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9-Azido Analogues of Three Sialic Acid Forms For Metabolic Remodeling of Cell-Surface Sialoglycans
9-Azido Analogues of Three Sialic Acid Forms For Metabolic Remodeling of Cell-Surface Sialoglycans
†
College of Chemistry and Molecular Engineering, ‡Beijing National Laboratory for
Molecular Sciences, §Peking−Tsinghua Center for Life Sciences,‖Academy for
⊥
Advanced Interdisciplinary Studies, Synthetic and Functional Biomolecules Center,
and #Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry
of Education, Peking University, Beijing 100871, China
Page S1
Table of Contents:
Scheme S1.……………………………………………………….........……………. S3
Figure S1……………………………………………………..………..……………. S3
Figure S2……………………………………………………..………..…………… S4
Figure S3……………………………………………………..………..…………… S4
Figure S4……………………………………………………..………..…………… S5
Figure S5……………………………………………………..………..…………… S6
Figure S6……………………………………………………..………..…………….S7
Figure S7……………………………………………………..………..…………….S8
Figure S8……………………………………………………..………..…………….S9
Experimental Procedures……………………………….…........…………....S10-S27
Table S1………………………………………………..………..…………….S28-S48
Supporting Reference……………………………………………….......………...S49
Page S2
Scheme S1. Synthesis of 9AzNeu5Gc
Figure S1: a, b, c, d) Representative scatter plots (FSC vs. SSC) and histograms of flow
cytometry analysis on Vero cells treated with 9AzNeu5Gc (a, c) or 9AzNeu5Ac (b, d) at
varied concentrations. The scatter plots were gated based on the FSC (forward scatter) and
SSC (side scatter) signals for cell debris discrimination. The gated cells were used to
analyze cell-surface fluorescence. This figure is related to Figure 1b in the main text.
Page S3
Figure S2. Evaluation of metabolic incorporation of 9AzNeu5Gc and 9AzNeu5Ac
in CHO (a), HeLa (b), and Jurkat cells (c). The cells were treated with 9AzNeu5Gc
or 9AzNeu5Ac at varied concentrations for 24 h, reacted with alkyne-PEG4-biotin and
stained with streptavidin−Alexa Fluor 488, followed by flow cytometry analysis.
MFI (mean fluorescence intensity) was normalized to that of control cells treated with
vehicle. Error bars represent mean ±SD from three independent experiments.
Figure S3. HeLa cells were treated with 9AzNeuAc or 9AzNeu5Gc at varied
concentrations for 24 h. The treated cells were reacted with DBCO-biotin and
stained with streptavidin−Alexa Fluor 647. The nuclei were stained with Hoechst
33342 (blue). The cells were imaged by confocal fluorescence microcopy. DIC,
differential interference contrast. Scale bar: 50 m.
Page S4
Figure S4. HPLC analysis of metabolic incorporation of 9-azido sialic acid analogs.
(a) CHO cells were treated with 9AzNeu5Ac, 9AzNeu5Gc, or 9AzKDN for 24 h.
(b) Vero cells were treated with 9AzNeu5Ac at varied concentrations for 24 h. (c)
MDCK II cells were cultured with 9AzNeu5Ac at varied concentrations for 24 h.
The treated cells were lysed, from which the proteins were precipitated and subjected
to acid hydrolysis to release ketosidically bound sialic acids. The released sialic
acids were then derivatized with DMB and analyzed by HPLC with fluorescence
detection. The symbol (*) indicates the peaks of DMB-azido sialic acid. The
dashed box indicates no peak of DMB-9AzNeu5Gc in the 9AzNeu5Ac-treated cells.
The quantified incorporation ratios were shown on the right.
Page S5
Figure S5. Evaluation of metabolic incorporation of 9AzKDN and 9AzNeu5Ac in
A375 (a), CHO (b), BJA-B K20 cultured with FBS in the medium (c), Daudi (d),
HeLa (e) and Vero cell (f). The cells were treated with 9AzKDN or 9AzNeu5Ac at
varied concentrations for 24 h, reacted with alkyne-PEG4-biotin and stained with
streptavidin−Alexa Fluor 488, followed by flow cytometry analysis. The zoomed
out bar graph inserted highlights 9AzKDN-labeling. MFI (mean fluorescence
intensity) was normalized to that of control cells treated with vehicle. Error bars
represent mean ±SD from three independent experiments.
