Molecular characterization and functional analysis of DIGIRR from golden pompano (Trachinotus ovatus)

Abstract

Mammalian single immunoglobulin (Ig) interleukin-1 receptor related molecule (SIGIRR), an important member of the Toll/interleukin-1 receptor (TIR) family, plays important balancing roles in the inflammatory responses. In the present study, the double Ig interleukin-1 receptor related molecule (DIGIRR), the homologous of SIGIRR, was characterized in golden pompano (Trachinotus ovatus) (termed as trDIGIRR). The full-length cDNA of trDIGIRR was 2,167 bp with an open reading frame (ORF) of 1,572 bp encoding 523 amino acids. The trDIGIRR contained several conserved domains including a signal peptide, two Ig domains, a transmembrane domain and a TIR domain, and shared high sequence identities with its teleost counterparts. Realtime qPCR analysis revealed that the trDIGIRR was distributed in all tissues examined, with high expressions in intestine, liver and head kidney. The expressions of trDIGIRR were induced by Vibrio alginolyticus, lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I:C) challenge. Further analysis revealed that trDIGIRR was mainly located in the cytoplasm. In addition, the co-immunoprecipitation (co-IP) assay identified that trDIGIRR could interact with myeloid differentiation factor 88 (MyD88), but not interact with TIR domain containing adaptor protein inducing interferon-β (TRIF). Our results provide basis for studying the immune role of fish DIGIRR.

Keywords: DIGIRR; Trachinotus ovatus; co-IP; immune response; subcellular localization.

Figure 1

The nucleotide and deduced amino acid sequence (A), secondary structure (B) and 3-D structure (C) of trDIGIRR. (A) The start codon was marked with a thick square, the stop codon was labeled with a black solid circle, the signal peptide was labeled with a double horizontal line, the Ig domain was marked with a square, the transmembrane region was marked with a horizontal line, the TIR domain was marked with a red background, and poly-A tail was italicized. Two in-frame stop condons were marked in yellow and the the polyadenylation signal (aataaa) upstream of the poly (A) tail was marked in blue. (B) The secondary structure of trDIGIRR was predicted using Protean software. (C) The 3-D structure of trDIGIRR was constructed using the SWISS-MODEL software.

Figure 2

Comparison of DIGIRR, SIGIRR predicted amino acid sequence (A), 3-D structure (B) and TIR domain key amino acid (C) in different vertebrates. The underlined part was the signal peptide and the red shaded part was the Ig domain. The yellow shaded part was transmembrane region. The green shaded part was TIR domain and the blue box (1-3) was the Box motif in the TIR domain. The 3-D structure was performed using Alpha fold 2 software. The GenBank accession numbers of each specie sequences were summarized in Table 2 .

Figure 3

The phylogenetic tree of DIGIRR and SIGIRR genes in different species. The phylogenetic tree was constructed using the Neighbor-joining method (NJ) with 10000 bootstrapping replications by MEGA 7.0 software. The sequence information of all species was shown in Table 2 .

Figure 4

Relative mRNA expression level of DIGIRR in spleen, HK, skin, muscle, gill, heart, liver, brain and intestine of healthy golden pompano (A), and the subcellular localization of trDIGIRR in HEK-293T cells (B). A: Bars that do not share a letter represent a significant difference (p < 0.05).

Figure 5

Expression patterns of trDIGIRR in spleen, head kidney (HK), liver, intestine and gill following the Vibrio alginolyticus, LPS and poly I:C challenge (A–E). The relative expression of target gene in each tissue was normalized to that of β-actin. The expression changes of the two genes were analyzed using the 2^-△△CT method and expressed as fold change. Results were means ± SE.*P<0.05, **P<0.01.

Figure 6

Co-immunoprecipitation analysis of the association between trDIGIRR with trMyD88 (A) or trTRIF (B).