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Structure and evolutionary history of a large family of NLR proteins in the zebrafish

Howe, Kerstin, Schiffer, Philipp H., Zielinski, Julia, Wiehe, Thomas, Laird, Gavin K., Marioni, John C., Soylemez, Onuralp, Kondrashov, Fyodor, Leptin, Maria·2016·Open Biology
DOI 10.1098/rsob.160009PMC PMC4852459

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Abstract

Multicellular eukaryotes have evolved a range of mechanisms for immune recognition. A widespread family involved in innate immunity are the NACHT-domain and leucine-rich-repeat-containing (NLR) proteins. Mammals have small numbers of NLR proteins, whereas in some species, mostly those without adaptive immune systems, NLRs have expanded into very large families. We describe a family of nearly 400 NLR proteins encoded in the zebrafish genome. The proteins share a defining overall structure, which arose in fishes after a fusion of the core NLR domains with a B30.2 domain, but can be subdivided into four groups based on their NACHT domains. Gene conversion acting differentially on the NACHT and B30.2 domains has shaped the family and created the groups. Evidence of positive selection in the B30.2 domain indicates that this domain rather than the leucine-rich repeats acts as the pathogen recognition module. In an unusual chromosomal organization, the majority of the genes are located on one chromosome arm, interspersed with other large multigene families, including a new family encoding zinc-finger proteins. The NLR-B30.2 proteins represent a new family with diversity in the specific recognition module that is present in fishes in spite of the parallel existence of an adaptive immune system.

