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Identification of Multiple Domains of Entamoeba histolytica Intermediate Subunit Lectin-1 with Hemolytic and Cytotoxic Activities.

ABSTRACT: Galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica have roles in the pathogenicity of intestinal amoebiasis. Igl1, the intermediate subunit lectin-1 of E. histolytica, has been shown to have both hemolytic and cytotoxic activities that reside in the C-terminus of the protein. To identify the amino acid regions responsible for these activities, recombinant proteins were prepared and used in hemolytic and cytotoxic assays. The results revealed that Igl1 has multiple domains with hemolytic and cytotoxic activities and that amino acids 787-846, 968-1028 and 1029-1088 are involved in these activities. The hemolytic activities of the fragments were partly inhibited by mannose, galactose and N-acetylgalactosamine, and glucose showed lower or negligible inhibitory effects for the activities. This is the first report of a protozoan protein with hemolytic and cytotoxic activities in multiple domains.

SUBMITTER: Kato K

PROVIDER: S-EPMC9316275 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Identification of Multiple Domains of Entamoeba histolytica Intermediate Subunit Lectin-1 with Hemolytic and Cytotoxic Activities.

Kato Kentaro K Tachibana Hiroshi H

International journal of molecular sciences 20220712 14

Galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica have roles in the pathogenicity of intestinal amoebiasis. Igl1, the intermediate subunit lectin-1 of E. histolytica, has been shown to have both hemolytic and cytotoxic activities that reside in the C-terminus of the protein. To identify the amino acid regions responsible for these activities, recombinant proteins were prepared and used in hemolytic and cytotoxic assays. The results revealed t ...[more]

PMID: 35887043

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Project description:BackgroundEntamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties.Methodology/principal findingsA prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene.Conclusions/significanceImmunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses.

Project description:Entamoeba histolytica is an enteric protozoan parasite that causes human amebic colitis and extraintestinal abscesses. As a prerequisite for parasite colonization and invasion, adherence of E. histolytica is predominantly mediated by galactose (Gal)- and N-acetyl-d-galactosamine (GalNAc)-inhibitable lectins. The intermediate subunit (Igl) of Gal-/GalNAc-inhibitable lectin is a cysteine-rich protein containing multiple CXXC motifs and is considered a key factor affecting trophozoite's pathogenicity. However, details of the function of Igl during parasite adherence remain unclear. Here, using segmentally expressed Igl proteins and a CHO cell model transfected with Igl fragments, we identified a carbohydrate-recognition domain (CRD)-like region between amino acids 989 and 1,088. Through single- and double-point mutations in the Igl segments, two core CXXC motifs responsible for carbohydrate recognition in the CRD-like region, which are highly conserved among several lectins, were confirmed. In addition to adhesion, the roles of CRD-like region and its core CXXC motifs in various pathogenic effects were further explored. To our knowledge, this is the first report showing an adhesion-related region in E. histolytica Igl. The identification and characterization of this CRD-like region provides further insights into molecular mechanisms underlying E. histolytica pathogenicity and also aids in the determination of a potential drug target in this parasite.ImportanceEntamoeba histolytica adhesion mainly depends on galactose (Gal)-/N-acetyl-d-galactosamine (GalNAc)-inhibitable lectins, subsequently triggering a series of amebic reactions. Among the three subunits of Gal-/GalNAc-inhibitable lectin, heavy subunit and intermediate subunit (Igl) have exhibited lectin activity, but that of Igl remains poorly understood. In this study, we confirmed a carbohydrate-recognition domain (CRD)-like limiting region in E. histolytica Igl and further identified its two core CXXC motifs responsible for carbohydrate recognition. Moreover, the role of Igl's CRD-like region and its CXXC motifs in hemolysis and pathogenic effects was explored. This is the first study to determine an adhesion-related region in E. histolytica Igl protein, providing a new reference direction for subsequent research studies. Since the potential homogeneity of galectin-2 in several mammals and Igl CRD-like region, it could be meaningful to relate the corresponding pathogeneses and phenotypes of these two proteins. Except for adhesion, studies on the involvement of Igl CRD-like region in different parasite-host interactions are also promising.

Dataset Information

Identification of Multiple Domains of Entamoeba histolytica Intermediate Subunit Lectin-1 with Hemolytic and Cytotoxic Activities.

Publications

Identification of Multiple Domains of <i>Entamoeba histolytica</i> Intermediate Subunit Lectin-1 with Hemolytic and Cytotoxic Activities.

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