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Yeast- and antibody-based tools for studying tryptophan C-mannosylation

Abstract

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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Fig. 1: Engineering tryptophan C-mannosylation into P. pastoris and the impact of this modification on protein stability and function.
Fig. 2: Mutagenesis of CeDPY19 and its substrates.
Fig. 3: Generation of mAbs that recognize tryptophan C-mannosylation.
Fig. 4: Structure of the 5G12 mAb–glycopeptide complex.
Fig. 5: 5G12-enchanced proteomic analysis of the mouse brain C-glycome.

Data availability

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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Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

A.J. performed all molecular biology, protein expression, biochemical assays and immunoprecipitations; M.A.J. performed the structural biology experiments; S.S. and R.M. assisted with the radioassays; R.W.B. and P.E.C. performed SPR; A.W.L. assisted with mAb sequencing; S.C. assisted with animal work; N.E.S. performed all MS experiments; E.D.G.-B. conceived the project; A.J. and E.D.G.-B. cowrote the manuscript.

Corresponding author

Correspondence to Ethan D. Goddard-Borger.

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Extended data

Extended Data Fig. 1 Expression of CeDPY19 mutants in P. pastoris.

Western blots demonstrating expression of all twelve CeDPY19 mutants in P. pastoris (n = 1 independent replicates).

Source data

Extended Data Fig. 2 Probing mAb selectivity by western blot.

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).

Source data

Extended Data Fig. 3 Determining mAb affinity by SPR.

Representative SPR sensorgrams used to determine the affinity of mAbs 6G4 and 7G12.

Extended Data Fig. 4 Stereo views of the glycopeptide in the 5G12:glycopeptide complex.

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.

Extended Data Fig. 5 Detection of modified proteins in mouse brain extract.

A representative western blot of protein extracted from mouse brain using the 5G12 mAb (one of n = 3 independent replicates).

Source data

Extended Data Fig. 6 Localisation of the tryptophan C-mannosylation site in Rxylt1.

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.

Supplementary information

Supplementary Information

Supplementary Figs. 1–14 and Tables 1–5.

Reporting Summary

Supplementary Data 1

Unprocessed western blots for Supplementary Fig. 1c.

Supplementary Data 2

Unprocessed gels for Supplementary Fig. 3b.

Supplementary Data 3

Unprocessed gels for Supplementary Fig. 14b.

Source data

Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 3

Unprocessed western blots and gels.

Source Data Extended Data Fig. 1

Unprocessed western blots.

Source Data Extended Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed western blots.

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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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