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

doi:10.12140/j.issn.1000-7423.2025.02.007

Abstract

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

CLC Number:

R382.311

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.

Figures/Tables 4

Fig. 1

Fig. 2

Fig. 3

Fig. 4

References 24

[1]	Zhang SX, Yang GB, Yang J, et al. Global, regional, and national burden of malaria, 1990-2021: Findings from the global burden of disease study 2021[J]. Decod Infect Transm, 2024, 2: 100030.
[2]	Arya A, Meena SS, Matlani M, et al. Trends in clinical features and severity of Plasmodium vivax malaria among children at tertiary care center in north India[J]. J Trop Pediatr, 2023, 69(6): fmad034.
[3]	Moxon CA, Gibbins MP, McGuinness D, et al. New insights into malaria pathogenesis[J]. Annu Rev Pathol, 2020, 15: 315-343.
[4]	曹伟, 王一, 张熙致, 等. 脑型疟辅助治疗研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(3): 361-373, 379.
	Cao W, Wang Y, Zhang XZ, et al. Research progress in adjunctive therapy of cerebral malaria[J]. Chin J Parasitol Parasit Dis, 2023, 41(3): 361-373, 379. (in Chinese).
[5]	Smith JD, Rowe JA, Higgins MK, et al. Malaria’s deadly grip: Cytoadhesion of Plasmodium falciparum-infected erythrocytes[J]. Cell Microbiol, 2013, 15(12): 1976-1983.
[6]	Carvalho BO, Lopes SCP, Nogueira PA, et al. On the cytoadhesion of Plasmodium vivax-infected erythrocytes[J]. J Infect Dis, 2010, 202(4): 638-647.
[7]	石天琪, 陈军虎. 间日疟原虫入侵网织红细胞相关蛋白的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(3): 396-401.
	Shi TQ, Chen JH. Research progress on reticulocyte binding proteins associated with Plasmodium vivax invasion of reticulocytes[J]. Chin J Parasitol Parasit Dis, 2022, 40(3): 396-401. (in Chinese).
[8]	Bernabeu M, Lopez FJ, Ferrer M, et al. Functional analysis of Plasmodium vivax VIR proteins reveals different subcellular localizations and cytoadherence to the ICAM-1 endothelial receptor[J]. Cell Microbiol, 2012, 14(3): 386-400.
[9]	Fernandez-Becerra C, Bernabeu M, Castellanos A, et al. Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection[J]. Proc Natl Acad Sci USA, 2020, 117(23): 13056-13065.
[10]	Rehn T, Lubiana P, Nguyen THT, et al. Ectopic expression of Plasmodium vivax vir genes in P. falciparum affects cytoadhesion via increased expression of specific var genes[J]. Microorganisms, 2022, 10(6): 1183.
[11]	Wang SH, Cheng JY, Tsai HH, et al. Conformational alteration in glycan induces phospholipase Cβ1 activation and angiogenesis[J]. J Biomed Sci, 2022, 29(1): 105.
[12]	Tunyasuvunakool K, Adler J, Wu Z, et al. Highly accurate protein structure prediction for the human proteome[J]. Nature, 2021, 596(7873): 590-596.
[13]	Edmunds NS, Genc AG, McGuffin LJ. Benchmarking of AlphaFold2 accuracy self-estimates as indicators of empirical model quality and ranking: A comparison with independent model quality assessment programmes[J]. Bioinformatics, 2024, 40(8): btae491.
[14]	Tran LH, Graulus GJ, Vincke C, et al. Nanobodies for the early detection of ovarian cancer[J]. Int J Mol Sci, 2022, 23(22): 13687.
[15]	Del Portillo HA, Ferrer M, Brugat T, et al. The role of the spleen in malaria[J]. Cell Microbiol, 2012, 14(3): 343-355.
[16]	Milner DA Jr. Malaria pathogenesis[J]. Cold Spring Harb Perspect Med, 2018, 8(1): a025569.
[17]	Del Portillo HA, Fernandez-Becerra C, Bowman S, et al. A superfamily of variant genes encoded in the subtelomeric region of Plasmodium vivax[J]. Nature, 2001, 410(6830): 839-842.
[18]	Carson M, Johnson DH, McDonald H, et al. His-tag impact on structure[J]. Acta Crystallogr D Biol Crystallogr, 2007, 63(3): 295-301.
[19]	Wiser MF. Knobs, adhesion, and severe falciparum malaria[J]. Trop Med Infect Dis, 2023, 8(7): 353.
[20]	da Veiga GTS, Moriggi MR, Vettorazzi JF, et al. Plasmodium vivax vaccine: What is the best way to go?[J]. Front Immunol, 2023, 13: 910236.
[21]	Lennartz F, Smith C, Craig AG, et al. Structural insights into diverse modes of ICAM-1 binding by Plasmodium falciparum-infected erythrocytes[J]. Proc Natl Acad Sci USA, 2019, 116(40): 20124-20134.
[22]	Chen SB, Wang Y, Kassegne K, et al. Whole-genome sequencing of a Plasmodium vivax clinical isolate exhibits geographical characteristics and high genetic variation in China-Myanmar border area[J]. BMC Genomics, 2017, 18(1): 131.
[23]	Na BK, Kim TS, Lin K, et al. Genetic polymorphism of Vir genes of Plasmodium vivax in Myanmar[J]. Parasitol Int, 2021, 80: 102233.
[24]	Hawadak J, Arya A, Chaudhry S, et al. Genetic diversity and natural selection analysis of VAR2CSA and Vir genes: Implication for vaccine development[J]. Genomics Inform, 2024, 22(1): 11.