Molecular evolution of vertebrate Toll-like receptors: evolutionary rate difference between their leucine-rich repeats and their TIR domains

Toll-like receptors (TLRs) that initiate an innate immune response contain an extracellular leucine rich repeat (LRR) domain and an intracellular Toll IL-receptor (TIR) domain. There are fifteen different TLRs in vertebrates. The LRR domains, which adopt a solenoid structure, usually have higher rates of evolution than do the TIR globular domains. It is important to understand the molecular evolution and functional roles of TLRs from this standpoint. Both pairwise genetic distances and Ka/Ks's (the ratios between non synonymous and synonymous substitution rates) were compared between the LRR domain and the TIR domain of 366 vertebrate TLRs from 96 species (from fish to primates). In fourteen members (TLRs 1, 2, 3, 4, 5, 6, 7, 8, 9, 11/12, 13, 14, 21, and 22/23) the LRR domains evolved significantly more rapidly than did the corresponding TIR domains. The evolutionary rates of the LRR domains are significantly different among these members; LRR domains from TLR3 and TLR7 from primates to fishes have the lowest rate of evolution. In contrast, the fifteenth member, TLR10, shows no significant differences; its TIR domain is not highly conserved. The present results suggest that TLR10 may have a different function in signaling from those other members and that a higher conservation of TLR3 and TLR7 may reflect a more ancient mechanism and/or structure in the innate immune response system. Gene conversions are suggested to have occurred in platypus TLR6 and TLR10. This study provides new insight about structural and functional diversification of vertebrate TLRs.     

Cellular recognition of tri-/di-palmitoylated peptides is independent from a domain encompassing the N-terminal seven leucine-rich repeat (LRR)/LRR-like motifs of TLR2

Toll-like receptors (TLRs) mediate microbial pattern recognition in vertebrates. A broad variety of agonists has been attributed to TLR2 and three TLRs, TLR4, TLR2, and TLR5, have been demonstrated to bind microbial products. Distinct agonists might interact with different subdomains of the TLR2 extracellular domain. The TLR2 extracellular domain sequence includes 10 canonical leucine-rich repeat (LRR) motifs and 8-10 additional and potentially functionally relevant LRR-like motifs. Thus, the transfection of TLR2 LRR/LRR-like motif deletion constructs in human embryonic kidney 293 cells and primary TLR2-deficient mouse fibroblasts was performed for analysis of the role of the regarding domains in specific pattern recognition. Preparations applied as agonists were highly purified soluble peptidoglycan, lipoteichoic acid, outer surface protein A from Borrelia burgdorferi, synthetic mycoplasmal macrophage-activating lipoprotein-2, tripalmitoyl-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4), dipalmitoyl-CSK4 (P2-CSK4), and monopalmitoyl-CSK4 (PCSK4) as well as lipopolysaccharide and inactivated bacteria. We found that a block of the N-terminal seven LRR/LRR-like motifs was not involved in TLR2-mediated cell activation by P3CSK4 and P2CSK4 ligands mimicking triacylated and diacylated bacterial polypeptides, respectively. In contrast, the integrity of the TLR2 holoprotein was compulsory for effective cellular recognition of other TLR2 agonists applied, including PCSK4. The formation of a functionally relevant subdomain by a region including the N-terminal seven LRR/LRR-like motifs rather than by single LRRs is suggested by our results. They further imply that TLR2 contains multiple binding domains for ligands that may contribute to the characterization of its promiscuous molecular pattern recognition.     

