Toxocara canis glycans influence antigen recognition by mouse IgG1 and IgM antibodies

Abstract

The impact of sugar moieties of Toxocara canis glycoprotein antigens on their recognition by infected mouse antibodies was investigated in this study. Native TES and recombinant Toxocara mucins generated in Pichia pastoris yeast as well as their deglycosylated forms were used in ELISA. TES and recombinant mucins were equally recognized by T. canis infected mouse IgG1 antibodies. IgM immunoglobulins predominantly recognized TES antigens. Among mucins recognition of Tc-MUC-4 was the most significant. Deglycosylation of antigens resulted in significant loss of IgM and IgG1 reactivity to TES, mucins, Tc-MUC-3 and Tc-MUC-4. The presence of sugar moieties had no influence on IgE binding to native or recombinant T. canis antigens. Our results suggest that glycans are involved in epitope formation what should be taken into consideration in production of recombinant helminth antigens for diagnostic purposes.

Reactivity of recombinant Toxocara canis TES‐30/120 in experimentally infected mice

“…Native and recombinant proteins might induce immune response with distinct kinetics, as protein folding is different in each organism . In addition, native TES is highly glycosylated, unlike our proteins, so it should not be expected to have the same reactivity as the native proteins in study; however, deglycosylation of native TES appears to increase sensitivity and specificity as reported by Roldán et al (2015) . This information helps explain the good performance of the antigens produced in E. coli , an expression platform incapable of performing protein glycosylation.…”

Immunoglobulin M antibodies are not specific for serodiagnosis of human toxocariasis

“…found that IgM antibodies from both human and mice infected with Toxocara recognize TES glycans, suggesting their possible use in the serodiagnosis of human toxocariasis. Likewise, Długosz and Wiśniewski found that murine IgM antibodies recognized preferentially glycosidic epitopes from both native and recombinant TES mucin antigens and deglycosylation of these antigens resulted in a significant loss of reactivity. Nevertheless, Mohamad et al .…”

Evaluation of Toxocara canis Glycosylated TES Produced in Pichia pastoris for Immunodiagnosis of Human Toxocariasis

“…Our main limitation to testing the deglycosylation hypothesis was that we did not manage to obtain a pure deglycosylated product, as we could still observe a glycosylated band on the SDS-PAGE (Figure 1). Nevertheless, considering that we applied a mixture of proteins in each test and that deglycosylated products were at a higher concentration (evidenced by a more intense band in SDS-PAGE, Figure 1), we should expect an increase or decrease in sensitivity, even with an "impure" product, as deglycosylated and glycosylated products should coat the microplate simultaneously [3,7]. Also, our deglycosylation methodology did not remove the O-glycosylation sites produced by Pichia pastoris, as the bands in Figure 2 were not at the same height (after deglycosylation) as the E. coli rTES-120 band and it continuous to interfere with the reaction; the presence of O-glycosylation could account for the difference between the sensitivities of E. coli and Pichia pastoris products, as suggested by Elefant and coauthors [18], Li and coauthors [19], and Irani and coauthors [20] studies, which collectively showed that glycosylation reduces sensitivity in serodiagnosis.…”

“…Another advantage is that P. pastoris can be cultivated in a lowcost medium at a broader pH range and lower temperatures-essential characteristics for industrial scale [6]. Finally, the last advantage over the E. coli system is the possibility of post-translational modifications, especially in the case of native TES, as the proteins are highly glycosylated and could respond to a part of the immune response [7]. Prior to this study, Fong and coauthors presented the recombinant TES-120 expressed in P. pastoris with high specificity, suggesting an unexplored potential for TES protein in Toxocara spp.…”