The evolution of zebrafish RAG2 protein is required for adapting to the elevated body temperature of the higher endothermic vertebrates

Ao Sun,Xianxia Cao,Xiaolong Liu,Taijiao Jiang,Hua Li,Ke Xu,Xiaoyan Zhu,Chenqi Xu,Yaohuang Shi,Tao Liang,Yanhong Ji,Xinyue Li,Haifeng Liu

doi:10.1038/s41598-020-61019-w

Abstract

The recombination activating gene (RAG or RAG1/RAG2 complex)-mediated adaptive immune system is a hallmark of jawed vertebrates. It has been reported that RAG originated in invertebrates. However, whether RAG further evolved once it arose in jawed vertebrates remains largely unknown. Here, we found that zebrafish RAG (zRAG) had a lower activity than mouse RAG (mRAG). Intriguingly, the attenuated stability of zebrafish RAG2 (zRAG2), but not zebrafish RAG1, caused the reduced V(D)J recombination efficiency compared to mRAG at 37 °C which are the body temperature of most endotherms except birds. Importantly, the lower temperature 28 °C, which is the best temperature for zebrafish growth, made the recombination efficiency of zRAG similar to that of mRAG by improving the stability of zRAG2. Consistent with the prementioned observation, the V(D)J recombination of Rag2KI/KI mice, which zRAG2 was substituted for mRAG2, was also severely impaired. Unexpectedly, Rag2KI/KI mice developed cachexia syndromes accompanied by premature death. Taken together, our findings illustrate that the evolution of zebrafish RAG2 protein is required for adapting to the elevated body temperature of the higher endothermic vertebrates.

Highlights

The recombination activating gene (RAG or RAG1/RAG2 complex)-mediated adaptive immune system is a hallmark of jawed vertebrates
Our findings suggest that the evolution of zebrafish RAG2 protein is required for adapting to the elevated body temperature of the higher endothermic vertebrates
The GFP reporter recombination assay for zRAG1/zebrafish RAG2 (zRAG2) in the NIH3T3 and HEK-293T cell lines showed that there were fewer GFP positive cells that had undergone V(D)J recombination in the zRAG1/zRAG2 group than in the mRAG1/mRAG2 group regardless of whether the cells were co-transfected with coding joint or signal joint plasmids (Fig. 1A,B and Fig. S1A,B,E)

Summary

Introduction

The recombination activating gene (RAG or RAG1/RAG2 complex)-mediated adaptive immune system is a hallmark of jawed vertebrates. The origin of RAG has been well studied, it has remained largely unknown whether RAG in jawed vertebrates from ectotherms to endotherms further evolved. When lower jawed vertebrates evolved into higher jawed vertebrates, they faced the transition in themselves from ectothermy to endothermy and the transition in environment from aquatic to terrestrial This process occurred along with numerous environmental changes associated with air, www.nature.com/scientificreports water, temperature, humidity, ion concentrations, pathogenic microorganisms and body temperature (Tb)[12,13]. For such great changes, we want to explore whether the adaptive immune system of jawed vertebrates, especially its hallmark RAG, evolved. Our findings suggest that the evolution of zebrafish RAG2 protein is required for adapting to the elevated body temperature of the higher endothermic vertebrates

Methods

Results

Conclusion