Domain combination of the vertebrate-like TLR gene family: implications for their origin and evolution

Domain shuffling, which is an important mechanism in the evolution of multi-domain proteins, has shaped the evolutionary development of the immune system in animals. Toll and Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate and adaptive immune systems. Draft genome sequences provide the opportunity to compare the Toll/TLR gene repertoire among representative metazoans. In this study, we investigated the combination of Toll/interleukin-1 receptor (TIR) and leucine-rich repeat (LRR) domains of metazoan Toll/TLRs. Before Toll with both domains occurred in Cnidaria (sea anemone, Nematostella vectensis), through domain combinations, TIR-only and LRR-only proteins had already appeared in sponges (Amphimedon queenslandica). Although vertebrate-like TIR (V-TIR) domain already appeared in Cnidaria, the vertebrate-like TLR (V-TLR) with both domains appeared much later. The first combination between V-TIR domain and vertebrate-like LRR (V-LRR) domain for V-TLR may have occurred after the divergence of Cnidaria and bilateria. Then, another combination for V-TLR, a recombination of both domains, possibly occurred before or during the evolution of primitive vertebrates. Taken together, two rounds of domain combinations may thus have co-shaped the vertebrate TLRs.     

Leucine-rich Repeat 11 of Toll-like Receptor 9 Can Tightly Bind to CpG-containing Oligodeoxynucleotides, and the Positively Charged Residues Are Critical for the High Affinity

TLR9 is a receptor for sensing bacterial DNA/CpG-containing oligonucleotides (CpG ODN). The extracellular domain (ECD) of human TLR9 (hTLR9) is composed of 25 leucine-rich repeats (LRR) contributing to the binding of CpG ODN. Herein, we showed that among LRR2, -5, -8, and -11, LRR11 of hTLR9 had the highest affinity for CpG ODN followed by LRR2 and -5, whereas LRR8 had almost no affinity. In vitro, preincubation with LRR11 more significantly decreased CpG ODN internalization, subsequent NF-κB activation, and cytokine release than with LRR2 and -5 in mouse peritoneal macrophages treated with CpG ODN. The LRR11 deletion mutant of hTLR9 conferred decreased cellular responses to CpG ODN. Single- or multiple-site mutants at five positively charged residues of LRR11 (LRR11m1-9), especially Arg-337 and Lys-367, were shown to contribute to hTLR9 binding of CpG ODN. LRR11m1-9 showed reduced inhibition of CpG ODN internalization and CpG ODN/TLR9 signaling, supporting the above findings. Prediction of whole hTLR9 ECD-CpG ODN interactions revealed that Arg-337 and Lys-338 directly contact CpG ODN through hydrogen bonding, whereas Lys-347, Arg-348, and His-353 contribute to stabilizing the shape of the ligand binding region. These findings suggested that although all five positively charged residues within LRR11 contributed to its high affinity, only Arg-337 and Lys-338 directly interacted with CpG ODN. In conclusion, the results suggested that LRR11 could strongly bind to CpG ODN, whereas mutations at the five positively charge residues reduced this high affinity. LRR11 may be further investigated as an antagonist of hTLR9.     

The polymorphism P315L of human toll-like receptor 1 impairs innate immune sensing of microbial cell wall components

As a pattern recognition receptor, TLR1 mediates innate immune responses to a variety of microbial cell wall components including bacterial lipoproteins. We have previously shown that the central region of the extracellular domain of human TLR1, comprising leucine-rich repeat (LRR) motifs 9-12, is required for the sensing of bacterial lipopeptides. In this study, we have investigated three nonsynonymous single nucleotide polymorphisms (SNPs) located in this region of TLR1 by generating these variants and examining receptor function. We have found that a variant of TLR1 based upon the SNP P315L, located in the loop of LRR motif 11 (LRR11), is greatly impaired in mediating responses to lipopeptides and a variety of other bacterial agonists for this receptor. Despite normal cell surface expression, the P315L variant also fails to bind to GD2.F4, a commonly used anti-TLR1 mAb. Although a number of amino acid substitutions at position 315 impair receptor function, the leucine substitution has the strongest deleterious effect. GD2.F4 inhibits agonist-induced activation of TLR1, supporting a crucial role for the loop of LRR11 in receptor function. These results also suggest that the P315L SNP may predispose certain individuals to infectious diseases for which the sensing of microbial cell components by TLR1 is critical to innate immune defense.     