Page S6
Figure S6: MS analysis of CMP-9AzKDN from CHO cells treated with 9AzKDN.
CHO cell was treated with 4 mM 9AzKDN for 24 h. The lysates were separated
using HPAEC-UV and the CMP-9AzKDN fraction corresponding to the peak eluting
at 29.4 min in figure 2a was collected and analyzed by MALDI-TOF mass
spectrometry using the cationic mode.
Page S7
Figure S7. HPAEC-UV/PAD analysis of CMP-9AzKDN from BJA-B K20 cells
treated with 4 mM 9AzKDN or vehicle for 24 h in medium containing FBS. Pure
CMP-9AzKDN was shown as the standard. Pure CMP-9AzKDN was co-injected
with the 9AzKDN-treated sample to further validate the CMP-9AzKDN peak.
Page S8
Figure S8. Competitive metabolic incorporation of azido sialic acid analogs with
Neu5Ac in CHO and PA-1 cells. (a) CHO cells were treated with 4 mM 9-azido
sialic acid analog (9AzKDN, 9AzNeu5Gc or 9AzNeu5Ac, respectively) and Neu5Ac
at varied concentration for 24 h. (b) PA-1 cells were treated with 4 mM 9-azido
sialic acid analog (9AzKDN or 9AzNeu5Ac, respectively) and Neu5Ac at varied
concentration for 24 h. The treated cells were reacted with alkyne-PEG4-biotin via
BTTAA-assisted CuAAC and stained with streptavidin−Alexa Fluor 488, followed by
flow cytometry analysis. MFI (mean fluorescence intensity) was normalized to that
of control cells treated with vehicle. Error bars represent mean ±SD from three
independent experiments.
Page S9
Experimental Procedures
Cell culture. CHO cells (Chinese hamster ovary cell), HeLa cells, MDCK Ⅱ cells,
A375 cell and Vero cells were grown in DMEM (Dulbecco’s modified Eagle’s
medium) supplemented with 10% FBS (fetal bovine serum), 100 units/mL penicillin
and 100 µg/mL streptomycin. PA-1 cells were incubated in MEM supplemented
with 10% FBS (fetal bovine serum), 100 units /mL penicillin and 100 µg/mL
streptomycin. BJA-B K20 cells (a gift from Prof. Michael Pawlita), Daudi cells and
Jurkat cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100
units /mL penicillin and 100 µg /mL streptomycin. All the cells were incubated at
37 oC under 5% CO2 in a water-saturated atmosphere. To obtain BJA-B K20 cell in
serum-free conditions, the cells were grown carefully for two passages in serum free
Page S10
medium (RPMI-1640 with 2 mM L-glutamine containing 1x Nutridoma SP, 50
units/mL penicillin, and 50 μg/mL streptomycin) to deplete endogenous sialic acid.
Flow cytometry analysis. After cells were incubated in 6-well plates with azido
sialic acids at the indicated concentrations for 24 h. The cells were harvested with
trypsin, transferred and distributed into 96-well V-bottomed plate, pelleted (800 g, 6
min, 4 oC), and washed with ice-chilled PBS containing 1% FBS for three times.
The pelleted cells were then suspended in PBS containing 0.5% FBS, 50 M alkyne-
PEG4-biotin, BTTAA-CuSO4 (50 M CuSO4, BTTAA: CuSO4 = 6:1), and 2.5 mM L-
sodium ascorbate at room temperature. After 5 min, the reaction was quenched by
adding 1 mM BCS (bathocuproine disulphonate). The cells were washed with cold
PBS containing 1% FBS for three times, and incubated with chilled PBS containing
1% FBS and 1 µg/mL Streptavidin-Alexa Fluor 488 for 30 min. After three washes
with chilled PBS containing 1% FBS, the cells were suspended in chilled PBS
containing 1% FBS and used for FACS (fluorescence-activated cell sorting) analysis
on a BD FACSCalibur Flow Cytometer system or BD AccuriTM C6 flow cytometer.
In-gel fluorescence scanning. The cells treated with sialic acid probes at the
indicated concentrations or vehicle for 24 hours were harvested by trypsin, washed
three times with ice-chilled PBS (800 g, 6 min). Then the pelleted cells were lysed
in modified RIPA lysis buffer (1% Nonidet P 40, 1% sodium deoxycholate, 0.1%
SDS, 50 mM triethanolamine, pH=7.4, 150 mM NaCl, EDTA-free Piercent HaltTM
protease inhibitor cocktail, 1 tablet/50 mL). Protein concentrations in the
homogenous lysis was normalized to 1 mg/ mL with lysis buffer. The normalized
samples were then reacted with 100 µM alkyne-Cy5 in a 60 µL reaction containing
premixed BTTAA-CuSO4 complex (50 µM CuSO4, BTTAA: CuSO4 in a 2:1 molar
ratio) and 2.5 mM L-sodium ascorbate (freshly prepared). The samples were
vortexed for 2 h at 4 °C and resolved on 10% SDS-PAGE gels. The gel was washed
in destaining solution (50% methanol, 40% H2O, 10% acetic acid) for 5 min, followed
Page S11
by washing in water for another 5 min. After that, the in-gel fluorescence was
scanned on a Typhoon FLA 9500 laser scanner (GE Healthcare, USA).
Page S12
37 °C, and dehydrated in 100% acetonitrile. The gel slices were rehydrated with 10
mM DTT in 50 mM ABC, and incubated for 45 min at 56 °C, then the slices were
incubated with 55 mM iodoacetamide in 50 mM ABC for 45 min at r.t. in the dark.
After dehydrated in 100% acetonitrile, gel pieces were rehydrated in a trypsin solution
Page S13
cells were cultured in the medium for another 48 hours before flow cytometry
analysis of cell-surface 9-Azido sialic acid as previously described.
Page S14
derivatization, dd H2O, DMB, β-mercaptoethanol and Na2S2O4 was added to adjust
the final concentration of acetic acid, DMB, β-mercaptoethanol, Na2S2O4 was 1.4
mM, 7 mM, 0.75 M and 18 mM, respectively. Then the DMB-derivatization
mixture was kept at 50 oC for 2.5 hours in the dark.9 The fluorescent DMB-sialic
acid derivatives were analyzed by RP-HPLC (Aglient 1260, XDB-C18 column, 5 µm,
4.6 × 250 mm) with a fluorescence detector (ex=373 nm, em=448 nm). The flow
rate was 0.8 mL/min and the elution gradient used was as followed: T (0 min) 84%
H2O + 9% CH3CN + 7% CH3OH, T (14 min) 84% H2O + 9% CH3CN + 7% CH3OH,
T (22 min) 64% H2O + 18% CH3CN + 18% CH3OH, T (28 min) 64% H2O + 18%
CH3CN + 18% CH3OH, T (29 min) 84% H2O + 9% CH3CN + 7% CH3OH, T (30
min) 84% H2O + 9% CH3CN + 7% CH3OH. The incorporation efficiency of azido
sialic acid into glycoproteins was quantified by integration of peak areas.
Page S15
A (140 mM NaOH in water) and B (140 mM NaOH + 600 mM NaOAc in water)
were applied in the separation process. To analyze CMP-sialic acids extracted from
CHO cells, the elution gradient used was as followed with a flow rate of 1 mL/min: T
(0 min) 20% B, T (10 min) 55% B, T (25min) 55% B, T (35 min) 80% B, T (40 min)
100% B, T (50 min) 100% B. To analyze CMP-sialic acids extracted from BJA-B
K20 cells, another elution gradient used was as followed with a flow rate of 1
mL/min: T (0 min) 4% B, T (45 min) 46% B, T (65min) 100% B, T (80 min) 100% B,
T (81 min) 4% B, T (90 min) 4% B.
