Blunt Snout Bream (Megalobrama amblycephala) MyD88 and TRAF6: characterisation, comparative homology modelling and expression

Abstract

MyD88 and TRAF6 play an essential role in the innate immune response in most animals. This study reports the full-length MaMyD88 and MaTRAF6 genes identified from the blunt snout bream (Megalobrama amblycephala) transcriptome profile. MaMyD88 is 2501 base pairs (bp) long, encoding a putative protein of 284 amino acids (aa), including the N-terminal DEATH domain of 78 aa and the C-terminal TIR domain of 138 aa. MaTRAF6 is 5474 bp long, encoding a putative protein of 542 aa, including the N-terminal low-complexity region, RING domain (40 aa), a coiled-coil region (64 aa) and C-terminal MATH domain (147 aa). Coding regions of MaMyD88 and MaTRAF6 genomic sequences consisted of five and six exons, respectively. Physicochemical and functional characteristics of the proteins were analysed. Alpha helices were dominant in the secondary structure of the proteins. Homology models of the MaMyD88 and MaTRAF6 domains were constructed applying the comparative modelling method. RT-qPCR was used to analyse the expression of MaMyD88 and MaTRAF6 mRNA transcripts in response to Aeromonas hydrophila challenge. Both genes were highly upregulated in the liver, spleen and kidney during the first 24 h after the challenge. While MyD88 and TRAF6 have been reported in various aquatic species, this is the first report and characterisation of these genes in blunt snout bream. This research also provides evidence of the important roles of these two genes in the blunt snout bream innate immune system.

Figure 1

Sequences and domain topology of MaMyD88 identified from the blunt snout bream transcriptome profile. (A) The nucleotide (upper row) and deduced amino acid (lower row) sequence are shown and numbered on the left. Start (ATG) and stop (TGA) codons are bolded and boxed. mRNA instability motifs (ATTTA) and a consensus polyadenylation signal sequence (AATAAA) are bolded and underlined. DEATH (Val11–Ile101) and TIR (Thr184–Pro284) domain are shaded in light- and dark-grey, respectively. The three highly conserved boxes within the TIR domain are double-underlined. Asterisk mark (*) indicates the stop codon; (B) The architecture of the domain topology of MaMyD88 protein, rendered by Simple Molecular Architecture Research Tool (SMART), showing cytoplasmic Toll/IL-1R homology (TIR) domain and a protein interaction module named DEATH domain.

Figure 2

Multiple alignment of the deduced MaMyD88 amino acid sequence with homologs from seven fish species: Cyprinus carpio (CcMyD88a, Accession No. ADC45019.2), Carassius carassius (CcaMyD88, Acc. No. AGO57937.1), Danio rerio (DrMyD88, Acc. No. NP_997979.2), Ictalurus punctatus (IpMyD88, Acc. No. NP_001187207.1), Plecoglossus altivelis altivelis (PaaMyD88, Acc. No. BAI68385.1), Oncorhynchus mykiss (OmMyD88, Acc. No. CDG03206.1) and Salmo salar (SsMyD88, Acc. No. NP_001130017.1). Asterisk marks (*) indicate identical amino acids. Sequences are numbered on the right, while the conserved substitutions are indicated by (:), semi-conserved substitutions by (.) and deletions by dashes. Arrows indicate DEATH and TIR domains and brackets indicate the three highly conserved boxes within the TIR domain.

Figure 3

Sequence and domain topology of MaTRAF6 identified from the blunt snout bream transcriptome profile. (A) The nucleotide (upper row) and deduced amino acid (lower row) sequence are numbered on the left. The potential N-linked glycosylation sites, start codon (ATG) and stop codon (TGA) are bolded and boxed. mRNA instability motifs (ATTTA) and the consensus polyadenylation signal sequence (AAAAAA) are bolded and underlined. The low complexity and coiled coil regions are single- and double-underlined, respectively. RING (Cys71–Asp109) and MATH (Trp379–Leu503) domains are shaded in light- and dark-grey, respectively. Asterisk mark (*) indicates the stop codon; (B) The architecture of the domain topology of MaTRAF6 protein, rendered by Simple Molecular Architecture Research Tool (SMART), showing the low-complexity region (pink), zinc finger type protein structural domain (RING), coiled-coil (green) and the meprin and TRAF homology (MATH) domain.

Figure 3

Sequence and domain topology of MaTRAF6 identified from the blunt snout bream transcriptome profile. (A) The nucleotide (upper row) and deduced amino acid (lower row) sequence are numbered on the left. The potential N-linked glycosylation sites, start codon (ATG) and stop codon (TGA) are bolded and boxed. mRNA instability motifs (ATTTA) and the consensus polyadenylation signal sequence (AAAAAA) are bolded and underlined. The low complexity and coiled coil regions are single- and double-underlined, respectively. RING (Cys71–Asp109) and MATH (Trp379–Leu503) domains are shaded in light- and dark-grey, respectively. Asterisk mark (*) indicates the stop codon; (B) The architecture of the domain topology of MaTRAF6 protein, rendered by Simple Molecular Architecture Research Tool (SMART), showing the low-complexity region (pink), zinc finger type protein structural domain (RING), coiled-coil (green) and the meprin and TRAF homology (MATH) domain.

Figure 4

Multiple amino acid sequence alignment of MaTRAF6 with TRAF6 homologs of five fish species: Ctenopharyngodon idella (CiTRAF6, Acc. no. AGI51678.1), Cyprinus carpio (CcTRAF6a, Acc. No. ADF56651.2 and ADM45856.1), Danio rerio (DrTRAF6, Acc. no. NP_001038217.1) and Carassius auratus auratus (CaaTRAF6, Acc. No. AHG97567.1). Asterisk marks (*) indicate identical amino acids. Sequences are numbered on the right, conserved substitutions are indicated by (:), semi-conserved by (.) and deletions by dashes. Low complexity, zinc finger type protein structural domain (RING), coiled-coil and meprin and TRAF homology (MATH) domains are indicated by arrows.

Figure 5

Neighbour-joining phylogenetic tree showing the relationships between the MaMyD88 protein and homologs in selected animals (accession numbers in the parentheses). The numbers at the branches indicate bootstrap values (1000 replications). The bar (0.05) indicates the genetic distance. MaMyD88 is marked by a black dot.

Figure 6

Neighbour-joining phylogenetic tree showing the relationships between the MaTRAF6 and homologs in selected animals (accession numbers in the parentheses). The numbers at the branches indicate bootstrap values (1000 replications). The bar (0.05) indicates the genetic distance. MaTRAF6 is marked by a black dot.

Figure 7

Three-dimensional structures of MaMyD88 (A) DEATH and (B) TIR domains, and of MaTRAF6 (C) RING and (D) MATH domains, predicted and rendered by SWISS-MODEL web server.

Figure 8

RT-qPCR results of gene expression profiles of MaMyD88 (A) and MaTRAF6 (B) in liver, spleen and kidney of blunt snout bream at 4, 12, 24, 72 and 120 h after challenge with A. hydrophila. Expression of genes in both control and experimental groups were normalised to the 18S rRNA as a reference gene. The control group expression level is designated as 1, so values >1 indicate up-regulation (a), whereas values <1 indicate downregulation (b). Each histogram represents the mean ± SE of three replicates. Statistically significant differences from the control group are marked as * p < 0.05 and ** p < 0.01.