Characterization of the galactose-specific binding activity of a purified soluble Entamoeba histolytica adherence lectin.
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 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 2.39 x 10(-8) M-1 with 5.97 x 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 x 10(5)/cell) rather than a significantly reduced dissociation constant (4.93 x 10(-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 micrograms/ml) to CHO cells was equivalent at 4 degrees C and 37 degrees C and unaltered by adding the microfilament inhibitor, Cytochalasin D (10 micrograms/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.(ABSTRACT TRUNCATED AT 250 WORDS)
Ravdin
J I
JI
Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio.
Murphy
C F
CF
D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D004748:Entamoeba histolytica / Q000235:genetics / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism*; D037102:Lectins / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D011863:Radioimmunoassay
D037102:Lectins; D005690:Galactose