Tryptophan C-mannosylation is an unusual co-translational protein modification performed by metazoans and apicomplexan protists. The prevalence and biological functions of this modification are poorly understood, with progress in the field hampered by a dearth of convenient tools for installing and detecting the modification. Here, we engineer a yeast system to produce a diverse array of proteins with and without tryptophan C-mannosylation and interrogate the modification’s influence on protein stability and function. This system also enabled mutagenesis studies to identify residues of the glycosyltransferase and its protein substrates that are crucial for catalysis. The collection of modified proteins accrued during this work facilitated the generation and thorough characterization of monoclonal antibodies against tryptophan C-mannosylation. These antibodies empowered proteomic analyses of the brain C-glycome by enriching for peptides possessing tryptophan C-mannosylation. This study revealed many new modification sites on proteins throughout the secretory pathway with both conventional and non-canonical consensus sequences.
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All relevant data are available from the authors upon request. Structure coordinates have been deposited in the Protein Data Bank (https://www.rcsb.org/) under accession code 6PLH. Proteomics data are available via ProteomeXchange60 (http://www.proteomexchange.org/) with identifiers PXD018401, PXD020336 and PXD021612. Some data were sourced from the UniProt (https://www.uniprot.org/) database (accession numbers D3YXG0, P11680, P59511, Q3UHD1, Q3UPR9, Q62217, Q69ZU6, Q6P4U0, Q80TI0, Q8VDX6, Q8VCC9). Source data are provided with this paper.
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We thank K. Wycherley, P. Masendycz and K. Mackwell at the Walter and Eliza Hall Institute Monoclonal Antibody Facility (WEHI-MAF) for their assistance in creating the mAbs reported in this paper. We also thank the beamline staff at the Australian Synchrotron for help with X-ray data collection as well as J. Newman and B. Marshall at the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Collaborative Crystallisation Centre (C3) for assistance in protein crystallization. This research was undertaken, in part, using the MX2 beamline at the Australian Synchrotron, part of ANSTO, and made use of the Australian Cancer Research Foundation (ACRF) detector. E.D.G.-B. would like to acknowledge support from the Walter and Eliza Hall Institute of Medical Research, National Health and Medical Research Council of Australia (NHMRC) project grants GNT1139546 and GNT1139549, the ACRF, a Victorian State Government Operational Infrastructure support grant and the support of the Brian M. Davis Charitable Foundation Centenary Fellowship.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Western blots demonstrating expression of all twelve CeDPY19 mutants in P. pastoris (n = 1 independent replicates).
Representative western blots using the collection of proteins with and without tryptophan C-mannosylation to map the epitope preferences of mAbs 10E9, 9E7, 6G4 and 7G12 (one of n = 3 independent replicates).
Representative SPR sensorgrams used to determine the affinity of mAbs 6G4 and 7G12.
Two stereo views of the glycopeptide antigen with tryptophan C-mannosylation that is bound to 5G12 with a Fo-Fc omit map contoured at 1.5σ.
A representative western blot of protein extracted from mouse brain using the 5G12 mAb (one of n = 3 independent replicates).
MS/MS spectra demonstrating the location of tryptophan C-mannosylation for Rxylt1, as well as a sequence alignment showing conservation of this site across the Euteleostomi clade.
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John, A., Järvå, M.A., Shah, S. et al. Yeast- and antibody-based tools for studying tryptophan C-mannosylation. Nat Chem Biol 17, 428–437 (2021). https://doi.org/10.1038/s41589-020-00727-w
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