Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells

Benjamin Schumann; Stacy Alyse Malaker; Simon Peter Wisnovsky; Marjoke Froukje Debets; Anthony John Agbay; Daniel Fernandez; Lauren Jan Sarbo Wagner; Liang Lin; Zhen Li; Junwon Choi; Douglas Michael Fox; Jessie Peh; Melissa Anne Gray; Kayvon Pedram; Jennifer Jean Kohler; Milan Mrksich; Carolyn Ruth Bertozzi

doi:10.1016/j.molcel.2020.03.030

Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells

Benjamin Schumann, Stacy Alyse Malaker, Simon Peter Wisnovsky, Marjoke Froukje Debets, Anthony John Agbay, Daniel Fernandez, Lauren Jan Sarbo Wagner, Liang Lin, Zhen Li, Junwon Choi, Douglas Michael Fox, Jessie Peh, Melissa Anne Gray, Kayvon Pedram, Jennifer Jean Kohler, Milan Mrksich, Carolyn Ruth Bertozzi

Research output: Contribution to journal › Article › peer-review

44 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	824-834.e15
Journal	Molecular cell
Volume	78
Issue number	5
DOIs	https://doi.org/10.1016/j.molcel.2020.03.030
State	Published - Jun 4 2020

Keywords

O-glycosylation
bioorthogonal
chemical proteomics
glycosyltransferase
isoenzyme
mucin

ASJC Scopus subject areas

Molecular Biology
Cell Biology

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10.1016/j.molcel.2020.03.030

Cite this

Schumann, B., Malaker, S. A., Wisnovsky, S. P., Debets, M. F., Agbay, A. J., Fernandez, D., Wagner, L. J. S., Lin, L., Li, Z., Choi, J., Fox, D. M., Peh, J., Gray, M. A., Pedram, K., Kohler, J. J., Mrksich, M., & Bertozzi, C. R. (2020). Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells. Molecular cell, 78(5), 824-834.e15. https://doi.org/10.1016/j.molcel.2020.03.030

Schumann, B, Malaker, SA, Wisnovsky, SP, Debets, MF, Agbay, AJ, Fernandez, D, Wagner, LJS, Lin, L, Li, Z, Choi, J, Fox, DM, Peh, J, Gray, MA, Pedram, K, Kohler, JJ, Mrksich, M & Bertozzi, CR 2020, 'Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells', Molecular cell, vol. 78, no. 5, pp. 824-834.e15. https://doi.org/10.1016/j.molcel.2020.03.030

@article{0a95b86cc12642fc960aefb89de46b5c,

title = "Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells",

abstract = "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.",

keywords = "O-glycosylation, bioorthogonal, chemical proteomics, glycosyltransferase, isoenzyme, mucin",

author = "Benjamin Schumann and Malaker, {Stacy Alyse} and Wisnovsky, {Simon Peter} and Debets, {Marjoke Froukje} and Agbay, {Anthony John} and Daniel Fernandez and Wagner, {Lauren Jan Sarbo} and Liang Lin and Zhen Li and Junwon Choi and Fox, {Douglas Michael} and Jessie Peh and Gray, {Melissa Anne} and Kayvon Pedram and Kohler, {Jennifer Jean} and Milan Mrksich and Bertozzi, {Carolyn Ruth}",

note = "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 Ram{\'o}n 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 . 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 Ram?n 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. B.S. M.F.D. S.P.W. S.A.M. L.L. L.J.S.W. J.J.K. M.M. and C.R.B. designed research; B.S. S.A.M. S.P.W. M.F.D. A.J.A. D.M.F. L.J.S.W. Z.L. L.L. D.F. J.P. and M.A.G. ran experiments; B.S. S.A.M. S.P.W. M.F.D. A.J.A. D.M.F. Z.L. L.L. M.M. and C.R.B. analyzed data; J.C. J.P. K.P. and J.J.K. made reagents and cell lines and contributed protocols; B.S. and C.R.B. wrote the paper with input from all authors. The authors declare no competing interests. Publisher Copyright: {\textcopyright} 2020 The Author(s)",

year = "2020",

month = jun,

day = "4",

doi = "10.1016/j.molcel.2020.03.030",

language = "English (US)",

volume = "78",

pages = "824--834.e15",

journal = "Molecular Cell",

issn = "1097-2765",

publisher = "Cell Press",

number = "5",

}

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 . 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 Ram?n 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. B.S. M.F.D. S.P.W. S.A.M. L.L. L.J.S.W. J.J.K. M.M. and C.R.B. designed research; B.S. S.A.M. S.P.W. M.F.D. A.J.A. D.M.F. L.J.S.W. Z.L. L.L. D.F. J.P. and M.A.G. ran experiments; B.S. S.A.M. S.P.W. M.F.D. A.J.A. D.M.F. Z.L. L.L. M.M. and C.R.B. analyzed data; J.C. J.P. K.P. and J.J.K. made reagents and cell lines and contributed protocols; B.S. and C.R.B. wrote the paper with input from all authors. The authors declare no competing interests. Publisher Copyright: © 2020 The Author(s)

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

UR - http://www.scopus.com/inward/record.url?scp=85085548431&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85085548431&partnerID=8YFLogxK

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 -

Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells

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