间日疟原虫VIR14蛋白与ICAM-1受体相互作用关键位点分析

doi:10.12140/j.issn.1000-7423.2025.02.007

中国寄生虫学与寄生虫病杂志 ›› 2025, Vol. 43 ›› Issue (2): 192-197.doi: 10.12140/j.issn.1000-7423.2025.02.007

间日疟原虫VIR14蛋白与ICAM-1受体相互作用关键位点分析

杨晚璇¹^,²()(), 沈海默², 陈绅波², 陈军虎¹^,²^,^*()()

1 杭州医学院基础医学与法医学院，浙江杭州 310013
2 中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心）；传染病溯源预警与智能决策国家重点实验室；国家卫生健康委员会寄生虫病原与媒介生物学重点实验室；世界卫生组织热带病合作中心；国家热带病国际研究中心，上海 200025

收稿日期:2025-01-27 修回日期:2025-03-27 出版日期:2025-04-30 发布日期:2025-04-27
通讯作者: * 陈军虎（ORCID：0000-0002-3765-0248），男，博士，研究员，从事重要媒传寄生虫感染与宿主免疫、诊断、疫苗研究。E-mail：chenjh@nipd.chinacdc.cn
作者简介:杨晚璇（ORCID：0009-0008-9626-6589），女，硕士研究生，从事疟原虫致病机制的研究。E-mail：881012022033@hmc.edu.cn
基金资助:
上海市国际科技合作基金(18490741100)

Analysis of the key sites of Plasmodium vivax VIR14 protein interacting with ICAM-1 receptor

YANG Wanxuan¹^,²()(), SHEN Haimo², CHEN Shenbo², CHEN Junhu¹^,²^,^*()()

1 School of Basic Medical Sciences and Forensic Medicine, Hangzhou Medical College, Hangzhou 310013, Zhejiang, China
2 National Institute of Parasitic Diseases, National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases; Chinese Center for Diseases Control and Prevention (Chinese Center for Tropical Diseases Research); NHC Key Laboratory of Parasite and Vector Biology; WHO Collaborating Centre for Tropical Diseases; National Centre for International Research on Tropical Diseases, Shanghai 200025, China

Received:2025-01-27 Revised:2025-03-27 Online:2025-04-30 Published:2025-04-27
Contact: * E-mail：chenjh@nipd.chinacdc.cn
Supported by:
Project of Shanghai Science and Technology Commission(18490741100)

摘要/Abstract

摘要：

目的了解间日疟原虫VIR14蛋白与细胞间黏附分子-1（ICAM-1）受体的结合情况并分析其关键位点。方法将重组质粒pET28a-VIR14转化至大肠埃希菌BL21（DE3），经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹（Western blotting）分析间日疟原虫重组VIR14蛋白的表达情况与纯度。通过表面等离子共振（SPR）技术验证VIR14蛋白或多肽与ICAM-1受体的结合情况。预测VIR14以及ICAM-1（28~480 aa）蛋白的结构，综合预测复合物建模分数（pTM）及局部距离差异测试分数（pLDDT），通过cluspro 2.0对接分析得分最高的VIR14和ICAM-1模型之间的相互作用。使用PDBePISA系统和pymol 3.0软件处理对接结果，分析对接位点。通过全球不同流行区数据分析结合位点氨基酸多态性。结合表位竞争抑制实验分析受体与配体相互作用的关键位点。结果 SDS-PAGE结果显示，成功表达并纯化相对分子质量（M_r）约为58 000的VIR14蛋白。SPR实验表明，不同浓度的VIR14蛋白与ICAM-1受体结合响应值呈浓度依赖上升，亲和力值为15.16 μmol/L。Alphafold2结构预测结果显示，VIR14蛋白最佳模型pLDDT和pTM评分分别为78.2和0.73；ICAM-1受体最佳模型pLDDT和pTM值分别为93.8和0.65。VIR14与ICAM-1蛋白之间形成极性键的区域集中在蛋白尾端（212~406 aa）。在含有预测结合位点的2条多肽中，仅P2多肽与ICAM-1结合，亲和力为48.99 μmol/L。竞争抑制实验表明，P2多肽能抑制VIR14蛋白与ICAM-1结合。将P2中的4个关键位点（ASP-310、ARG-314、LYS-316、ASP-318）突变为丙氨酸以改变它们的侧链后，P2丧失了抑制能力。P2多肽在全球338个地理株中高度保守。结论间日疟原虫VIR14蛋白能与ICAM-1受体紧密结合，4个关键氨基酸位点发挥了重要作用，且在全球种群中具有高度保守性。

关键词: 间日疟原虫, VIR蛋白, 表面等离子共振, Alphafold2, 遗传多样性

Abstract:

Objective To investigate the binding of Plasmodium vivax VIR14 protein to intercellular cell adhesion molecule-1 (ICAM-1) receptor and identify key binding sites. Methods The recombinant plasmid pET28a-VIR14 was transformed into Escherichia coli BL21 (DE3). The expression levels and purity of VIR14 protein were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Binding of VIR14 protein or peptides to ICAM-1 was verified using surface plasmon resonance (SPR). Structures of VIR14 and ICAM-1 (28-480 aa) were predicted, and complex modeling scores (pTM and pLDDT) were integrated. Docking analysis was performed using ClusPro 2.0 to evaluate the interactions between top-scoring VIR14 and ICAM-1 models. Docking sites were analyzed using PDBePISA and Pymol 3.0. Amino acid polymorphisms at binding sites were examined using global isolate data. Key interaction sites were analyzed via epitope competition inhibition assays. Results SDS-PAGE confirmed successful expression and purification of VIR14 protein, with a relactive molecular mass (M_r) of 58 000. SPR showed concentration-dependent binding of VIR14 to ICAM-1, with an affinity of 15.16 μmol/L. According to structural predictions from Alphafold2, the optimal model of the VIR14 protein had a pLDDT score of 78.2 and a pTM score of 0.73. In contrast, the optimal model of the ICAM-1 receptor showed a pLDDT score of 93.8 and a pTM score of 0.65. The regions where polar bonds formed between the VIR14 and ICAM-1 proteins were predominantly located in the C-terminal region of the protein, specifically between the 212 and 406 amino acids. Among the two peptides containing the predicted binding sites, only peptide P2 bound to ICAM-1, with an affinity of 48.99 μmol/L. Competitive inhibition experiments indicated that P2 could inhibit the binding of VIR14 protein to ICAM-1. After replacing the side chains of the four key residues (ASP-310, ARG-314, LYS-316, and ASP-318) with alanine, P2 lost its inhibitory function. P2 was highly conserved across 338 P. vivax global isolates. Conclusion The VIR14 protein of P. vivax exhibits tight binding to the ICAM-1 receptor, with four amino acid residues playing essential roles in this interaction. These key sites demonstrate high conservation across P. vivax global isolates.

Key words: Plasmodium vivax, VIR protein, Surface plasmon resonance, Alphafold2, Genetic diversity

中图分类号:

R382.311

杨晚璇, 沈海默, 陈绅波, 陈军虎. 间日疟原虫VIR14蛋白与ICAM-1受体相互作用关键位点分析[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(2): 192-197.

YANG Wanxuan, SHEN Haimo, CHEN Shenbo, CHEN Junhu. Analysis of the key sites of Plasmodium vivax VIR14 protein interacting with ICAM-1 receptor[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2025, 43(2): 192-197.

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间日疟原虫VIR14蛋白与ICAM-1受体相互作用关键位点分析

Analysis of the key sites of Plasmodium vivax VIR14 protein interacting with ICAM-1 receptor

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