Distinctive Structural Hallmarks and Biological Activities of the Multiple Cathelicidin Antimicrobial Peptides in a Primitive Teleost Fish This information is current as of October 1, 2021. Xu-Jie Zhang, Xiang-Yang Zhang, Nu Zhang, Xia Guo, Kai-Song Peng, Han Wu, Long-Feng Lu, Nan Wu, Dan-Dan Chen, Shun Li, Pin Nie and Yong-An Zhang J Immunol published online 15 April 2015 http://www.jimmunol.org/content/early/2015/04/15/jimmun Downloaded from ol.1500182 Supplementary http://www.jimmunol.org/content/suppl/2015/04/15/jimmunol.150018 Material 2.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published April 15, 2015, doi:10.4049/jimmunol.1500182 The Journal of Immunology Distinctive Structural Hallmarks and Biological Activities of the Multiple Cathelicidin Antimicrobial Peptides in a Primitive Teleost Fish Xu-Jie Zhang,*,† Xiang-Yang Zhang,*,† Nu Zhang,*,† Xia Guo,*,† Kai-Song Peng,*,‡ Han Wu,* Long-Feng Lu,*,† Nan Wu,* Dan-Dan Chen,*,† Shun Li,* Pin Nie,* and Yong-An Zhang* Cathelicidin antimicrobial peptides (CAMPs) represent a crucial component of the innate immune system in vertebrates. Although widely studied in mammals, little is known about the structure and function of fish CAMPs. Further to the previous findings, two more cathelicidin genes and multiple transcripts from rainbow trout were identified in the present study. Interestingly, we found that trout have evolved energy-saving forms of cathelicidins with the total deletion of the characteristic cathelin-like domain. Sequence Downloaded from analysis revealed that salmonid CAMPs have formed a special class of antimicrobial peptides in vertebrates with three distinctive hallmarks: the N terminus is intensified by positive charges, the central region consists of repetitive motifs based on RPGGGS, and the C terminus is lowly charged. Immunofluorescence localization of trout CAMPs demonstrated that these peptides expressed mainly at the mucosal layer of gut. Meanwhile, signals around sinusoids were also detected in head kidney. Moreover, the biological activities of trout CAMPs were proved to be mediated by the N terminus. Additionally, the repetitive motifs characteristically existing in Salmonidae increased the structural flexibilities of peptides and further increased the antibacterial and IL-8–stimu- http://www.jimmunol.org/ lating activities. Unlike most a helical and cytotoxic mammalian CAMPs, trout CAMPs, mainly consisting of b-sheet and random coil, exhibited no cytotoxic activities. The distinctive structural features of trout CAMPs provide new insights into the under- standing of the evolution of CAMPs in vertebrates. Moreover, the high bacterial membrane selectivity of trout CAMPs will help to design excellent peptide antibiotics. The Journal of Immunology, 2015, 194: 000–000. ntimicrobial peptides represent an evolutionarily old Cathelicidins are likely present in all vertebrates (2, 6, 7). The component of the innate immune system in all organisms discovery of three cathelicidin-like members in the ancient ver- (1, 2). They form the first line of host defense against tebrate hagfish (Myxine glutinosa) implies that cathelicidins al- A by guest on October 1, 2021 infectious microorganisms prior to stimulating the adaptive im- ready existed ∼450 million years ago (8). Until now, only one mune system of animals (1–3). The increasing resistance of bac- cathelicidin antimicrobial peptide (CAMP) has been found in teria to conventional antibiotics stimulates the exploitation of primates (2). Nevertheless, at least six CAMPs have been dis- antimicrobial peptides as candidates for new antibiotics (3, 4). coveredinothermammalsbelongingtoEuungulata,suchas Among them, cathelicidins and defensins represent the major and cattle, goat, sheep, and pig, whereas four have been discovered in well-studied antimicrobial peptide families in animals (2, 5). birds, three in reptiles, and two in amphibians (2, 9, 10). As in fish, three CAMPs have been identified in Atlantic cod (Gadus morhua), whereas two or one has been discovered in several other *Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; species (5, 7, 11). In vivo studies conducted in mice have sug- †University of Chinese Academy of Sciences, Beijing 100049, China; and gested that the duplication and divergence of cathelicidins are ‡ College of Animal Science and Technology, Anhui Agricultural University, Hefei beneficial to host defense against infections (12, 13). 