School of Marine Science and Engineering, Qingdao Agricultural University, Qingdao, Shandong Province, 266109, China; Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, Shandong Province, 266109, China.
School of Marine Science and Engineering, Qingdao Agricultural University, Qingdao, Shandong Province, 266109, China.
Fish Shellfish Immunol. 2024 Apr;147:109467. doi: 10.1016/j.fsi.2024.109467. Epub 2024 Feb 28.
LEAP2 (liver expression antimicrobial peptide 2), is an antimicrobial peptide widely found in vertebrates and mainly expressed in liver. LEAP2 plays a vital role in host innate immunity. In teleosts, a number of LEAP2 homologs have been reported, but their in vivo effects on host defense are still limited. In this study, a LEAP2 homolog (SsLEAP2) was identified from black rockfish, Sebastes schlegelii, and its structure, expression as well as biological functions were analyzed. The results showed that the open reading frame of SsLEAP2 is 300 bp, with a 5'- untranslated region (UTR) of 375 bp and a 3' - UTR of 238 bp. The deduced amino acid sequence of SsLEAP2 shares the highest overall identity (96.97%) with LEAP2 of Sebastes umbrosus. SsLEAP2 possesses conserved LEAP2 features, including a signal peptide sequence, a prodomain and a mature peptide, in which four well-conserved cysteines formed two intrachain disulphide domain. The expression of SsLEAP2 was highest in liver and could be induced by experimental infection with Listonella anguillarum, Edwardsiealla piscicida and Rock bream iridovirus C1 (RBIV-C1). Recombinant SsLEAP2 (rSsLEAP2) purified from Escherichia coli was able to bind with various Gram-positive and Gram-negative bacteria. Further analysis showed that rSsLEAP2 could enhance the respiratory burst activity, and induce the expression of immune genes including interleukin 1-β (IL-1β) and serum amyloid A (SAA) in macrophages; additionally, rSsLEAP2 could also promote the proliferation and chemotactic of peripheral blood lymphocytes (PBLs). In vivo experiments indicated that overexpression of SsLEAP2 could inhibit bacterial infection, and increase the expression level of immune genes including IL-1β, tumor necrosis factor ligand superfamily member 13B (TNF13B) and haptoglobin (HP); conversely, knock down of SsLEAP2 promoted bacterial infection and decreased the expression level of above genes. Taken together, these results suggest that SsLEAP2 is a novel LEAP2 homolog that possesses apparent antibacterial activity and immunoregulatory property, thus plays a critical role in host defense against pathogens invasion.
LEAP2（肝表达抗菌肽 2）是一种广泛存在于脊椎动物中的抗菌肽，主要在肝脏中表达。LEAP2 在宿主先天免疫中起着至关重要的作用。在硬骨鱼中，已经报道了许多 LEAP2 同源物，但它们对宿主防御的体内作用仍然有限。在本研究中，从青石斑鱼（Sebastes schlegelii）中鉴定出一种 LEAP2 同源物（SsLEAP2），并分析了其结构、表达和生物学功能。结果表明，SsLEAP2 的开放阅读框为 300bp，5'-非翻译区（UTR）为 375bp，3'-UTR 为 238bp。SsLEAP2 的推导氨基酸序列与 Sebastes umbrosus 的 LEAP2 具有最高的整体同一性（96.97%）。SsLEAP2 具有保守的 LEAP2 特征，包括信号肽序列、前导肽和成熟肽，其中四个保守的半胱氨酸形成两个链内二硫键结构域。SsLEAP2 在肝脏中的表达最高，可被鳗弧菌、迟缓爱德华氏菌和虹彩病毒 C1（RBIV-C1）实验感染诱导。从大肠杆菌中纯化的重组 SsLEAP2（rSsLEAP2）能够与各种革兰氏阳性和革兰氏阴性细菌结合。进一步分析表明，rSsLEAP2 可以增强巨噬细胞的呼吸爆发活性，并诱导白细胞介素 1-β（IL-1β）和血清淀粉样蛋白 A（SAA）等免疫基因的表达；此外，rSsLEAP2 还可以促进外周血淋巴细胞（PBLs）的增殖和趋化性。体内实验表明，过表达 SsLEAP2 可以抑制细菌感染，并增加包括白细胞介素 1-β、肿瘤坏死因子配体超家族成员 13B（TNF13B）和触珠蛋白（HP）在内的免疫基因的表达水平；相反，敲低 SsLEAP2 则促进细菌感染，并降低上述基因的表达水平。总之，这些结果表明 SsLEAP2 是一种新型的 LEAP2 同源物，具有明显的抗菌活性和免疫调节特性，因此在宿主抵抗病原体入侵的防御中起着关键作用。
