Two-Step Chemoenzymatic Detection of N-Acetylneuraminic Acid-α(2-3)-Galactose Glycans

Article abstract of DOI:10.1021/jacs.6b07132

Sialic acids are typically linked α(2-3) or α(2-6) to the galactose that located at the non-reducing terminal end of glycans, playing important but distinct roles in a variety of biological and pathological processes. However, details about their respective roles are still largely unknown due to the lack of an effective analytical technique. Herein, a two-step chemoenzymatic approach for the rapid and sensitive detection of N-acetylneuraminic acid-α(2-3)-galactose glycans is described.

Full text of DOI:10.1021/jacs.6b07132

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Communication
Two-step Chemoenzymatic Detecting N-
Acetylneuraminic acid-#(2-3)-galactose Glycans
Liuqing Wen, Kuan Jiang, Yuan Zheng, Mingzhen Zhang, Shukkoor Muhammed
Kondengaden, Shanshan Li, Kenneth Huang, Jing LI, Jing Song, and Peng George Wang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b07132 • Publication Date (Web): 23 Aug 2016
Downloaded from http://pubs.acs.org on August 24, 2016
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Two-step Chemoenzymatic Detecting N-Acetylneuraminic ac-
id-α(2-3)-galactose Glycans
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Liuqing Wen,† Kuan Jiang,†§ Yuan Zheng,† Mingzhen Zhang,# Shukkoor Muhammed Kondenga-
den,† Shanshan Li,† Kenneth Huang,† Jing Li,†§ Jing Song,† Peng George Wang†§*
§State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300071,
China
†Department of Chemistry, #Institute for Biomedical Sciences, Georgia State University, Atlanta, GA 30303, USA
Supporting Information
non-natural functionalities, chemoenzymatic labeling gly-
ABSTRACT: Sialic acids are typically linked α2-3 or α2-6 to
cans relies on the substrate-specific glycosyltransferases,
which transfer non-natural sugars that contain bioorthogo-
nal functional groups onto target glycan in vitro.8 Chemoen-
zymatic method does not rely on cell’s biosynthetic machin-
ery and therefore can be employed in any desired biological
contexts where feeding cells with non-natural sugar analogs
is not possible, such as human tissue extracts.8b As glycosyl-
transferase-mediated reaction and click chemistry reaction
proceed high specificity and efficiency, chemoenzymatic
labeling provides a higher sensitivity and selectivity com-
pared to other analytical methods such as antibodies, lectins,
and metabolic labeling. In this communication, we report a
two-step chemoenzymatic method that takes advantage of
the galactose that located at the non-reducing terminal end
of glycans, playing important but distinct roles in a variety of
biological and pathological processes. However, details about
their respective roles are still largely unknown due to the
lack of an effective analytical technique. Herein, a two-step
chemoenzymatic approach for the rapid and sensitive detec-
tion
of
N-acetylneuraminic
acid-α(2-3)-Galactose
(Neu5Acα(2-3)Gal) glycans was described.
N-acetylneuraminic acid (Neu5Ac) is the most widespread
form of sialic acid and almost the only form found in hu-
mans.1 N-glycolylneuraminic acid (Neu5Gc) and ketodeox-
ynonulosonic acid (Kdn) are common in other vertebrates
but rarely present in humans.1 Neu5Ac is present essentially
in all human tissues and always attaches to the galactose
residue at the nonreducing terminal end of glycans through
α2-3 or α2-6 linkage.2 It is well established that Neu5Acα(2-
3)Gal and Neu5Acα(2-6)Gal glycans play crucial but distinc-
tive roles in diverse biological and pathological processes
including immune responses, cell–cell and cell–pathogen
interactions.3 However, studies are hindered due to the lack
of an effective method to analyze such glycans or glycopro-
teins.
the
substrate
promiscuity of
a
β-(1,4)-N-
acetylgalactosaminyltransferase from Campylobacter jejuni
(CgtA) and click chemistry reaction to rapidly and sensitively
detect Neu5Acα(2-3)Gal glycans (Scheme 1).
Scheme 1. Two-step Chemoenzymatic detecting Neu5Acα(2-3)Gal
glycans by CgtA with UDP-GalNAz.
