Huang Pao-Hsian, Chen Jyh-Yih, Kuo Ching-Ming
Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, 23-10 Dahuen Road, Jiaushi, Ilan 262, Taiwan, ROC.
Mol Immunol. 2007 Mar;44(8):1922-34. doi: 10.1016/j.molimm.2006.09.031. Epub 2006 Oct 25.
Hepcidins are antimicrobial peptides that play important roles in resisting pathogenic infection. Through hybridization of a phage library, the cDNA sequences of three hepcidin-like antimicrobial peptides (named TH1-5, TH2-2, and TH2-3) in tilapia, Oreochromis mossambicus, were determined. The complete hepcidin cDNA sequences of TH1-5, TH2-2, and TH2-3 were respectively composed of 478, 533, and 583 bases, and contained a translated region of 88, 86, and 91 amino acids. An evolutionary assay of the three deduced amino acid sequences, which share eight cysteines at identical conserved positions, showed that tilapia TH2-3 is similar to Japanese flounder (Paralichthys olivaceus) JF2, tilapia TH2-2 is similar to Japanese flounder JF1, and tilapia TH1-5 is similar to seabream (Chrysophrys major) hepcidin. The predicted molecular weights of TH1-5, TH2-2, and TH2-3 are 9.5, 9.4, and 9.8 kDa, respectively. The predicted signal peptide cleavage sites in TH1-5 is between codons 24 and 25, in TH2-2, it is between codons 22 and 23, and in TH2-3, it is between codons 24 and 25. The structural models of tilapia hepcidins, constructed using the crystal structures of bass (Morone chrysopsx M. saxatilis) hepcidin as a respective template, showed that the positional cysteine residues form disulfide bonds with tilapia hepcidin, and the cysteines likely form disulfide bonds with the bass hepcidin cysteine. The tissue-specific, lipopolysaccharide (LPS) stimulation-specific, and polyinosinic-polycytidylic acid (poly I:poly C) stimulation-specific expressions of tilapia hepcidin mRNA were determined by a comparative reverse-transcription polymerase chain reaction. Results of the tissues distribution analysis revealed high expression levels of hepcidin messenger RNA (mRNA) in the liver and head kidneys for TH1-5. TH2-3 had high mRNA expression after LPS challenge in comparison to TH2-2 and TH1-5 in fish injected with 10mug/ml LPS. TH1-5 had high mRNA expression after poly I:poly C challenge in comparison to TH2-2 and TH2-3. Immunohistochemical analysis with the polyclonal antiserum of tilapia hepcidin TH1-5 (using a rabbit polyclonal antibody) showed that the peptide was localized in the spleen and head kidneys. Synthesized TH1-5 and TH2-3 peptides showed antimicrobial activity against several bacteria in this study, while the synthesized TH 2-2 peptide did not.
铁调素是一类抗菌肽，在抵抗病原体感染中发挥重要作用。通过噬菌体文库杂交，确定了莫桑比克罗非鱼（Oreochromis mossambicus）中三种铁调素样抗菌肽（命名为TH1-5、TH2-2和TH2-3）的cDNA序列。TH1-5、TH2-2和TH2-3的完整铁调素cDNA序列分别由478、533和583个碱基组成，包含88、86和91个氨基酸的翻译区。对三个推导氨基酸序列进行的进化分析表明，它们在相同保守位置共有8个半胱氨酸，罗非鱼TH2-3与牙鲆（Paralichthys olivaceus）JF2相似，罗非鱼TH2-2与牙鲆JF1相似，罗非鱼TH1-5与黑鲷（Chrysophrys major）铁调素相似。TH1-5、TH2-2和TH2-3的预测分子量分别为9.5、9.4和9.8 kDa。TH1-5中预测的信号肽切割位点在密码子24和25之间，TH2-2中在密码子22和23之间，TH2-3中在密码子24和25之间。以鲈鱼（Morone chrysopsx M. saxatilis）铁调素的晶体结构为模板构建的罗非鱼铁调素结构模型表明，位置半胱氨酸残基与罗非鱼铁调素形成二硫键，这些半胱氨酸可能与鲈鱼铁调素半胱氨酸形成二硫键。通过比较逆转录聚合酶链反应测定了罗非鱼铁调素mRNA的组织特异性、脂多糖（LPS）刺激特异性和聚肌苷酸-聚胞苷酸（poly I:poly C）刺激特异性表达。组织分布分析结果显示，TH1-5的铁调素信使RNA（mRNA）在肝脏和头肾中表达水平较高。在注射10μg/ml LPS的鱼中，与TH2-2和TH1-5相比，LPS刺激后TH2-3的mRNA表达较高。与TH2-2和TH2-3相比，poly I:poly C刺激后TH1-5的mRNA表达较高。用罗非鱼铁调素TH1-5的多克隆抗血清（使用兔多克隆抗体）进行免疫组织化学分析表明，该肽定位于脾脏和头肾。在本研究中，合成的TH1-5和TH2-3肽对几种细菌具有抗菌活性，而合成的TH2-2肽则没有。
