Glycan Engineering in Living Cells

Literature seminar #3
M2 Yuki Yamanashi
2021/08/19 (Thu)Overview: Development of live-cell glycoengineering tools utilizing the power of chemical biology

proteins,
lipids, ...etc.

glycoengineering
tools
glycans on/insideliving cells- Exoenzymatic engineering- Metabolic engineering- Genetic engineering...etc.

modified glycans- Imaging- Structure-function relationships- Therapeutics...etc.

2

Contents-

Introduction- The role of glycans in living organisms- The structure of glycans- Previous development of glycoengineering tools-

Development of next-generation glycoengineering tools
1. Bump-and-Hole engineering
2. Two-step glycan editing-

Perspectives-

Summary

3

Contents-

Introduction- The role of glycans in living organisms- The structure of glycans- Previous development of glycoengineering tools-

Development of next-generation glycoengineering tools
1. Bump-and-Hole engineering
2. Two-step glycan editing-

Perspectives-

Summary4

Glycans are major building blocks of life.

- templated synthesis- linear- templated synthesis- linear5- non-templated synthesis- branched

Scott, E. and Munkley, J. Int. J. Mol. Sci. 2019, 20, 1389-1418.

Glycans play crucial roles in a myriad of biological processes.

6- Glycans on cell surface- cell-cell comunication- membrane protein trafficking- pathogen invasion- immune response-

Glycans inside cells- properties of proteins(e.g., stability, conformations)- transcriptional regulation...etc.

Hart, G. W. and Copeland, R. J. Cell 2010, 143, 672–676.

A growing number of glycan-associated diseases are discovered from 1990s.(1)7

Human disorders with a major genetic defect in glycosylation pathways

glycolipid
proteoglycan
glycoprotein-

Cancer metastasis
Autoimmune diseases
Alzheimer’s diseases
Diabetes
Viral immune escape
Kidney diseases...etc.

Varki, A. Glycobiology 2017, 27, 3–49.

A growing number of glycan-associated diseases are discovered from 1990s.(2)8Inflammatory bowel disease (IBD) and core fucosylation on T cells

Asn
GDP-Fuc

Fut8
GDP

Asn
OH OH

=

OH
O

Me
OH

OH
OH

O

OH
HO
OH
AcHN

L-Fucose

O
OH

CO2H
OH

HO
HO

O
OH

OH

HO2C
OH

OH
O

OH
OH

- Fut8 expression was abnormally increased in the inflamed area, resulting in high core fucosylation level.
- Fut8 KO inhibited IBD pathogenesis via inhibition of transportation of receptors to the lipid raft.
- Fut8 inhibition can be a new therapeutic strategy.
Fujii, H., Shinzaki, S., et al. Gastroenterology 2016, 150, 1620–1632.

Glycans composed of many kinds of monosaccharides.

9

The 10 most abundant monosaccharide units in human cells
OH
O

HO
HO

OH

OH

OH

OH

O

HO
HO

NHAc

O

OH OH
O

O

HO
HO
OH

OH

OH

HO
OH

O
OH

HO
NHAc

OH

D-Glc

D-GlcNAc

D-GlcA

D-Gal

D-GalNAc

2.5%

31.8%

0.3%

24.8%

4.8%

OH
HO
HO

OH OH

OH OH

OH
O

Me
OH

O

OH
HO
OH
AcHN

OH
OH

CO2H

O
OH

D-Man

L-Fuc

18.9%

Neu5Ac

7.2%

8.3%

OH

HO
HO

O
OH

OH

HO2C
OH

D-Xyl

0.1%

OH
O

OH
OH

L-IdoA

0.1%

Werz, D. B., Ranzinger, R. et al. ACS Chem. Biol. 2007, 2, 685–691.

Glycosylation of proteins and lipids occur in the ER and Golgi apparatus.
Major types of glycans in humans

The synthetic pathway of N-glycans

REVIEWS

8
6S

NS

NS

3S
6S

S

2S

S

4S

Free GAG
Hyaluronan

4S

Chondroitin or dermatan sulfate

S or T

O-glycansN-glycans

GPI-anchoredglycoproteins

S or T

O-glycansNS or T

Chapter 1

EGF or TSR

N

S or T

4S

S or T
S or T

Glycosphingolipids

NH

P

Downloaded on 8/4/2021 10:24:55 AM.
shed on 20 March 2017 on https://pubs.rsc.org | doi:10.1039/9781782623823-00001

Glycoproteins

View Onl

Proteoglycans

Heparan sulfate
6S

10

3S

S or T

3S

P

Fuc

Glc

GlcNAcMan

S or T

Gal

GlcA

IdoA

Sialic acid

S or T

GalNAcGlcN

Inositol

Xyl

O-GlcNAc
glycoproteinsFig. 1 | Major types of glycosylation in humans. Glycans can be covalently attached to proteins and lipids to form
glycoconjugates; glycans in these compounds are classified according to the linkage to the lipid, glycan or protein
moieties. Glycoproteins consist of glycans and glycan chains linked to nitrogen and oxygen atoms of amino acid residues
and are thus termed N-glycans and O-glycans, respectively. N-glycans consist of N-acetylglucosamine (GlcNAc) attached
by a β1-glycosidic linkage to the nitrogen atom of the amino group of Asn (N) at the consensus glycosylation motif
Asn-X-Ser/Thr (in which X denotes any amino acid except for Pro). These branched and highly heterogeneous N-glycan
structures consist of a core glycan containing two GlcNAc residues and three mannose (Man) residues. Perhaps the mostdiverse form of protein glycosylation is O-glycosylation, in which glycans attach to the oxygen atom of the hydroxyl groups
of Ser (S) or Thr (T) residues. O-glycans can be further subclassified on the basis of the initial sugar attached to the protein
and the additional sugar structures added to the initial glycan. For example, mucin-type O-glycosylation denotes that theinitial glycan is N-acetylgalactosamine (GalNAc); mucin-type glycans can be further classified on the basis of the glycans
attached to the initial GalNAc6. Other types of O-glycans, such as O-linked fucose (Fuc) and O-linked Man, often occur in
specific proteins or protein domains, such as epidermal growth factor (EGF) repeats, thrombospondin type I repeats (TSR)or dystroglycan. N-glycans and O-glycans are often capped with negatively charged sialic acid. O-GlcNAc is a unique typeof O-glycosylation that is synthesized by O-GlcNAc transferase; it occurs in the cytosol and nucleus. Proteoglycans
represent a major class of glycoproteins that are defined by long glycosaminoglycan (GAG) chains attached to proteinsthrough a tetrasaccharide core consisting of glucuronic acid (GlcA)–galactose (Gal)–Gal–xylose (Xyl); this carbohydratecore is attached to the hydroxyl group of Ser at Ser-Gly-X-Gly amino acid motifs. Proteoglycan GAGs can be further
classified according to the number, composition and degree of sulfation of their repeating disaccharide units; common
GAGs include heparan sulfate, chondroitin sulfate and dermatan sulfate. Glycosylphosphatidylinositol (GPI)-anchoredglycoproteins represent another major class of glycoconjugates. These glycoproteins are linked at the carboxyl terminus
through a phosphodiester linkage to phosphoethanolamine attached to a trimannosyl-nonacetylated glucosamine(Man3-GlcN) core; the GlcN residue is linked to phosphatidylinositol, which is embedded in the cell membrane.
Glycosphingolipids are a class of glycoconjugate in which glycans, such as Gal or glucose (Glc), are attached to cellular
membrane lipids. Another major class of glycans is represented by GAGs that are not attached to protein cores, such as- It is estimated more than half of humanproteins are glycosylated.
- The two major classes- N-glycans: at Asn- O-glycans: at Ser/Thr

