Molecular cloning of proteasome activator PA28-β subunit of large yellow croaker (Pseudosciana crocea) and its coordinated up-regulation with MHC class I α-chain and β2-microglobulin in poly I:C-treated fish

Antigenic peptides presented on MHC class I molecules to cytotoxic T-cells are generated in the cytosol by the 20S proteasome. Two activators PA28-α and PA28-β, which are inducible by interferon-γ (IFN-γ), activate the latent 20S proteasome, thus playing an important role in the processing of MHC class I antigen. Molecular properties and function in the MHC class I antigen processing of PA28 have been well studied and documented in mammals while little is known in fish. In the present study, we reported the cloning of a PA28-β gene homologue from the spleen of large yellow croaker (Pseudosciana crocea), an economically important marine fish (LycPA28-β). The full-length cDNA of LycPA28-β is 1133 nucleotides (nt) encoding a protein of 245 amino acids (aa), with a putative molecular weight of 27.7 kDa. The deduced protein shares 76, 69, 61, 60, 59, 57 and 57% sequence identity to sequences found in zebrafish, flounder, pig, rat, mouse, cattle and human, respectively. The deduced LycPA28-β contains a PA28-β subunit-specific insert in the region corresponding to the KEKE motif of the known PA28-α (Region B), a conserved activation loop (Region C) and a highly homologous C-terminal region among all three PA28 subunits (Region E), and a characteristic proline-rich motif (Region A) and a potential protein kinase C recognition site (Region D). Western blot analysis of various tissues indicated that LycPA28-β was constitutively expressed in kidney, liver, spleen and intestine, and weakly expressed in muscle tissue, but not detected in gills, heart and brain. The LycPA28-β expression was significantly up-regulated in kidney, liver, spleen, intestine and muscle tissues, and also induced in gills after 72 h of treatment with a viral micmic, polyinosinic polycytidynic acid (poly I:C). The transcriptional analysis of LycPA28-β and MHC class I α-chain (α-chain) and β2-microglobulin (β2m) in spleens of poly I:C-induced large yellow croaker was further performed by RT-PCR. The results showed that the expression of LycPA28-β and class I α-chain and β2m genes was coordinately up-regulated by poly I:C, suggesting that induction of the MHC class I antigen processing and presentation pathway may be required for the antiviral immune response triggered poly I:C in large yellow croaker.