TY - JOUR
T1 - Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells
AU - Schumann, Benjamin
AU - Malaker, Stacy Alyse
AU - Wisnovsky, Simon Peter
AU - Debets, Marjoke Froukje
AU - Agbay, Anthony John
AU - Fernandez, Daniel
AU - Wagner, Lauren Jan Sarbo
AU - Lin, Liang
AU - Li, Zhen
AU - Choi, Junwon
AU - Fox, Douglas Michael
AU - Peh, Jessie
AU - Gray, Melissa Anne
AU - Pedram, Kayvon
AU - Kohler, Jennifer Jean
AU - Mrksich, Milan
AU - Bertozzi, Carolyn Ruth
N1 - Funding Information:
The authors thank Katrine T. Schjoldager and Hans H. Wandall (both University of Copenhagen, Denmark) for HepG2-T1-KO and HepG2-T2-KO cells and helpful discussions. We thank Lawrence Tabak (National Institutes of Health, Bethesda, MD) for full-length human GalNAc-T2 in the plasmid pCMV-NTAP, Tyler J. Stewart and Matthew Renfrow for advice on GalNAc-T expression, Michael C. Bassik (Stanford University, USA) for the K-562-spCas9 cell line, Jonathan S. Weissman (University of California, San Francisco, USA) for lentiviral plasmids, Jon Agirre (University of York) for help on crystal structure optimization using Privateer, and Ramón Hurtado-Guerrero (University of Zaragoza, Spain) for helpful discussions. We thank David Spiciarich, Yi-Chang Liu, and Christina M. Woo for help with designing experiments. We thank the Cell Services Science Technology Platform at the Francis Crick Institute for support in cell culture. We thank Mia I. Zol-Hanlon for Graphical Abstract image design. The authors are grateful for generous funding by Stanford University , Stanford ChEM-H , University of California, Berkeley , and Howard Hughes Medical Institute . A portion of this work was performed at the Stanford ChEM-H Macromolecular Structure Knowledge Center. This work was supported by the National Institutes of Health ( R01 CA200423 to C.R.B. and R21 DK112733 to J.J.K.), the Defense Threat Reduction Agency (GRANT11631647 to M.M.) and the Francis Crick Institute (to B.S. and Z. L.) which receives its core funding from Cancer Research UK ( FC001749 ), the UK Medical Research Council ( FC001749 ), and the Wellcome Trust ( FC001749 ). Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. Beamline 5.0.1 of the Advanced Light Source, a U.S. DOE Office of Science User Facility under Contract No. DE-AC02-05CH11231, is supported in part by the ALS-ENABLE program funded by the National Institutes of Health, National Institute of General Medical Sciences , grant P30 GM124169-01 . B.S. was supported by a Feodor Lynen Fellowship by the Alexander von Humboldt Foundation . S.P.W. was supported by a Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research . S.A.M. was supported by a National Institute of General Medical Sciences F32 Postdoctoral Fellowship ( F32-GM126663-01 ). M.F.D. was supported by an NWO Rubicon Postdoctoral Fellowship. A.J.A. was supported by a Stanford ChEM-H undergraduate scholarship. J.P. was supported by National Institutes of Health Postdoctoral Fellowship 5F32CA224985 . M.A.G. and K.P. were supported by the National Science Foundation Graduate Research Fellowship. M.A.G. was supported by the Stanford ChEM-H Chemistry/Biology Interface Predoctoral Training Program . K.P. was supported by a Stanford Graduate Fellowship .
PY - 2020/6/4
Y1 - 2020/6/4
N2 - Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.
AB - Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.
KW - O-glycosylation
KW - bioorthogonal
KW - chemical proteomics
KW - glycosyltransferase
KW - isoenzyme
KW - mucin
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U2 - 10.1016/j.molcel.2020.03.030
DO - 10.1016/j.molcel.2020.03.030
M3 - Article
C2 - 32325029
AN - SCOPUS:85085548431
VL - 78
SP - 824-834.e15
JO - Molecular Cell
JF - Molecular Cell
SN - 1097-2765
IS - 5
ER -