Lectin binding has been the primary method to analyze si-
alylated glycans,4 but lectins often suffer from weak binding
affinity, limited specificity, and cross-reactivity. In recent
years, the development of bioorthogonal chemistry provides
a powerful tool for probing glycans, proteins and lipids.5
Bioorthogonal functional groups (azide and alkyne) carried
The Neu5Acα(2-3)Gal epitope localized on cell surface is
well known to be the receptor of many infectious microbes
such as the influenza virus.2 Abnormal Neu5Acα(2-3)Gal
expression has frequently been observed in many carcino-
mas.3b Traditionally, lectin binding using Maackia amurensis
leukoagglutinin (MAL I) and hemagglutinin (MAH or MAL II)
is the main method for Neu5Acα(2-3)Gal detection. However,
MAL I only bind terminal Neu5Acα(2-3)Galβ(1-4)GlcNAc
by
N-acetylmannosamine or Neu5Ac analogues were meta-
bolically incorporated into glycans, allowing the covalent
conjugation by click chemistry reaction with either fluores-
cent tags for visualization, or affinity probes for enrichment
of sialylated glycans and glycoproteins.6 Chemical approach
to tag sialylated glycans has also been suggested.7 Neverthe-
less, these methods suffer from low detection sensitivity and
efficiency, toxicity of labeling regents, and the inability to
detect complicated glycan structures.
trisaccharide in N
-glycans.9 MAH binds preferentially trisac-
charide Neu5Acα(2-3)Galβ(1-3)GalNAc in
O
-glycans.10 They
also bind some nonsialylated structures such as SO4-3-Galβ(1-
3)GalNAc.11 Moreover, it was reported that Maackia amuren-
As a complementary strategy to remodel glycans with
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sis lectins require a high minimum agglutinating concentra-
Table 1. Substrate specificity of CgtA with UDP-GalNAz to-
wards sialylated oligosaccharides
tion (up to 125 ug to 500 ug),12 and therefore a long incuba-
tion time (typically overnight) is necessary for glycoprotein
detection.13 Thus, the development of an simple, rapid and
sensitive method for detecting Neu5Acα(2-3)Gal glycans
remains an unmet need.
1
2
3
4
5
6
Entry
1
Substrate
RA (%)a
100
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8
2
104.3
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3
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100.6
113.4
96.5
Figure 1. CgtA recognizes Neu5Acα(2-3)Gal with UDP-GalNAc or
UDP-GalNAz
6
7
0.08
0.28
Neu5Acα(2-3)Gal disaccharide is the outer core compo-
nent of many Campylobacter jejuni strains.14 CgtA is respon-
sible for the extension of Neu5Acα(2-3)Gal with GalNAc resi-
due (Figure 1).14 We reasoned that CgtA might tolerate sub-
stitution at the C-2 position of GalNAc, allowing for the in-
troduction of an azide group for further click chemistry reac-
tion (Figure 1). Substrate specificity study using Ganglio-
oligosaccharide GM3 (entry 1, Table 1) shows that both UDP-
GalNAz and UDP-GalNAc are efficient substrate of CgtA
(Figure S1). Kinetic analysis revealed a kcat/Km value of 1.34
nM-1min-1 for UDP-GalNAc, and 1.51 nM-1min-1 for UDP-
GalNAz. Indeed, treatment of GM3 with CgtA and UDP-
GalNAz overnight led to a complete conversion of GM3. The
product was confirmed by MALDI-TOF-MS and NMR (see
supporting information).
8
9
0.31
0.41
0.25
10
aRA: relative activity. See SI for experimental details.
NAc
Glc Glc-
hydrolyzes α2-3-linked Neu5Ac.17 After the treatment of
NanC, a slight migration change compare to native feutin
was observed on SDS-PAGE gel (Figure 2A). The α2-6-linked
Neu5Ac was confirmed by biotinylated Sambucus nigra ag-
glutinin (B-SNA) (Figure 2A). Native fetuin or NanC-treated
fetuin was labeled by CgtA with UDP-GalNAz at 37°C for 1
hour, while other control groups were performed in parallel.
Following copper-free click reaction (DIBO-alkyne, 10 uM),
the proteins were analyzed by western blot using streptavi-
din-linked horseradish peroxidase (S-HRP). Strong fluores-
cence in labeling group was observed, while all the control
groups failed to be labeled (Figure 2A), demonstrating that
the designed scheme could be used to specifically label
Neu5Acα(2-3)Gal glycans on glycoprotein. The labeled fetuin
was further treated with peptide
which remove -Glycans from glycoprotein, and detected by
S-HRP. Fluorescence labeling in the PNGF-treated sample
still can be observed (Figure 2A, bottom graph), indicating
that both N-glycans and O-glycans were labeled. Meanwhile,
the probe of same amount of fetuin with biotinylated MAL II
is unsuccessful. Thus, our chemoenzymatic approach pro-
vides a more credible detection strategy for Neu5Acα(2-3)Gal
glycans and enable the highly sensitive detection of glycopro-
teins.
