc in primary embryonic mouse fibroblasts (Abarca-Heidemann et al., 2002). 0145-305X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dci.2013.11.019 ⇑ Corresponding author. Tel.: +1 (519) 888 4567x32665. E-mail address: bdixon@uwaterloo.ca (B. Dixon). Developmental and Comparative Immunology 44 (2014) 86–93 Contents lists available at ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci