Glycan moieties in Entamoeba histolytica ubiquitin are immunodominant

Abstract

The ubiquitin‐proteasome system plays a central role performing several functions to maintain parasite homeostasis. We have reported the partial characterization of N‐linked glycosylation profile in E. histolytica ubiquitin (EhUb). Here we examined the immunogenicity and antigenicity of carbohydrates in EhUbiquitin. Rabbits were immunized with purified EhUbiquitin or purified recombinant rUb expressed by E. coli. Using Western Blot, we explored the immunogenicity and antigenicity of protein portion and carbohydrates moiety. Interestingly, immunized rabbits produced antibodies to both Ub glycoprotein and rUb; but antibodies against carbohydrates were immunodominant, rather than antibodies to the protein moiety of EhUbiquitin. In addition, we observed that antibodies to protein moiety are not conserved in serum unless antigen is continually administrated. Conversely, anti‐Ub glycoprotein antibodies are well maintained in circulation. In humans, infection with Entamoeba histolytica induces strong IgG anti‐Ub response. The human antibodies recognize both, the protein moieties and the glycosylated structure. Entamoeba histolytica ubiquitin is immunogenic and antigenic. The glycan moieties are immunodominant and induces IgG. These data open the door to use carbohydrates as potential targets for diagnose tests, drugs and vaccine to prevent this parasitic disease.

Ubiquitin of Entamoeba histolytica induces antibody response in patients with invasive amoebiasis

“…In a previous study, we found antibodies to Ehub in the sera of six patients with invasive amoebiasis. 9 This was unexpected because ubiquitin is an evolutionarily conserved molecule with high sequence similarity between species; the E. histolytica ubiquitin differs in only six amino acid residues from the homologue consensus in humans and lower organisms. 20 To rule out a fortuitous finding, we decided to expand our study.…”

“…Escherichia coli expressing recombinant ubiquitin from E. histolytica HM-1: IMSS rEhub was obtained as we reported previously. 9 Briefly, RNA isolated from axenic E. histolytica HM-1: IMSS trophozoites were used for cDNA synthesis. The PCR product was cloned and expressed in Escherichia coli BL21 transformed with pET-28a vector containing the Ehub protein fused with 6X Histidine tag.…”

“…To develop the coloured reaction, the substrate H 2 O 2 and the chromogen 3,3 diaminobenzidine tetrahydro chloride were used. 8,9,18 3 | RESULTS…”

“…We showed that the glycan moieties are immunodominant and induce antibodies, immunizing animals with purified Ehub or purified recombinant E. histolytica recombinant ubiquitin (rEhub) expressed by E. coli. 8,9 Inhabitants of areas endemic to E. hystolytica have high seroprevalence of antibodies to amoebas, even if they have not suffered invasive amoebiasis. Antibodies from animals with experimental amoebic liver abscess recognize the membrane proteins of E. histolytica trophozoites.…”

“…[10][11][12] Previously, we found that human infection with Entamoeba histolytica induces strong IgG anti-Ehub response. 9 This work analysed the dominance antibodies to the glycosylated-Ehub or non-glycosylated rEhub protein by using sera from patients with ALA, and healthy people living in endemic areas of amoebiasis with high seroprevalence of antibodies to amoeba. An additional control included patients with alcoholic liver cirrhosis to rule out the cross-reaction of liver damage-induced autoantibodies to ubiquitin or Ehub.…”