Having demonstrated that CgtA accepts UDP-GalNAz as
substrate, we next tested its substrate specificity towards
sialylated oligosaccharides with UDP-GalNAz. Many sialylat-
ed oligosaccharides containing α2-3-, α2-6-, or α2-8-linked
sialic acid (Table 1 and Table S1) were synthesized using the
methods reported previously.15 We found that CgtA requires
only linear disaccharide structure of Neu5Acα(2-3)Gal (Table
1, entries 1 to 5) or Neu5Gcα(2-3)Gal (Table S1, entry 2) when
using UDP-GalNAz as donor. Meanwhile, only very low rela-
tive activity towards the structure containing α2-6-linked
sialic acid or without sialic acid was detected (Table 1, Entries
6 to 10; Table S1, entries 3 to 5). Indeed, no observable prod-
uct or by-product (UDP) could be found on TLC after the
incubation of these compounds with CgtA and UDP-GalNAz
overnight. These findings indicated the potential of CgtA for
application in selective labeling Neu5Acα(2-3)Gal glycans.
N-glycosidase F (PNGF),
N
To test the practicality of the described strategy on protein
labeling, we used fetal bovine fetuin as an example. Fetal
bovine fetuin is a well-studied model protein for sialylated
glycans analysis and commercially available. It contains three
N-glycosylation and three
O-glycosylation sites, on which
Neu5Ac attached to the terminal galactose residues through
α2-3- or α2-6-linkage.16 To perform a control, fetal bovine
fetuin was treated with a sialidase (NanC), which specifically
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—
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—
—
UDP-GalNAz
UDP-GalNAc
CgtA
+
+
+
—
—
—
B
CgtA
C
D
+
UDP-GalNAz
UDP-GalNAc
CgtA
A
+
+
CgtA
+
-
UDP-GalNAz
CgtA
—
+
—
—
+
+
—
+
+
+
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+
—
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+
1
2
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—
—
—
+
+
+
+
—
—
—
—
+
+
+
+
PNGF
AF488
DAPI
—
—
+
NanC
—
—
NanC
kDa
55
CBB
kDa
kDa
170
170
5
6
7
8
55
55
B-SNA
S-HRP
70
70
35
S-HRP
S-HRP
35
—
—
+
PNGF
+
Overlay
9
10
11
β
-Actin
β-Actin
S-HRP
CBB
Figure 2. (A) Chemoenzymatic detection Neu5Acα(2-3)Gal glycans on fetal bovine fetuin. CBB: Coomassie brilliant blue staining. S-HRP: strep-
tavidin-linked horseradish peroxidase. B-SNA: biotinylated SNA. NanC: The sample was treated with NanC before performing labeling reaction.
PNGF: the labeled fetuin was further treated with PNGF (bottom graph). (B) Chemoenzymatic detection of Neu5Acα(2-3)Gal glycoproteins from
HEK293T cell lysates. (C) Chemoenzymatic detection of Neu5Acα(2-3)Gal glycoproteins on cell surface of HEK 293T. (D) The imaging of cell-
surface Neu5Acα(2-3)Gal glycans on live HeLa cells (Green) using fluorescence microscopy. Nuclei were stained with 4',6-diamidino-2-
phenylindole (DAPI; blue). See SI for experimental details.
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We next determined whether the approach could be used
to track Neu5Acα(2-3)Gal glycoproteins in complex samples.
Cell lysates from human embryonic kidney 293 (HEK293T)
cells was incubated with CgtA and UDP-GalNAz at 37°C for 1
hour, while control groups were performed parallel. Follow-
ing the Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC)
reaction with diazo-biotin-alkyne, biotinylated cell lysates
was detected by western blot using S-HRP (Figure 2B).
Strong fluorescence labeling in cell lysates was observed,
while only background level of nonspecific labeling in NanC-
47
1.5%
7.4%
Catalytic activity
Transporter activity
Receptor activity
Binding
treated group and other control groups was observed (Figure
2B), highlighting further the specificity of the designed strat-
egy towards Neu5Acα(2-3)Gal glycans. Cell surface
Neu5Acα(2-3)Gal glycans were selectively labeled by incubat-
ing suspension HEK293T living cells with CgtA and UDP-
GalNAz at 37°C for 30 min. Following the biotinylation by
CuAAC reaction, strong fluorescence labeling was observed
compared to control groups in western blot (Figure 2C).
