Subunit structure of the galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica

W A Petri,M D Chapman,T Snodgrass,B J Mann,J Broman,J I Ravdin

doi:10.1016/s0021-9258(19)81714-3

Abstract

The galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica is a cell surface protein which mediates parasite adherence to human colonic mucus, colonic epithelial cells, and other target cells. The amebic lectin was purified in 100-micrograms quantities from detergent-solubilized trophozoites by monoclonal antibody affinity chromatography. The adherence lectin was purified 500-fold as judged by radioimmunoassay. The nonreduced lectin had a molecular mass of 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of pH 6.2. The amebic lectin reduced with beta-mercaptoethanol consisted of 170- and 35-kDa subunits. Both subunits could be labeled on the cell surface with 125I, and both were metabolically labeled with [3H]glucosamine. The amino termini of the subunits had unique amino acid sequences, and polyclonal antisera to the heavy subunit did not cross-react with the light subunit. The yield of phenylthiohydantoin derivatives from the second and third positions in the sequence of the heavy and light subunits gave a molar ratio of one 170- to one 35-kDa subunit. Antibodies directed to the heavy subunit inhibited amebic adherence to Chinese hamster ovary cells by 100%, suggesting that the heavy subunit is predominantly responsible for mediating amebic adherence.

Highlights

Itable adherence lectin of Entamoeba histolytica is a histolytica, resulting in at least 60,000 deaths worldwide each cell surface protein which mediates parasite adherenyceear [1, 2]
By using Chinese hamster ovary (CHO)' cells as subunits. Both subunits could be labeled on the cell target cells, amebic adherence was studied in the absence of surface with "'1, and bothwere metabolically labeled CHO cell lysis at 4 "C
The amino termini of the sub- mediated by an amebic adhesion protein with carbohydrateunits had unique amino acid sequences, and polyclonabilnding properties: galactose (Gal) or N-acetyl-D-galactosantisera to the heavy subunit did not cross-reawcitth amine (GalNAc)completely inhibited E. histolytica adherence thelightsubunit.The yield ofphenylthiohydantoin to CHOcells, whereas other simple carbohydrates had no derivatives from the second and third positions in the effect [8]

Summary

Introduction

Itable adherence lectin of Entamoeba histolytica is a histolytica, resulting in at least 60,000 deaths worldwide each cell surface protein which mediates parasite adherenyceear [1, 2]. By using Chinese hamster ovary (CHO)' cells as subunits Both subunits could be labeled on the cell target cells, amebic adherence was studied in the absence of surface with "'1, and bothwere metabolically labeled CHO cell lysis at 4 "C. The amino termini of the sub- mediated by an amebic adhesion protein with carbohydrateunits had unique amino acid sequences, and polyclonabilnding properties: galactose (Gal) or N-acetyl-D-galactosantisera to the heavy subunit did not cross-reawcitth amine (GalNAc)completely inhibited E. histolytica adherence thelightsubunit.The yield ofphenylthiohydantoin to CHOcells, whereas other simple carbohydrates had no derivatives from the second and third positions in the effect [8]. Gal or GalNAc prevented the subsequent sequence of theheavy and light subunitgsave a molar contact-dependent lysis of the CHO cells at 37 "C(8-12). This ratio of one 170- to one 35-kDa subunit. Monoclonal and polyclonal antibodies tions occurring at theorganismal level

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