{ "abstract": "Plasmodium infection induces extensive remodeling of host erythrocytes through parasite-exported proteins that interact with host cell components. Among these, tryptophan-rich antigens (TRAgs) are conserved across Plasmodium species and have been proposed as vaccine candidates, but their molecular functions remain largely unexplored. In this study, we characterized Plasmodium knowlesi tryptophan-rich antigen 40.1 (PkTRAg40.1) to investigate its localization, biochemical properties, host interactions, and immunogenicity. Recombinant PkTRAg40.1 showed no detectable binding to intact human erythrocytes in flow cytometry-based binding assays. Biochemical characterization demonstrated that native PkTRAg40.1 was predominantly associated with detergent-resistant insoluble fractions and was detected as multiple higher-molecular-weight forms. Protease protection assays further showed that native PkTRAg40.1 was accessible to protease under the experimental conditions tested. Consistent with these observations, immunofluorescence assays revealed that PkTRAg40.1 colocalized with skeleton-binding protein 1 (PkSBP1), supporting its localization to Sinton and Mulligan’s clefts (SMCs), parasite-derived membranous structures. Pull-down assays and MALDI-TOF/MS identified spectrin alpha as a host binding partner, and biolayer interferometry confirmed its specific interaction with the spectrin alpha α12–16 fragment (KD = 4.71 ± 0.62 μM). Serological analysis of P. knowlesi patient samples revealed that PkTRAg40.1 is immunogenic, exhibiting 50% sensitivity and 92.9% specificity, and also displayed cross-reactivity with sera from Plasmodium vivax-infected patients. Collectively, these findings identify PkTRAg40.1 as a TRAg associated with parasite-derived membranous structures that interacts with the host erythrocyte cytoskeleton, thereby expanding current understanding of the molecular functions of Plasmodium TRAgs. Author summary: Malaria parasites extensively remodel infected red blood cells by exporting proteins that interact with host cell components. Tryptophan-rich antigens (TRAgs) are conserved across Plasmodium species and are considered important for parasite biology, but their molecular functions remain unclear. Although previous studies have suggested that some TRAgs may associate with parasite-induced membrane structures and host cell structural components, the biological significance of these observations has yet to be established. We characterized PkTRAg40.1 from the zoonotic malaria parasite P. knowlesi to investigate its localization, host interactions, and immunogenicity. We found that recombinant PkTRAg40.1 showed no measurable binding to intact human erythrocytes in flow cytometry-based binding assays. Instead, PkTRAg40.1 localized to parasite-induced membranous structures within infected erythrocytes. Biochemical analyses identified spectrin alpha, a major component of the erythrocyte membrane skeleton, as a host binding partner. Patient sera further showed that PkTRAg40.1 is immunogenic and is recognized by antibodies from P. knowlesi-infected patients. To our knowledge, this is the first study to identify an interaction between a P. knowlesi TRAg and a host erythrocyte structural protein. These findings provide new molecular insight into the interaction of PkTRAg40.1 with host membrane and cytoskeletal components and provide a framework for future studies investigating the biological functions of TRAg proteins.", "abstract_shown": true, "affiliations": [ "Department of Tropical Medicine, School of Medicine, Kangwon National University, Chuncheon, Republic of Korea", "Department of Epidemiology and Tropical Diseases, Faculty of Public Health, Universitas Diponegoro, Semarang, Indonesia", "Department of Parasitic Diseases and Vector Control, National Institute for Medical Research, Dar es Salaam, Tanzania", "Department of Pathogen Biology and Immunology, School