Comprehensive survey and genomic characterization of Toll-like receptors (TLRs) in channel catfish, Ictalurus punctatus: identification of novel fish TLRs

A comprehensive survey of channel catfish Toll-like receptors (TLRs) was undertaken following a genomic PCR approach based on degenerate primers. Twenty different TLRs were identified in channel catfish. Channel catfish TLR sequences were characterized by phylogenetic analysis based on their conserved Toll/interleukin-1 receptor domain and by in-depth analysis of leucine-rich repeat (LRR) motifs of the ligand binding extracellular domain (ECD). The catfish have representatives of all the TLR types defined in vertebrates with the exception of TLR6, TLR10, TLR11, TLR12, TLR13, TLR15, TLR23, and TLR24. Additionally, two new types were discovered: TLR25 and TLR26. TLR25 is also present in cyprinids, cichlids, plecoglossids, and adrianichthyids, suggesting its presence early in fish evolution. To date, TLR26 was found only in channel catfish. Like TLR18–23, TLR25 and TLR26 were not found in any other vertebrate classes and appear to be fish specific. Data mining using the catfish TLR sequences revealed that in addition to ictalurids and cyprinids, TLR4 is also present in salmonids. TLR19 and TLR20 were both found in ictalurids, cyprinids, and salmonids, demonstrating a wider range than previously known. The LRR structure within ECDs appeared generally well conserved. TLR7 demonstrated a very high identity to human TLR7 strongly suggesting that ligand specificity maybe conserved. Finally, expression profiling confirmed that most TLRs are widely expressed in a diversity of tissues and revealed marked differences of expression level.     

Sequence variability and protein domain architectures for bovine Toll-like receptors 1, 5, and 10

The mammalian Toll-like receptors (TLRs) play an important role in the recognition of invading pathogens and the modulation of innate immune responses. The primary objective of this study was to characterize single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (indels) within bovine TLRs 1, 5, and 10, thereby facilitating future TLR signaling and association studies relevant to bovine innate immunity. Comparative sequence analysis for 10 bovine breeds derived from Bos taurus and Bos indicus revealed 98 polymorphisms (92 SNPs and 6 indels), with at least 14 nonsynonymous SNPs located within predicted TLR domains considered to be of functional significance. Of the 98 polymorphisms detected, 94 are reported here for the first time. Notably, 2 nonsynonymous SNPs were determined to modulate the prediction of a novel leucine-rich repeat (LRR) domain within B. indicusTLR5. Prediction and comparison of TLR protein domain architectures for multiple species revealed seven conserved regions of LRR patterning associated with the three genes investigated.     

Functions of toll-like receptors: lessons from KO mice

The innate immune response is a first-line defense system in which individual Toll-like receptors (TLRs) recognize distinct pathogen-associated molecular patterns (PAMPs) and exert subsequent immune responses against a variety of pathogens. TLRs are composed of an extracellular leucine-rich repeat (LRR) domain and a cytoplasmic domain that is homologous to that of the IL-IR family. Upon stimulation, TLR recruits a cytoplasmic adaptor molecule MyD88, then IL-IR-associated kinase (IRAK), and finally induces activation of NF-kappaB and MAP kinases. However, the responses to TLR ligands differ, indicating the diversity of TLR signaling pathways. Besides MyD88, several novel adaptor molecules have recently been identified. Differential utilization of these adaptor molecules may provide the specificity in the TLR signaling.     

Functional diversification of the toll-like receptor gene family

Phylogenetic analyses supported the hypothesis that the vertebrate toll-like receptors (TLRs) include two very ancient groups that arose by gene duplication prior to the divergence of protostomes and deuterostomes: (1) the TLR1 family (including mammalian TLR1, TLR2, TLR6, and TLR10); and (2) a clade including the remainder of mammalian TLRs. Correlating data on ligand type, subcellular localization, and gene expression in leukocytes and other tissues with the phylogeny provided evidence that certain major functional specializations within the TLRs occurred after ancient gene duplication events and that these traits have been retained through further events of gene duplication. For example, the recognition of bacterial lipoproteins appears to have arisen in the ancestor of the TLR1 family and continues to characterize members of that family whose ligands are known. Likewise, expression on the endosomal membrane and the recognition of nucleic acids appears to have been arisen in the ancestor of the TLR7 family and some related TLRs. On the other hand, gene expression patterns across tissues appear to have been much more volatile over the evolution of the vertebrate TLRs, since genes may show expression profiles similar to those of distantly related genes but dissimilar to those of closely related genes. Thus, the vertebrate TLRs provide an example of a multi-gene family in which gene duplication has been followed by extensive changes in certain aspects of gene function, while others have been conserved throughout vertebrate history. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Crystal structure of a plant leucine rich repeat protein with two island domains

