3种灵长类动物SAGE1与INTS3相互作用保守性的验证

doi:10.3969/j.issn.1674-8115.2024.04.004

摘要/Abstract

摘要：

目的·研究恒河猴（Rhesus）、狨猴（Marmoset）、倭狐猴（Mouse Lemur）这3种代表性灵长类动物的癌-睾丸抗原（cancer-testis antigen，CTA）家族成员之一的肉瘤抗原1（sarcoma antigen 1，SAGE1）的进化，及其与整合体复合物（integrator complex，INT）亚基INTS3之间的相互作用的保守性。方法·首先在HEK293T细胞中利用免疫共沉淀（co-immunoprecipitation，Co-IP）的方法验证3种灵长类动物的SAGE1和INTS3相互作用。随后在互作蛋白截短的片段上分别添加His-SUMO和GST标签，并在大肠埃希菌中进行表达，经过系列纯化步骤得到目标蛋白。通过表面等离子体共振（surface plasmon resonance，SPR）实验测定目标蛋白之间的结合强弱，借助广角激光散射凝胶排阻层析（size exclusion chromatography with multi-angle light scattering，SEC-MALS）技术检测复合物的结合比例，并利用分子对接技术预测SAGE1和INTS3截短体的结合模型。分析人类INTS3和SAGE1互作界面，挑选在灵长类中也相对保守的关键互作氨基酸位点，将其突变后再次使用Co-IP分析INTS3和SAGE1的结合情况。结果·通过Co-IP方法，确定3种灵长类动物中INTS3和SAGE1全长蛋白之间存在相互作用。截短体蛋白经过外源诱导表达和多步纯化，获得高纯度蛋白样品；经过SPR对其进行结合和解离检测分析，得到了它们的解离常数在微摩尔级别；并且SEC-MALS结果显示INTS3与SAGE1的结合比例为2∶1。使用分子对接预测SAGE1和INTS3截短体结合模型，发现该复合物具有典型的“蝴蝶型结构”，由INTS3二聚体组成“蝴蝶身体”，由SAGE1组成“蝴蝶头部”，结合界面中疏水相互作用构成了主要的互作模式。分析突变后的SAGE1与INTS3全长蛋白相互作用，它们之间的结合明显减弱。结论·恒河猴、狨猴、倭狐猴中INTS3与SAGE1存在相互作用，并且相互作用具有高度的保守性。

关键词: 肉瘤抗原1, 整合体复合物亚基3, 相互作用

Abstract:

Objective ·To investigate the evolution of sarcoma antigen 1 (SAGE1), a member of the cancer-testis antigen (CTA) family, in three representative primate species—Macaca mulatta (Rhesus), Callithrix jacchus (Marmoset), and Microcebus murinus (Mouse Lemur), as well as the conservation of its interaction with INTS3, a subunit of the integrator complex. Methods ·The interaction between INTS3 and SAGE1 in these primates and the interaction between INTS3 and human SAGE1 mutants were first validated in HEK293T cells by using co-immunoprecipitation (Co-IP). Truncated proteins were then tagged with His-SUMO and GST, respectively, and expressed in Escherichia coli, followed by a series of purification steps to obtain the target proteins. Surface plasmon resonance (SPR) was used to measure the binding affinity of the target proteins, and size exclusion chromatography with multi-angle light scattering (SEC-MALS) was used to determine the stoichiometry of the complex. Additionally, molecular docking was employed to predict the binding model of the truncated SAGE1 and INTS3. Mutations were performed on human SAGE1 key interface residues to analyze their binding to INTS3 by Co-IP. Results ·The interaction between the full-length human-derived INTS3 and the full-length SAGE1 from the three primate species was confirmed by Co-IP. Truncated proteins were purified through multiple steps of tandom chromatography. The dissociation constants of the three pairs of truncated INTS3-SAGE1 were measured via SPR, and the SEC-MALS results demonstrated that the binding ratio of INTS3-SAGE1 was 2∶1. Furthermore, molecular docking predicted a structural model of the truncated INTS3-SAGE1 complex and the binding interface was extensively constituted with hydrophobic contacts assisted by some hydrophilic interactions. The interaction between the mutant SAGE1 and INTS3 full-length protein was significantly weakened. Conclusion ·There is a conserved interaction between INTS3 and SAGE1 across Rhesus, Marmoset, and Mouse lemur.

