interleukin 17 , first known as cytotoxic t lymphocyte associated antigen ( ctla ) 8 , is originated from a t - cell derived factor with cytokine - like activity [ 1 , 2 ] . with a ubiquitous expression in different tissues , this protein , nowadays known as il17a , has a sequence composition different from all the other cytokine families [ 1 , 3 ] . il17a , along with five functional homodimers ( il17b - f ) , one heterodimer ( il17a / f ) , and 5 receptors ( il17ra - re ) , composes the il17 family , which is important to adaptive immunity responses , namely , as mediator of chronic inflammation and autoimmune diseases [ 36 ] . there is a wide range of genes that are targeted by il17 , such as proinflammatory and hematopoietic cytokines , genes associated with acute phase response , and antimicrobial substances [ 3 , 7 ] . this protein is also part of a subset of cd4 t helper ( th ) cells known as th17 which are able to establish a connection between innate and adaptive immune responses , being a complement to th1 and th2 defense mechanisms . furthermore , the production of il17a is important for host defense against extracellular pathogens ( fungi , viruses , bacteria , and parasites ) assisting in neutrophils recruitment and activation and also promoting antimicrobial peptides [ 812 ] . studies in mice [ 1215 ] and humans [ 1618 ] highlighted the importance of il17 expressing cells for immunity against several diseases , and low expression levels of il17 and il17ra make organisms more susceptible to disease , including those caused by extracellular pathogens such as francisella tularensis . f. tularensis is highly pathogenic gram negative intracellular bacteria included by the center of disease control and prevention ( cdc ) in category a of bioterrorism ( http://emergency.cdc.gov/agent/agentlist.asp ) . able to cause the zoonotic disease tularemia , this microorganism has several known hosts , from mammals to protozoans ; however transmission to humans is normally associated with direct contact with lagomorphs , rodents , and some arthropods [ 15 , 2022 ] . in lagomorphs and rodents , f. tularensis has the ability to cause septicemia while in humans the outcome of infection is a multisystem organ failure . there are several reports of f. tularensis infections in leporids , mainly in rabbits ( european rabbit and cottontails ) [ 2426 ] and hares [ 24 , 26 , 27 ] and despite an apparent period of stasis ( 20062010 ) there were some recently documented outbreaks of tularemia in europe [ 22 , 28 ] . the order lagomorpha includes two families , leporidae ( rabbits and hares ) with eleven genera and ochotonidae ( pikas ) with only one genus , ochotona . together with rodents , lagomorphs form the clade glires , a sister group of euarchonta that includes primates [ 30 , 31 ] . along with mouse , the european rabbit had been used as a research model for several human diseases , development of therapeutics and vaccines . several studies have suggested that the european rabbit may be a better research model than mouse [ 3337 ] . with the exception of humans and mouse , there is a big gap of information on il17a in other mammalian groups , including leporids . thus , considering the important biological role of the european rabbit immune response against several diseases , including tularemia , we performed a genetic characterization of il17a in four leporid genera ( oryctolagus , brachylagus , sylvilagus , and lepus ) . samples of european rabbit ( oryctolagus cuniculus cuniculus and oryctolagus cuniculus algirus ) , pygmy rabbit ( brachylagus idahoensis ) , brush rabbit ( sylvilagus bachmani ) , and european brown hare ( lepus europaeus ) were provided by the cibio lagomorpha tissue collection . genomic dna ( gdna ) was extracted using the easyspin genomic dna minipreps tissue kit ( citomed , torun , poland ) according to the manufacturer 's instructions . total rna was extracted by using the rneasy mini kit also according to the manufacturer 's instructions ( qiagen , hilden , germany ) from one specimen of european rabbit and one of european brown hares . complementary dna ( cdna ) was synthesized using oligo(dt ) as primers and superscript iii reverse transcriptase ( invitrogen , carlsbad , ca , usa ) . the european rabbit and american pika il17a sequences were retrieved from public databases ( accession numbers are given in bold in figure 1 ) . pcr amplification was performed with the multiplex pcr kit ( qiagen ) by using two pairs of primers designed according to the retrieved sequences ( for genomic dna f1-cgtccaacctcagttgatc + r1-cactgtaccatctatcctgc and f2-ccttcatttactcccattcg + r2-catccatcacatggcctaa ; for cdna the combination of primers f1 + r2 was used ) . sequencing was performed on an abi prism 310 genetic analyzer ( pe applied biosystems , foster city , ca , usa ) and pcr products were