Figure 1.2

An abbreviated biosynthetic scheme for protein N-glycosylation in
mammalian cells. First, the synthesis of Man5GlcNAc2–P–P–dolichol
is completed on the cytoplasmic face of the ER membrane. It is thenflipped to the ER lumen and extended to form Glc3Man9GlcNAc2–P–P–
Reily, dolichol.
C., Stewart,
et al.
Nat. 2Rev.
Nephrol.to2019,15, 346–366.
After T.
GlcJ.
is transferred
the nascent
polypep3Man
9GlcNActide,
theN-linkedglycans
matureinastepwisefashionintheand
Chaffey, P. K., Guan, X. et al. Chemical Biology of Glycoproteins, RSC 2017,ER
1–19.

11
Glycansarebiosynthesized
by
more
than
250
glycosyltransferases(GTs).
Research article
Cell Biology Physics of Living Systems

Research article

B-antigen- Glycans are synthesized by glycosyltransferases (GTs).
- Each GTs have relatively low specificity, but the variability is tightly controlled by compartmentalization.
- The detailed mechanism is still unknown.
Jaiman, A. and Thattai, M. Elife 2020, 9, 1–22.

Because of their high complexity, the study of glycans is quite difficult.

12

Heterogeneity

Structural diversity
D-Glc – D-Glc(8 patterns)
O

OH
HO
HO

O4
O

β

O

O

OH
O

6

3

(b)

O

2O

O

α

theoretical number of different oligomers
Oligomersize

DNAProtein

Glycan

1

4

20

20

2

16

400

1360

3

64

8,000

126,0804

256

160,000

13,495,040

5

1024

3,200,000

1,569,745,920

6

4096

64,000,000

192,780,943,360(c)Werz, D. B., Ranzinger, R. et al. ACS Chem. Biol. 2007, 2, 685–691.
Čaval, T., de Haan, N. et al. Curr. Opin. Struct. Biol. 2021, 68, 135–141.

Many live-cell glycoengineering tools have been developed.

13

Genetic glycoengineering in mammalian cells*GT: glycosyltransferase

Exoenzymatic engineering
addition of non-natural units
precise control of glycans
high efficiency
identification of a suitable GT is necessary

today’s
topic

Metabolic engineering
addition of non-natural units
precise control of the monosaccharide of interest
low off-target effectlow efficiency (competition with natural substrates)

Genetic engineeringa variety of reliable approaches
precise control of GTs
low glycan- and protein-selectivity
developmental defects and embryonic lethality
addition of new functionalities is difficultof glycoengineering strategies. Basic principles for approaches available to modulate the cellular glycosylation processes and the
Griffin,M.etc.)
E. and Hsieh-Wilson, L. C. Cell Chem. Biol. 2016, 23, 108–121.
ed. Extracellularly, glycans may be modulated by more or less selective endo-/exo-glycosidases (sialidases, galactosidases,
PNGase,zymatic labeling methods utilizing, e.g., glycosyltransferases (GTs) may be applied to install natural or unnatural substrates on cell
Narimatsu, Y., Büll, C. et al. J. Biol. Chem. 2021, 296, 100448.

Contents-

Introduction- The role of glycans in living organisms- The structure of glycans- Previous development of glycoengineering tools-

Development of next-generation glycoengineering tools
1. Bump-and-Hole engineering
2. Two-step glycan editing-

Perspectives-

Summary14

Next-generation
glycoengineering
tools
have beendeveloped.
Bump-and-HoleEngineering
Identifies
Specific
Substratesengineeringof Glycosyltransferases in 2.
LivingCells
1. Bump-and-Hole
Two-stepglycan editing(Metabolicengineering)
Graphical Abstract

Authors

15(Exoenzymatic engineering)

Benjamin Schumann,
Stacy Alyse Malaker,
Simon Peter Wisnovsky, ...,
Jennifer Jean Kohler, Milan Mrksich,Carolyn Ruth Bertozzi

Correspondenceb.schumann@imperial.ac.uk (B.S.),
bertozzi@stanford.edu (C.R.B.)

In Brief
Schumann et al. establish a cellularglycosyltransferase bump-and-holesystem to chemically tag the substrates
of individual isoenzymes of the GalNAc-T
family.

→ Highlights
More precise control of glycotransferasesd→ More precise control of glycans

GalNAc-T glycosyltransferase bump-and-hole engineering

Choi, J., Wagner,
L. J. S., et al. J. Am. Chem. Soc. 2019, 141, 13442–13453.
retains structure and substrate specificity
Schumann, B., Malaker, S. A. et al. Mol. Cell 2020, 78, 824-834.e15.

Tang, F.; Zhou, M. et al. Nat. Chem. Biol. 2020, 16, 766–775.

1. Bump-and-Holeengineering
Bump-and-HoleEngineering Identifies Specific
Substratesengineeringof Glycosyltransferases in 2.
LivingCells
1. Bump-and-Hole
Two-stepglycan editing(Metabolicengineering)
Graphical Abstract

Authors

16(Exoenzymatic engineering)

Benjamin Schumann,
Stacy Alyse Malaker,
Simon Peter Wisnovsky, ...,
Jennifer Jean Kohler, Milan Mrksich,Carolyn Ruth Bertozzi

Correspondenceb.schumann@imperial.ac.uk (B.S.),
bertozzi@stanford.edu (C.R.B.)

In Brief
Schumann et al. establish a cellularglycosyltransferase bump-and-holesystem to chemically tag the substrates
of individual isoenzymes of the GalNAc-T
family.

→ Highlights
More precise control of glycotransferasesd→ More precise control of glycans

GalNAc-T glycosyltransferase bump-and-hole engineering

Choi, J., Wagner,
L. J. S., et al. J. Am. Chem. Soc. 2019, 141, 13442–13453.
retains structure and substrate specificity
Schumann, B., Malaker, S. A. et al. Mol. Cell 2020, 78, 824-834.e15.

Tang, F.; Zhou, M. et al. Nat. Chem. Biol. 2020, 16, 766–775.

2

Validation of the activity of a certain GalNAc-transferase subtype was difficult.