230036, China In mammals, CAMPs are produced by epithelial cells and Received for publication January 23, 2015. Accepted for publication March 17, 2015. lymphocytes (14, 15) and are mainly expressed at bone marrow This work was supported by National Basic Research Program of China Grant 2014CB138601 and by National Natural Science Foundation of China Grant and the epithelial surfaces of skin, gastrointestinal tract, 31172431. and lung (15–17). CAMPs are first produced as inactive pre- The sequences presented in this article have been submitted to GenBank (http:// cursors, which consist of a signal peptide followed by a cathelin- www.ncbi.nlm.nih.gov/genbank/) under accession numbers KP347620–KP347627. like domain (CLD), as well as a C-terminal mature peptide The structures of trout CAMPs have been submitted to the Protein Model DataBase (http://bioinformatics.cineca.it/PMDB/) under identification numbers PM0079827 containing 12–80 residues (2, 6, 18). The CLD shows high and PM0079829–PM0079833. interspecies sequence homology, whereas the mature peptide Address correspondence and reprint requests to Prof. Yong-An Zhang, Institute shows high diversity both inter- and intraspecies (2, 6, 19). The of Hydrobiology, Chinese Academy of Sciences, 7 Donghu South Road, Wuhan active mature peptide is released via proteolytic processing with 430072, China. E-mail address: [email protected] specific proteases (2, 18). The released CAMPs are small, cat- The online version of this article contains supplemental material. ionic, and amphipathic peptides (2, 5, 6). Although varying in Abbreviations used in this article: CAMP, cathelicidin antimicrobial peptide; CD, circular dichroism; CLD, cathelin-like domain; EF-1a, elongation factor 1a; MHB, amino acid sequence and size, most mammalian CAMPs are Mueller–Hinton broth; pAb, polyclonal Ab; PBL, peripheral blood leukocyte; PI, linear peptides consisting of 23–37 residues and adopting am- propidium iodide; qPCR, quantitative real-time PCR; RTG, rainbow trout gonadal; phipathic a helical structures in the environments mimicking SEM, scanning electronic microscopy; TFE, 2,2,2-trifluoroethanol. biological membranes (6). These CAMPs exhibit a broad spec- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 trum of antimicrobial activities against bacteria, viruses, and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500182 2 STRUCTURES AND ACTIVITIES OF TROUT CAMPs fungi (2, 6, 18). In addition to their direct antimicrobial effects, ExPASy proteomics server (http://www.expasy.org/). Multiple sequence mammalian CAMPs possess other modulatory activities, such as alignment was generated using ClustalW2 at the European Molecular LPS neutralizing (20, 21) and cytokine stimulating (22, 23). Biology Laboratory–European Bioinformatics Institute Web site (http:// www.ebi.ac.uk/). Although CAMPs have been widely studied in mammals, re- search on fish CAMPs has been predominantly limited to gene Expression patterns of trout cathelicidin genes identification and antibacterial activity test. No information is Three healthy trout (50–70 g) were euthanized and total RNA was isolated available about the structure/activity relationships of fish CAMPs. from the blood, head kidney, spleen, thymus, gut, skin, gill, liver, heart, However, the special living environment and evolutionary status muscle, and brain. Meanwhile, two groups of healthy trout were injected make fish an interesting host to study the functions of CAMPs. i.p. with LPS (from Escherichia coli 0111:B4; Sigma-Aldrich; 1 mg/ml in PBS) or PBS (Invitrogen), 100 ml per fish, and total RNA was isolated from Thus, the aim of the present study was two-fold: first, to unearth the head kidney and gut of three fish at 0 h, 12 h, 24 h, 3 d, and 7 d more cathelicidin genes and transcripts in rainbow trout (Onco- postinjection. The first-strand cDNA was synthesized as described above. rhynchus mykiss), a commercially farmed fish throughout the The transcripts of rtCATH-2a and rtCATH-2b were amplified with the world; and second, to further investigate the structure and multi- primer set rtCATH-2rtF/rtCATH-2rtR (Table I) by 38 cycles and then function of these CAMPs. We found that trout possess at