Identification of Entamoeba histolytica thiol-specific antioxidant as a GalNAc lectin-associated protein.
Entamoeba histolytica is a human intestinal parasite that causes amebic dysentery. A cell surface amebic adhesin, the galactose and N-acetyl-D-galactosamine inhibitable (GalNAc) lectin mediates amebic adherence to and contact-dependent killing of host cells. Previous work has suggested that the GalNAc lectin transduces signals via protein interactions with its short cytoplasmic domain. We used a yeast two-hybrid system to screen an E. histolytica cDNA library for proteins that interact with the GalNAc lectin cytoplasmic domain. One isolate was the E. histolytica thiol-specific antioxidant (TSA). TSA is an enzyme that detoxifies hydrogen peroxide. TSA did not interact in yeast two-hybrid experiments with a mutant version of the lectin cytoplasmic domain, confirming the specificity of the lectin-TSA interaction. Furthermore, mutational analyses of the TSA isolate demonstrated that an in-frame five amino acid sequence introduced between amino acids 61-62 yielded a TSA mutant that did not interact with the lectin cytoplasmic domain upon expression in the yeast two-hybrid system. The association of TSA and GalNAc lectin was further supported by co-immunoaffinity purification. Confocal microscopy demonstrated co-localization of TSA and GalNAc lectin at sites of ameba:host cell contact. Recruitment of TSA by the GalNAc lectin suggests a novel mechanism of parasite defense against reactive oxygen intermediates generated by host peripheral mononuclear cells.
Hughes
Molly A
MA
Department of Internal Medicine, Division of Infectious Diseases, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA. bjm2r@virginia.edu
Lee
Constance W
CW
Holm
Christopher F
CF
Ghosh
Salil
S
Mills
Andrew
A
Lockhart
Lauren A
LA
Reed
Sharon L
SL
Mann
Barbara J
BJ
D000116:Acetylgalactosamine / Q000378:metabolism*; D000818:Animals; D018938:Caco-2 Cells; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional / Q000379:methods; D009154:Mutation; D009504:Neutrophils / Q000378:metabolism; D010544:Peroxidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054464:Peroxiredoxins; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
C434500:Igl2 protein, Entamoeba histolytica; D037102:Lectins; D015800:Protozoan Proteins; D010544:Peroxidases; D054464:Peroxiredoxins; D000116:Acetylgalactosamine