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Protection against invasive amebiasis by a single monoclonal antibody directed against a lipophosphoglycan antigen localized on the surface of Entamoeba histolytica.

Marinets A, Zhang T, Guillén N, Gounon P, Bohle B, Vollmann U, Scheiner O, Wiedermann G, Stanley SL, Duchêne M - J. Exp. Med. (1997)

Bottom Line: Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A.Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess.Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

View Article: PubMed Central - PubMed

Affiliation: Institute for Specific Prophylaxis and Tropical Medicine, A-1095 Vienna, Austria.

ABSTRACT
A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

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Metabolic labeling experiments. (A) Labeling with [3H]glucose. Lane 1, total PBS washed amebas; lane 2, fraction E, immunoprecipitated with antibody EH5; lane 3, fraction E, immunoprecipitated with  isotype-matched control antibody BIP 1; lane 4, fraction E, immunoprecipitated with amebiasis patient's serum. (B) Labeling with [32P]orthophosphate. Lane 1, fraction E immunoprecipitated with antibody EH5;  lane 2, fraction E immunoprecipitated with amebiasis patient's serum;  lane 3, fraction E immunoprecipitated with control antibody BIP 1. At  the left side, molecular masses from a protein marker are given for better  orientation. However, these cannot serve as size markers for molecules  such as lipophosphoglycans.
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Figure 2: Metabolic labeling experiments. (A) Labeling with [3H]glucose. Lane 1, total PBS washed amebas; lane 2, fraction E, immunoprecipitated with antibody EH5; lane 3, fraction E, immunoprecipitated with isotype-matched control antibody BIP 1; lane 4, fraction E, immunoprecipitated with amebiasis patient's serum. (B) Labeling with [32P]orthophosphate. Lane 1, fraction E immunoprecipitated with antibody EH5; lane 2, fraction E immunoprecipitated with amebiasis patient's serum; lane 3, fraction E immunoprecipitated with control antibody BIP 1. At the left side, molecular masses from a protein marker are given for better orientation. However, these cannot serve as size markers for molecules such as lipophosphoglycans.

Mentions: The patterns in the immunoblots resembled patterns that had been previously observed for lipophosphoglycan antigens (19, 20). To test whether the EH5 antigen copurified with lipophosphoglycans, whole E. histolytica SFL-3 trophozoites were delipidated, and glycolipids (fraction E) were extracted. An immunoblot of the fractions demonstrated that most of the EH5 antigen was extracted into fraction E (results not shown). To identify some of the components present in the EH5 antigen, a metabolic labeling experiment was performed. SFL-3 trophozoites were either labeled with [3H]glucose or [32P]orthophosphate. The radiolabeled amebas were washed and delipidated, and fraction E was obtained and resuspended in RIPA buffer. The preparation was preadsorbed with protein G–Sepharose to remove unspecific complexes. The samples were then incubated either with antibody EH5, with isotype-matched control antibody BIP 1 (31), or as a positive control with the serum from an amebiasis patient in a 1:500 dilution. Immune complexes were bound to protein G–Sepharose, precipitated, and analyzed by SDS-PAGE followed by fluorography or autoradiography. Fig. 2 A shows the results for the labeling with [3H]glucose. Only the patient's serum (Fig. 2 A, lane 4) and antibody EH5 (Fig. 2 A, lane 2), but not the control antibody BIP 1 (Fig. 2 A, lane 3), precipitated the antigen. This showed that radiolabeled glucose was incorporated into the EH5 antigen. Fig. 2 B shows the results for the labeling with [32P]orthophosphate. Whole amebas were labeled very heavily (data not shown). Again, antibody EH5 (Fig. 2 B, lane 1) and the patient's serum (Fig. 2 B, lane 2), but not the control antibody BIP 1 (Fig. 2 B, lane 3) precipitated a phosphate-labeled antigen. Thus, EH5 antigen also incorporated radiolabeled phosphate.


