= Emerging. More than 5 years before clinical availability. |
= Expected to be clinically available in 1 to 4 years. |
= Clinically available now. |
Topic: Proteomics
Authors: Martina Pirro (1), Esmee Schoof (1), Yassene Mohammed (1), Sandra van Vliet (2), Yoann Rombouts (3), Peter van Veelen (1), Manfred Wuhrer (1) and Paul Hensbergen (1)
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Short Abstract O-glycosylation is generally initiated by the transfer of a N-acetylgalactosamine to Ser/Thr residues of proteins, forming the Tn antigen. This truncated surface glycan is expressed at high levels by tumor cells and is associated with higher metastatic behaviour and poor prognosis of patients. The Tn antigen is recognised by the C-type macrophage galactose lectin (MGL), which induces the activation of immunosuppressive responses. Here, we investigated the MGL binding proteins in Jurkat cells. The optimization of pull-down assays and subsequent glycoproteomic analysis by mass spectrometry, allowed us to identify 20 cell surface proteins as novel MGL-ligands. |
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Long Abstract Introduction Glycosylation is one of the most common post-translational modifications occurring on eukaryotic proteins (Reis et al. 2010). In general, O-glycosylation is initiated by the transfer of a N-acetylgalactosamine (GalNAc) to serine (Ser) or threonine (Thr) residues of proteins (Reis et al. 2010), forming the Thomsen/nouvelle (Tn) antigen. In normal cells, this glycan is elongated with other carbohydrate residues, for example a galactose to form the core 1 T antigen. This step is mediated by the enzyme T-synthase with the help of its chaperone Cosmc. Mutations in cosmc are responsible for the abortive elongation of O-glycans and higher expression of the Tn antigen on human tumor cells (Ju et al. 2008). Moreover, this surface truncated glycan is associated with higher metastatic behaviour and consequent poor prognosis of patients. This outcome may be mediated by the specific binding of the Tn antigen to the C-type macrophage galactose lectin (MGL), expressed on tolerogenic dendritic cells (DC) and macrophages. Since previous studies demonstrated that MGL-mediated recognition of aberrant glycosylation is associated with activation of immunosuppressive responses and tumor immune tolerance (Rabinovich and Croci 2012), we investigated the MGL binding proteins carrying Tn. For this purpose, we optimized a protocol which makes use of Fc-coupled MGL as a bait in pull-down assays, followed by mass spectrometry-based proteomics, to analyze specific ligands from cellular extract of the Jurkat T cell leukemia cell line. This cell line represents an interesting cellular model because it expresses high levels of Tn antigen due to a single nucleotide deletion in cosmc. First of all, this approach confirmed CD45 and CD43 as MGL binders. However, this approach also allowed the identification of 20 cell surface proteins as novel MGL ligands. For most of these proteins the peptides carrying the Tn antigen could be identified, explaining the interaction with MGL. Van Vliet et al. (van Vliet et al. 2006) showed that CD45 is involved in apoptotic signalling in the Jurkat cell line, mediated by the binding of MGL-Fc and the simultaneous TCR (T cell receptor) activation. The effect of MGL binding to the newly identified ligands warrants further investigation. The deeper understanding of their role in inducing immune reactions might give new opportunities in the design of glycopeptides to target MGL as an anti-cancer immunotherapeutic tool. Methods Results Conclusions & Discussion |
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References & Acknowledgements: Ju, T., G. S. Lanneau, T. Gautam, Y. Wang, B. Xia, S. R. Stowell, M. T. Willard, W. Wang, J. Y. Xia, R. E. Zuna, Z. Laszik, D. M. Benbrook, M. H. Hanigan, and R. D. Cummings. 2008. 'Human tumor antigens Tn and sialyl Tn arise from mutations in Cosmc', Cancer Res, 68: 1636-46. Rabinovich, G. A., and D. O. Croci. 2012. 'Regulatory circuits mediated by lectin-glycan interactions in autoimmunity and cancer', Immunity, 36: 322-35. Reis, C. A., H. Osorio, L. Silva, C. Gomes, and L. David. 2010. 'Alterations in glycosylation as biomarkers for cancer detection', J Clin Pathol, 63: 322-9. van Vliet, S. J., S. I. Gringhuis, T. B. Geijtenbeek, and Y. van Kooyk. 2006. 'Regulation of effector T cells by antigen-presenting cells via interaction of the C-type lectin MGL with CD45', Nat Immunol, 7: 1200-8.
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Description | Y/N | Source |
Grants | yes | LUMC |
Salary | yes | LUMC |
Board Member | no | |
Stock | no | |
Expenses | no |
IP Royalty: no
Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |