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The World of Protozoa, Rotifera, Nematoda and Oligochaeta

Ref ID : 4365

Jonathan I. Ravdin and Cheryl F. Murphy; Characterization of the Galactose-Specific Binding Activity of a Purifed Soluble Entamoeba histolytica Adherence Lectin. J.Protozool. 39(2):319-323, 1992

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We studied galactose (Gal)-specific binding of the soluble purified 260-kDa Entamoeba histolytica adherence protein into glycosylation deficient Chinese hamster ovary (CHO) cell mutants. Our goal was to further define the lectin's functional activity and carbohydrate receptor specificity. The adherence protein was purified by acid elution from an immunoaffinity column; however, exposure of the surface membrane lectin on viable trophozoites to identical acid pH conditions had no effect on carbohydrate binding activity. Saturable Gal-specific binding of soluble lectin to parental CHO cells was demonstrated at 4 degrees C by radioimmunoassay; the dissociation coefficient (Kd) was 2.39x10E-8 M(-1) with 5.97x10E4 lectin receptors present per CHO cell. Gal-specific binding of lectin to Lec2 CHO cells mutants, which have increased N- and O-linked terminal Gal residues on cell surface carbohydrates, was increased due to an enhanced number of receptors (2.41x10E5/cell) rather than a significantly reduced dissociation constant (4.93x10E-8 M(-1)). At 4 degrees C, there was no measurable Gal-specific binding of the adherence protein to the Lec1 and 1dlD. Lec1 CHO mutants, which contain surface carbohydrates deficient in terminal Gal residues. Binding of lectin (20 µg/ml) to CHO cells was equivalent at 4 degrees C and 37 degrees C and unaltered by adding the microfilament inhibitor, Cytochalasin D (10 µg/ml). Gal-specific binding of the lectin at 4 degrees C was calcium independent and reduced by 81% following adsorption of only 0.2% of the lectin to CHO cells. In summary, these findings indicate that the purified E. histolytica adherence lectin demonstrates saturable Gal-specific binding to beta 1-6 branched-N-linked and not O-linked galactose terminal cell surface carbohydrates; however, apparently only a small percentage of purified amebic lectin molecules actually possess galactose binding activity.