17

H. C. Hang, C. R. Bertozzi / Bioorg.(GalNAc-T)
Med. Chem. 13 (2005) 5021–5034
O-glycans and GalNAc-transferases()TD$FIG][ Review
GalNAcTrends in Cell Biology Ma(a)High density
O-GalNAc

P T T D S T T PA P T T K K

Low density
O-GalNAce.g., GalNAc-T7 / T10

P T T D S T T PA P T T K K“Initiating” GalNAc-Ts

e.g., GalNAc-T1 / T2

O-glycosylation
Initiation“Finishing” GalNAc-TsP T T D S T T PA P T T K K(b)

O-glycan capping

Sialic acid ST3Gal
ST6Galadditionure 1. Mucin-type O-linked glycosylation. (A) Architecture of membrane-bound and secreted mucins that crosslink into oligomers. Blue ribbons
esent protein backbones. Red spheres represent O-linked glycans. (B) Initiation of mucin-type O-linked glycosylation by the ppGalNAcTs, which- GalNAc transrefases (GalNAc-Ts) are one of the largent glycosyltransferaseGalactose
families.
extensionze UDP-GalNAc as the nucleotide sugar donor and modify protein substrates.
of
core
1
4
- Because of its redundancy, the precise role of each subtype was unclear.
poly-lactosamineCore extension
d the development of chemical tools for understandNAc linkage to Thr/Ser was ultimately
determined byextension of core 2 / 4

this family of glycosyltransferases in biology.

Weissman and Hinrichsen in 1969 with a purified a-N13
O-glycanHang,
C. andliver.
Bertozzi,
C. R. Bioorg. Med. Chem.
2005,sugars
13, 5021–5034.
acetylgalactosaminidase
fromH.bovine
Gill, D. J., Clausen, H. et al. Trends Key:
Cell Biol. GalNAc
2011, 21, 149–158.

Bump-Hole engineering enabled subtype-specific labeling.

18

Bump-and-Hole engineeringtarget enzyme(WT)target protein

clickable tag+ “bump”-UDP-

other subtypes-UDPmutant enzyme

-

-

-UDP

-UDP

-

Normal metabolic labelingsmall clickable tag(N3, alkyne, etc.)
-UDP
-UDP

“Bumped” substrates is notrecognized by native enzymes→ Low false-positive signals

-UDP
-UDP

-UDP

Mutant enzymes do notrecognize→ High S/N ratio.

Mutagenesis of gatekeeper residues changed substrate specificity of GalNAc-T2 in vitro.
Structure of GalNAc-T2

of WT/mut enzymes
Journal ofSubstrate
the Americanspecificity
Chemical Societyof the American Chemical Society

Article

WT

19

mut

37 ℃, 1 h
R = Mebumped substrate
WT

mutdentiﬁcation of gatekeeper residues. (A) Residues within 5 Å of GalNAc methyl carbon for GalNAc-T2 (PDB ID 4D0T). Five of sevens in close proximity to the GalNAc methyl contain side chains; of these, H359 and D224 coordinate Mn2+, while I253, L310, and F361
Figure 3. Screening GalNAc-T2 for an orthogonal enzyme−substrate pair. (A) Scheme for glycosylation reaction with Peptide-1, GalNng hydrophobic residues. (B) Space-ﬁlling model of gatekeeper
residues within
5 Å of GalNAc
in glycosylated
GalNAc-T2Peptide-1.
(PDB IDBlue
4D0T).
UDP-GalNAc
or UDP-GalNAc
analogmethyl
to form
T indicates the Thr glycosylation site used by GalNAo acid sequences of human GalNAc-T1−GalNAc-T20 surrounding
residues
a high degree
Glycopeptidepotential
formation gatekeeper
by wild-type andmutantdemonstrate
GalNAc-T2. UDP-GalNAc
and ofPeptide-1 were incubated with GalNAc-T2 at 37 °C fn with 13 isoenzymes containing Ile/Leu and 18 containingIleatresidues
homologous
to
GalNAc-T2
positions
253/310
or
253,
Choi,
J.,
Wagner,
L.
J.
S.,
et
al.
J.
Am.
Soc.
2019, 141,product13442–13453.
reaction was quenched by addition of aqueous EDTA (150 mM, pH = 8.0). PercentChem.
conversion
to glycopeptide
was quantiﬁed

gatekeeper residues

The same mutation was effective for other subtypes, without changing the site specificity in vitro.

NAc

Selectivity of other subtypes

Glycosylation site specificity (LC-MS/MS)

Journal of the American Chemical Society

20

Art

Article

WT

mut

WT
mut

bumpedsubstrate

Figure 6. Glycosylation Choi,
of natural
substrates
wild-typeengineered
GalNAc-T
pairs. (A) SchemJ.,peptide
Wagner,L. J.byS.,
et al.andJ. Am.
Chem.
Soc. isoenzyme−substrate
2019, 141, 13442–13453.

Caged compound was converted to GalNAc-UDP analog in living cells.
Substrate delivery using caged substrate analog
Articlell
Figure 4. Substrate Delivery to the Cytosol of
Living Cells

steps are indicated by crosse
erase GALE interconverts
21
UDP-GalNAc.
(B) HPAEC-PAD traces of ext
cells stably expressing WT-AG
fed with the indicated compou
indicate retention times of sta
reference runs. The product oftion of 1 by GALE, compoundarrowhead. Data are of one e
repeated for compound 5 insiently transfected with AGX1 cstably transfected K-562 cells.
to 8 is suppressed in GALE-d
expressing mut-AGX1 and fed
carrying a control single guidreference trace of compound 1
one representative out of two
ments.
See also Figure S3.

(A) Schematic of substrate delivery. Non-permissive
steps are indicated by crossed arrows. The epimerase GALE interconverts UDP-GlcNAc and
UDP-GalNAc.
(B) HPAEC-PAD traces of extracts from HEK293T
cells stably expressing WT-AGX1 or mut-AGX1 andfed with the indicated compounds. Dashed boxes
indicate retention times of standards in separate
reference runs. The product of potential epimerization of 1 by GALE, compound 8, is marked with anarrowhead. Data are of one experiment and wererepeated for compound 5 in HEK293T cells transiently transfected with AGX1 constructs, as well as
stably transfected K-562 cells. Insert: epimerization
to 8 is suppressed in GALE-deficient K-562 cells
expressing mut-AGX1 and fed with 5, but not cells
assess any potential background protein labeling when UDP- cocarrying a control single guide RNA (sgRNA). A(Ch
analogcould
when 1 was
whenorfunctional
bump-areference GalNAc
trace of compound
1 is 1
shown.
Datanot
are ofbe biosynthesized
one representativeout of two independent
experiTp
biosynthesized
but BH-GalNAc-Ts
were absent.
To preclude
mut-AGX1,
and BH-GalN
ments.
any labeling due to epimerization of 1 to UDP-GlcNAc
sent (Figuresanalog
5C andvenS
See also Figure S3.

isomer(UDP-GlcNAc analog)
bumped substrate-an
8, we first used K-562 GALE-KO cells for waslabeling.
Cellsforwererequiredlabeling(UDP-GalNAc
analog)supplemented with GalNAc to compensate
for the loss probe
of beeour experiments
Expression of mutAGX (F383A) and knockout
of
GALE
was
necessary.
mig
UDP-GalNAc biosynthesis (Figure S3C). GalNAc-T
expression
sylation rather
than nonwhen
bump-and-holepairs (5,was induced(Figure S4A),
cells were fed with
caged GalNAcby
Compound 5 was the only substrate
offunctional
mutAGX.
modification(Qin et al.,mut-AGX1,
and
BH-GalNAc-Ts)
were
premo
1-phosphate analog 5, and the cell surface
was wasreactedwith inte
cenceof similar
sent (Figures Schumann,
5C and S4F).
Mut-AGX1
B., Malaker,
S. A.
Mol. Cellof
2020,
78, 824-834.
lecclickable Alexa488-picolyl
azide
in et
theal.presencea non-mem-Selective metabolic labeling was achieved in living cells.

