Fish and Shellfish Immunology 93 (2019) 17–27 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article Unraveling the evolutionary origin of ELR motif using fish CXC chemokine CXCL8 T Krishnakant Gangelea, Minal Jamsandekara, Amit Mishrab, Krishna Mohan Poluria,* a Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, Uttarakhand, India b Cellular and Molecular Neurobiology Unit, Indian Institute of Technology Jodhpur, Jodhpur, 342011, Rajasthan, India ARTICLE INFO ABSTRACT Keywords: Chemokines are chemotactic proteins involved in host defense through the migration of immune-regulatory cells Chemokines to the site of infection. Interleukin-8 (CXCL8/IL8) is the most studied “ELR-CXC chemokine/neutrophil acti- Interleukin-8 vating chemokine (NAC) that regulate neutrophil trafficking during infections and inflammation by binding to CXCL8 its cognate G-protein coupled receptors CXCR1/CXCR2. The “ELR” motif of NAC chemokines is essential for the Neutrophil activating chemokines CXCR1/CXCR2 receptor activation. In order to understand the evolutionary origin of “ELR” motif in the CXC Molecular evolution chemokines, a thorough evolutionary study of CXCL8 gene from various fishes and primates was performed. Functional divergence Phylogenetic analysis revealed that the CXCL8 gene can be classified into four distinct lineages (CXCL8-L1a, CXCL8-L1b, CXCL8-L2, and CXCL8-L3), where CXCL8-L1a is the fastest evolving lineage and CXCL8-L3 is the slowest. Selection analysis suggested that The “ELR/DLR” motif containing branches (gadoid and coelacanth) are positively selected. The probable evolutionary trend of “ELR” motif suggested that this motif in ancestor CXCL8 is evolved from the GGR of Lamprey (Agnatha), followed by duplication giving rise to two main motifs in CXCL8 “NXH” in L3 lineage and “ELR/DLR” in L1a/L1b lineages. Although, structural analysis suggested that the overall topology of the CXCL8 proteins is similar, differences do exist at the individual structural elements among the members of different lineages. Functional distance analysis suggested that the CXCL8-L3 lineage is more distant compared to the CXCL8-L1a and L1b lineages from the inferred ancestor. Functional divergence analysis between different lineages suggested that most of the selected residues are important for receptor or glycosaminoglycan binding. Such a functional diversification can be attributed to the novel set of functions adopted by CXCL8 in various species. 1. Introduction single ancestor of two descendent species, and indeed such orthologous evolutionary relationships for multigene families is very crucial to Molecular evolution of proteins is a complex and continuous process elucidate the origin of the gene/gene families [8,9]. A general trend that is dependent on various constraints such as molecular structure, observed among the protein families is that the proteins involved in the functions, and stability. During the process of molecular evolution, a host defense mechanism experience a higher rate of evolution com- particular gene undergoes selective genetic variations along the axis of pared to the rest [10]. time and functional constraints to achieve better fitness [1,2]. The Chemokines are small molecular weight ubiquitous chemotactic enhanced fitness could be the consequence of either positive selection proteins involved in host defense through the migration of im- or diversifying selection of a particular change in the protein and could munoregulatory cells to the site of infection during the infection/in- have differential phenotypic characteristics [3,4]. Phenotypic varia- flammation [11,12]. They bridge the innate and adaptive immune tions in the proteins can be classified as fluctuations in function, systems to facilitate elevated immune response [13,14]. The root of structure, stability, and binding etc [5,6]. The functional and structural chemokine evolution lies in the early ancestor of vertebrates that diversity of proteins can originate from gene duplication events, as the evolved around 650 mya. They are one of the important genes that resultant gene could get neofunctionalized, pseudofunctionalized or evolved with the advent of the adaptive immune system in vertebrates might have faced deletion in one of the descendant at the time of [15]. Chemokines are broadly categorized into four groups based on the speciation [7]. Orthologous genes are those which originated from a position of the N-terminal cysteine residues such as CC, CXC, CX3C, and * Corresponding author. Department of Biotechnology, Indian Institute of Technology Roorkee (IIT-Roorkee), Roorkee, 247667, Uttarakhand, India. E-mail addresses: [email protected], [email protected] (K.M. Poluri). https://doi.org/10.1016/j.fsi.2019.07.034 