sequenced in both directions . haplotype phases of the sequences obtained were reconstructed with the program phase , built into the software dnasp . multiple sequence comparison by log - expectation ( muscle ; http://www.ebi.ac.uk/ )   was used for sequence alignment . the number of nucleotide differences per site between sequences was estimated in mega6   with the following options : bootstrap method ( 1000 replicates ) , p - distance as model , and pairwise deletion for gaps / missing data treatment . a maximum likelihood approach was used to estimate the phylogenetic relationships between the il17a nucleotide sequences by using mega6 ; the best - fit nucleotide substitution model was predicted by the same software and 1000 bootstrap replicates were used . the secondary structure of il17a was predicted using psipred ( http://bioinf.cs.ucl.ac.uk/psipred/ ) [ 42 , 43 ] and diaminoacid neural network application ( dianna ) ( http://clavius.bc.edu/~clotelab/dianna/ ) . both methods predict protein cysteines that create disulfide bonds , but while psipred uses position specific iterated - blast ( psi - blast ) to obtain evolutionary information used to predict the secondary structure of the query protein , dianna is a neural network that recognizes cysteines in an oxidized state ( sulfur covalently bonded ) distinguishing them from those in a reduced state . in this study we amplified and sequenced the il17a gene for four leporids species ( european rabbit , european brown hare , brush rabbit , and pygmy rabbit ) . for european rabbit ( o. c. cuniculus ) and european brown hare , both genomic and cdna sequences were identical and only one of the sequences is presented ; however both sequences have been assigned different accession numbers . these sequences were further compared to sequences of il17a from another lagomorph , american pika ( ochotona princeps ) , and from representatives of the most relevant mammalian groups ( e.g. , artiodactyla , carnivores , chiroptera , primates , rodents , etc . ) , il17a is located in the forward strand of chromosome 12 and has a similar structure to other mammals with three coding exons . the il17a cdna sequence obtained in this work for lepus europaeus showed a similar structure . in humans , il17a codes for a protein with 155 amino acids ( aa ) and has the ability to bind with high affinity to il17ra and il17rc [ 6 , 45 , 46 ] . the interaction between interleukins and their receptors is crucial for their function and signaling and any changes in the amino acid composition may induce alterations in the protein conformation . in humans and rodents these interactions sites are described [ 6 , 45 ] and include leu52 , ile54 , ser61 , ser70-tyr72 , arg75 , arg84 , arg88-val94 , trp96 , leu103 , his114 , his115 , asn117 , ser118 , gln122-glu124 , leu128 , arg130 , phe139 , and pro155-met160 ( figure 1 ) . in leporids , il17a codes for a protein with 153 aa and we observed that the sites that likely interact with the receptors are quite conserved . indeed , from the thirty - three amino acids involved in the linkage between il17a and il17ra , eighteen are conserved : twelve are maintained between mammals and the other six , despite being different , do not alter the charge or the polarity . for the remaining fifteen amino acids , only three are differently charged , seven have distinct polarity , and five have both different charge and polarity ( table 1 ) . between leporids these sites are highly conserved , but a mutation was observed that is located in the external coil of the il17a in a site where this protein interacts with il17ra ( figure 2 ) . this mutation , 88arg > pro , occurs in the european rabbit and in the european brown hare , while in brush rabbit and the american pika the amino acid present is a proline as in most mammals . some studies showed that arg > pro mutations have crucial effects in the protein function [ 4749 ] . indeed , the 332arg > pro mutation in human trim5 restricts infection by hiv-1 ( human immunodeficiency virus-1 )   while the 132arg > pro mutation in the helicase protein of coronavirus infectious bronchitis virus was lethal to infectivity in vitro . additionally , this mutation alters the physiochemical properties of the amino acid by changing from a basic polar and positively charged arginine to a nonpolar and neutral proline . disulfide bounds and n - glycosylation sites ( asn - x - ser / thr / cys motifs where x can be any amino acid except proline ) are important for the protein structure , stability , and function [ 5052 ] . disulphide bounds occur between cysteines side chains and these linkages are also important for protein protection . in human and rodents , il17a has a cysteine knot fold characterized by two sets of paired -strands ( 1/2 and 3/4 ) interconnected by two disulfide bounds between strands 2 and 4 linked