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. 2024 Apr;14(4):532-544.
doi: 10.1002/2211-5463.13769. Epub 2024 Feb 6.

Characterization of fish-specific IFNγ-related binding with a unique receptor complex and signaling through a novel pathway

Affiliations

Characterization of fish-specific IFNγ-related binding with a unique receptor complex and signaling through a novel pathway

Yasuhiro Shibasaki et al. FEBS Open Bio. 2024 Apr.

Abstract

Unlike mammals, fish express two type II interferons, IFNγ and fish-specific IFNγ (IFNγ-related or IFNγrel). We previously reported the presence of two IFNγrel genes, IFNγrel 1 and IFNγrel 2, which exhibit potent antiviral activity in the Ginbuna crucian carp, Carassius auratus langsdorfii. We also found that IFNγrel 1 increased allograft rejection; however, the IFNγrel 1 receptor(s) and signaling pathways underlying this process have not yet been elucidated. In this study, we examined the unique signaling mechanism of IFNγrel 1 and its receptors. The phosphorylation and transcriptional activation of STAT6 in response to recombinant Ginbuna IFNγrel 1 (rgIFNγrel 1) was observed in Ginbuna-derived cells. Binding of rgIFNγrel 1 to Class II cytokine receptor family members (Crfbs), Crfb5 and Crfb17, which are also known as IFNAR1 and IFNGR1-1, respectively, was detected by flow cytometry. Expression of the IFNγrel 1-inducible antiviral gene, Isg15, was highest in Crfb5- and Crfb17-overexpressing GTS9 cells. Dimerization of Crfb5 and Crfb17 was detected by chemical crosslinking. The results indicate that IFNγrel 1 activates Stat6 through an interaction with unique pairs of receptors, Crfb5 and Crfb17. Indeed, this cascade is distinct from not only that of IFNγ but also that of known IFNs in other vertebrates. IFNs may be classified by their receptor and signal transduction pathways. Taken together, IFNγrel 1 may be classified as a novel type of IFN family member in vertebrates. Our findings provide important information on interferon gene evolution in bony fish.

Keywords: IFNγ‐related; STAT; cytokine receptor; signal transduction; type II interferon.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Activation of STAT6 in IFNγrel 1‐stimulated GTS9 cells. (A) GTS9 cells were treated with 100 ng·mL−1 of rgIFNγrel 1. Cellular proteins were extracted at the indicated times. The cell lysates were loaded onto an SDS/PAGE gel under reducing conditions. Phosphorylated and total STAT6 protein and tubulin were detected with anti‐STAT6‐Y641, anti‐Stat6, and anti‐tubulin antibodies, respectively, as described in the EXPERIMENTAL PROCEDURES. (B) GTS9 cells were transfected with a construct containing the Cε STAT6 optimal binding element fused to luciferase (pGL4.15‐STAT6). The transfected cells were treated with various concentration of rgIFNγrel 1 for 12 h. Measurement of luciferase activity was done as described in the EXPERIMENTAL PROCEDURES. Each value represents the mean of three independent experiments and error bars represent standard deviations. An asterisk indicates statistical significance using one‐way ANOVA followed by Tukey's post hoc test (**P < 0.01).
Fig. 2
Fig. 2
Transcriptional activation in response to Ginbuna crucian carp IFNγrel 1 in GTS9 cells. (A) GTS9 cells were transfected with a FLAG‐STAT6 or mock vector and pGL4.15‐STAT6. Transfected cells were treated with various concentrations of rgIFNγrel 1 for 12 h. Preparation of cell extracts and measurement of luciferase activity were done as described in the EXPERIMENTAL PROCEDURES. Each value represents the mean of three independent experiments and error bars represent standard deviations. (B) GTS9 cells transiently transfected with FLAG‐STAT6 or mock vector were exposed with or without 10 ng·mL−1 rgIFNγrel 1 for 6 h. After stimulation, mRNA expression of the IFNγrel 1 inducible gene, isg15, was examined by real‐time PCR. The expression levels were normalized to that of the ef1a gene and the values were calculated relative to the expression of each transfectant stimulated without rgIFNγrel 1 (mean ± standard error; n = 3). An asterisk indicates statistical significance using (A) one‐way ANOVA followed by Tukey's post hoc test and (B) a T‐test (*P < 0.05).
Fig. 3
Fig. 3
Screening of IFNγrel 1 receptor candidate. HEK293T cells transiently expressing FLAG‐zCrfb1, FLAG‐zCrfb2, FLAG‐zCrfb7, FLAG‐zCrfb8, FLAG‐zCrfb12, FLAG‐zCrfb14, V5‐zCrfb4, V5‐zCrfb5, V5‐zCrfb6, V5‐zCrfb9, V5‐zCrfb13, V5‐zCrfb15, V5‐zCrfb16, or V5‐zCrfb17 were exposed to 100 ng·mL−1 of rgIFNγrel 1 for 15 min. Mock vector, pcDNA6/V5‐His A, and p3XFLAG‐CMV™‐14 were used as negative controls. Binding was detected by FCM with an anti‐gIFNγrel 1 antibody.
Fig. 4
Fig. 4
Crfb5 and Crfb17 are functional IFNγrel 1 receptor. HEK293T cells transiently expressing (A) V5‐gCrfb5 or (B) FLAG‐Crfb17 were treated with 100 ng·mL−1 of rgIFNγrel 1 for 15 min. Mock vector, pcDNA6/V5‐His A, and p3XFLAG‐CMV™‐14 were used as negative controls. Binding was detected by FCM using an anti‐gIFNγrel 1 antibody. (C) Ginbuna carp‐derived GTS9 cell lines transiently expressing mock vectors, Crfb5, Crfb17, or Crfb5 plus Crfb17 were treated with or without 10 ng·mL−1 rgIFNγrel 1 for 6 h. After stimulation, the expression of isg15 was measured by real‐time PCR. The expression levels were normalized to that of the Ef1a gene, and the values were calculated relative to the expression of each transfectant without rgIFNγrel 1 treatment (mean ± standard error; n = 4). An asterisk indicates statistical significance using one‐way ANOVA followed by Tukey's post hoc test (*P < 0.05).
Fig. 5
Fig. 5
Detection by chemical crosslinking. GTS9 cell lines transiently expressing mock vectors, or Crfb5 and Crfb17, were untreated or treated with 100 ng·mL−1 rgIFNγrel 1 for 1 h and then crosslinked with BS3. Cell lysates were immunoprecipitated with FLAG antibody and immunoblotted with (A) anti‐V5 antibody or (B) anti‐FLAG antibody. Arrow indicates heterodimeric receptor formation.
Fig. 6
Fig. 6
Schematic model for the IFNγrel and IFNγ signaling. Model for the (A) IFNγrel and (B) IFNγ signaling in zebrafish, grass carp and Ginbuna crucian carp.

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