Adherence Lectin Research Articles

Isolation of the galactose-binding lectin that mediates the in vitro adherence of Entamoeba histolytica.

Entamoeba histolytica adheres to human colonic mucus, colonic epithelial cells, and other target cells via a galactose (Gal) or N-acetyl-D-galactosamine (GalNAc) inhibitable surface lectin. Blockade of this adherence lectin with Gal or GalNAc in vitro prevents amebic killing of target cells. We have identified and purified the adherence lectin by two methods: affinity columns derivatized with galactose monomers or galactose terminal glycoproteins, and affinity columns and immunoblots prepared with monoclonal antibodies that inhibit amebic adherence. By both methods the adherence lectin was identified as a 170-kD secreted and membrane-bound amebic protein. The surface location of the lectin was confirmed by indirect immunofluorescence. Purified lectin competitively inhibited amebic adherence to target cells by binding to receptors on the target Chinese hamster ovary cells in a Gal-inhibitable manner.

Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica.

Establishment of adherence by Entamoeba histolytica is mediated by a 170-kD Gal/GalNAc inhibitable lectin and is required for cytolysis and phagocytosis of mammalian target cells. We studied the biochemical mechanisms of the in vitro interaction between rat and human colonic mucins and axenic E. histolytica trophozoites. Crude mucus prevented amebic adherence to Chinese hamster ovary (CHO) cells by up to 70%. Purification of the colonic mucins by Sepharose 4B chromatography, nuclease digestion, and cesium chloride gradient centrifugation resulted in a 1,000-fold enrichment of the inhibitory mucins. Purified rat mucin inhibited amebic adherence to and cytolysis of homologous rat colonic epithelial cells. Oxidation and enzymatic cleavage of rat mucin Gal and GalNAc residues completely abrogated mucin inhibition of amebic adherence. The binding of rat 125I-mucin to amebae was galactose specific, saturable, reversible, and pH dependent. A monoclonal antibody specific for the 170-kD amebic Gal/GalNAc lectin completely inhibited the binding of rat 125I-mucin. Rat mucin bound to Affigel affinity purified the amebic lectin from conditioned medium. Colonic mucin glycoproteins act as an important host defense by binding to the parasite's adherence lectin, thus preventing amebic attachment to and cytolysis of host epithelial cells.

Recognition of the galactose- or N-acetylgalactosamine-binding lectin of Entamoeba histolytica by human immune sera.

Cure of amebic liver abscess is associated with resistance to recurrent invasive amebiasis and the development of a humoral and cell-mediated immune response. We determined whether human immune sera contain blocking antibody for the 170-kilodalton (kDa) galactose or N-acetylgalactosamine (Gal/GalNAc)-binding lectin of Entamoeba histolytica. By Western blot (immunoblot) of whole amebae subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all eight immune sera studied here prominently recognized a 170-kDa amebic protein. Western blot of the purified Gal/GalNAc lectin with pooled human immune sera (PHIS) confirmed that the 170-kDa band was the adherence lectin. Immunoprecipitation of [35S]methionine-metabolically-labeled amebae with the antilectin monoclonal antibody H8-5 and with PHIS demonstrated that the 170-kDa lectin was the major antigen recognized by PHIS. The in vitro adherence of E. histolytica trophozoites to CHO cells at 4 degrees C was inhibited by prior exposure of amebae to greater than or equal to 1.0% PHIS. The humoral response to the Gal/GalNAc-binding lectin of the parasite may contribute to the development of protective immunity against invasive amebiasis.

The interaction of human T‐lymphocytes and Entamoeba histolytica: killing of virulent amoebae by lectin‐dependent lymphocytes

Clinical and experimental studies indicate that following invasive disease due to Entamoeba histolytica, development of human cell-mediated immune mechanisms may provide protective immunity. Activated, human monocyte-derived macrophages in vitro can kill virulent axaenic amoebic trophozoites. This study describes the interaction of lectin-stimulated T-lymphocytes and E. histolytica trophozoites (virulent strain HM1-IMSS). Amoebae progressively killed unstimulated nonimmune T-lymphocytes over 18 h incubation with no effect on amoebic viability. T-lymphocytes, stimulated with phytohaemagglutinin (PHA), were progressively cytotoxic for virulent HMI amoebae over 18 h incubation, but were also reduced in viability themselves. Lymphocyte cytotoxicity for amoebae was absent if PHA was removed before or added only during the assay. PHA-stimulated T-lymphocytes killed amoebae at cell ratios of lymphocytes to amoebae as low as 50:1 and cytotoxicity was antibody-independent. PHA-stimulated T-lymphocytes, depleted of T8-bearing cells by complement-mediated lysis, were unable to kill amoebae. Adherence of PHA-stimulated T-lymphocytes to amoebae was greater than with unstimulated T-lymphocytes. Inhibition of the amoebic adherence lectin with N-acetyl-D-galactosamine decreased lymphocyte-amoebic adherence and resulted in increased lymphocyte amoebicidal activity and lymphocyte survival. Suspension of amoebae with or without adherent PHA-stimulated T-lymphocytes in a 10% dextran solution indicated that cytotoxicity was contact dependent. In summary, PHA-stimulated T-lymphocytes of the T8-phenotype can kill virulent axaenic E. histolytica trophozoites through a contact-dependent, antibody-independent mechanism.