Leucine rich repeat(LRR)domain,characterized by a repetitive sequence pattern rich in leucine residues,is a universal protein-protein interaction motif present in all life forms.LRR repeats interrupted by sequences of 30 70 residues(termed island domain,ID)have been found in some plant LRR receptor-like kinases(RLKs)and animal Toll-like receptors(TLR7-9).Recent studies provide insight into how a single ID is structurally integrated into an LRR protein.However,structural information on an LRR protein with two IDs is lacking.The receptor-like protein kinase 2(RPK2)is an LRR-RLK and has important roles in controlling plant growth and development by perception and transduction of hormone signal.Here we present the crystal structure of the extracellular LRR domain of RPK2(RPK2-LRR)containing two IDs from Arabidopsis.The structure reveals that both of the IDs are helical and located at the central region of the single RPK2-LRR solenoid.One of them binds to the inner surface of the solenoid,whereas the other one mainly interacts with the lateral side.Unexpectedly,a long loop immediately following the N-terminal capping domain of RPK2-LRR is presented toward and sandwiched between the two IDs,further stabilizing their embedding to the LRR solenoid.A potential ligand binding site formed by the two IDs and the solenoid is located at the C-terminal side of RPK2-LRR.The structural information of RPK2-LRR broadens our understanding toward the large family of LRR proteins and provides insight into RPK2-mediated signaling.     

Identification of a unique TLR2-interacting peptide motif in a microbial leucine-rich repeat protein

Pathogenesis of many bacterially-induced inflammatory diseases is driven by Toll-like receptor (TLR) mediated immune responses following recognition of bacterial factors by different TLRs. Periodontitis is a chronic inflammation of the tooth supporting apparatus often leading to tooth loss, and is caused by a Gram-negative bacterial consortium that includes Tannerella forsythia. This bacterium expresses a virulence factor, the BspA, which drives periodontal inflammation by activating TLR2. The N-terminal portion of the BspA protein comprises a leucine-rich repeat (LRR) domain previously shown to be involved in the binding and activation of TLR2. The objective of the current study was to identify specific epitopes in the LRR domain of BspA that interact with TLR2. Our results demonstrate that a sequence motif GC(S/T)GLXSIT is involved in mediating the interaction of BspA with TLR2. Thus, our study has identified a peptide motif that mediates the binding of a bacterial protein to TLR2 and highlights the promiscuous nature of TLR2 with respect to ligand binding. This work could provide a structural basis for designing peptidomimetics to modulate the activity of TLR2 in order to block bacterially-induced inflammation.     

Comparison of linkage disequilibrium and haplotype diversity on macro- and microchromosomes in chicken

Background

Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.

Results

The study presents association between TLR gene mutations and increased susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further in silico analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.

Conclusion

The most critical positions that may alter the pathogen recognition ability of TLR were: the 9^th amino acid position in LRR motif (TLR1–LRR10) and 4^th residue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.     

Preparation of the Extracellular Domain of Recombinant Human Toll-like Receptor 6

Toll-like receptors (TLRs) mediate immune responses upon recognition of a variety of ligands. To further elucidate the function of TLRs, it is important to identify novel ligands and their action mechanisms including polymer assembly. In this study, we propose an efficient method for preparation of the extracellular domain of human Toll-like receptor 6 (TLR6_ED) in Escherichia coli using the bubbling cultivation method. Our preparation method improved the level of expression of TLR6_ED into a soluble fraction as compared with typical cultivation using a rotary shaker. Circular dichroism (CD) experiments confirmed the structural formation of TLR6_ED with secondary structure contents similar to leucine-rich repeat (LRR) modules. In addition, we also provided a procedure for preparing this recombinant protein using Sf9 insect cells, which ensures preservation of some key posttranslational modifications often lacking in bacteria-expressed proteins. These materials would be useful for analyzing novel molecules that bind directly to TLR6, complex formations with other regulators including TLR2 and TLR4, and the functional effects of N-linked glycosylation.     

