Yeast- and antibody-based tools for studying tryptophan C-mannosylation
Alan John, Michael A Jarva, Sayali Shah, Runyu Mao, Stephane Chappaz, Richard W Birkinshaw, Peter E Czabotar, Alvin W Lo, Nichollas E Scott, Ethan D Goddard-Borger
Nature Chemical Biology | NATURE RESEARCH | Published : 2021
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 modifi..View full abstract
Awarded by National Health and Medical Research Council of Australia (NHMRC)
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.