Intermediate Subunit of the Gal/GalNAc Lectin of Entamoeba histolytica Is a Member of a Gene Family Containing Multiple CXXC Sequence Motifs

Abstract

Killing by Entamoeba histolytica requires parasite adherence to host galactose-and N-acetyl-D-galactosamine (Gal/GalNAc)-containing cell surface receptors. A 260-kDa heterodimeric E. histolytica Gal/GalNAc lectin composed of heavy (Hgl) and light (Lgl) subunits has been previously described. Here we present the cloning and characterization of Igl, a 150-kDa intermediate subunit of the Gal/GalNAc lectin. Igl, Hgl, and Lgl colocalized on the surface membrane of trophozoites. Two unlinked copies of genes encoding Igl shared 81% amino acid sequence identity (GenBank accession no. AF337950 and AF337951). They encoded cysteine-rich proteins with amino-and carboxy-terminal hydrophobic signal sequences characteristic of glycosylphosphatidylinositol (GPI)-anchored membrane proteins. The igl genes lacked carbohydrate recognition domains but were members of a large family of amebic genes containing CXXC and CXC motifs. These data indicate that Igl is part of the parasite's multimolecular Gal/GalNAc adhesin required for host interaction.Carbohydrate-protein interactions initiate the contact-dependent cytotoxicity for which Entamoeba histolytica was named. Parasite recognition of host galactose (Gal) and Nacetyl-D-galactosamine (GalNAc) residues initiates trophozoite adherence to human colonic mucin, colonic epithelium, neutrophils and erythrocytes, certain bacteria, and a variety of cultured cell lines (3-7, 16, 19-22, 27, 36-38). Contact-dependent killing of target cells is Ͼ90% inhibited by Gal and GalNAc (34,37,41). Additionally, Chinese hamster ovary (CHO) cell glycosylation-deficient mutants lacking terminal Gal/GalNAc residues on N-and O-linked sugars are nearly totally resistant to amebic adherence and cytolytic activity (23,24,39).The E. histolytica 260-kDa Gal/GalNAc lectin is a heterodimer of transmembrane heavy (170 kDa) (Hgl) and GPIanchored light (35 or 31 kDa) (Lgl) glycoproteins linked by disulfide bonds. It was originally identified by galactose affinity chromatography and with adherence-inhibitory monoclonal antibodies (MAbs) (30,43). Both Hgl and Lgl are encoded by gene families (28,35). Antibodies that block or augment parasite Gal/GalNAc binding activity map to the cysteine-rich region (amino acids 356 to 1143) of Hgl (25), and this region (when expressed in Escherichia coli) contains a functional carbohydrate recognition domain (14, 33). The cytoplasmic tail of Hgl has homology to the cytoplasmic domain of ␤2 and ␤7 integrins, including regions implicated in binding of the intracellular signaling molecules Shc and Grb2. Overexpression of the cytoplasmic tail results in a dominant negative effect on endogenous lectin activity, with decreased adherence, cytotoxicity, and in vivo virulence (44).The 150-kDa lectin intermediate subunit (Igl) was originally identified as a trophozoite surface antigen recognized by MAbs which block trophozoite adherence to mammalian cells in vitro (9)(10)(11)42). The EH3015 MAb specific for Igl significantly inhibits adherence of amebae to erythrocytes and CHO cells, erythrop...

Characterization ofEntamoeba histolyticaIntermediate Subunit Lectin-Specific Human Monoclonal Antibodies Generated in Transgenic Mice Expressing Human Immunoglobulin Loci

“…Western immunoblot analysis has also shown that MAb EH3015 recognizes 150-and 170-kDa molecules in the H-302: NIH strain of E. histolytica (48). The present study revealed that the 170-kDa band is Igl1 and the 150-kDa band is Igl2, although the molecular mass calculated from the DNA sequence is larger for Igl2 than for Igl1 (9).…”

“…The immunization of hamsters with native Igl can inhibit amebic liver formation (11). There are two isoforms of Igl, which have 1,101 and 1,105 amino acids and are referred to as Igl1 and Igl2, respectively (9). The Igls are known to be cysteinerich proteins containing multiple CXXC motifs, but the association between inhibitory effects and each Igl isoform is not well understood.…”

Involvement of Raft-Like Plasma Membrane Domains of Entamoeba histolytica in Pinocytosis and Adhesion

“…The adhesion lectin is comprised of a transmembrane heavy subunit (Hgl; 170 kDa) disulfide linked to a GPI-anchored light subunit (Lgl; 31 to 35 kDa) (14,35). This heterodimer associates noncovalently with a GPI-anchored intermediate subunit (Igl; 150 kDa) (9,43). Hgl has been shown to contain a carbohydrate recognition domain specific for Gal/GalNAc and, more recently, a cytoplasmic tail with sequence homology to those of ␤2 and ␤7 integrins (14,68).…”

VH3 Gene Usage in Neutralizing Human Antibodies Specific for theEntamoeba histolyticaGal/GalNAc Lectin Heavy Subunit

“…The amebic lectin is a 260-kDa hetrodimeric glycoprotein composed of 170-kDa heavy and 35-or 31-kDa light subunits (32). In addition, a 150-kDa intermediate subunit of the lectin also contributes to adherence (8,12). Immunization of experimental animals with these lectin subunits provides protection from liver abscess formation (10,25,27,34,50).…”