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Characterization of the Galactose‐Specific Binding Activity of a Purified Soluble Entamoeba histolytica Adherence Lectin
Author(s) -
RAVDIN JONATHAN I.,
MURPHY CHERYL F.
Publication year - 1992
Publication title -
the journal of protozoology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 0022-3921
DOI - 10.1111/j.1550-7408.1992.tb01322.x
Subject(s) - chinese hamster ovary cell , lectin , biochemistry , galactose , cd69 , receptor , ficolin , affinity chromatography , microbiology and biotechnology , c type lectin , cell surface receptor , mutant , entamoeba histolytica , biology , chemistry , mannan binding lectin , in vitro , enzyme , cytotoxic t cell , il 2 receptor , gene
. We studied galactose (Gal)‐specific binding of the soluble purified 260‐kDa Entamoeba histolytica adherence protein to 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 immunoaffnity 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°C by radioimmunoassay; the dissociation coefficient (Kd was 2.39 × 10 −8 M −1 with 5.97 × 10 4 lectin receptors present per CHO cell. Gal‐specific binding of lectin to Lec2 CHO cell mutants, which have increased N‐ and O‐linked terminal Gal residues on cell surface carbohydrates, was increased due to an enhanced number of receptors (2.41 × 10 5 /cell) rather than a significantly reduced dissociation constant (4.93 × 10 −8 M −1 ). At 4°C, there was no measurable Gal‐specific binding of the adherence protein to the Lec and IdlD.Lecl CHO mutants, which contain surface carbohydrates deficient in terminal Gal residues. Binding of lectin (20 μg/ml) to CHO cells was equivalent at 4°C and 37°C and unaltered by adding the microfilament inhibitor, Cytochalasin D (10 μg/ml). Gal‐specific binding of the lectin at 4°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 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.