with methanol (150 L), the suspension was centrifuged (1,2000 x g, 2 min), the
supernatant was used for CMP-9AzKDN analysis with MALDI-TOF mass
Binding of SubAB to BJA-B K20 cell-surface sialic acids. BJA-B K20 cells
cultured in serum-free medium for two passages was treated with 2 mM sialic acid or
9-azido sialic acid for 48 hours, then the cells was harvested by centrifugation (400 x
g, 5 min) and counted. Equal amount of cells were pelleted and washed twice with
PBS. The pellet was suspended in serum-free culture medium (RPMI 1640 with 2
mM L-glutamine containing 1x Nutridoma SP, 50 U/mL penicillin, and 50 μg/mL
streptomycin) containing 1g/mL OG-SubAB (~3 x106 cell/mL), the suspension was
kept at 37oC for 30 mins, then pelleted and washed twice with PBS. After fixation in
PBS containing 4% paraformaldehyde (25 oC, 10 min), the OG-SubAB labeled cells
was washed and suspended in PBS, the Oregon Green fluorescence intensity was
quantified by FACS on a BD FACSCalibur Flow Cytometer system.
Page S17
Chemical Synthesis
5-acetoxyacetamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid
(Neu5GcAc, 2)
Page S18
1
H NMR of Neu5GcAc (2)
13
C NMR of Neu5GcAc (2)
Page S19
1-Methyl-5-acetoxyacetamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic
acid (1-Met-Neu5GcAc, 3)
Page S20
1
H NMR of 1-Met-Neu5GcAc (3)
13
C NMR of 1-Met-Neu5GcAc (3)
Page S21
1-Methyl-5-acetoxyacetamido-9-tosyl-3,5,9-trideoxy-D-glycero-D-galacto-2-
nonulosonic acid (1-Met-9-Ts-Neu5GcAc, 4)
Page S22
1
H NMR of 1-Met-9-Ts-Neu5GcAc (4)
13
C NMR of 1-Met-9-Ts-Neu5GcAc (4)
Page S23
5-acetoxyacetamido-9-azido-3,5,9-trideoxy-D-glycero-D-galacto-2-nonulosonic
acid (9-N3-Neu5GcAc, 5)
53.9, 52.2, 39.2, 20.1. HRMS (ESI): Calcd for C13H20N4O10Na [M+Na] +
415.10716, found 415.10774.
Page S24
1
H NMR of 9-N3-Neu5GcAc (5)
13
C NMR of 9-N3-Neu5GcAc (5)
Page S25
9-Azido-9-deoxy-N-glycolylneuraminic acid (9AzNeu5Gc, 6)
51.9, 39.0. HRMS (ESI): Calcd for C11H18N4O9Na [M+Na] + 373.09660, found
373.09702.
Page S26
1
H NMR of 9AzNeu5Gc (6)
13
C NMR of 9AzNeu5Gc (6)
Page S27
Table S1: Identification of sialoglycoproteins with three 9-azido sialic acid
analogs in PA-1 cells
phosphate receptor
nonspecific isozyme
related protein 1
phosphate receptor
related protein 8
phosphatase zeta
phosphatase gamma
Page S29
Ectonucleotide ENPP1 2 107 63 27 √ √ √
pyrophosphatase/phosphodiesterase
family member 1
receptor
receptor 1
glycoprotein 1
Polypeptide N- GALNT2 4 98 61 13 √ √
acetylgalactosaminyltransferase 2
Alpha-mannosidase 2 MAN2A1 1 95 50 27 √ √ √
membrane protein 2
Prominin-1 PROM1 0 89 54 11 √ √ √
Page S30
Leucyl-cystinyl aminopeptidase LNPEP 0 84 99 16 √ √ √
transporter 2
related protein 4
molecule
oligosaccharide 1,2-alpha-
mannosidase
