Protein evolution of Toll-like receptors 4, 5 and 7 within Galloanserae birds

Background Toll-like receptors (TLR) are essential activators of the innate part of the vertebrate immune system. In this study, we analysed the interspecific variability of three TLR (bacterial-sensing TLR4 and TLR5 and viral-sensing TLR7) within the Galloanserae bird clade, investigated their phylogeny, assessed their structural conservation and estimated site-specific selection pressures. Results Physiochemical properties varied according to the TLR analysed, mainly with regards to the surface electrostatic potential distribution. The predicted ligand-binding features (mainly in TLR4 and TLR5) differed between the avian proteins and their fish and mammalian counterparts, but also varied within the Galloanserae birds. We identified 20 positively selected sites in the three TLR, among which several are topologically close to ligand-binding sites reported for mammalian and fish TLR. We described 26, 28 and 25 evolutionarily non-conservative sites in TLR4, TLR5 and TLR7, respectively. Thirteen of these sites in TLR4, and ten in TLR5 were located in functionally relevant regions. The variability appears to be functionally more conserved for viral-sensing TLR7 than for the bacterial-sensing TLR. Amino-acid positions 268, 270, 343, 383, 444 and 471 in TLR4 and 180, 183, 209, 216, 264, 342 and 379 in TLR5 are key candidates for further functional research. Conclusions Host-pathogen co-evolution has a major effect on the features of host immune receptors. Our results suggest that avian and mammalian TLR may be differentially adapted to pathogen-derived ligand recognition. We have detected signatures of positive selection even within the Galloanserae lineage. To our knowledge, this is the first study to depict evolutionary pressures on Galloanserae TLR and to estimate the validity of current knowledge on TLR function (based on mammalian and chicken models) for non-model species of this clade. Electronic supplementary material The online version of this article (doi:10.1186/s12711-014-0072-6) contains supplementary material, which is available to authorized users.

length ranging from 16 aa to 21 aa [See Additional file 1, Table S3]. Also the number of predicted LRRs remarkably varied among orthologous TLRs [See Additional file 1, Table   S3]. Both N-terminal and C-terminal caps of the most external LRRs (forming LRRNT and LRRCT, respectively) were present only in TLR7, while in TLR4 and TLR5 only the Cterminal caps were predicted, uniformly in all investigated species.

Section S2 Evolution of TLR secondary structures within the Galloanserae lineage
The secondary structure variability detected in the present study in the Galloanserae TLRs was low. The ECDs did not differ from the intracellular regions in the level of variability of the predicted secondary structures. Nevertheless, regions between residues 225-311, 412-438 and 628-636 in TLR4 and 407-431 and 623-626 in TLR5 were variable among individual species. TLR7 was the most conservative of the TLRs investigated. Although the predicted variability seems only minor, in TLR4 the first two regions comprise sites known in mammals to be involved in MD-2 binding, TLR4-TLR4 dimerization, and even LPS binding (Ohto et al. 2012). These results, thus, suggest that some of the protein conformation variability within the Galloanserae clade could be associated with potential differences in the mode of ligand binding.

Section S3 Variability in features of predicted ligand-binding residues
In TLR4 the amino acid binding features at the functional sites known in mammals remained probably unchanged in 19 out of 29 sites (66%). Similarly, in TLR5 32 out of 56 (57%) functional sites remained physiochemically similar. The difference between zebrafish functional sites identified by Yoon et al. (2012) and their avian counterparts is dramatic (physiochemical similarity only in 23 out of 45) and further increased by missing amino acids. This indicates that flagellin binding is probably not completely identical in fish and amniotes. Nonetheless, if we do not include sites that were recognised only in zebrafish and 2 focus only on those predicted by Andersen-Nissen et al. (2007) for mammals, then 10 out of the 11 (91%) functional sites are conservative. Very similar to this is the situation in TLR7 where 7 out of 9 functional sites show physiochemical conservatism. Little (TLR5) or no (TLR7) interspecific polymorphism in the mammal-predicted ligand-binding residues within the Galloanserae lineage, however, suggests that the ligand-binding properties in both these proteins may be reasonably conserved between birds and mammals. Intriguingly, in TLR4 the residues 449 (= HoSaTLR4-F440) and 472 (= HoSaTLR4-F463) that have been previously reported as key for LPS binding (Resman et al. 2009) are altered in birds when compared to mammals. At all these sites nonpolar F residues are required in humans (Resman et al. 2009).
In contrast, in Galloanserae birds there is uniformly aromatic hydrophilic Y at site 449 and tiny hydrophilic S at site 472. This means that either the model proposed by Resman et al. (2009) is incorrect or, more likely, that there is a substantial difference between birds and mammals in TLR4-MD-2-LPS binding. Moreover, there are several sites in TLR4 (268,397,428,445 and 454) at which Galliformes systematically differ from Anserifomes. Most of these residues (268, 397 and 445) were found in humans to be involved in interaction with LPS (Park et al. 2009). This suggests differences in LPS binding between Galliformes and Anseriformes. Again, we found only little interspecific variability in the predicted key binding residues within the two individual avian orders. Only limited polymorphism has been revealed on two well-known human functional SNP sites (Arbour et al. 2000;Rallabhandi et al. 2006). At the site of avian 303 which is identical to human D299G SNP position aspartic acid (D) was invariantly present (except for AnAnTLR4-N303). Residues on position 408 (equivalent to HoSaTLR4-T399I SNP) were variable, with proline being present in Galliform birds and alanine being present in Anseriform birds.  Table   S5]. Residues 343 in TLR4 and 180 and 379 in TLR5 seem to be potentially more evolutionarily relevant since the substitutions on these sites significantly alter amino acid properties and may, hence, affect ligand binding.

3
The ConSurf analysis [See Additional file 1, Table S9]  interface. In TLR7 sites 39, 99, 383 and 665 were detected in Galloanserae birds and mammals, no sites were consistently identified in Galloanserae and other birds and sites 3, 565 and 700 that were identified in the present study were located in close proximity to some 4 other positively selected sites identified in other taxa. Out of these, sites none was located in the predicted ligand-binding region.
Based on the evidence summarised above we may propose several sites that should be investigated in closer detail. Mainly the sites 270, 343,444 and 471 in TLR4 and 180,183,209,216,264,342 and 379 in TLR5 are clear key candidates for further research on functional significance of selection acting on TLRs in birds. Since no selection on this site has been evidenced in mammals or other birds, we may hypothesise that the site 268 in TLR4 may be of some special importance in the evolution of Galloanserae birds. Although we do not know the functional importance of the sites, based solely on congruence of the selection analyses in birds and mammals special attention should be also paid to positions [244][245][246][273][274][275]314,342,422,525,[532][533]