Buckley KM and Rast JP (2012), Dynamic evolution of toll-like receptor multige...

ECB-ART-42457

Front Immunol 2012 Jan 01;3:136. doi: 10.3389/fimmu.2012.00136.

Show Gene links Show Anatomy links

Dynamic evolution of toll-like receptor multigene families in echinoderms.

Buckley KM , Rast JP .

???displayArticle.abstract???
The genome sequence of the purple sea urchin, Strongylocentrotus purpuratus, a large and long-lived invertebrate, provides a new perspective on animal immunity. Analysis of this genome uncovered a highly complex immune system in which the gene families that encode homologs of the pattern recognition receptors that form the core of vertebrate innate immunity are encoded in large multigene families. The sea urchin genome contains 253 Toll-like receptor (TLR) sequences, more than 200 Nod-like receptors and 1095 scavenger receptor cysteine-rich domains, a 10-fold expansion relative to vertebrates. Given their stereotypic protein structure and simple intron-exon architecture, the TLRs are the most tractable of these families for more detailed analysis. A role for these receptors in immune defense is suggested by their similarity to TLRs in other organisms, sequence diversity, and expression in immunologically active tissues, including phagocytes. The complexity of the sea urchin TLR multigene families is largely derived from expansions independent of those in vertebrates and protostomes, although a small family of TLRs with structure similar to that of Drosophila Toll can be traced to an ancient eumetazoan ancestor. Several other echinoderm sequences are now available, including Lytechinus variegatus, as well as partial sequences from two other sea urchin species. Here, we present an analysis of the invertebrate deuterostome TLRs with emphasis on the echinoderms. Representatives of most of the S. purpuratus TLR subfamilies and homologs of the mccTLR sequences are found in L. variegatus, although the L. variegatus TLR gene family is notably smaller (68 TLR sequences). The phylogeny of these genes within sea urchins highlights lineage-specific expansions at higher resolution than is evident at the phylum level. These analyses identify quickly evolving TLR subfamilies that are likely to have novel immune recognition functions and other, more stable, subfamilies that may function more similarly to those of vertebrates.

???displayArticle.pubmedLink??? 22679446
???displayArticle.pmcLink??? PMC3367398
???displayArticle.link??? Front Immunol

Genes referenced: LOC100887844 LOC100890978 LOC115923479 LOC115925415

???attribute.lit??? ???displayArticles.show???

	Figure 1. The TLR subfamily in sea urchins. The protein domain structure for each of the TLR subfamilies is shown. Among the sccTLR sequences, the number of LRRs varies from 21 to 25. TLRs in subfamilies Ib and Ie vary in the total number of LRRs (LRRs that are not present in all sequences are shown in light gray). The divergent TIR sequences that are present in the mccTLRs, intron-containing, and short TLRs are shown in light blue (see Figure 2). The numbers of TIR domains from each group and subfamily that are present in the S. purpuratus and L. variegatus genomes are shown below the diagram structures.
	Figure 2. Phylogeny of sea urchin TLR sequences. The TIR domains from the S. purpuratus (red branches) and L. variegatus (blue branches) TLRs were used to construct a neighbor-joining tree in MEGA 5.0 (Tamura et al., 2011) using Poisson corrected distances. Alignment positions containing gaps were eliminated completely from the analysis. Bootstrap values are shown and are based on 1000 replicates. Each of these clades is also reconstructed using other methods (maximum likelihood and maximum parsimony; data not shown). The subfamily for each clade is designated on the right. A more detailed version of this tree is shown in Figure A1 in Appendix.
	Figure 3. TLR TIR domains are more conserved than the LRR regions. The diversity of the amino acid sequences for each of the subfamilies that contain more than eight complete sequences was analyzed as a measure of sequence entropy (Durbin et al., 1998). In the graphs shown, the light blue line indicates the average diversity over a sliding window of 10 amino acids, and the black line shows the average diversity of each of the protein domains marked on the x-axis. In each of the subfamilies, the TIR domains exhibit greater conservation than the ectodomains, and there is significant sequence variation within the LRR domains.
	Figure 4. Analysis of positive selection within TLR subfamilies. (A) The majority of residues subject to positive selection are located in the putative convex face of the ectodomain. Residues under positive selection were identified in each subfamily containing more than eight complete, non-pseudogene sequences using PAML (Ia, Ib, Ic, IIa, III, and IV; Yang, 2007). Sequence alignments can be found in Files S1-S6 in Supplementary Material. Each of the LRRs is shown as a single row with individual amino acids indicated as squares. Consensus hydrophobic LRR residues are shown in gray and the conserved asparagines residues are indicated in green (Bell et al., 2003; Kang and Lee, 2011). Amino acids under positive selection are shown in red; insertions into the LRR framework are indicated by yellow squares. The predicted consensus structure is indicated below the squares: the first 10 amino acids form the β-strands on the convex surface of the ectodomains, while the rest of the LRR forms the loop structures. In subfamily IIa, no individual residues were found to be likely under positive selection. (B) Positive selection within the Ia subfamily. A generalized TLR structure is shown based on known solenoid structures of LRR-containing proteins. The majority of positively selected positions are located within the β-strands (red dots), and a few residues are also located on the front face of the TLR just beyond the β-strands (yellow dots). One positively selected residue is found on the outside of the LRRs (blue dot), and there are four residues under positive selection that are not located within the LRRs (two in the LRR-NT domain, and one on either side of the transmembrane domain; green dots).
	Figure 5. TLR subfamilies are differentially expressed. RNA-Seq was used to analyze gene expression from larvae (A), immune cells (B), and gut (C). Phagocytic coelomocytes and gut were isolated from an animal 12 h after intracoelomic injection of a gut bacteria preparation. Larvae were exposed to V. diazotrophicus for 0, 6, 12, and 24 h, and collected for transcriptome sequencing. RPKM values were calculated for each of the TIR domains; the average RPKM for each group is shown. The sccTLR subfamilies correspond to those shown in Figure 2. Note that the scales are different for each graph. The divergent TLRs are indicated as follows: M, mccTLRs; S, short; Int, intron-containing (Figure 1). In phagocytic coelomocytes, TLRs from the Ib, IIa, and X subfamilies, as well as the mccTLR genes are most highly expressed. In contrast, the III TLRs are primarily expressed in the gut tissue. The TLRs are expressed at lower levels in the larvae, and little change in TLR expression in the larvae is observed in response to bacterial challenge. The predominantly expressed families at the larval stage are Id and VI, are different than those expressed in coelomocytes.
	Figure A1. Complete phylogeny of the sea urchin TLR sequences. The TIR domains of the TLR sequences from S. purpuratus and L. variegatus were used to construct the tree shown, which is a more detailed version of the tree in Figure 2. Bootstrap values greater than 50 are shown. Red indicates clades that are specific to S. purpuratus, blue clades contain only sequences from L. variegatus, and black clades contain sequences from both species. Each of the boxes (1–4) is shown in greater detail as indicated. The sequences are labeled by scaffold number and the position of the open reading frame (scaffold_start_stop). More information about the sequences can be found in Tables S1 and S2 in Supplementary Material. Subfamily designations are indicated on the right of each tree and correspond to those shown in Figure 2.

References [+] :

Alcaide, Molecular evolution of the toll-like receptor multigene family in birds. 2011, Pubmed