An anionic synthetic sugar containing 6‐SO₃‐NAcGlc mimics the sulfated cruzipain epitope that plays a central role in immune recognition

Abstract

Cruzipain (Cz), the major cysteine proteinase of Trypanosoma cruzi, is a glycoprotein that contains sulfated high‐mannose‐type oligosaccharides. We have previously determined that these sulfate groups are targets of specific immune responses. In order to evaluate the structural requirements for antibody recognition of Cz, a systematic structure–activity study of the chemical characteristics needed for antibody binding to the Cz sulfated epitope was performed by immunoassays. With this aim, different synthesized molecules were coupled to the proteins BSA and aprotinin and confronted with (a) mouse sera specific for Cz and its carboxy‐terminal (C‐T) domain, (b) antibodies raised in rabbits immunized with Cz and its C‐terminal domain and (c) IgGs purified from human Chagas disease sera. Our results indicate that a glucosamine containing an esterifying sulfate group in position O‐6 and an N‐acetyl group was the preferred epitope for the immune recognition of sera specific for Cz and its C‐T domain. Although to a minor extent, other anionic compounds bearing sulfate groups in different positions and number as well as different anionic charged groups including carboxylated or phosphorylated monosaccharides, disaccharides and oligosaccharides were recognized. In conclusion, we found that synthetic anionic sugar conjugates containing N‐acetyl d‐glucosamine‐6‐sulfate sodium salt (GlcNAc6S) competitively inhibit the binding of affinity purified rabbit anti‐C‐T IgG to the C‐T extension of Cz. Extending these findings to the context of natural infection, immune assays performed with Chagas disease serum confirmed that the structure of synthetic GlcNAc6S mimics the N‐glycan‐linked sulfated epitope displayed in the C‐T domain of Cz.

A method to identify trace sulfated IgG N-glycans as biomarkers for rheumatoid arthritis

“…It was believed that sulfation of N-glycans can significantly alter biological recognition and/or facilitate rapid clearance of the protein from the body 37 – 40 . Especially, N-glycans with sulfo-modifications constitute important recognition codes in cell adhesion, e.g., a glucosamine containing sulfate group in position O-6 and an N-acetyl group was the preferred epitope for the immune recognition 31 , 41 – 43 . The effect of sulfated N-glycans on IgG is yet to be known.…”

Involvement of sulfates from cruzipain, a major antigen of Trypanosoma cruzi, in the interaction with immunomodulatory molecule Siglec-E

“…Recently, we found that synthetic anionic sugar conjugates containing GlcNAc6S competitively inhibit the binding of affinity-purified rabbit anti-C-T IgG to the C-terminal extension of Cz. Interestingly, extending these findings to the context of natural infection, immune assays performed with Chagas disease serum confirmed that the structure of synthetic GlcNAc6S mimics the N-glycan-linked sulfated epitope displayed in the C-T domain of Cz [59]. However, no studies about the possible participation of these groups enhancing host-parasite interaction via Siglec-E binding have been studied so far.…”

Cruzipain Sulfotopes-Specific Antibodies Generate Cardiac Tissue Abnormalities and Favor Trypanosoma cruzi Infection in the BALB/c Mice Model of Experimental Chagas Disease

“…We have demonstrated that a glucosamine containing an esterifying sulfate group in position O-6 and an N -acetyl group was the preferred epitope for the immunorecognition of sera specific for Cz and its C-T. In addition, in the context of natural infection, immune assays performed with ChD serum confirmed that the structure of synthetic anionic sugar conjugates containing N -acetyl d -glucosamine-6-sulfate (GlcNAc-6-SO3) mimics the N -glycan-linked sulfated epitope displayed in the C-T of natural Cz ( Couto et al., 2012 ). Interestingly, human IgG2 antibody levels specific for sulfated structures (IgG2-SO 3 ) on Cz are inversely correlated with the severity of ChD, indicating that antibodies specific for sulfated moieties might play a relevant biomarker role in the progression of Chagas heart disease ( Acosta et al., 2008 ; Couto et al., 2012 ).…”

“…Data from our laboratory have identified the presence of sulfate-bearing glycosylations in the high immunogenic C-T domain as targets of specific immune responses both in BALB/c model and in chronic ChD patients ( Acosta et al., 2008 ; Couto et al., 2012 ). We have shown that sulfated moieties are responsible for eliciting IgG2b isotype murine responses to Cz and that subjects chronically infected with T. cruzi mount specific humoral immune responses to sulfated glycoproteins.…”