of Medicine, Yangzhou University, Yangzhou, China", "Department of Parasitology and Tropical Medicine, Inha University School of Medicine, Incheon, Republic of Korea", "Department of Obstetrics and Gynecology, Kangwon National University Hospital, Chuncheon, Republic of Korea", "Department of Pharmacology, School of Medicine, Kangwon National University, Chuncheon, Republic of Korea", "Department of Physiology, School of Medicine, Kangwon National University, Chuncheon, Republic of Korea", "Department of Parasitology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia", "Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom", "Institute of Medical Sciences, Kangwon National University, Chuncheon, Republic of Korea" ], "article_type": "Research Article", "arxiv": null, "author_total": 18, "authors": [ { "affs": [ 0 ], "name": "Wang-Jong Lee" }, { "affs": [ 1 ], "name": "Fadhila Fitriana" }, { "affs": [ 2 ], "name": "Jadidan Hada Syahada" }, { "affs": [ 3 ], "name": "Johnsy Mary Louis" }, { "affs": [ 4 ], "name": "Hyun-Young Sim" }, { "affs": [ 5 ], "name": "Ernest Mazigo" }, { "affs": [ 6 ], "name": "Hojong Jun" }, { "affs": [ 7 ], "name": "Fauzi Muh" }, { "affs": [ 8 ], "name": "Feng Lu" }, { "affs": [ 9 ], "name": "Seok Ho Cha" }, { "affs": [ 10 ], "name": "Se Jin Lee" }, { "name": "Sunghun Na" }, { "name": "Wanjoo Chun" }, { "name": "Won Sun Park" }, { "name": "Yee Ling Lau" }, { "name": "Robert W. Moon" }, { "name": "Eun-Taek Han" }, { "name": "Jin-Hee Han" } ], "bibtex": "https://research.draconitic.com/paper/73v56c8zd0jf3.bib", "cited_by": 0, "doi": "10.1371/journal.pntd.0014700", "fulltext_url": "https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014700", "id": "73v56c8zd0jf3", "issns": [ "1935-2735" ], "landing_url": "https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014700", "language": null, "licence": "CC-BY-4.0", "licence_label": "CC BY 4.0", "licence_source": "journal", "licence_url": "https://creativecommons.org/licenses/by/4.0/", "metadata_licence": null, "native_subjects": [ [ "openalex", "2739|Malaria Research and Control" ], [ "plos", "/Biology and life sciences/Biochemistry/Proteins/Cytoskeletal proteins/Spectrins" ], [ "plos", "/Biology and life sciences/Biochemistry/Proteins/Recombinant proteins" ], [ "plos", "/Biology and life sciences/Cell biology/Cellular structures and organelles/Cell membranes" ], [ "plos", "/Biology and life sciences/Cell biology/Cellular structures and organelles/Cell membranes/Membrane proteins" ], [ "plos", "/Biology and life sciences/Cell biology/Cellular types/Animal cells/Blood cells/Red blood cells" ], [ "plos", "/Biology and life sciences/Cell biology/Cellular types/Animal cells/Blood cells/Red blood cells/Erythrocyte membrane" ], [ "plos", "/Biology and life sciences/Organisms/Eukaryota/Protozoans/Parasitic protozoans/Malarial parasites" ], [ "plos", "/Biology and life sciences/Parasitology/Parasite groups/Apicomplexa/Plasmodium" ] ], "openalex": "W7213471394", "pdf_url": "https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0014700&type=printable", "pmcid": null, "pmid": null, "published": "2026-09-17", "publisher": "Public Library of Science", "ris": "https://research.draconitic.com/paper/73v56c8zd0jf3.ris", "sources": [ { "first_seen": "2026-10-30T12:17:49Z", "native_id": "10.1371/journal.pntd.0014700", "source": "plos", "url": "https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014700" } ], "subjects": [ { "field": "biology", "origin": "plos:cell biology", "subfield": "cell-biology" } ], "title": "Plasmodium knowlesi tryptophan-rich antigen 40.1 binds human erythrocyte spectrin alpha", "title_text": "Plasmodium knowlesi tryptophan-rich antigen 40.1 binds human erythrocyte spectrin alpha", "type": "peer-reviewed", "updated_at": "2026-10-30T12:17:49Z", "url": "https://research.draconitic.com/paper/73v56c8zd0jf3", "venue": "PLOS Neglected Tropical Diseases", "versions": [] }