Molecular cloning of proteasome activator PA28-beta subunit of large yellow croaker (Pseudosciana crocea) and its coordinated up-regulation with MHC class I alpha-chain and beta 2-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-alpha and PA28-beta, which are inducible by interferon-gamma (IFN-gamma), 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-beta gene homologue from the spleen of large yellow croaker (Pseudosciana crocea), an economically important marine fish (LycPA28-beta). The full-length cDNA of LycPA28-beta 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-beta contains a PA28-beta subunit-specific insert in the region corresponding to the KEKE motif of the known PA28-alpha (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-beta 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-beta 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-beta and MHC class I alpha-chain (alpha-chain) and beta(2)-microglobulin (beta(2)m) in spleens of poly I:C-induced large yellow croaker was further performed by RT-PCR. The results showed that the expression of LycPA28-beta and class I alpha-chain and beta(2)m 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.
Liu
Guozhu
G
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, 178 Daxue Road, Xiamen 361005, PR China.
Zheng
Wenbiao
W
Chen
Xinhua
X
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D029941:Fish Proteins / Q000235:genetics* / Q000302:isolation & purification; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D010473:Perciformes / Q000235:genetics* / Q000276:immunology; D011070:Poly I-C / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics* / Q000302:isolation & purification; D015854:Up-Regulation / Q000235:genetics / Q000276:immunology; D001613:beta 2-Microglobulin / Q000096:biosynthesis* / Q000235:genetics
D029941:Fish Proteins; D015395:Histocompatibility Antigens Class I; D021122:Protein Subunits; D001613:beta 2-Microglobulin; D046988:Proteasome Endopeptidase Complex; D011070:Poly I-C