title: Structural analysis of the light subunit of the Entamoeba histolytica galactose-specific adherence lectin.
abstract: Adherence of Entamoeba histolytica trophozoites to colonic mucins and resistance to lysis by the membrane attack complex of complement are mediated by a galactose- and N-acetyl-D-galactosamine-specific cell-surface lectin. This lectin is a heterodimeric glycoprotein of heavy (170 kDa) and light (35/31 kDa) subunits. In this work, the amino acid sequence and membrane anchor of the light subunit were analyzed. The light subunit cDNA encoded a protein with a calculated molecular mass of 32 kDa containing two potential sites for N-linked glycosylation and putative amino- and carboxyl-terminal signal sequences characteristic of glycosylphosphatidylinositol (GPI)-anchored proteins. No classical carbohydrate-binding domains common to C- or S-type eukaryotic lectins were detected by sequence analysis of either the heavy or light subunits, leaving the location of the ligand-binding site of the lectin unknown. Analysis of restriction enzyme-digested E. histolytica DNA by Southern blotting was consistent with the presence of more than one light subunit gene. Two light subunit isoforms of 31 and 35 kDa were identified by SDS-polyacrylamide gel electrophoresis analysis of affinity-purified lectin, and the isoforms were shown on two-dimensional gel analysis to form distinct 170/35- and 170/31-kDa heterodimers. The amino acid compositions and cyanogen bromide peptide patterns of the two light subunit isoforms were nearly identical. The 35-kDa isoform labeled more efficiently than the 31-kDa isoform with [3H]glucosamine, while only the 31-kDa isoform labeled with [3H]myristate and [3H]palmitate. Nitrous acid deamination released lipid from the 31-kDa isoform, which co-migrated on thin layer chromatography with acylphosphatidylinositol, a component of some GPI anchors. Gas chromatography and mass spectrometry of the deamination product from the 31-kDa subunit identified both myo- and chiro-inositols, supporting the presence of a GPI membrane 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.
authors.affiliation: Department of Microbiology, University of Virginia School of Medicine, Charlottesville 22908.
authors.forename: J J
authors.initials: JJ
authors.lastname: McCoy
authors.forename: B J
authors.initials: BJ
authors.lastname: Mann
authors.forename: T S
authors.initials: TS
authors.lastname: Vedvick
authors.forename: Y
authors.initials: Y
authors.lastname: Pak
authors.forename: D B
authors.initials: DB
authors.lastname: Heimark
authors.forename: W A
authors.initials: WA
authors.lastname: Petri
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003488:Cyanogen Bromide; D016054:DNA, Protozoan; D015180:Electrophoresis, Gel, Two-Dimensional; D004748:Entamoeba histolytica / Q000378:metabolism*; D005690:Galactose; D008401:Gas Chromatography-Mass Spectrometry; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006868:Hydrolysis; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D009608:Nitrous Acid / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010449:Peptide Mapping; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010739:Phospholipase D / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D016054:DNA, Protozoan; D017261:Glycosylphosphatidylinositols; D037102:Lectins; D009227:Myristic Acids; D010169:Palmitic Acids; D015800:Protozoan Proteins; D019814:Myristic Acid; C078014:N-acetylgalactosamine inhibitable lectin 35-kDa subunit, Entamoeba histolytica; D019308:Palmitic Acid; D010727:Phosphoric Diester Hydrolases; D010739:Phospholipase D; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D003488:Cyanogen Bromide; D009608:Nitrous Acid; D005690:Galactose