Then, several other randomly selected human cancer cell
lines including A549, HeLA, and HepG2 were chemoen-
zymatically labeled using the same strategy (see supporting
information). The labeled samples were further treated with
PNGF, resulting in significant fluorescence reduction, indi-
cating that Neu5Acα(2-3)Gal mainly attach to N-glycans in
these cell lines.
11.8%
22.1%
32.4%
8
6
25%
Enzyme regulator
activity
Structural molecule
activity
Figure 3. (A) Global identification of cell surface Neu5Acα(2-3)Gal
glycoproteins from HEK293T. SAR: streptavidin agarose resin.
(B)Total 61 proteins were probed. NP:
-glycosylation protein. (C) Molecular function of the identified
N-glycosylation protein. OP:
O
sialoglycoproteins.
flow cytometry (Figure S9).
Finally, Neu5Acα(2-3)Gal sialoglycoproteins that exist on
the cell surface of HEK 293T were globally identified using
the described strategy (Figure 3A). It is well established that
cell surface glycoproteins containing Neu5Acα(2-3)Gal gly-
cans play important roles in living cells, where these proteins
are the potential therapeutic targets. However, there has
been relatively little study of profiling such sialoglycopro-
teins due to the lack of an effective enrichment method. Alt-
hough lectin affinity chromatography has been explored to
enrich sialoglycoproteins,19 this method was limited by the
weak binding affinity and limited specificity of lectins. Alter-
natively, avidin-biotin complex is the strongest known non-
covalent interaction between a protein and ligand (Kd = 10-14
~10-15 M), making avidin-biotin binding to be an ideal system
for protein enrichment.20 In this work, the biotinylated cell
surface proteins of HEK293T were captured using streptavi-
din agarose resin (SAR). After digestion with trypsin, the
peptide fragments were analyzed by LS-MS. After filtering
the non-specific binding proteins in control group, 61 pro-
We next investigated the potential application of the de-
scribed strategy for Neu5Acα(2-3)Gal glycans imaging and
quantification. The determination of the expression level of
Neu5Acα(2-3)Gal glycans is very important to understand
sialic-acid-related microbe infection and carcinogenesis.18
Adherent HeLa cells were labeled by CgtA and UDP-GalNAz
at 37°C for 30 min. After copper-free click reaction (DIBO-
biotin, 30 uM), a fluorescent reporter was subsequently in-
stalled by incubation with streptavidin-linked Alexa Fluor
488 (10 ug/ml). Membrane-associated fluorescence was ob-
served for cells treated with both CgtA and UDP-GalNAz,
whereas no fluorescence labeling was detected for control
cells labeled in the absence of CgtA, confirming the specifici-
ty of the in situ chemoenzymatic reaction (Figure 2D). The
fluorescence intensity, which reflects the expression level of
Neu5Acα(2-3)Gal glycans proportionally, was determined by
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teins were identified (Table S2). 53 probed proteins are the
reported glycoproteins (Figure 3B), highlighting the feasibil-
ity of the described strategy. Among of these reported glyco-
1
2
3
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5
6
7
8
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proteins, 47 proteins contain
N-glycans and six proteins con-
tain both - and -glycans. This data is also well in accord
N
O
with the western blot observation (Figure 2C). Molecular
function analysis using the Protein ANalysis THrough Evolu-
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of the total probed proteins. These are consistent with the
well-known functions of cell surface sialoglycoproteins.6f
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In conclusion, on the basis of a glycosyltransferase that
could specifically recognize Neu5Acα(2-3)Gal with UDP-
GalNAz and site-specific click chemistry reaction, we have
developed the first strategy for the rapid and sensitive detect-
ing Neu5Acα(2-3)Gal glycans. This method is far superior to
the traditional lectin-based methods to detect Neu5Acα(2-
3)Gal, which are limited by their inherent disadvantages.
This method also allows that the global analysis of
Neu5Acα(2-3)Gal glycoproteins is achievable, providing a
powerful tool for sialic-acid-related research. Moreover, sub-
strate specificity study indicated that the described strategy
can be also used to probe Neu5Gcα(2-3)Gal glycans, which
are currently detected by polyclonal monospecific antibody.2
Future studies will enable the exploration of new glycosyl-
transferase for use in more glycans detecting.
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ASSOCIATED CONTENT
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Materials, experimental methods, and supporting figures and
tables (PDF)
AUTHOR INFORMATION
Corresponding Author
pwang11@gsu.edu
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We are grateful to Dr. Xi Chen from University of California—Davis
for generously providing us with CgtA plasmid. This work was sup-
ported by NIH grants (R01 GM085267 and R01 AI083754) and the
National Natural Science Foundation of China (Grants 21102076,
91013013, and 31100587).
Reference
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