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Molecular cloning of a 30-kilodalton lysine-rich surface antigen from a nonpathogenic Entamoeba histolytica strain and its expression in a pathogenic strain.

ABSTRACT: A monoclonal antibody (MAb), 318-28, that specifically reacts with a 30-kDa antigen present on membrane surfaces of all nonpathogenic (NP) Entamoeba histolytica strains tested and which did not react with pathogenic (P) strains was used for the isolation of the cDNA coding for this antigen from an expression library of an NP E. histolytica strain. The deduced amino acid composition was rich in lysine residues (14.5%), with some sequence similarity to a polyadenylate-binding protein. Southern and Northern (RNA) blot analyses, as well as amplifications of DNA segments by PCR, indicate that a very similar gene (identity of 96.5%) exists in P strains of E. histolytica. Unexpectedly, the NP-specific antigen was also identified by MAb 318-28 on the surfaces of a cloned, xenically cultivated and well-characterized P strain (BNI:0591) that was recently isolated from a human liver abscess. Binding of the MAb, both to the cell surfaces and to Western blots (immunoblots), was abolished, however, upon axenization of the BNI:0591 cultures. Oligonucleotide primers, designed to anneal only to specific DNA sequences of the NP 30-kDa protein gene copy, amplified a DNA segment from P strain BNI:0591 which was identical in sequence to that of the NP 30-kDa protein gene. Our findings indicate that a P strain of E. histolytica can possess and express, under certain growth conditions, an antigen that is usually detected only in NP strains.

SUBMITTER: Bracha R

PROVIDER: S-EPMC173090 | biostudies-other | 1995 Mar

REPOSITORIES: biostudies-other

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Molecular cloning of a 30-kilodalton lysine-rich surface antigen from a nonpathogenic Entamoeba histolytica strain and its expression in a pathogenic strain.

Bracha R R Nuchamowitz Y Y Mirelman D D

Infection and immunity 19950301 3

A monoclonal antibody (MAb), 318-28, that specifically reacts with a 30-kDa antigen present on membrane surfaces of all nonpathogenic (NP) Entamoeba histolytica strains tested and which did not react with pathogenic (P) strains was used for the isolation of the cDNA coding for this antigen from an expression library of an NP E. histolytica strain. The deduced amino acid composition was rich in lysine residues (14.5%), with some sequence similarity to a polyadenylate-binding protein. Southern and ...[more]

PMID: 7868264

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Project description:Adherence of Entamoeba histolytica trophozoites to colonic mucin, epithelium, and other target cells is mediated by the amebic Gal/GalNAc lectin. We constructed in vitro expression vectors containing full-length (residues 1 to 1280), cysteine-poor (1 to 353 and 1 to 480), and cysteine-rich (356 to 1143 and 480 to 900) fragments of the gene encoding the heavy subunit of the adherence lectin, hgl2. In vitro transcription followed by translation using a nuclease-treated rabbit reticulocyte lysate system was carried out. Immunoreactivity of in vitro-translated Hgl2 was confirmed by immunoprecipitation with lectin-specific monoclonal antibodies (MAbs) 1G7 and 8A3, which recognize linear epitopes. Protein disulfide isomerase (PDI) refolding of Hgl2 enhanced immunoreactivity (P < 0.05) with the conformationally dependent MAb 3F4. Binding of PDI-refolded full-length (P < 0.001) and cysteine-rich (P = 0.005) Hgl2 to CHO cells was galactose dependent and competitively inhibited by native hololectin (50% inhibitory concentration of 39.6 ng/ml). The cysteine-poor region (1 to 353) did not bind CHO cells. Both full-length (1 to 1280) and cysteine-rich (356 to 1143) Hgl2 bound the glyconeoconjugate GalNAc(19)BSA in a GalNAc-specific manner. The smaller cysteine-rich fragment (480 to 900) also exhibited GalNAc-specific binding but to a lesser extent (P < 0.05) than residues 1 to 1280 and 356 to 1143. Neither the cysteine-poor fragment (1 to 480), luciferase (protein control), nor control translation reactions (without hgl2 lectin mRNA) bound GalNAc(19)BSA. Binding to GalNAc(19)BSA was shown to be dependent on the concentration of GalNAc(19)BSA coated in each well or (35)S-lectin added (K(D) = 0.85 +/- 0.37 pM). Binding was competitively inhibited by the terminal GalNAc-containing glycoprotein asialofetuin (P < 0.005). Taken together, these data provide direct evidence that the cysteine-rich region of the Gal/GalNAc lectin heavy subunit contains one or more carbohydrate-binding domains.

Project description:BackgroundAmoebiasis is the third most common parasitic cause of morbidity and mortality, particularly in countries with poor hygienic settings. There exists an ambiguity in the diagnosis of amoebiasis, and hence there arises a necessity for a better diagnostic approach. Serine-rich Entamoeba histolyticaprotein (SREHP), peroxiredoxin and Gal/GalNAc lectin are pivotal in E. histolyticavirulence and are extensively studied as diagnostic and vaccine targets. For elucidating the cellular function of these proteins, details regarding their respective quaternary structures are essential. However, studies in this aspect are scant. Hence, this study was carried out to predict the structure of these target proteins and characterize them structurally as well as functionally using appropriate in-silicomethods.MethodsThe amino acid sequences of the proteins were retrieved from National Centre for Biotechnology Information database and aligned using ClustalW. Bioinformatic tools were employed in the secondary structure and tertiary structure prediction. The predicted structure was validated, and final refinement was carried out.ResultsThe protein structures predicted by i-TASSER were found to be more accurate than Phyre2 based on the validation using SAVES server. The prediction suggests SREHP to be an extracellular protein, peroxiredoxin a peripheral membrane protein while Gal/GalNAc lectin was found to be a cell-wall protein. Signal peptides were found in the amino-acid sequences of SREHP and Gal/GalNAc lectin, whereas they were not present in the peroxiredoxin sequence. Gal/GalNAc lectin showed better antigenicity than the other two proteins studied. All the three proteins exhibited similarity in their structures and were mostly composed of loops.DiscussionThe structures of SREHP and peroxiredoxin were predicted successfully, while the structure of Gal/GalNAc lectin could not be predicted as it was a complex protein composed of sub-units. Also, this protein showed less similarity with the available structural homologs. The quaternary structures of SREHP and peroxiredoxin predicted from this study would provide better structural and functional insights into these proteins and may aid in development of newer diagnostic assays or enhancement of the available treatment modalities.

Dataset Information

Molecular cloning of a 30-kilodalton lysine-rich surface antigen from a nonpathogenic Entamoeba histolytica strain and its expression in a pathogenic strain.

Publications

Molecular cloning of a 30-kilodalton lysine-rich surface antigen from a nonpathogenic Entamoeba histolytica strain and its expression in a pathogenic strain.

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