Tripeptidyl-peptidase 1 TPP1 2 78 82 8 √ √
Apolipoprotein E APOE 15 78 67 32 √
Sodium/potassium-transporting ATP1B1 2 76 66 13 √ √ √
protein 44
member 3
Page S31
Integrin alpha-3 ITGA3 0 64 76 12 √ √ √
glycoprotein 2
CPVL
regulator
containing protein 2
Plexin-B2 PLXNB2 0 60 63 9 √ √ √
phosphatase alpha
transporter 1
Beta-galactosidase GLB1 9 54 53 17 √ √
subunit alpha-2/delta-1
Plexin-D1 PLXND1 0 53 21 2 √ √
N-acetylgalactosaminyltransferase 7 GALNT7 0 52 41 12 √ √ √
UFO
Frizzled-2 FZD2 4 52 57 10 √ √
Page S32
Pituitary tumor-transforming gene 1 PTTG1IP 11 52 66 15 √
protein-interacting protein
Anoctamin-6 ANO6 0 51 30 5 √ √ √
Polypeptide N- GALNT1 0 51 43 26 √ √ √
acetylgalactosaminyltransferase 1
subunit a isoform 1
5~-nucleotidase NT5E 0 50 48 5 √ √ √
protein 1
VIP36
Page S33
CD109 antigen CD109 0 44 23 2 √ √
transporter
Ephrin-B1 EFNB1 1 42 48 9 √ √ √
Alpha-1,6-mannosyl-glycoprotein 2- MGAT2 0 40 31 5 √ √ √
beta-N-
acetylglucosaminyltransferase
47
Vasorin VASN 0 37 36 9 √ √ √
Tetraspanin-6 TSPAN6 0 37 41 4 √ √
transporter 1
subunit alpha
Page S34
Uncharacterized protein KIAA0319- KIAA0319L 0 34 18 4 √ √
like
Osteopetrosis-associated OSTM1 0 33 41 8 √ √ √
transmembrane protein 1
protein 1
factor
Sortilin SORT1 0 32 32 8 √ √ √
Glucosylceramidase GBA 3 32 41 4 √ √
factor receptor
dipeptidase 2
Semaphorin-4D SEMA4D 0 30 20 6 √ √ √
Keratinocyte-associated KCT2 0 29 34 6 √ √ √
transmembrane protein 2
Xylosyltransferase 2 XYLT2 0 28 6 4 √ √
Page S35
Membrane cofactor protein CD46 0 27 28 8 √ √ √
sialyltransferase 1
Alpha-N-acetylglucosaminidase NAGLU 0 26 24 4 √ √
Plexin-A1 PLXNA1 0 26 21 1 √ √
Endoglin ENG 0 25 19 5 √ √ √
Alpha-galactosidase A GLA 1 25 24 3 √ √
receptor-related protein 1
N-acetylglucosamine-1- NAGPA 0 24 24 0 √ √
phosphodiester alpha-N-
acetylglucosaminidase
phosphatase F
Fibulin-1 FBLN1 0 23 43 5 √ √ √
Ectonucleotide ENPP4 0 23 16 3 √ √
pyrophosphatase/phosphodiesterase
family member 4
Protocadherin-1 PCDH1 0 23 14 1 √ √
Semaphorin-7A SEMA7A 2 23 25 5 √ √
Page S36
Sodium/myo-inositol cotransporter SLC5A3 1 23 12 2 √ √
containing protein 9
N-acetylglucosamine-6-sulfatase GNS 2 22 45 10 √ √ √
Alpha-1,3-mannosyl-glycoprotein 2- MGAT1 0 22 10 1 √ √
beta-N-
acetylglucosaminyltransferase
transporter 2
related protein 6
Neogenin NEO1 0 21 13 3 √ √
Alpha-(1,3)-fucosyltransferase FUT4 0 20 13 1 √ √
Beta-1,4-galactosyltransferase 5 B4GALT5 0 20 18 3 √ √
domain-containing protein 10
Exostosin-like 2 EXTL2 0 20 12 1 √ √
acetylglucosaminyltransferase
Beta-1,4-galactosyltransferase 3 B4GALT3 0 20 8 0 √ √
Page S37
Receptor tyrosine-protein kinase ERBB2 0 19 27 1 √ √
erbB-2
phosphatase eta
Beta-1,4-galactosyltransferase 1 B4GALT1 0 19 8 0 √ √
Neuroligin-3 NLGN3 0 19 7 0 √ √
phosphatase kappa
