STRUCTURAL-ANALYSIS OF THE LIGHT SUBUNIT OF THE ENTAMOEBA-HISTOLYTICAGALACTOSE-SPECIFIC ADHERENCE LECTIN

Adherence of Entamoeba histolytica trophozoites to colonic mucins and resistance to lysis by the membrane attack complex of complement are m ediated by a galactose- and N-acetyl-D-galactosamine-specific cell-sur face lectin. This lectin is a heterodimeric glycoprotein of heavy (170 kDa) and light (35/31 kDa) subunits. In this work, the amino acid seq uence and membrane anchor of the light subunit were analyzed. The ligh t subunit cDNA encoded a protein with a calculated molecular mass of 3 2 kDa containing two potential sites for N-linked glycosylation and pu tative amino- and carboxyl-terminal signal sequences characteristic of glycosylphosphatidylinositol (GPI)-anchored proteins. No classical ca rbohydrate-binding domains common to C- or S-type eukaryotic lectins w ere detected by sequence analysis of either the heavy or light subunit s, leaving the location of the ligand-binding site of the lectin unkno wn. Analysis of restriction enzyme-digested E. histolytica DNA by Sout hern blotting was consistent with the presence of more than one light subunit gene. Two light subunit isoforms of 31 and 35 kDa were identif ied by SDS-polyacrylamide gel electrophoresis analysis of affinity-pur ified lectin, and the isoforms were shown on two-dimensional gel analy sis to form distinct 170/35- and 170/31-kDa heterodimers. The amino ac id compositions and cyanogen bromide peptide patterns of the two light subunit isoforms were nearly identical. The 35-kDa isoform labeled mo re efficiently than the 31-kDa isoform with [H-3] glucosamine, while o nly the 31-kDa isoform labeled with [H-3]myristate and [H-3]palmitate. Nitrous acid deamination released lipid from the 31-kDa isoform, whic h co-migrated on thin layer chromatography with acylphosphatidylinosit ol, a component of some GPI anchors. Gas chromatography and mass spect rometry of the deamination product from the 31-kDa subunit identified both myo- and chiro-inositols, supporting the presence of a GPI membra ne anchor. The covalent association of a transmembrane protein with a GPI-anchored protein, as suggested by the cDNA sequences of the lectin heavy and light subunits, is novel and suggests unique roles for the two subunits in the pathogenesis of amebiasis.