RPP13 is a simple locus in Arabidopsis thaliana for alleles that specify downy mildew resistance to different avirulence determinants in Peronospora parasitica

Disease resistance (R) genes are found in plants as either simple (single allelic series) loci, or more frequently as complex loci of tandemly repeated genes. These different loci are likely to be under similar evolutionary forces from pathogens, but the contrast between them suggests important differences in mechanisms associated with DNA structure and recombination that generate and maintain R gene diversity. The RPP13 locus in Arabidopsis represents an important paradigm for studying the evolution of an R gene at a simple locus. The RPP13 allele from the accession Nd-1, designated RPP13-Nd, confers resistance to five different isolates of the biotrophic oomycete, Peronospora parasitica (causal agent of downy mildew), and encodes an NBS-LRR type R protein with a putative amino-terminal leucine zipper. The RPP13-Rld allele, cloned from the accession Rld-2, encodes a different specificity. Comparison of three RPP13 alleles revealed a high rate of amino acid divergence within the LRR domain, less than 80% identity overall, compared to the remainder of the protein (> 95% identity). We also found evidence for positive selection in the LRR domain for amino acid diversification outside the core conserved beta-strand/beta-turn motif, suggesting that more of the LRR structure is available for interaction with target molecules than has previously been reported for other R gene products. Furthermore, an amino acid sequence (LLRVLDL) identical in an LRR among RPP13 alleles is conserved in other LZ NBS-LRR type R proteins, suggesting functional significance.     

LRRC19, a novel member of the leucine-rich repeat protein family, activates NF-κB and induces expression of proinflammatory cytokines

We have identified a new functional transmembrane receptor, LRRC19 (leucine-rich repeat containing 19), that belongs to the LRR protein family. LRRC19’s central core has four analogous LRR repeating modules in a juxtaposed array and a casein kinase (CK2) phosphorylation site in the cytoplasmic domain. LRRC19 mRNA was found in the kidney, spleen and intestine of adult mice using both RT-PCR and in situ hybridization. LRRC19 does not contain a cytoplasmic Toll/IL-1 receptor (TIR) domain but was able to activate NF-κB and induce production of proinflammatory cytokines. LRRC19 shares a close evolutionary relationship with multiple Toll-like receptors (TLRs), especially TLR3. Importantly, the TLR3 ligand, as well as other TLR ligands, significantly promoted the expression of proinflammatory cytokines and the activation of NF-κB by LRRC19. Thus, LRRC19 may play an important role in inducing innate immune responses in certain tissues such as the kidney.     

Characterization of gene structure and expression of two toll-like receptors from Japanese flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We cloned the cDNAs and genes of two different types of toll-like receptors from Japanese flounder. The results of homology searches suggested that these genes (designated JF-TLR2 and JF-TLR22) are homologues of human TLR2 and fugu TLR22, respectively. The cDNAs of JF-TLR2 and JF-TLR22 encoded 818 and 961 amino acid residues, respectively. JF-TLR2 and JF-TLR22 contained two distinct structural/functional motifs of the TLR family, such as a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The genes of JF-TLR2 and JF-TLR22 consisted of 12 exons (4.9 kb in total length) and four exons (4.3 kb in total length), respectively. The first exon of each gene is a non-coding exon. Southern blot hybridization indicated that both JF-TLR2 and JF-TLR22 exist as single copies in the genome. These genes were mainly expressed in peripheral blood leukocytes (PBLs) and weakly expressed in PBL-rich organs such as kidney, spleen and gill. Expression of these genes was induced by both peptidoglycan and polyI:C, although the number of JF-TLR-expressing cells were not changed after induction. All of these results suggest that they are involved in the innate immune system.