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Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates

Tuberculosis is still one of the most deadly infectious diseases worldwide, and the use of conjugated antigens, obtained by combining antigenic oligosaccharides, such as the lipoarabinomannane (LAM), with antigenic proteins from Mycobacterium tuberculosis (MTB), has been proposed as a new strategy f...

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Autores principales: Bavaro, Teodora, Tengattini, Sara, Piubelli, Luciano, Mangione, Francesca, Bernardini, Roberta, Monzillo, Vincenzina, Calarota, Sandra, Marone, Piero, Amicosante, Massimo, Pollegioni, Loredano, Temporini, Caterina, Terreni, Marco
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152100/
https://www.ncbi.nlm.nih.gov/pubmed/28661444
http://dx.doi.org/10.3390/molecules22071081
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author Bavaro, Teodora
Tengattini, Sara
Piubelli, Luciano
Mangione, Francesca
Bernardini, Roberta
Monzillo, Vincenzina
Calarota, Sandra
Marone, Piero
Amicosante, Massimo
Pollegioni, Loredano
Temporini, Caterina
Terreni, Marco
author_facet Bavaro, Teodora
Tengattini, Sara
Piubelli, Luciano
Mangione, Francesca
Bernardini, Roberta
Monzillo, Vincenzina
Calarota, Sandra
Marone, Piero
Amicosante, Massimo
Pollegioni, Loredano
Temporini, Caterina
Terreni, Marco
author_sort Bavaro, Teodora
collection PubMed
description Tuberculosis is still one of the most deadly infectious diseases worldwide, and the use of conjugated antigens, obtained by combining antigenic oligosaccharides, such as the lipoarabinomannane (LAM), with antigenic proteins from Mycobacterium tuberculosis (MTB), has been proposed as a new strategy for developing efficient vaccines. In this work, we investigated the effect of the chemical glycosylation on two recombinant MTB proteins produced in E. coli with an additional seven-amino acid tag (recombinant Ag85B and TB10.4). Different semi-synthetic glycoconjugated derivatives were prepared, starting from mannose and two disaccharide analogs. The glycans were activated at the anomeric position with a thiocyanomethyl group, as required for protein glycosylation by selective reaction with lysines. The glycosylation sites and the ex vivo evaluation of the immunogenic activity of the different neo-glycoproteins were investigated. Glycosylation does not modify the immunological activity of the TB10.4 protein. Similarly, Ag85B maintains its B-cell activity after glycosylation while showing a significant reduction in the T-cell response. The results were correlated with the putative B- and T-cell epitopes, predicted using a combination of in silico systems. In the recombinant TB10.4, the unique lysine is not included in any T-cell epitope. Lys30 of Ag85B, identified as the main glycosylation site, proved to be the most important site involved in the formation of T-cell epitopes, reasonably explaining why its glycosylation strongly influenced the T-cell activity. Furthermore, additional lysines included in different epitopes (Lys103, -123 and -282) are also glycosylated. In contrast, B-cell epitopic lysines of Ag85B were found to be poorly glycosylated and, thus, the antibody interaction of Ag85B was only marginally affected after coupling with mono- or disaccharides.
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spelling pubmed-61521002018-11-13 Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates Bavaro, Teodora Tengattini, Sara Piubelli, Luciano Mangione, Francesca Bernardini, Roberta Monzillo, Vincenzina Calarota, Sandra Marone, Piero Amicosante, Massimo Pollegioni, Loredano Temporini, Caterina Terreni, Marco Molecules Article Tuberculosis is still one of the most deadly infectious diseases worldwide, and the use of conjugated antigens, obtained by combining antigenic oligosaccharides, such as the lipoarabinomannane (LAM), with antigenic proteins from Mycobacterium tuberculosis (MTB), has been proposed as a new strategy for developing efficient vaccines. In this work, we investigated the effect of the chemical glycosylation on two recombinant MTB proteins produced in E. coli with an additional seven-amino acid tag (recombinant Ag85B and TB10.4). Different semi-synthetic glycoconjugated derivatives were prepared, starting from mannose and two disaccharide analogs. The glycans were activated at the anomeric position with a thiocyanomethyl group, as required for protein glycosylation by selective reaction with lysines. The glycosylation sites and the ex vivo evaluation of the immunogenic activity of the different neo-glycoproteins were investigated. Glycosylation does not modify the immunological activity of the TB10.4 protein. Similarly, Ag85B maintains its B-cell activity after glycosylation while showing a significant reduction in the T-cell response. The results were correlated with the putative B- and T-cell epitopes, predicted using a combination of in silico systems. In the recombinant TB10.4, the unique lysine is not included in any T-cell epitope. Lys30 of Ag85B, identified as the main glycosylation site, proved to be the most important site involved in the formation of T-cell epitopes, reasonably explaining why its glycosylation strongly influenced the T-cell activity. Furthermore, additional lysines included in different epitopes (Lys103, -123 and -282) are also glycosylated. In contrast, B-cell epitopic lysines of Ag85B were found to be poorly glycosylated and, thus, the antibody interaction of Ag85B was only marginally affected after coupling with mono- or disaccharides. MDPI 2017-06-29 /pmc/articles/PMC6152100/ /pubmed/28661444 http://dx.doi.org/10.3390/molecules22071081 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Bavaro, Teodora
Tengattini, Sara
Piubelli, Luciano
Mangione, Francesca
Bernardini, Roberta
Monzillo, Vincenzina
Calarota, Sandra
Marone, Piero
Amicosante, Massimo
Pollegioni, Loredano
Temporini, Caterina
Terreni, Marco
Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates
title Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates
title_full Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates
title_fullStr Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates
title_full_unstemmed Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates
title_short Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates
title_sort glycosylation of recombinant antigenic proteins from mycobacterium tuberculosis: in silico prediction of protein epitopes and ex vivo biological evaluation of new semi-synthetic glycoconjugates
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152100/
https://www.ncbi.nlm.nih.gov/pubmed/28661444
http://dx.doi.org/10.3390/molecules22071081
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