Identification of the 150-kDa surface antigen of Entamoeba histolytica as a galactose- and N  -acetyl- d -galactosamine-inhibitable lectin

Abstract

A monoclonal antibody that reacts with a 150-kDa protein of Entamoeba histolytica on Western immunoblotting under nonreducing conditions inhibits the adherence and cytotoxicity of the ameba to mammalian cells in vitro. Affinity purification of solubilized trophozoites using the monoclonal antibody and electrophoresis yielded three glycoproteins with molecular masses of 150, 170, and 260 kDa, suggesting the existence of either a common epitope or the close association of these proteins. The 260-kDa fraction was identified as the well-known galactose (Gal)- and N-acetyl-D-galactosamine (GalNAc)-inhibitable lectin. The 150- and 170-kDa fractions seemed to exist as part of a 380-kDa native protein with an isoelectric point of pH 6.9. The N-terminal amino acid sequence of the 150-kDa protein was unique, indicating that the protein was not a degraded product of the 260-kDa lectin. By gel filtration, the 260-kDa lectin and the 150/170-kDa protein could be separated. When Chinese hamster ovary cells were pretreated with the fraction consisting of the 150/170-kDa protein the adherence of trophozoites to Chinese hamster ovary cells was competitively inhibited to a level equivalent to that observed for the 260-kDa lectin. The inhibitory effect was lost in the presence of Gal and GalNAc but was not influenced by the presence of glucose. These results demonstrate that the 150/170-kDa protein is a Gal/GalNAc-inhibitable lectin. The existence of a sugar-binding domain in the protein was confirmed by Gal-affinity chromatography.

Evaluation of Recombinant Fragments of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit for Serodiagnosis of Amebiasis

“…This observation is coincident with the previous data in which affinity-purified native protein was recognized by sera from symptomatic and asymptomatic individuals in a Western immunoblot analysis (8).…”

“…In the previous study, we examined the reactivity of sera from ameba-infected patients to affinity purified Igl by Western immnoblot analysis. The native Igl was recognized by all sera from not only symptomatic patients but also asymptomatic cyst passers (8). However, the possibility that copurified Hgl might affect the reactivity of Igl could not be excluded.…”

“…Hgl is a transmembrane protein that assumes a heterodimeric conformation that conforms with glycosylphosphatidylinositol (GPI)-anchored 31/35-kDa light subunit by disulfide bonds (15). We recently identified a GPI-anchored 150-kDa intermediate subunit (Igl) of lectin, which is noncovalently associated with Hgl (4,8). A mouse monoclonal antibody specific for Igl significantly inhibits adherence and cytotoxicity of trophozoites to mammalian cells, erythrophagocytosis, and liver abscess formation in hamsters (5,7,23).…”

Rab5-associated Vacuoles Play a Unique Role in Phagocytosis of the Enteric Protozoan Parasite Entamoeba histolytica

“…One intriguing hypothesis to explain why PPV formation is specifically induced by red blood cells is that PPV is required for the degradation and/or detoxification of the content of red blood cells. It was previously reported that amoebae recognizes surface glycans with Gal␤1-4GlcNAc terminal glycosphingolipid on red blood cells (64) by a Gal/GalNAc-inhibitable lectin (21,22). The Gal␤1-4GlcNAc terminal glycosphingolipid is absent on the surface of latex beads, yeast, and E. coli (65,66).…”

“…Phagocytosis has been implicated to be closely associated with the pathogenesis of the amoeba because phagocytosis-deficient amoeba mutants were shown to be avirulent (20). Although a number of amoebic molecules involved in attachment, phagocytosis, and degradation of microorganisms and host cells have been identified including galactose/N-acetylgalactosamine (Gal/GalNAc)-inhibitable lectin (21,22), cytoskeletal proteins and their associated regulatory molecules (23)(24)(25), cysteine proteinases (CP), 1 and pore-forming peptides (i.e. amoebapores) (26,27), the molecular mechanism of phagocytosis in this parasite remains largely unknown.…”