Liu Guozhu, Zheng Wenbiao, Chen Xinhua
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, 178 Daxue Road, Xiamen 361005, PR China.
Mol Immunol. 2007 Feb;44(6):1190-7. doi: 10.1016/j.molimm.2006.06.024. Epub 2006 Aug 9.
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.
在MHC I类分子上呈递给细胞毒性T细胞的抗原肽是由20S蛋白酶体在细胞质中产生的。两种激活剂PA28-α和PA28-β可被干扰素-γ（IFN-γ）诱导，激活潜伏的20S蛋白酶体，从而在MHC I类抗原的加工过程中发挥重要作用。PA28在MHC I类抗原加工中的分子特性和功能在哺乳动物中已得到充分研究和记录，而在鱼类中却知之甚少。在本研究中，我们报道了从大黄鱼（Pseudosciana crocea）脾脏中克隆出PA28-β基因的同源物（LycPA28-β），大黄鱼是一种具有重要经济价值的海洋鱼类。LycPA28-β的全长cDNA为1133个核苷酸（nt），编码一个由245个氨基酸（aa）组成的蛋白质，推测分子量为27.7 kDa。推导的蛋白质与斑马鱼、比目鱼、猪、大鼠、小鼠、牛和人的序列分别具有76%、69%、61%、60%、59%、57%和57%的序列同一性。推导的LycPA28-β在对应于已知PA28-α的KEKE基序的区域（区域B）、保守的激活环（区域C）以及所有三个PA28亚基中高度同源的C末端区域（区域E）含有一个PA28-β亚基特异性插入片段，还有一个特征性的富含脯氨酸基序（区域A）和一个潜在的蛋白激酶C识别位点（区域D）。对各种组织的蛋白质免疫印迹分析表明，LycPA28-β在肾脏、肝脏、脾脏和肠道中组成性表达，在肌肉组织中弱表达，但在鳃、心脏和大脑中未检测到。在用病毒模拟物聚肌苷酸胞嘧啶核苷酸（poly I:C）处理72小时后，LycPA28-β在肾脏、肝脏、脾脏、肠道和肌肉组织中的表达显著上调，在鳃中也被诱导表达。通过RT-PCR进一步对poly I:C诱导的大黄鱼脾脏中的LycPA28-β、MHC I类α链（α链）和β2-微球蛋白（β2m）进行转录分析。结果表明，poly I:C协同上调了LycPA28-β、I类α链和β2m基因的表达，这表明在大黄鱼中，poly I:C触发的抗病毒免疫反应可能需要诱导MHC I类抗原加工和呈递途径。