K562 cells(GALE-KO)

ArticlemutGalNAc-T1/2
mutAGX

5

transfection
24 hlabeling
20 h

click
(CF680)collect cellsurface proteins
FACS

FACS

Article22

SDS-PAGE
SDS-PAGE

ll
ArticleFigure 4. Substrate Delivery to the Cytosol of
Living Cells(A) Schematic of substrate delivery. Non-permissive
steps are indicated by crossed arrows. The epimerase GALE interconverts UDP-GlcNAc and
mutAGXUDP-GalNAc.
UDP-GalNAc analog
mutfrommut
WTHEK293T(B) HPAEC-PAD traces of mut
extracts
mutGalNAc-T
cells stably expressing WT-AGX1 or mut-AGX1 andprotein-GalNAc analog
mut
WT
mut
fed with the indicated compounds. Dashed boxes
indicate retention times of standards in separate
reference runs. The product of potential epimerization of 1 by GALE, compound 8, is marked with anarrowhead. Data are of one experiment and were
- More than 15-fold fluorescence was detected from labeled cells.
repeated for compound 5 in HEK293T cells tran- There were slight difference between band patterns from GalNAc-T1
and GalNAc-T2.
siently transfected with AGX1 constructs, as well as
stably transfected K-562 cells. Insert: epimerization
Schumann, B., Malaker, S. A. et al. Mol. Cell 2020, 78, 824-834.

Bumped GalNAc analog was recognized by downstream enzymes.
n = 1 or 2
mutGalNAc-Tn
mutAGX
HepG2 cells
transfection(GalNAcTn-KO)
24 h

23

LC-MS/MS5labeling
20 hcollect cell culture
supernatants
click (acid-cleavable biotin linker);(secretome)
digestion;
LC-MS/MS

Figure 5. Selective Bioorthogonal Labeling of the Living Cell Surface with Bump-and-Hole Engineered GalN

previous
data(A) GalNAc-T and AGX1 co-expression construct and workflow of cell surface labeling. Red star depicts
a fluorophore.
(B) Labeling analysis of K-562 GALE-KO cells by flow cytometry of MB488-picolyl azide labeled and intracellular VSV-G-st
individual values from three independent experiments, mean ± SEM of MB488 median fluorescence intensity of VSV-G-p
performed by two-tailed ratio paired t test.
this method(C) Labeling analysis by in-gel fluorescence of PNGase F-treated lysates from metabolically labeled K-562 cells. In-gel fluore
from one gel, and expression analyses are from one separate western blot. Data are representative of three independent e(D) Schematic of glycoprotein enrichment and on-bead digest. The bifunctional molecule 10 bears an acid-labile diphenyl- Known glycosylation
were detected (bumped analog was recognized by downstream enzymes).
(E) Exemplary MS data: mass spectrum (HCD) of a fully elaborated glycopeptide from SERPIN5A (site Thr39) and further e
Subtype-specificglycosylation
clearlyEngineered
observed. GalNAc-Tsling -of the
Living CellSTC2
Surfacewith(Thr28)and Bump-and-Hole
APOE was(Ser308).
truct and workflow of cell(F)surface
labeling.
Red data
star ondepictsa fluorophore.
Upper panel:previous
ApoAI220-230
glycosylation in GalNAc-T1 and T2 KO HepG2 cells (Schjoldager et al., 2015
Schumann, B., Malaker, S. A. et al. Mol. Cell 2020, 78, 824-834.

Substrates of Glycosyltransferases in Living Cells
Short summary: Glyco-engineering by bump-and-hole engineering
Graphical Abstract24

Authors
Benjamin Schumann,
Stacy Alyse Malaker,
Simon Peter Wisnovsky, ...,
Jennifer Jean Kohler, Milan Mrksich,Carolyn Ruth Bertozzi

Correspondenceb.schumann@imperial.ac.uk (B.S.),
bertozzi@stanford.edu (C.R.B.)

In Brief
Schumann et al. establish a cellularglycosyltransferase bump-and-holesystem to chemically tag the substrates
of individual isoenzymes of the GalNAc-T
family.

-Highlights

The first glycotransferase
bump-and-hole system in the living cell was established.
GalNAc-T glycosyltransferase bump-and-hole engineering
This system allowed isoenzyme-specificretains structure and substratelabelingspecificity in the endogenous environments.
ddBumped UDP-GalNAc analogs can be delivered to the
Choi, J., Wagner, L. J. S., et al. J. Am. Chem. Soc. 2019, 141, 13442–13453.
living cell

Schumann, B., Malaker, S. A. et al. Mol. Cell 2020, 78, 824-834.e15.

2. Two-step
glycan editing
Bump-and-HoleEngineering Identifies Specific
Substratesengineeringof Glycosyltransferases in 2.
LivingCells
1. Bump-and-Hole
Two-stepglycan editing(Metabolicengineering)
Graphical Abstract

Authors

25(Exoenzymatic engineering)

Benjamin Schumann,
Stacy Alyse Malaker,
Simon Peter Wisnovsky, ...,
Jennifer Jean Kohler, Milan Mrksich,Carolyn Ruth Bertozzi

Correspondenceb.schumann@imperial.ac.uk (B.S.),
bertozzi@stanford.edu (C.R.B.)

In Brief
Schumann et al. establish a cellularglycosyltransferase bump-and-holesystem to chemically tag the substrates
of individual isoenzymes of the GalNAc-T
family.

→ Highlights
More precise control of glycotransferasesd→ More precise control of glycans

GalNAc-T glycosyltransferase bump-and-hole engineering

Choi, J., Wagner,
L. J. S., et al. J. Am. Chem. Soc. 2019, 141, 13442–13453.
retains structure and substrate specificity
Schumann, B., Malaker, S. A. et al. Mol. Cell 2020, 78, 824-834.e15.

Tang, F.; Zhou, M. et al. Nat. Chem. Biol. 2020, 16, 766–775.

