Neutralizing monoclonal antibody epitopes of the Entamoeba histolytica galactose adhesin map to the cysteine-rich extracellular domain of the 170-kilodalton subunit

Abstract

Entamoeba histolytica adheres to human colonic mucins and colonic epithelial cells via a galactose-binding adhesin. The adhesin is a heterodimeric glycoprotein composed of 170-and 35-kDa subunits. Fragments of the hgll gene encoding the 170-kDa subunit were expressed as recombinant fusion proteins in Escherichia coli and reacted with anti-adhesin monoclonal antibodies (MAbs) or pooled human immune sera. The MAbs tested recognize seven distinct epitopes on the 170-kDa subunit and have distinct effects on the adherence and complement-inhibitory activities of the adhesin. All seven MAbs reacted with a fusion protein containing the cysteine-rich domain of the protein. Pooled human immune sera reacted with the same cysteine-rich domain as the MAbs and also with a construct containing the first 596 amino acids. Reactivity of three MAbs with the surface of intact trophozoites confirmed that the cysteine-rich domain was located extracellularly. The location of individual epitopes was fine mapped by constructing carboxy-terminal deletions in the cysteine-rich region of the fusion protein. The locations of adherence-enhancing and-inhibiting epitopes were partially distinguished, and the epitopes where complement-inhibitory MAbs bound were demonstrated to be near the adhesin's area of sequence identity with the human complement inhibitor CD59.

Identification of a Gal/GalNAc Lectin in the Protozoan Hartmannella vermiformis as a Potential Receptor for Attachment and Invasion by the Legionnaires' Disease Bacterium

“…3 B showed that the antiserum immunoprecipitated a 170-kD protein which was tyrosine dephosphorylated after 5 min of infection. The identity of the 170-kD lectin was further confirmed by Western blotting of total amebal lysates using a mouse mAb which recognizes the Gal/GalNAc lectin of E. histolytica (28). The mAb bound to a 170-kD protein of H. vermiformis (Fig.…”

“…The cells were pelleted and resuspended in fresh serum-free medium at 10 7 CFU/ml. The amebas were incubated in triplicate on ice in the presence of the mAbs 1G7, H85, BC6, 7F4, 8C12 (28,32,33), or a nonspecific mouse IgG mAb for 45 min, followed by 15 min at 37ЊC. Prewarmed L. pneumophila was added to amebas, and the infection was allowed to proceed for 2 h at 37ЊC.…”

Two cysteine protease inhibitors, EhICP1 and 2, localized in distinct compartments, negatively regulate secretion in Entamoeba histolytica

“…To verify the distinct localization of EhICP1 and EhICP2, the amoeba lysates were subjected to differential centrifugation at 5000 · g and 100 000 · g, and immunoblot analyses with antibodies against EhICP1, EhICP2, CP5, Gal/GalNAc lectin heavy subunit [31], and NifU (a cytosolic scaffold protein necessary for the biosynthesis of iron-sulfur clusters [30]). EhICP2 was detected in both the 100 000 · g pellet and the 5000 · g pellet fractions (and also the 5000 · g supernatant), similar to CP5, while EhICP1 was associated with the 100 000 · g supernatant, similar to NifU.…”

“…The supernatant was subsequently ultracentrifuged at 100 000 · g for 20 min at 4°C. These supernatants and pellets were subjected to immunoblot analyses with anti-EhICP1, anti-EhICP2, anti-NifU [30], anti-CP5, and anti-Hgl IgGs the last of (a gift by Barbara J. Mann and William A. Petri, Jr.) [31].…”

Complement Resistance of Parasites

“…The smaller part (35 kDa) of the adhesin is GPI-anchored like the human C regulators DAF and protectin [54], but the C regulating activity of the adhesin resides in a cysteine-rich domain of the transmembrane 170 kDa subunit. A section of the 170 kDa subunit has been suggested to show antigenic cross-reactivity and limited sequence similarity with human protectin [53,55].…”