Key words: sarcoma antigen 1 (SAGE1), integrator complex subunit 3 (INTS3), interaction

中图分类号:

李晓畅, 李明月, 雷鸣, 武健. 3种灵长类动物SAGE1与INTS3相互作用保守性的验证[J]. 上海交通大学学报（医学版）, 2024, 44(4): 444-453.

LI Xiaochang, LI Mingyue, LEI Ming, WU Jian. Conservation of protein interaction between SAGE1 and INTS3 identified in 3 different types of primates[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(4): 444-453.

图/表 4

图1 SAGE1与INTS6在进化上的相似性Note： A. Evolution tree of INTS6. B. The SAGE1 full-length sequence and C-terminal sequence domain pattern map of four species of human, rhesus, marmoset and mouse lemur, and the full-length domain pattern map of human INTS3 and INTS6. C. Sequence alignment between human SAGE1CTD and INTS6CTD. D. Sequence alignment between human SAGE1CTD and three different primates SAGE1CTD. E. Sequence alignment between human INTS3CTD and three different primates INTS3CTD.

Fig 1 Evolutionary similarity between SAGE1 and INTS6

图2 SAGE1与INTS3的免疫共沉淀实验、蛋白纯化及解离常数测定Note： A. Immunoprecipitation of full-length INTS3 and full-length SAGE1 in Rhesus. B. Immunoprecipitation of full-length INTS3 and full-length SAGE1 in Marmoset. C. Immunoprecipitation of full-length INTS3 and full-length SAGE1 in Lemur. D. Purification of human INTS3CTD. E. Purification of Rhesus SAGE1CTD. Gel filtration chromatography results shown on the left; SDS-PAGE analysis of fractions across the major peak shown on the right. F. Purification of Marmoset SAGE1CTD. G. Purification of Lemur SAGE1CTD. H. Rhesus SAGE1CTD binding to INTS3CTD in a concentration-dependent manner with a slow kinetics. I. Marmoset SAGE1CTD binding to INTS3CTD in a concentration-dependent manner with a slow kinetics. J. Lemur SAGE1CTD binding to INTS3CTD in a concentration-dependent manner with a slow kinetics. Co-IP—Co-immunoprecipitation; SPR—surface plasmon resonance.

Fig 2 Co-immunoprecipitation experiment, protein purification and dissociation constant determination of SAGE1 and INTS3

图3 INTS3CTD-SAGE1CTD 复合物体外组装与SEC-MALS分析Note： A. Rhesus INTS3CTD-SAGE1CTD complex assemble in vitro. B. Marmoset INTS3CTD-SAGE1CTD complex assemble in vitro. C. Lemur INTS3CTD-SAGE1CTD complex assemble in vitro. D. SEC-MALS analysis of purified Rhesus INTS3CTD-SAGE1CTD complex. E. SEC-MALS analysis of purified Marmoset INTS3CTD-SAGE1CTD complex. F. SEC-MALS analysis of purified Lemur INTS3CTD-SAGE1CTD complex. MW—molecular weight.

Fig 3 In vitro INTS3CTD-SAGE1CTD assembly and SEC-MALS analysis

图4 INTS3CTD-SAGE1CTD 结构预测与相互作用界面分析、INTS3与人类突变体SAGE1免疫共沉淀实验Note： A. The Human INTS3CTD-INTS6CTD structure published on PDB (id: 7BV7) and its interaction interface. H-bond is marked by black dash line. B. The Human INTS3CTD-SAGE1CTD model created by molecular docking and its interaction interface. C. Immunoprecipitation of full-length INTS3 and full-length SAGE1 mutant in Human. D. The Rhesus INTS3CTD-SAGE1CTD model created by molecular docking and its interaction interface. E. The Marmoset INTS3CTD-SAGE1CTD model created by molecular docking and its interaction interface. F. The Lemur INTS3CTD-SAGE1CTD model created by molecular docking and its interaction interface.

Fig 4 Structure prediction and interaction interface analysis of INTS3CTD-SAGE1CTD, and co-immunoprecipitation experiment of INTS3 with human mutant SAGE1

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