Neuroligin-2 NLGN2 0 19 0 0 √
N-acetylglucosamine-1- GNPTG 0 18 13 10 √ √ √
Nucleobindin-1 NUCB1 1 18 13 1 √ √
protein 4
transporter
Attractin ATRN 0 17 25 5 √ √ √
Page S38
HLA class I histocompatibility HLA-A 0 17 0 0 √
Alpha-1,6-mannosylglycoprotein 6- MGAT5 0 16 13 4 √ √
beta-N-
acetylglucosaminyltransferase A
domain-containing protein 15
Nidogen-1 NID1 0 16 13 4 √ √
Occludin OCLN 2 16 17 4 √ √
Semaphorin-4C SEMA4C 0 16 13 1 √ √
Tetraspanin-13 TSPAN13 0 16 22 1 √ √
Clusterin CLU 0 15 18 1 √ √
domain-containing protein 9
Tetraspanin-3 TSPAN3 0 15 26 3 √ √
Lactadherin MFGE8 0 15 11 0 √ √
acetylgalactosaminyltransferase-
like protein 3
Tenascin TNC 2 15 16 0 √ √
Beta-mannosidase MANBA 1 14 20 6 √ √ √
N-acetylglucosamine-1- GNPTAB 0 14 11 3 √ √
phosphotransferase subunits
alpha/beta
Page S39
Receptor expression-enhancing REEP5 1 14 13 2 √ √
protein 5
containing protein 7
protein 2
Plexin-B1 PLXNB1 0 14 16 0 √ √
Sodium/potassium-transporting ATP1A2 0 14 24 0 √ √
Tetraspanin-18 TSPAN18 0 14 9 0 √ √
Beta-1,3-galactosyltransferase 6 B3GALT6 0 14 4 1 √
member 1
Frizzled-1 FZD1 0 13 19 6 √ √ √
Leukosialin SPN 0 13 8 4 √ √
containing protein 4
Galectin-1 LGALS1 1 12 7 6 √ √ √
phosphodiesterase 3b
Calumenin CALU 1 12 18 1 √ √
Glycerophosphoinositol GDPD2 0 12 9 1 √ √
inositolphosphodiesterase GDPD2
Page S40
Peptidyl-glycine alpha-amidating PAM 0 12 6 1 √ √
monooxygenase
Alpha-N-acetylgalactosaminidase NAGA 0 12 14 0 √ √
domain-containing protein 23
immunoglobulin-like domains
protein 1
acetylglucosaminyl-transferase
Latrophilin-3 LPHN3 0 11 7 2 √ √
Syndecan-2 SDC2 0 11 9 2 √ √
4 protein
domain-containing protein 17
nucleotidase 1
Tetraspanin-9 TSPAN9 0 11 9 0 √ √
Gremlin-1 GREM1 2 11 3 3 √
Latrophilin-1 LPHN1 0 11 3 0 √
Page S41
Protein HEG homolog 1 HEG1 0 10 22 10 √ √ √
Ephrin-B2 EFNB2 0 10 15 2 √ √
Syndecan-1 SDC1 0 10 10 3 √ √
Tetraspanin-31 TSPAN31 0 10 12 1 √ √
superfamily member 21
subfamily G member 2
macrophage protein 2
protein 3
Sialin SLC17A5 0 10 7 0 √ √
superfamily member 16
Agrin AGRN 0 10 4 1 √
transmembrane domain-containing
protein 1
Melanotransferrin MFI2 0 10 3 0 √
M6-b
taurine transporter
Granulins GRN 1 9 8 0 √ √
antigen 3
Page S42
Major facilitator superfamily domain- MFSD8 0 9 8 0 √ √
containing protein 8
channel protein 1
acetylglucosaminyltransferase 2
hydroxylase
Osteopontin SPP1 0 8 7 3 √ √
Porimin TMEM123 0 8 6 1 √ √
containing protein 1
protein 2
Gamma-glutamyltranspeptidase 1 GGT1 0 8 13 0 √ √
Mucolipin-1 MCOLN1 0 8 7 0 √ √
Cadherin-13 CDH13 0 8 4 1 √
Ephrin-B3 EFNB3 0 8 3 0 √
Page S43
H(+)/Cl(-) exchange transporter 3 CLCN3 0 8 2 0 √
13A3
Cadherin-5 CDH5 0 7 18 1 √ √
group 5 member B
ester hydrolase
Alpha-1,3-mannosyl-glycoprotein 4- MGAT4B 0 7 2 0 √
beta-N-