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Figure 1. Structure of the fish-specific NLR-B30.2 protein family members. (a) Overview of an alignment of the entire set of 368 predicted NLR-B30.2 proteins in the zebrafish, based on a Clustal–Omega alignment. The original alignment (electronic supplementary material) was edited by hand to improve the alignments of the N-terminal repeats and the LRRs. The colour coding is a random assignment of colours to amino acids created in Jalview. Gaps were manually introduced in the alignment at the positions of introns (marked by a grey arrowhead below the alignment), except between the C-terminal extensively duplicated LRRs in group 2b and the extensively duplicated N-terminal repeats in groups 2b and 3b. A gap was inserted between the LRRs 6 and 7 of groups 2b and 3b to allow the conserved C-terminal LRR and the B30.2 domains of groups 1, 2a and 3a to be positioned immediately after the 1–6 LRRs in these groups. Further large gaps are created, because some positions are prone to variable and often long insertions or internal duplications (marked by red stars below the alignment). Some domains are present only in subsets of the genes; these include death-fold domains in group 1 and 2a, a B30.2 domain and various N-terminal peptide repeats. Group 2 consists of two subgroups, the large and very homogeneous group 2a and the smaller group 2b, which is restricted to a cluster on chromosome 22. Group 3 has several subgroups that differ in their N-terminal peptides, their LRRs, or their B30.2 domains. *unassigned: the set of incomplete genes that lacked a NACHT domain could not be assigned to a group. (b) A schematic representation of the protein domains in each group, on the same scale as in the alignment above. Each box represents an exon. A defining motif for each group is the sequence of the Walker A motif, for which the consensus over the whole set is G[IV]AG[IV]AGK[TS]. All four groups share the Fisna domain, the NACHT domain and the LRRs. Please refer to the alignment file (electronic supplementary material) for the many details not captured in this simplified figure.
Figure 1. Structure of the fish-specific NLR-B30.2 protein family members. (a) Overview of an alignment of the entire set of 368 predicted NLR-B30.2 proteins in the zebrafish, based on a Clustal–Omega alignment. The original alignment (electronic supplementary material) was edited by hand to improve the alignments of the N-terminal repeats and the LRRs. The colour coding is a random assignment of colours to amino acids created in Jalview. Gaps were manually introduced in the alignment at the positions of introns (marked by a grey arrowhead below the alignment), except between the C-terminal extensively duplicated LRRs in group 2b and the extensively duplicated N-terminal repeats in groups 2b and 3b. A gap was inserted between the LRRs 6 and 7 of groups 2b and 3b to allow the conserved C-terminal LRR and the B30.2 domains of groups 1, 2a and 3a to be positioned immediately after the 1–6 LRRs in these groups. Further large gaps are created, because some positions are prone to variable and often long insertions or internal duplications (marked by red stars below the alignment). Some domains are present only in subsets of the genes; these include death-fold domains in group 1 and 2a, a B30.2 domain and various N-terminal peptide repeats. Group 2 consists of two subgroups, the large and very homogeneous group 2a and the smaller group 2b, which is restricted to a cluster on chromosome 22. Group 3 has several subgroups that differ in their N-terminal peptides, their LRRs, or their B30.2 domains. *unassigned: the set of incomplete genes that lacked a NACHT domain could not be assigned to a group. (b) A schematic representation of the protein domains in each group, on the same scale as in the alignment above. Each box represents an exon. A defining motif for each group is the sequence of the Walker A motif, for which the consensus over the whole set is G[IV]AG[IV]AGK[TS]. All four groups share the Fisna domain, the NACHT domain and the LRRs. Please refer to the alignment file (electronic supplementary material) for the many details not captured in this simplified figure.CC BY · PubMed Central
Figure 2. The Fisna domain and its homologues in mammalian proteins. (a) Alignment of sequences identified in mammalian genomes using an HMM search with PF14484 and examples of zebrafish Fisna domains. (b) Secondary structure predictions for selected examples.
Figure 2. The Fisna domain and its homologues in mammalian proteins. (a) Alignment of sequences identified in mammalian genomes using an HMM search with PF14484 and examples of zebrafish Fisna domains. (b) Secondary structure predictions for selected examples.CC BY · PubMed Central
Figure 3. Relationships between NLR-B30.2 genes in different fishes. Phylogenetic tree resulting from a recursive phylogenetic analysis. Expansions of groups in zebrafish are indicated in yellow with numbers of genes per group displayed. Bootstrap values are given above each branch where higher than 50% and pp values (from MrBayes) below each branch, where they are higher than 50% and the topology is congruent.
Figure 3. Relationships between NLR-B30.2 genes in different fishes. Phylogenetic tree resulting from a recursive phylogenetic analysis. Expansions of groups in zebrafish are indicated in yellow with numbers of genes per group displayed. Bootstrap values are given above each branch where higher than 50% and pp values (from MrBayes) below each branch, where they are higher than 50% and the topology is congruent.CC BY · PubMed Central
Figure 4. Evolutionary history of NLR genes. A reduced dendrogram of metazoa based on the NCBI taxonomy database displaying key events in the evolution of NLR genes as described in the main text. See electronic supplementary material for phylograms and figure S6.
Figure 4. Evolutionary history of NLR genes. A reduced dendrogram of metazoa based on the NCBI taxonomy database displaying key events in the evolution of NLR genes as described in the main text. See electronic supplementary material for phylograms and figure S6.CC BY · PubMed Central
Figure 5. Location of the NLR genes in the genome in assemblies Zv9 and GRCz10. (a) The chromosomes containing NLR-B30.2 genes are shown in the outer circle (note that corrections of the genome between Zv9 and the GRCz10 have changed the lengths of some of the chromosomes. The genes were annotated on Zv9 and lifted over to the GRCz10 path where possible as the GRCz10 gene set did not become available until May 2015. The members of the four groups of NLR genes are shown as radial lines within the circles with group 1 in the outermost and group 4 in the inner ring. Each gene is connected by a black line to its most closely related paralogue, based on the number of amino acid substitutions per site calculated in MEGA5 (Poisson correction model). Most genes are most closely related to a near neighbour, resulting in a line reaching towards the centre and returning to nearly its origin (for example, the group 2 genes on chromosome 22). The changes in the assembly have led to many genes that were closely related but resided on different chromosomes in Zv9 being located in closer proximity in GRCz10. (b) Normalized location of NLR-B30.2 genes on chromosomes. Each chromosome is shown as a horizontal line of 100% length, and the NLR genes are plotted at their relative positions along the chromosome. Apart from the genes on chromosome 4 (marked in blue), all other genes are found within the first or last quarter of the chromosome.
Figure 5. Location of the NLR genes in the genome in assemblies Zv9 and GRCz10. (a) The chromosomes containing NLR-B30.2 genes are shown in the outer circle (note that corrections of the genome between Zv9 and the GRCz10 have changed the lengths of some of the chromosomes. The genes were annotated on Zv9 and lifted over to the GRCz10 path where possible as the GRCz10 gene set did not become available until May 2015. The members of the four groups of NLR genes are shown as radial lines within the circles with group 1 in the outermost and group 4 in the inner ring. Each gene is connected by a black line to its most closely related paralogue, based on the number of amino acid substitutions per site calculated in MEGA5 (Poisson correction model). Most genes are most closely related to a near neighbour, resulting in a line reaching towards the centre and returning to nearly its origin (for example, the group 2 genes on chromosome 22). The changes in the assembly have led to many genes that were closely related but resided on different chromosomes in Zv9 being located in closer proximity in GRCz10. (b) Normalized location of NLR-B30.2 genes on chromosomes. Each chromosome is shown as a horizontal line of 100% length, and the NLR genes are plotted at their relative positions along the chromosome. Apart from the genes on chromosome 4 (marked in blue), all other genes are found within the first or last quarter of the chromosome.CC BY · PubMed Central
Figure 6. Genomic positions of genes for multi-Zn-finger proteins, fintrims and NLR-B30.2 proteins. (a) Frequency of genes encoding proteins with the indicated number of Zn-finger domains. Total number of Zn-finger encoding gene predictions: 1102. (b) The genomic locations of Zn-finger encoding genes is plotted on a circos diagram representing the Zv9 assembly for five subgroups defined by the number of Zn-finger domains. Inner circles (light to dark blue): 1–3, 4–5, 6–7, 8–9, 10–12 and more than 12 domains; outer circle (red): NLR genes. (c) Spatial distribution of NLR genes, trim genes and genes with at least 10 Zn-finger domains in the GRCz10 assembly (for gene liftover, see figure 5).
Figure 6. Genomic positions of genes for multi-Zn-finger proteins, fintrims and NLR-B30.2 proteins. (a) Frequency of genes encoding proteins with the indicated number of Zn-finger domains. Total number of Zn-finger encoding gene predictions: 1102. (b) The genomic locations of Zn-finger encoding genes is plotted on a circos diagram representing the Zv9 assembly for five subgroups defined by the number of Zn-finger domains. Inner circles (light to dark blue): 1–3, 4–5, 6–7, 8–9, 10–12 and more than 12 domains; outer circle (red): NLR genes. (c) Spatial distribution of NLR genes, trim genes and genes with at least 10 Zn-finger domains in the GRCz10 assembly (for gene liftover, see figure 5).CC BY · PubMed Central