Received 24 May 2019; Received in revised form 12 July 2019; Accepted 12 July 2019 Available online 13 July 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved. K. Gangele, et al. Fish and Shellfish Immunology 93 (2019) 17–27 C[16]. Among these, the CXC chemokine group has been divided into 2.2. Phylogenetic analysis two subgroups, ELR(+)CXC and ELR(−)CXC chemokines depending upon the presence of ELR motif, preceding the first cysteine residue at The output of multiple sequence alignment of CXCL8 genes from the N-terminal [17,18]. In mammals, these two subgroups play dif- Clustal Omega was used as an input data for molecular evolutionary ferent functional roles [19]. ELR(+)CXC chemokines can activate and genetic analysis (MEGA6) [38]. The phylogenetic tree of fish CXCL8 recruit neutrophils, whereas ELR(−)CXC members can only induce the was constructed using neighbor-joining method based on p-distance migration of lymphocytes but cannot activate neutrophil cells [19,20]. and substitution type as an amino acid in MEGA6. The reliability of the Humans possess seven ELR(+)CXC chemokines, among which CXCL8/ tree was assessed by the bootstrap method using 2000 bootstrap re- IL8/Interleukin-8 is the most prominent member [21]. CXCL8 binds to plications with 95% site coverage that resulted in bootstrap proportion cell surface glycosaminoglycans (GAGs) and regulates their functions for each internal branch of the tree. Further, FigTree3.0 program was by activating two G-protein coupled receptors (GPCRs) CXCR1 and used to create the colored graphical representation of the phylogenetic CXCR2 [22,23]. It has a chemotactic property towards several types of tree [39]. known immune cells [24]. It plays an important role in wound healing, angiogenesis, neovascularization, metastasis and inflammation 2.3. Conservation analysis [25–27]. Differential expression of CXCL8 is involved in several pa- thological conditions such as chronic obstructive pulmonary disease The conservation profile of CXCL8 protein was calculated to obtain (COPD), human obstructive pancreatitis, psoriasis, rheumatoid ar- the conserved nature of each residue using the lineage wise aligned thritis, cystic fibrosis, and many cancers [28,29]. protein sequences as input by the WebLogo server (https://weblogo. Indeed, chemokines play a major role in the immune defense me- berkeley.edu/logo.cgi). chanism of primitive vertebrate species such as fish as they do not have a very well developed adaptive immune system and are greatly de- 2.4. Selection analysis pendent on their innate immune system [14,15]. Although the ELR motif is a conserved feature of avian and mammalian ELR(+)CXC 2.4.1. Site selection analysis chemokines (CXCL8/IL8 protein), this motif is absent in the teleost fish The codon-based maximum likelihood (codeml) program in the except for haddock (Melanogrammus aeglefinus), and Atlantic cod PAML package was used to study the positive selection in the CXCL8 (Gadus morhua) [30]. The first non-mammalian CXCL8-like sequence gene from all existing species of fish. Recombination possibilities were was discovered in the lamprey in 1999, since then several studies have assessed using the GARD program of the Datamonkey server [40]. For reported a large number of CXC chemokines in the teleost fish, in- selection analyses, codon alignment and neighbor-joining (NJ) tree cluding some novel CXC chemokines whose structural and functional were used as the input data. Codeml selection analysis was performed homologs are absent in humans [31–33]. Phylogenetically, CXCL8 like using M0 (one ratio), M1 (nearly neutral), M2 (positive selection), M7 chemokines in fish were categorized into three groups, CXCL8-L1, (beta) and M8 (beta and ω) for the site selection whereas two ratio CXCL8-L2, and novel type lineage as CXCL8-L3 [34,35]. Earlier studies models such as alternate and null models for branch and branch site reported that some teleost CXCL8 have substitution form of ELR motif [41]. M0 model had a constant ω-ratio; whereas in M1 and M2 model, such as DLR/NLH and revealed that they are able to activate the neu- ω0 is varied between 0 and 1 and ω1 is fixed at 1. M7 and M8 models trophils [30,36,37]. Plenty of studies have been performed to under- have distributed ω values. Likelihood ratio test (LRT) of M1 with M2 stand the structure and function of CXC chemokine from human and and M7 with M8 was performed to find out the most significant model. mouse although fragmentary information is available from the primi- p-values for the test were determined by taking the log likelihood dif- tive species. In an effort to unravel the evolutionary origins of chemo- ference between the two models, multiplying by 2 and checking