Development of an in vitro TurboID labeling assay for the detection of protoRAG-interacting proteins in the amphioxus

Abstract

The recombination-activating gene (RAG)-mediated V(D)J rearrangement mechanism is essential for generating the diversity of the adaptive immune system in jawed vertebrates. The RAG1 and RAG2 proteins form a tetrameric complex that recognizes and cleaves pairs of recombination signal sequences (RSSs). In jawed vertebrates, the N-terminal region of RAG1 and the C-terminal region of RAG2 are critical for the regulation of physiological function and enhancement of recombination accuracy. Moreover, the intracellular activity of RAG1/2 is rigorously regulated and is thought to represent the aspect shaped by host coevolution. However, these regulatory mechanisms remain incompletely understood, impeding our deeper understanding of the physiological functions of RAG. Previous studies have shown that jawed vertebrate RAG evolved from invertebrate RAG-like (RAGL) transposases and underwent key structural changes during evolution. RAGL genes have been identified in multiple invertebrate species, but from Branchiostoma belcheri (Bb), the RAG-like protein in Bb amphioxus (BbRAG1L) and the RAG2-like protein in Bb amphioxus (BbRAG2L) are among the most extensively characterized to date. Lancelets (amphioxus) represent the most basal extant chordates (cephalochordates) that diverged from the other two chordate lineages (urochordates and vertebrates). Consequently, amphioxus has been considered a “living fossil” for studying the origins and evolution of adaptive immunity. Homologs of the core structural domain of RAG1 were discovered in the amphioxus genome in 2005, and the homologs were subsequently shown to exhibit recombinase activity in cooperation with murine-derived RAG2. A large number of transposon genes were then identified in the amphioxus genome, followed by the identification of target site duplication (TSD) and terminal inverted repeat (TIR) sequences in the amphioxus genome. Notably, BbRAG2L lacks the C-terminal region present in murine RAG2, including the acidic hinge and PHD domain. BbRAG1L coupled with BbRAG2L displays significant transposition activity in vitro and in human cell lines. In contrast, vertebrate RAG exhibits limited transposon activity in vitro and extremely low transposon activity in vivo. This leads to the question of how RAG lost its transposase activity during evolution. Structural comparison and functional experiments of BbRAGL (protoRAG) and vertebrate RAG have revealed that vertebrates undergo essential adaptive changes to eliminate the primitive transposase activity of protoRAG. Research on BbRAGL has therefore provided strong support for the hypothesis that RAG originated from a domesticated transposon. Given the similarity in both structure and function between BbRAG1L and vertebrate RAG and the fact that BbRAGL proteins are the only RAGL proteins that can currently be efficiently expressed in vertebrate cells, characterizing the binding partners of BbRAGL involved in amphioxus is of great significance for understanding the regulation of BbRAGL and its adaptation and evolution. Parallel comparison of BbRAGL and RAG interaction networks may further clarify how BbRAGL transitioned from a transposase to a recombinase during host coevolution and reveal general principles of vertebrate intracellular RAG regulation. In recent years, proximity labeling techniques have greatly facilitated approaches to identifying RAG cofactors. For example, RAG1 fused to biotin ligase RAG1 fused to a biotin ligase found in Escherichia coli (BirM) has been used to define dynamic interaction networks of RAG-associated proteins. Inspired by these advances, we sought to apply a similar approach to identify the BbRAGL cofactors in amphioxus and build a regulatory network of BbRAGL that could shed light on its relationship with host evolution. However, endogenous BbRAGL expression is undetectable in the adult amphioxus (data not shown), and the generation of transgenic amphioxus lines expressing proximity-based labeling proteins remains technically challenging. These constraints make it difficult to analyze native cofactors of endogenous BbRAGL in vivo. Because BbRAGL can be expressed in vertebrate cell lines, we instead developed an in vitro proximity-labeling strategy. We fused TurboID (TbID), an engineered biotin ligase, to the N-terminus of BbRAG1L or BbRAG2L (Figure 1A), purified the fusion proteins, and incubated them with protein lysates prepared from the hepatic cecum and colons of adult B. belcheri amphioxus in vitro to label the interacting proteins. The proteins were finally analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Figure 1B). A pTT5 vector was used to express plasmids encoding maltose-binding protein (MBP) and TurboID with or without full-length BbRAG1L or BbRAG2L, yielding pTT5-MBP-TurboID, pTT5-MBP-TurboID-BbRAG1L, and pTT5-MBP-TurboID-BbRAG2L. Plasmids expressing MBP-BbRAG1L and MBP-BbRAG2L were also generated.

1. Introduction

The recombination-activating gene (RAG)-mediated V(D)J rearrangement mechanism is essential for generating the diversity of the adaptive immune system in jawed vertebrates. The RAG1 and RAG2 proteins form a tetrameric complex that recognizes and cleaves pairs of recombination signal sequences (RSSs). In jawed vertebrates, the N-terminal region of RAG1 and the C-terminal region of RAG2 are critical for the regulation of physiological function and enhancement of recombination accuracy. Moreover, the intracellular activity of RAG1/2 is rigorously regulated and is thought to represent the aspect shaped by host coevolution. However, these regulatory mechanisms remain incompletely understood, impeding our deeper understanding of the physiological functions of RAG.

Previous studies have shown that jawed vertebrate RAG evolved from invertebrate RAG-like (RAGL) transposases and underwent key structural changes during evolution. RAGL genes have been identified in multiple invertebrate species, but from Branchiostoma belcheri (Bb), the RAG-like protein in Bb amphioxus (BbRAG1L) and the RAG2-like protein in Bb amphioxus (BbRAG2L) are among the most extensively characterized to date. Lancelets (amphioxus) represent the most basal extant chordates (cephalochordates) that diverged from the other two chordate lineages (urochordates and vertebrates). Consequently, amphioxus has been considered a “living fossil” for studying the origins and evolution of adaptive immunity. Homologs of the core structural domain of RAG1 were discovered in the amphioxus genome in 2005, and the homologs were subsequently shown to exhibit recombinase activity in cooperation with murine-derived RAG2. A large number of transposon genes were then identified in the amphioxus genome, followed by the identification of target site duplication (TSD) and terminal inverted repeat (TIR) sequences in the amphioxus genome. Notably, BbRAG2L lacks the C-terminal region present in murine RAG2, including the acidic hinge and PHD domain. BbRAG1L coupled with BbRAG2L displays significant transposition activity in vitro and in human cell lines. In contrast, vertebrate RAG exhibits limited transposon activity in vitro and extremely low transposon activity in vivo. This leads to the question of how RAG lost its transposase activity during evolution. Structural comparison and functional experiments of BbRAGL (protoRAG) and vertebrate RAG have revealed that vertebrates undergo essential adaptive changes to eliminate the primitive transposase activity of protoRAG. Research on BbRAGL has therefore provided strong support for the hypothesis that RAG originated from a domesticated transposon.

Given the similarity in both structure and function between BbRAG1L and vertebrate RAG and the fact that BbRAGL proteins are the only RAGL proteins that can currently be efficiently expressed in vertebrate cells, characterizing the binding partners of BbRAGL involved in amphioxus is of great significance for understanding the regulation of BbRAGL and its adaptation and evolution. Parallel comparison of BbRAGL and RAG interaction networks may further clarify how BbRAGL transitioned from a transposase to a recombinase during host coevolution and reveal general principles of vertebrate intracellular RAG regulation.