A genomic view of the NOD-like receptor family in teleost fish: identification of a novel NLR subfamily in zebrafish

Abstract

Background: A large multigene family of NOD-like receptor (NLR) molecules have been described in mammals and implicated in immunity and apoptosis. Little information, however, exists concerning this gene family in non-mammalian taxa. This current study, therefore, provides an in-depth investigation of this gene family in lower vertebrates including extensive phylogenetic comparison of zebrafish NLRs with orthologs in tetrapods, and analysis of their tissue-specific expression.

Results: Three distinct NLR subfamilies were identified by mining genome databases of various non-mammalian vertebrates; the first subfamily (NLR-A) resembles mammalian NODs, the second (NLR-B) resembles mammalian NALPs, while the third (NLR-C) appears to be unique to teleost fish. In zebrafish, NLR-A and NLR-B subfamilies contain five and six genes respectively. The third subfamily is large, containing several hundred NLR-C genes, many of which are predicted to encode a C-terminal B30.2 domain. This subfamily most likely evolved from a NOD3-like molecule. Gene predictions for zebrafish NLRs were verified using sequence derived from ESTs or direct sequencing of cDNA. Reverse-transcriptase (RT)-PCR analysis confirmed expression of representative genes from each subfamily in selected tissues.

Conclusion: Our findings confirm the presence of multiple NLR gene orthologs, which form a large multigene family in teleostei. Although the functional significance of the three major NLR subfamilies is unclear, we speculate that conservation and abundance of NLR molecules in all teleostei genomes, reflects an essential role in cellular control, apoptosis or immunity throughout bony fish.

Figure 1

Phylogenetic comparison of vertebrate NLR molecules. Amino acid sequences of the NACHT domains (between the GxxGxGKS/T motif and the 'FAAFY' signature of human NOD2 or equivalent region in other NLRs) of vertebrate NLRs were aligned using CLUSTALW. Trees were constructed from these multiple alignments using the Maximum evolution and Neighbor-joining methods within the MEGA 3.1 program, using Poisson correction and complete deletion of gaps. Maximum evolution trees are shown. The resulting trees were bootstrapped 1000 times (shown as percentages). [A] Zebrafish NLRs were compared to human NLRs to estimate orthology. [B] The NALP subfamily was analyzed in more detail by comparing all zebrafish and Xenopus tropicalis predicted NALP-like molecules to human NALPs. [C] The NOD/NLR-A subfamilies of zebrafish, frog, chicken and humans were compared. DR = Danio rerio; XT = Xenopus tropicalis; GG = Gallus gallus; HS = Homo sapiens.

Figure 2

Schematic diagram depicting the deduced protein structures of zebrafish NLRs. Zebrafish NLR subfamily-A have structures similar to the NOD subfamily in mammals, with NOD1/NLR-A1 possessing one CARD motif while NOD2/NLR-A2 possesses two. While only the NACHT domain was identified for most members of the NLR-B subfamily, one member of this group (NLR-B2) was predicted with a putative N-terminal CARD effector domain. All NLR-C subfamily members were predicted to have an N-terminal effector domain, a central NACHT domain and a LRR domain. In addition, some of the NLR-C molecules were identified with a C-terminal B30.2 domain. The predicted effector domains of molecules within the NLR-C subfamily varied; some had a pyrin (P) effector domain, while others had a distinct uncharacterized effector domain (X). B30.2 domains are also found in other important immune related molecules such as certain TRIMs and the Pyrin molecule, whose structures are also shown. C = card domain, P = pyrin domain, X = other domain, N = NACHT domain, L = LRR region, B = B30.2/PRY-SPRY domain, R = ring finger domain, BB = B-box, CC = coiled coil.

Figure 3

Many N-terminal effector domains of the predicted zebrafish NLR-C molecules are recognized as pyrin/PAAD-DAPIN domains based on the HMM logo. Some examples are shown, with conserved amino acids [A]. Other N-terminal sequences were observed for NLR-C molecules, which were confirmed in the EST databases at TIGR [B]. TC326097 encodes a pyrin domain, whereas TC353741 and TC343155 represent undefined N-terminal domains such as those denoted by 'X' in Figure 2.

Figure 4

A. Schematic representation of the approximate positions of NLR encoding EST sequences relative to predicted NLR-C proteins. TIGR database accession numbers for the ESTs are given. N1 = X effector domain beginning with sequence MAEERV, N2 = recognized Pyrin effector domain, N3 = X effector domain beginning with sequence MEDTHS. B. Full cDNA sequence for EST CK126487 that spans a region from the NACHT domain to the 3'UTR of an NLR-C gene. LRR signatures are indicated with a wavy underline, and the signature for the B30.2 domain is boxed. Features for polyadenlyation (AATAAA) and mRNA instability (ATTTA) in the 3'UTR are double or single underlined respectively, and the stop codon (tga) is shown in bold italics.

Figure 5

RT-PCR analysis of the NLR gene family in intestine (1 and 2), liver (3 and 4) and spleen (5 and 6) of two individual naïve zebrafish. ARP expression was amplified to verify cDNA synthesis. Negative controls were performed using templates from cDNA synthesis reactions without reverse transcriptase (7). Genomic DNA (8) was amplified to verify primer efficiency and to show the difference in size of genomic amplicons compared to cDNA amplicons.