Two-step editing will reduce the heterogeneity of glycans on living cells.
The heterogeneity of N-glycans

Two-step glycan editing strategy
ARTICLES

NATURE CHEMICAL BIOLOGY
Complex-type

Non-core-fucosylated

NATURE CHEMICAL BIOLOGY
FucHigh-mannose Hybrid

Man

GlcNAc( )n( )n( )n
( )n

Gal

( )n
( )n

Core-fucosylated

Biantennary

Bisecting

Triantennary( )n( )n( )n( )n( )n ( )n( )n ( )n( )n( )n( )n( )n( )n( )n( )n( )n( )n( )n

( )n
( )n

Neu5Ac n = 0 or 1

Complex-type

Non-core-fucosylated

High-mannose Hybrid( )n( )n( )n( )n ( )n
( )n

Gal

Core-fucosylated

Biantennary

Bisecting

Triantennary( )n( )n( )n( )n( )n ( )n( )n ( )n( )n( )n( )n( )n( )n( )nCell-surface N -glycans( )n( )n( )n( )n

( )n
( )n

Endo-F3 WT

Non-core-fucosylated( )n( )n( )n
( )n

or 1

)n
)n

26

( )n
( )n

Selectively‘delete’Selectively‘delete’Core-fucosylated( )n( )n( )n( )nNon-core-fucosylated( )n( )n( )n( )n( )n( )n

COOH

N

O
AcHN
β4
O
HO
COOH
N
O
AcHN
β4
HO

N3

Endo-F3 mutant
Az-SCT-ox

Non-core-fucosylated

Selectively‘delete’Endo-M WT- The heterogeneity of glycans often results inambiguousconclusions about
the glycosylation- Core-fucosylated
Core-fucosylated
Non-core-fucosylated
involved functions of cell membrane.

( )n ( )n( )n ( )n

( )n
( )n

β2

HO

α6

β2

α3

O
β4 HO

O
N

Endo-M mutant
Az-SCT-oxO“insert”

Precisely‘insert’Az-SCT-ox (1)Cell-surface N -glycans
Selectively‘delete’( )n( )n( )n( )nCell-surface N -glycans
O

N3

Endo-F3 WTCore-fucosylated( )n ( )n( )n ( )nCell-surface N -glycans

Precisely‘insert’“delete”

Endo-M WT( )n( )n( )n( )n( )n( )n( )n( )n( )n( )n( )n( )n( )n
( )n

( )n
( )n

Core-fucosylated( )n( )n( )n ( )n( )n ( )nN3--N3 N3-

Non-core-fucosylated-N3 N3--N3Cell-surface N -glycans

N3--N3 N3--N3 N3-Core-fucosylated-N3 N 3-N3( )n( )n( )n( )n( )n( )n( )n( )n( )n( )n

( )n
( )n

Cell-surface N -glycans( )nFig. 1 | Cell-surface N-glycan editing in a subtype-selective manner by a two-step strategy. Left path: selective editing of core-fucosylated N-glycans by a()n step with WT Endo-F3 and an ‘insert’ step with Endo-F3 mutant. Right path: selective editing of non-core-fucosylated N-glycans by a ‘delete’ step‘delete’with WT Endo-M and an ‘insert’ step with Endo-M mutant.

the adaptability of this two-step editing strategy for various glycoproteins.
To test the compatibility of this approach with other cells, weapplied this N-glycan editing to cells such as the SK-BR-3 and H838

core-fucosylated N-glycan subclass through fluorescence resonanceenergy transfer (FRET) technology, which has been used successfully to image glycans of a specific membrane protein40. As shownin Supplementary Fig. 10, the sialic acid motif after glyco-editing

Tang, F.; Zhou, M. et al. Nat. Chem. Biol. 2020, 16, 766–775.

WT/mut endo-glycosidases catalyze glycoprotein “delete/insert” reaction.
Glycoengineering by WT endo-glycosidases (“delete”)

27

View Article Online

Review Article

Chem Soc Rev

WT enzyme (e.g., Endo-F3)
WT

glycoprotein(glycan-GlcNAc-Asn)

X = OH
View Article Online

Chem Soc Rev
Glycoengineering by mutant endo-glycosidases(“insert”)

mutant enzyme (e.g., Endo-F3 D165A)

mut

Fig. 3The use of ENGases to catalyse: (a) glycoprotein remodelling; (b) convergent glycopeptide synthesis.

Table 1

A selection of the most well-studied ENGases

X = oxazoline

substrate(glycan-oxazoline)

glycoprotein(GlcNAc-Asn)

EnzymeSourceGH family

Glycan structure hydrolysed

Glycans with corefucose hydrolysed?

Catalytic
residues

Glycosynthasemutants

Endo-A

Arthrobacter protophormiae

85High mannose/hybrid

No

E173a
N171a

N171Aa
E173QaFairbanks, A. J. Chem. Soc. Rev. 2017, 46, 5128–5146.
aThe biosynthesis of the glycan portions of N-linked glycoproteins is not under direct genetic control; rather a complex
sequence of enzymatically catalysed events leads to the formationof diverse N-glycan structures. Firstly the enzyme oligosaccharyltransferase (OST) mediates the transfer of a 14-mer dolichol phosphate bound oligosaccharide (Glc3Man9GlcNAc2) to particular

Therefore all recombinant therapeutic glycoproteins, including
mAbs, are currently manufactured and administered as mixturesof materials in which different oligosaccharide structures arelinked to the same peptide chain.9
Access to pure single glycoforms of glycopeptides and glycoproteins has consequently become a major scientific objective.10

Supplementary
Tables:Many endo-glycosidases with different selectivity
havebeen discovered.

Substrate selectivity of endo-glycosidases

28

Supplementary Table 1. Substrate specificities of Endo-glycosidasesa [ref]EnzymeGH

Biantennary

Fucosylated

Triantennary

Fucosylated

High

Hybridcomplex-type

Biantennarycomplex-type

Triantennarymannosetype

familyEndo-A

85

-

-

-

-

+++

+

Endo-M

85

+

-

-

-

++

+

-

++

+

-

-

+

+

-

-

-

-

2,6-branched
Endo-Om85++-

triantennary(+)

Fig. 1 Chemical structures and Glycobiology representations of typical N-glycans showing the ENGase cleavage site: (a) high mannose; (b) hybrid;(c) complex biantennary with core fucose.

This journal is © The Royal Society of Chemistry 2017

Chem. Soc. Rev., 2017, 46, 5128--5146 | 5129

Endo-H18--

Endo-D

85

Truncated

Truncatedcore (+++)

core (+++)

Endo-CC

85

+

-

-

-

+

-

Endo-CE

85

-

-

-

+

+

Endo-BH

85

-

-

-

-

+

+

Endo-Sb
b

18

+

+

-

-

-

-

Endo-S2

18

+

+

-

-

+

+

Endo-F1

18

-

-

-

-

+

+

Endo-F2

18

+

+

-

-

-

-

Endo-F318+ (weak)++++ (weak)

+++

-

-

a

Truncatedcore (+)

Each N-glycan icon in the table represents a typical structure of a certain subtype but does not cover all of the- Endo-F3: selective for core-fucosylated N-glycans(400-foldhigherthan
non-fucosylated
ones)
subtype N-glycan
structures. Endo-S
and Endo-S2hydrolyze
IgG antibody Fc N-glycan ina much more higher rateother glycoproteins and glycopeptides. Mark of “-” represents no hydrolytical activity; Mark of “+”- Endo-M: selective for non-corfe-fucosylated thanN-glycansbrepresents positive hydrolytical activity and marks of “++” and “+++” represent relatively higher hydrolytical

Fairbanks, A. J. Chem. Soc. Rev. 2017, 46, 5128–5146.
Tang, F.; Zhou, M. et al. Nat. Chem. Biol. 2020, 16, 766–775.
[ref] 1. Waddling, C. A., Plummer, T. H., Tarentino, A. L. & Van Roey, P. Structural Basis for the Substrate Specificity
activity.

