The cysteine-rich region of the Entamoeba histolytica adherence lectin (170-kilodalton subunit) is sufficient for high-affinity Gal/GalNac-specific binding in vitro

Adherence of Entamoeba histolytica trophozoites to colonic mucin, epitheliu m, and other target cells is mediated by the amebic Gal/GalNAc lectin. We c onstructed in vitro expression vectors containing full-length (residues 1 t o 1280), cysteine-poor (1 to 353 and 1 to 480), and cysteine-rich (356 to 1 143 and 480 to 900) fragments of the gene encoding the heavy subunit of the adherence lectin, hgl2. In vitro transcription followed by translation usi ng a nuclease-treated rabbit reticulocyte lysate system was carried out. Im munoreactivity of in vitro-translated Hgl2 was confirmed by immunoprecipita tion with lectin-specific monoclonal antibodies (MAbs) 1G7 and 8A3, which r ecognize linear epitopes. Protein disulfide isomerase (PDI) refolding of Hg l2 enhanced immunoreactivity (P < 0.05) with the conformationally dependent MAb 3F4. Binding of PDI-refolded full-length (P < 0.001) and cysteine-rich (P = 0.005) Hgl2 to CHO cells was galactose dependent and competitively in hibited by native hololectin (50% inhibitory concentration of 39.6 ng/ml), The cysteine-poor region (1 to 353) did not bind CHO cells. Both full-lengt h (1 to 1280) and cysteine-rich (356 to 1143) Hgl2 bound the glyconeoconjug ate GalNAc(19)BSA in a GalNAc-specific manner. The smaller cysteine-rich fr agment (480 to 900) also exhibited GalNAc-specific binding but to a lesser extent (P < 0.05) than residues 1 to 1280 and 356 to 1143. Neither the cyst eine-poor fragment (1 to 480), luciferase (protein control), nor control tr anslation reactions (without hgl2 lectin mRNA) bound GalNAc(19)BSA. Binding to GalNAc(19)BSA was shown to be dependent on the concentration of GalNAc( 19)BSA coated in each well or S-35-lectin added (K-D = 0.85 +/- 0.37 pM). B inding was competitively inhibited by the terminal GalNAc-containing glycop rotein asialofetuin (P < 0.005), Taken together, these data provide direct evidence that the cysteine-rich region of the Gal/GalNAc lectin heavy subun it contains one or more carbohydrate-binding domains.