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Molecules 2017, 22(7), 1081;

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

Department of Drug Sciences, University of Pavia, via Taramelli 12, I-27100 Pavia, Italy
Department of Biotechnology and Life Sciences, University of Insubria, via J.H. Dunant 3, I-21100 Varese, Italy
The Protein Factory, Interuniversity Centre Politecnico of Milano and University of Insubria, via Mancinelli 7, I-20131 Milano, Italy
Microbiology and Virology Unit, IRCCS San Matteo Hospital Foundation, viale Camillo Golgi 19, I-27100 Pavia, Italy
Department of Biomedicine and Prevention and Animal Technology Station, University of Rome “Tor Vergata”, via Montpellier 1, I-00133 Roma, Italy
Infection Disease Unit, Internal Medicine and Medical Therapy Department, University of Pavia, via Aselli 43/45, I-27100 Pavia, Italy
These authors equally contributed to the present paper.
Author to whom correspondence should be addressed.
Received: 25 May 2017 / Revised: 23 June 2017 / Accepted: 23 June 2017 / Published: 29 June 2017
(This article belongs to the Special Issue Synthesis and Biological Applications of Glycoconjugates)
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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. View Full-Text
Keywords: neo-glycoproteins; glycoconjugate vaccines; MTB recombinant antigens; epitope neo-glycoproteins; glycoconjugate vaccines; MTB recombinant antigens; epitope

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Bavaro, T.; Tengattini, S.; Piubelli, L.; Mangione, F.; Bernardini, R.; Monzillo, V.; Calarota, S.; Marone, P.; Amicosante, M.; Pollegioni, L.; Temporini, C.; Terreni, M. Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates. Molecules 2017, 22, 1081.

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