Selective editing and imaging of core-fucosylated N-glycans is achieved.

29

CHO cells (ovary)
SK-Br-3ARTICLES
cells
FBS+ medium
without “delete” step e (breast cancer)c

NATURE CHEMICAL BIOLOGYa(“delete”)

ARTICLES

NATURE CHEMICAL BIOLOGY
a

c

e

b

d

f

(“insert”)

CHO cells (ovary)
FBS– medium
dwith “re-delete” step fb

H838 cells(lung cancer)

Supplementary Figure 3. Cell surface N-glycan editing by Endo-F3 enables selective
glycoengineering of core-fucosylated N-glycan to replace the native heterogeneous glycoforms
with a labeled homogeneous N-glycan.

Endo-F3 wild type(“re-delete”)

g

h
15

i

128

1

2

3

4

1

2

3

4

Post-Endo-F3

Endo-F3 D165A

128

Az-SCT-ox

15h

Pre-Endo-F3

g 10

countscence intensity

kDa
250

i 130
100

Tang, F.; Zhou, M. et al. Nat.+ Chem.
Biol.702020, 16, 766–775.
– + –Selective editing and imaging of non-core-fucosylated N-glycans is achieved.

ARTICLESARTICLESaa

CHO cells(ovary)30

HEK293T cells(kidney)
c
c

e 150

(“delete”)b(“insert”)d

CHO cells (ovary) High Five insect cells(ovary)without“insert” step dbNumber ofNumber
cells of cells

e 150
100

100
50

50
0
10
0
10

mentary Figure 15. Cell surface N-glycan editing by Endo-MFig.enables
selective
3 | Selective
editing and imaging of non-core-fucosylated N-glycans on a living cell surface
mutant.
a,c,d, Confocal microscopy imaging of glyco-edited CHO cells (a), HEK293T cells (c) an
gineering of non-core-fucosylated N-glycan to replace the native
heterogeneous
Non-enzymatic control group of CHO cells without Endo-M N175A in the second ‘insert’ step. eFig.
3 | Selective editing
andediting.
imagingGreenof non-core-fucosylated
N-glycansa living cell surface
rms with a labeled homogeneous N-glycan.
Endo-M-catalyzed
N-glycancurve, unedited cells;
red curve,onnon-enzymaticcontmutant.
Confocal microscopy
imaging
CHO cells
HEK293T
cells (c) an
Zhou,
M.ofetglyco-edited
al. Nat. Chem.
Biol.(a),2020,
16, 766–775.
Data area,c,d,representativeofTang,three F.;
individual
experiments.

N-glycan editing reduced the heterogeneity of glycans structure-selectively.

31

Glycan-edited CHO cell

Heterogeneity of glycans (MALDI-TOF MS)
f

×105

Unedited Lec4 N-glycan profile

1.0
0.5
0
×105

Edited by Endo-F3 and CT-ox

Intensity

1.0
0.5
0
×105

selective editing forfucosylated glycans→ less heterogenous

Edited by Endo-F3/A/M and CT-ox

1.0
0.5
0
×105

Edited by Endo-F3/A/M and GN2M3-ox

editing for
amost all glycans→ homogenous

1.0
0
1,500

1,750

2,000

2,250

2,500

2,750

3,000

3,250

3,500

3,750

m/z

erogeneous to homogeneous N-glycan editing on Lec4 CHO cells. a, Schematic of selective glyco-editing on Lec4 CHO cells. b–e, LSCM
Tang,F.; Zhou,M. et al.groupNat. Chem.
Biol.
2020, 16, 766–775.
o-edited cells under different conditions: glycan editing and labeling in a 5% FBS-containing PBS
buffer(b); control
withoutEndo-F3

Dimerization and internalization of OPRD1 was highly affected by N-glycans although agonist recognition was not.

ARTICLES
Dimerization of OPRD1a

F3 mutant/
Az-SCT-ox

F3

NATURE CHEMICAL BIOLOGYInternalization
ofNATURE
OPRD1CHEMICAL BIOLOGY

PNGf

c

WT

a

WT

CT

SCT

32

ARTICLES
F3

PNG

0 min

monomer→

35 kDa1

2

3

4

IB: anti-HA tag on OPRD1

NATURE CHEMICAL BIOLOGY

b

WT

c

F3

F3
N18/33Q
N18/33Q
crosslinked
crosslinked

60,000Competitive cAMP assay
665/620 ratio × 10,000

50 kDaWT

Forskolin 30 µM

75 kDa40,000120 min

20,000bRD1

N18/33Q
N18/33Q
kedcrosslinked

D3C2
D3C2-F3
D3C2-SCT

40,0001

2

3

4

IB: anti-HA tag on OPRD1
–10

–5

0
log[5-leu concentration (µM)]

55

D3C2-F30.8

0.014 ***

0.7
EC50 (nM)
0.6

***

0.5

20,000

0
–15

D3C2

0.9

0.4
0.3
0.2
0.1

*

*

*

***

*

**

****

**

***

0.020

1.3

“delete”
only

1.0

60,000

1.4

5

1.1

Forskolin 30 µM

Ratio

4

–10
–5
0
log[5-leu concentration (µM)]

1.3
1.2

Competitive cAMP assay
665/620 ratio × 10,000

35 kDa35 kDaInternalization/membrane
fluorescent ratio
without agonist

0
1.4
–15

Agonist recognition of OPRD1
75 kDa75 kDa50kDa
50 kDa

D3C2
D3C2-F3
D3C2-SCT

**

**

1.1
1.0

PNGaseF
0.9
D3C2-SCT
Endo-F30.8
WT0.7
SCT 0.015
0.6
CT
0.5

“delete”+“insert”

