The Cysteine-Rich Region of the Entamoeba histolytica Adherence Lectin (170-Kilodalton Subunit) Is Sufficient for High-Affinity Gal/GalNAc-Specific Binding In Vitro

AUTOR(ES)

Pillai, Dylan R.

FONTE

American Society for Microbiology

RESUMO

Adherence of Entamoeba histolytica trophozoites to colonic mucin, epithelium, and other target cells is mediated by the amebic Gal/GalNAc lectin. We constructed in vitro expression vectors containing full-length (residues 1 to 1280), cysteine-poor (1 to 353 and 1 to 480), and cysteine-rich (356 to 1143 and 480 to 900) fragments of the gene encoding the heavy subunit of the adherence lectin, hgl2. In vitro transcription followed by translation using a nuclease-treated rabbit reticulocyte lysate system was carried out. Immunoreactivity of in vitro-translated Hgl2 was confirmed by immunoprecipitation with lectin-specific monoclonal antibodies (MAbs) 1G7 and 8A3, which recognize linear epitopes. Protein disulfide isomerase (PDI) refolding of Hgl2 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 inhibited 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-length (1 to 1280) and cysteine-rich (356 to 1143) Hgl2 bound the glyconeoconjugate GalNAc19BSA in a GalNAc-specific manner. The smaller cysteine-rich fragment (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 cysteine-poor fragment (1 to 480), luciferase (protein control), nor control translation reactions (without hgl2 lectin mRNA) bound GalNAc19BSA. Binding to GalNAc19BSA was shown to be dependent on the concentration of GalNAc19BSA coated in each well or 35S-lectin added (KD = 0.85 ± 0.37 pM). Binding was competitively inhibited by the terminal GalNAc-containing glycoprotein asialofetuin (P < 0.005). Taken together, these data provide direct evidence that the cysteine-rich region of the Gal/GalNAc lectin heavy subunit contains one or more carbohydrate-binding domains.

Documentos Relacionados

• Sequence of a cysteine-rich galactose-specific lectin of Entamoeba histolytica.
• Neutralizing monoclonal antibody epitopes of the Entamoeba histolytica galactose adhesin map to the cysteine-rich extracellular domain of the 170-kilodalton subunit.
• Protection of Gerbils from Amebic Liver Abscess by Vaccination with a 25-mer Peptide Derived from the Cysteine-Rich Region of Entamoeba histolytica Galactose-Specific Adherence Lectin
• A cysteine-rich domain of the “mannose” receptor mediates GalNAc-4-SO4 binding
• VH3 Gene Usage in Neutralizing Human Antibodies Specific for the Entamoeba histolytica Gal/GalNAc Lectin Heavy Subunit