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

Abstract

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.

Identification of the galactose-adherence lectin epitopes of Entamoeba histolytica that stimulate tumor necrosis factor-alpha production by macrophages.

“…Soluble amebic proteins were prepared after freeze-thaw lysis of trophozoites (26). Purification of the native Gal-lectin by mAb affinity chromatography has been described (19). In brief, octyl glucosidesolubilized amoebae were applied to a column consisting of purified anti-lectin mAbs H85, 7F4, 5B8, 3F4, and 6D2.…”

“…The native amebic Gal-lectin is a 260-kDa heterodimer glycoprotein composed of 170-kDa heavy and 31-to 35-kDa light subunits linked by disulfide bonds (19). The cysteine-rich extracellular domain of the 170-kDa heavy subunit mediates E. histolytica adherence to mammalian cells and colonic mucins (20).…”

The Mouse Model of Amebic Colitis Reveals Mouse Strain Susceptibility to Infection and Exacerbation of Disease by CD4+ T Cells

“…Log-phase growth trophozoites were iced and spun (900 ϫ g for 5 min) before intracecal inoculation. E. histolytica lectin was purified as described (16). Soluble amebic Ag (SAA) was obtained from the supernatant of axenic trophozoites washed in sterile HBSS, lysed by freeze-thaw, and spun (10,000 ϫ g for 10 min).…”

Cytopathogenicity of Entamoeba Histolytica to Human Intestinal Epithelial Cells: Inhibition by Monoclonal Antibodies and Sera From Amoebic Patients

“…Cytopathogenicity of E. histolytica trophozoites to intestinal epithelial cell (Henle -407) monolayers: A, control epithelial cell monolayers; E. histolytica trophozoites used were; B, untreated; C, pretreated with normal peritoneal fluid (NPF); D, pretreated with monoclonal antibody (MoAb C8); E, pretreated with sera from patients of amoebic liver abscess (ALA); F, pretreated with sera from healthy subjects; G, pretreated with antiamoebic HIS. (13)(14)(15). This may precisely be the reason for partial inhibition of adhesion/cytopathogenicity of amoebae to target cells.…”

“…The proteolytic enzymes like 56 kDa major neutral protease (9), several cysteine proteinases (10) and electron-dense granules with collagenelytic activity (11) have also been suggested to be associated with the invasive (virulence) potential of the amoebic trophozoites. In addition, amoebic lectins (12,13), 220 kDa protein (14), 112 kDa adhesin (15) (MoAb C8); e, sera from patients of amoebic liver abscess; f , sera from healthy subjects. Each set in duplicate wells stained with methylene blue was photographed.…”