between four conserved cysteines ( cys100cys150 and cys105cys152 ) [ 45 , 54 , 55 ] . in addition to these cysteines two other cysteines are common to all mammals , cys36 and cys135 . for the european rabbit , the psipred predicted secondary structure and the dianna predicted disulfide bonds results are in agreement with those obtained and described for human and rodents [ 45 , 54 ] . when compared to other mammals , there is an extra cysteine ( cys19 ) in leporids located in the signal peptide . the rat and the european hedgehog also have an extra cysteine located in different sites of the signal peptide ( cys11 and cys3 , resp . ) . given that the signal peptide is cleaved in order for the protein to become active , this extra cysteine should not have an impact on the il17a structure . n - glycosylation is a crucial factor for the modulation of protein activity ; therefore , alteration on these sites may interfere with recognition of targets , including receptors , and consequently affects the biological activity of the proteins and also their ability to diffuse through the organism [ 56 , 57 ] . detection of putative n - glycosylation sites indicated that this n - glycosylation site is present in the majority of mammals , including rodents and lagomorphs . other putative n - glycosylation sites were detected and include asn56 in lagomorphs , pig , and cattle , asn51 in american pika and armadillo , and asn49 in the lesser hedgehog tenrec . the killer whale and the african bush elephant have no putative n - glycosylation sites . the implications of the absence / presence of n - glycosylation sites in il17a are unknown ; however some studies indicate that presence / removal of glycans in some proteins do not alter their folding or function , although a decrease in the protein dynamics is observed [ 50 , 52 , 57 ] . comparison of the nucleotide sequences ( table 2 ) indicated that , in leporids , the european rabbit and the european brown hare il17a sequences are the least divergent ( 0.011 ) while the european rabbit and the pygmy rabbit il17 sequences are the most divergent ( 0.026 ) . between the european rabbit and american pika , the genetic diversity obtained was 0.1120.115 . for the remaining mammals the highest divergence occurs for the lesser hedgehog tenrec ( 0.312 ) and the lowest divergence for the flying lemur ( 0.145 ) . the comparison of the nucleotide diversity of several interleukins in the european rabbit suggested that it could represent a better animal model for research . for il17a , similar results were obtained , with the human sequence being more closely related to the european rabbit ( 0.169 ) than to mouse or rat il17a sequences ( 0.236 ) . this is further supported by a maximum likelihood tree inferred for il17a mammalian sequences ( figure 3 ) . overall , the genomic organization , the location of the cysteine residues , and the presence of n - glycosylation sites are highly conserved in leporids . nevertheless , a single mutation was detected within the interaction site with il17ra which may induce crucial changes in il17a structure , function , stability , signaling , and conformation . further functional and structural studies should be performed to fully understand the impact of this specific mutation . the lowest divergence between the european rabbit and human il17a sequences reinforces the hypothesis that the european rabbit might be a more suitable animal model for studies in the human innate immunity .
in leporids , il17a had been implicated in the host defense against extracellular pathogens , such as francisella tularensis that infects hares and rabbits and causes the zoonotic disease tularemia . here , we studied il17a from five lagomorphs , european rabbit , pygmy rabbit , brush rabbit , european brown hare , and american pika . 
 we observed that this protein is highly conserved between these species , with a similarity of 9799% in leporids and ~88% between leporids and american pika . 
 the exon / intron structure , n - glycosylation sites , and cysteine residues are conserved between lagomorphs . however , at codon 88 , one of the interaction sites between il17a and its receptor il17ra , there is an arg > pro mutation that only occurs in european rabbit and european brown hare . 
 this could induce critical alterations in the il17a structure and conformation and consequently modify its function . 
 the differences observed between leporids and humans or rodents might also represent important alterations in protein structure and function . 
 in addition , as for other interleukins , il17a sequences of human and european rabbit are more closely related than the sequences of human and mouse or european rabbit and mouse . 
 this study gives further support to the hypothesis that european rabbit might be a more suitable animal model for studies on human il17 .