Protection against invasive amebiasis by a single monoclonal antibody directed against a lipophosphoglycan antigen localized on the surface of Entamoeba histolytica.

Marinets A, Zhang T, Guillén N, Gounon P, Bohle B, Vollmann U, Scheiner O, Wiedermann G, Stanley SL, Duchêne M - J. Exp. Med. (1997)

Metabolic labeling experiments. (A) Labeling with [3H]glucose. Lane 1, total PBS washed amebas; lane 2, fraction E, immunoprecipitated with antibody EH5; lane 3, fraction E, immunoprecipitated with  isotype-matched control antibody BIP 1; lane 4, fraction E, immunoprecipitated with amebiasis patient's serum. (B) Labeling with [32P]orthophosphate. Lane 1, fraction E immunoprecipitated with antibody EH5;  lane 2, fraction E immunoprecipitated with amebiasis patient's serum;  lane 3, fraction E immunoprecipitated with control antibody BIP 1. At  the left side, molecular masses from a protein marker are given for better  orientation. However, these cannot serve as size markers for molecules  such as lipophosphoglycans.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199119&req=5

Figure 2: Metabolic labeling experiments. (A) Labeling with [3H]glucose. Lane 1, total PBS washed amebas; lane 2, fraction E, immunoprecipitated with antibody EH5; lane 3, fraction E, immunoprecipitated with isotype-matched control antibody BIP 1; lane 4, fraction E, immunoprecipitated with amebiasis patient's serum. (B) Labeling with [32P]orthophosphate. Lane 1, fraction E immunoprecipitated with antibody EH5; lane 2, fraction E immunoprecipitated with amebiasis patient's serum; lane 3, fraction E immunoprecipitated with control antibody BIP 1. At the left side, molecular masses from a protein marker are given for better orientation. However, these cannot serve as size markers for molecules such as lipophosphoglycans.
Mentions: The patterns in the immunoblots resembled patterns that had been previously observed for lipophosphoglycan antigens (19, 20). To test whether the EH5 antigen copurified with lipophosphoglycans, whole E. histolytica SFL-3 trophozoites were delipidated, and glycolipids (fraction E) were extracted. An immunoblot of the fractions demonstrated that most of the EH5 antigen was extracted into fraction E (results not shown). To identify some of the components present in the EH5 antigen, a metabolic labeling experiment was performed. SFL-3 trophozoites were either labeled with [3H]glucose or [32P]orthophosphate. The radiolabeled amebas were washed and delipidated, and fraction E was obtained and resuspended in RIPA buffer. The preparation was preadsorbed with protein G–Sepharose to remove unspecific complexes. The samples were then incubated either with antibody EH5, with isotype-matched control antibody BIP 1 (31), or as a positive control with the serum from an amebiasis patient in a 1:500 dilution. Immune complexes were bound to protein G–Sepharose, precipitated, and analyzed by SDS-PAGE followed by fluorography or autoradiography. Fig. 2 A shows the results for the labeling with [3H]glucose. Only the patient's serum (Fig. 2 A, lane 4) and antibody EH5 (Fig. 2 A, lane 2), but not the control antibody BIP 1 (Fig. 2 A, lane 3), precipitated the antigen. This showed that radiolabeled glucose was incorporated into the EH5 antigen. Fig. 2 B shows the results for the labeling with [32P]orthophosphate. Whole amebas were labeled very heavily (data not shown). Again, antibody EH5 (Fig. 2 B, lane 1) and the patient's serum (Fig. 2 B, lane 2), but not the control antibody BIP 1 (Fig. 2 B, lane 3) precipitated a phosphate-labeled antigen. Thus, EH5 antigen also incorporated radiolabeled phosphate.

Bottom Line: Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A.Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess.Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

View Article: PubMed Central - PubMed

Affiliation: Institute for Specific Prophylaxis and Tropical Medicine, A-1095 Vienna, Austria.

ABSTRACT
A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

Show MeSH
Related in: MedlinePlus