Internalization/membrane
fluorescent ratiowith 1 µM agonist

1.2

Ratio

dimer→

0.4

*

*
WT
PNGase F
Endo-F3
CT

0.3
0.2
0.1

Fig. 5 | N-glycan editing of the OPRD1 receptor on Lec4 CHO cell membrane deciphers theroleofblot analysis of 0HA-tagged0
15
30 glycosylation.
45
60
75
90a, Western
105 120
0
15
30
45
60
75
90
Min
Min
D3C2-SCTOPRD1 during glyco-editing. Lane 4,D3C2
wild-typeD3C2-F3
OPRD1. Lane
3, OPRD1 after PNGase-F digestion. Lane 2, OPRD1
after WT Endo-F3. Lane1, OPRD1 after
EC (nM)the Endo-F3
0.014
0.020
0.015
*PNGase
F: cuta, between
Asn OPRD1-Lec4
and glycan
two-step glyco-editing using
mutantand Az-SCT-ox(0.5!mM). b, Westernof assaycrosslinked
OPRD1
and
mutant.
WT antibody.
Fig. 6 | blot
OPRD1analysis
internalization
by time-lapse
monitoringon itsconfocalimagingLanewith an1,anti-HA
Imaging of glyco-edited
cells at 0 and 120!min without agonist. Images are representative of three individual experiments. Scale bars, 20!μm. b, Internalization rate analysis based
OPRD1. Lane 2, OPRD1 after Endo-F3 digestion. Lane 3, OPRD1 after Endo-F3 digestion, followed by crosslinking with a bifunctional linker (BS3, Thermo

3

0

50

4

5

Tang, F.; Zhou, M. et al. Nat. Chem. Biol. 2020, 16, 766–775.

on internalized/membrane fluorescence ratios (repeated twice with similar results). Data are presented as mean!±!s.e.m. of n!=!15 cells, and were analyzed

Short summary: Glyco-engineering by two-step exogenous enzyme reaction-

33

N-glycan selective editing was achieved by two step enzymatic reaction.
The heterogeneity of live-cell surface glycans became much lower after reaction.
This system may be practical tools to elucidate the functions of a certain type of glycosylation.
Tang, F.; Zhou, M. et al. Nat. Chem. Biol. 2020, 16, 766–775.

Contents-

Introduction- The role of glycans in living organisms- The structure of glycans- Previous development of glycoengineering tools-

Development of next-generation glycoengineering tools
1. Bump-and-Hole engineering
2. Two-step glycan editing-

Perspectives-

Summary34

displayed on the surface of living cells

Perspective:
Glycobiology
is receiving
increased attention.
Graphical abstract
Authors
NATURECHEMICAL
BIOLOGY
Correspondence

Immune cell
Immuneryan.flynn@childrens.harvard.edu
cell
1/2

ITIMIn brief

ITIM

Sialylated
proteins& lipids

PD-L1

ITIM

Identification of stable mammalian RNAs
decorated with glycan structures opens
up a new dimension for regulatory controlof RNA localization andPD-1
function by posttranscriptional modification.

SiglecARTICLES
Cleavedglycans a

PD-L1

Flynn, R. A., Pedram, K. et al. Cell 2021, 184, 3109-3124.e22.

ArticleProtein degradation with glycan liganddaCancer cell or APC

LYTAC

e

d

d

d

RNA-glycan conjugates, glycoRNAs, are conserved, small,noncoding RNAs
GlycoRNAs possess N-glycans that are highly sialylated andTargetfucosylatedg
Tar

GlycoRNAs are displayed on the cell surface and can bind
Siglec receptors

etCI-M6PR
get
Tar

Flynn et al., 2021, Cell 184, 3109–3124
June 10, 2021 ª 2021 Elsevier Inc.
https://doi.org/10.1016/j.cell.2021.04.023

T-Sia 1c+

Conjugate
HER2 EMT6
i.p. injections
orthotopic(10 or 15 mg kg–1)
injection

Endpoint:
1.5
80 andP =Mooney,
0.013
Wang, H.
D. J. Nat. Chem. 2020,12, 1102–1114.
tumor volume
or ulceration

60

Cancer cell or APCb

ARTICLESa

LectinOAc
AcO binding80
O
P = 0.013
domainAcO

0
Tras

Conjugate
HER2+ EMT6
i.p. injections
orthotopicO
d kg–1)(10 or 15 mg
injection

13 steps

AcO
40

20

OAc
O

AcO
AcO

b

100

60

OEt
O P OEt

f

0

40

1.2

CatalyticOAc
domainHN100
0

8 10 13

O

17 O

HN c
O

PBS

c 0

Tras

Tras

NA-647

T-Sia 2

T-Sia 2

ll

0.9

O

50 2 (15 mg kg–1)
T-Sia

0

HO

1.8
1.5

e

1.2

2. TMSBr, CH2Cl2
0.9
Days
then K2CO3

M6Pn-NCA
T-Sia 2 (10 mg kg–1)

20

Days

0.6
1. Ni(COD)2, bpy
OH–1
T-Sia 2 (10 mg kg )THF, 48 h, RT PBS
NATUREBIOLOGY
0.3
O P OH CHEMICAL–1
Poly(M6Pn-co-Ala
Tras
T-Sia 2 (15 mg kg
)
Me
OH
0
M6Pn = 20–90
T-Sia 2O
HO
O

Endpoint:
tumor volume
or ulcerationO

8 10 13 17

0.6

H

H
N

0

0.3
0

O

O
N

HER2+
H
EMT6
Me
s.c.
injectionf––– PBSO

T-Sia 2 i.p.
5
10
20
30
injection
Time (d after tumor injection)

0P=
Fig. 1 | Targeted desialylation of Siglec ligands with the antibody–enzyme conjugate T-Sia 2 as a modality for immune therapy. a,b, PD-1 (a) and 0.0010

Degradation

1.8

Cancercell or APC degradation for cancer therapy
Chemoenzymatic
glycanCancer cell or APC

Synthetic, clickable sugars that label glycoproteins andglycolipids also label RNAs

Percentage cytotoxicity

Highlightsb

100

Tumor volume (cm3)SiglecITSM

PNA binding(exposed galactose)

SHP
1/2

PD-1

Immune cell

(R.A.F.),(C.R.B.) ITSM
SHP
ITSMbertozzi@stanford.edu

II binding
sialic acid)

ITIM

desialylation therapy

inhibitor therapy

Siglec signaling

Immune cell
ITSM

Metaboliclabeling
for cancer therapydTargeted
Checkpoint

Tumor volume (cm3)bPercentage cytotoxicity

PD-1 signaling

ARTICLES

Ryan A. Flynn, Kayvon Pedram,
Stacy A. Malaker, ..., Peter W. Villalta,
Jan E. Carette, Carolyn
R. BertozzicPercentage survival

Glycan-modified
RNA
a

35

n

40

f to recruitment+ Control (ex vivo
d binding. Engagemente0
CI-M6PR
Siglecs(b) areS.
receptors
that typicallysuppressimmunecell function
ligandof PD-1
receptors
LectinBanik,
M., Pedram,
K. etal. Nature
2020,
584, on291–297.
Gray,
M.and
A.,Siglec
Stanczak,M.leads
A.
et al. Nat. Chem. Biol. 2020,
16, 1376–1384.
0