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Metadata

Authors
Howe, K., Schiffer, P. H., Zielinski, J., Wiehe, T., Laird, G. K., Marioni, J. C., Soylemez, O., Kondrashov, F., & Leptin, M.
Affiliations
  • Wellcome Trust Sanger Institute, Cambridge, UK
  • Institut für Genetik, Universität zu Köln, Köln, Germany
  • Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG) 88 Dr. Aiguader, 08003 Barcelona, Spain
ORCID
Venue
Open Biology
Publisher
The Royal Society
Published
2016-04
Volume
6
Issue
4
Pages
160009
ISSN
2046-2441
DOI
10.1098/rsob.160009
PMC
PMC4852459
Cited by
137
References
3
Language
en
Licence
royalsociety.org/journals/ethics-policies/data-sharing-mining/ (tdm)
Funders
  • AGAUR program (2014 SGR 0974)
  • Wellcome Trust
  • National Human Genome Research Institute (HG002659)
  • MINECO (Sev-2012-0208)
  • European Research Council (335980_EinME)
  • EMBL
  • Deutsche Forschungsgemeinschaft (SFB 670 “Zellautonome Immunität”, SFB 680 "Molecular basis of evolutionary innovatio, SPP1819)
  • Howard Hughes Medical Institute (55007424)
  • Volkswagen Foundation

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