acetylglucosaminyltransferase B
Carboxypeptidase M CPM 0 7 2 0 √
Frizzled-6 FZD6 0 7 2 0 √
subunit a isoform 2
Battenin CLN3 0 6 9 0 √ √
Exostosin-like 3 EXTL3 0 6 10 0 √ √
Page S44
Fibroblast growth factor-binding FGFBP3 0 6 7 0 √ √
protein 3
phosphatase mu
Tissue factor F3 0 6 7 0 √ √
cofactor NHE-RF2
Phosphoinositide-3-kinase-interacting PIK3IP1 0 6 1 0 √
protein 1
receptor 1
glycine transporter 1
transporter 2
protein homolog
Tetraspanin-4 TSPAN4 0 6 1 0 √
Page S45
Tumor necrosis factor receptor TNFRSF10B 0 6 1 0 √
subunit beta
Prostasin PRSS8 0 5 5 1 √ √
Sialidase-1 NEU1 0 5 6 1 √ √
protein 5
Glycerophosphodiester GDPD5 0 5 5 0 √ √
phosphodiesterase domain-
containing protein 5
protein 1
transporter 1
Tetraspanin-14 TSPAN14 0 5 7 0 √ √
Cathepsin B CTSB 2 5 11 5 √
marker
Claudin-12 CLDN12 0 5 1 0 √
CMP-N-acetylneuraminate-beta- ST3GAL2 0 5 2 0 √
galactosamide-alpha-2,3-
sialyltransferase 2
Page S46
Proteinase-activated receptor 1 F2R 0 5 2 0 √
exchanger
sialyltransferase
N-sulphoglucosamine SGSH 2 4 20 3 √
sulphohydrolase
Frizzled-5 FZD5 0 4 6 0 √
membrane protein 4
type receptor 2
transporter 1
Anosmin-1 KAL1 0 2 10 1 √
Endosialin CD248 0 2 6 1 √
mannosidase
Protocadherin-18 PCDH18 0 2 7 0 √
member 3
Arylsulfatase E ARSE 0 1 0 42 √
Page S47
Protein disulfide-isomerase TMX3 TMX3 1 1 1 7 √
protein 12
protein 1
regulated by oncogenes
Semenogelin-1 SEMG1 0 0 0 5 √
phosphatase delta
Page S48
Supporting reference
(1) Izumi, M., Shen, G. J., Wacowich-Sgarbi, S., Nakatani, T., Plettenburg, O., and Wong,
C. H. (2001) Microbial glycosyltransferases for carbohydrate synthesis: alpha-2,3-
sialyltransferase from Neisseria gonorrheae. J. Am. Chem. Soc. 123, 10909-10918.
(2) Pearce, O. M., and Varki, A. (2010) Chemo-enzymatic synthesis of the carbohydrate
antigen N-glycolylneuraminic acid from glucose. Carbohydr. Res. 345, 1225-1229.
(3) Kong, D. C. M., and von Itzstein, M. (1997) The chemoenzymatic synthesis of 9-
substituted 3,9-dideoxy-D-glycero-D-galacto-2-nonulosonic acids. Carbohydr. Res. 305, 323-
329.
(4) Han, S., Collins, B. E., Bengtson, P., and Paulson, J. C. (2005) Homomultimeric
complexes of CD22 in B cells revealed by protein-glycan cross-linking. Nat. Chem. Biol. 1,
93-97.
(5) Yu, H., Yu, H., Karpel, R., and Chen, X. (2004) Chemoenzymatic synthesis of CMP-
sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of
three microbial CMP-sialic acid synthetases. Bioorg. Med. Chem. 12, 6427-6435.
(6) Mbua, N. E., Li, X., Flanagan-Steet, H. R., Meng, L., Aoki, K., Moremen, K. W.,
Wolfert, M. A., Steet, R., and Boons, G. J. (2013) Selective Exo-Enzymatic Labeling of N-
Glycans on the Surface of Living Cells by Recombinant ST6Gal I. Angew. Chem., Int. Ed.
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