10

20

PBS
30

40

9T-Sia 2

sialidase treated)determination of O-GlcNAc protein stoichiom
Cite This: ACS Chem. Biol. 2020, 15, 1059−1066
Onlineexpected,
we observed an Read
increasein O-GlcNAc36 l
Cell Chemical BiologyVarious tools for glycan engineering is
Perspective:
growing.
Review
synuclein determinedby a mass
shift assay upon ex
Direct manipulation of glycans
Protein-selective
manipulationofglycans
Metrics & More
Article Recommendations
Supporting Inf
ACCESS
RFP(13)and nEPEA(4),but not the *sıcatalytica
mutant nEPEA(4,K852A) (Figure 5B). Notably, ex
untargeted RFP(13) resulted in a dramatic increase
gure 4. Quantitative proteomics and glycoproteomics of the globalof O-GlcNAcylation across the global HEK293T ce
GlcNAc proteome from α-syn KO HEK293T cells. (A)uantitative proteomics of enriched O-GlcNAcylated proteins fromthat was signiﬁcantly reduced in the nEPEA(4) sam
syn KO HEK293T cells after co-expression of nEPEA(4)together, these data demonstrate the high sele
A

B

C

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Please cite this article as: Griffin and Hsieh-Wilson, Glycan Engineering for Cell and Developmental Biology, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2015.12.007

ghlighted in blue) and JunB-Flag-EPEA (highlighted in red)mpared to expression of JunB-Flag-EPEA alone (control). (B)nn diagram and list of unambiguous glycosite assignments for
ABSTRACT: O-Linked β-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in ce
nB-Flag-EPEA. (C) Venn diagram and list of unambiguous
throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc lev
M. E. and for
Hsieh-Wilson,
L. C. Cell Chem.
2016,protein
23, 108–121.
proteinin cells
would accelerate studies of this essential modiﬁcation. Here, we report a generalizable strategy forcositeGriffin,
assignments
Nup62-Flag-EPEA.
TheBiol.
targetwas
Fig
nEPEA:
nanobody
which
GlcNAcinto
selectedtarget proteins in cellsWith
using aperiodate
nanobody astreatmenta proximity-directing
agent fused
to O-GlcNAc
trans
a
c
binds
α-Synuclein
Biotinfusion protein in Fusion
expressed with the indicated OGT
α-synKOof a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequenceglyc
EPE
K20
K88
O N
OGT yielded nanobody-OGT
constructs
that
selectively
delivered
O-GlcNAc
toaseriesoftaggedtargetproteins
(e
HOOC
5,000
EK293T cells, immunoprecipitated, and analyzed
by andMS.
Only
C
Nup62).
Truncationofthetetratricopeptide
repeatdomainasin
OGT(4)
increased
selectivity
forthetarget
prote
(a)
O
O
AcHN
nanobodybyreducing
globalelevationof
O-GlcNAclevelsinthecell.
Quantitative
chemicalproteomics
conﬁrmed–anilineonoglycosylated peptides withunambiguous
assignments
andaBiotinHO
NH
O-GlcNAc to the 4,000
target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) aldeor fu
Oreplicates are shown. Three
O biological+anilineM count of >2 across
HOOC
produced a similar glycosite proﬁle on the target protein JunB and Nup62. Finally, we demonstrate the ability to se
bypr
endogenous α-synuclein
3,000for O-GlcNAcylation in HEK293T cells. These ﬁrst proximity-directed OGT constructs
O
ological replicates wereperexperiment.
AcHNperformedO
strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteom
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BRIEF COMMUNICATIONS

Figure 9. De novo Display of Cell-Surface Glycans(A) NeuAc-containing glycans and HS GAGs were chemically appended to a ketone-functionalized polymer and inserted into the plasma membrane using aphospholipid tail.
(B) CS GAGs were displayed on the cell surface by functionalization onto ketone-containing liposomes.
(C) Genetic incorporation of the HaloTag protein allowed for the immobilization of HS GAGs modified with a chlorohexyl linker.

erythrocytes (which lack A- and B-type antigens) to allow for lipid
insertion of the glycomimetics in the membrane. The newly
2 blood cells displaying the artificial A- and B-type
remodeledglycolipids acquired a strong immune response to anti-A andanti-B antibodies, respectively. This proof-of-concept approachdemonstrated that de novo glycan display through the passive
insertion of lipid-anchored glycans into membranes could be
used to control biological responses.
More recently, the immunoevasive properties of tumorigenic
cells were examined through membrane insertion of synthetic
glycopolymers. Many cancer cells develop mechanisms suchas increased surface sialylation to avoid detection by naturalkiller (NK) cells in the innate immune response (Büll et al.,2014; Fuster and Esko, 2005). Using glycan-functionalized polymers containing a terminal phospholipid, different carbohydrates were anchored to the surface of cancer cells (Figure 9A)(Hudak et al., 2013). Cells were then co-incubated with NK cells
to determine if the exogenous glycans recapitulated the naturally
observed avoidance of NK-mediated cell death. Only sialylated
NeuAc-containing polymers significantly attenuated cytotoxicity, and the polymer-displaying cells also were found to prevent

Mean channel
fluorescencemembranes using approaches such as lipid insertion, liposomal
fusion, or protein conjugation (Frame et al., 2007; Huang et al.,2014; Hudak et al., 2013; Paszek et al., 2014; Pulsipher et al.,2014, 2015). De novo display methods provide excellent control
over the glycan structure of interest and enable the presentationof both small and large glycans at the cell surface. Moreover, thisapproach is not constrained by the substrate specificity of natural enzymes, thus expanding the scope of glycan engineering tocomplex structures unattainable by other methods. However,exogenous sugars are typically displayed alongside the native
glycan population, which could obscure the biological effectsof the newly added carbohydrates. To address this complication, de novo glycan display methods can be used in combination with genetic depletion approaches to minimize the contributions of interfering endogenous glycans (Huang et al., 2014). The
versatility of the technique also allows for the display of a wide
range of carbohydrate-based structures, including glycomimetics such as synthetic glycopolymers, glycans appended tosimplified proteins, or even the glycan component of glycoproteins alone (Huang et al., 2014; Hudak et al., 2013; Paszek
et al., 2014; Pulsipher et al., 2014, 2015). Despite the use of mini-

Zeng, Y., Ramya, T. N. C. et al. Nat. Methods 2009,has
6, been207–209.
implicated in numerous biological processes and(e.g., phosphorylation
2020,and15,ubiquitinylation)
1059–1066.

Summary: Glycan Engineering in Living Cells- Introduction- Glycans play crucial roles in a myriad of biological processes.
- A growing number of glycan-associated diseases are discovered.
- Because of their high complexity, the study of glycans is quite difficult.
- 1. Bump-and-hole- The first glycotransferase bump-and-hole system in the living cell was established.
- This system allowed isoenzyme-specific labeling in the endogenous environments.
- 2. Two-step exogenous enzyme reaction- N-glycan selective editing was achieved by two step enzymatic reaction.
- The heterogeneity of live-cell surface glycans became much lower after reaction.
- This system may be practical tools to elucidate the functions of a certain type of glycosylation.
- Perspective- Glycobiology is receiving increased attention.
- Various tools for glycan engineering is growing.

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