Site-specific click chemistry-mediated labeling of

Site-specific click chemistry-mediated labeling of antibody glycans using metabolic and
enzymatic approaches
Robert Aggeler, Ruth Deveny, Courtenay Hart, Tamara Nyberg, Carolyn Peterson and Brian Agnew
Molecular Probes-Life Technologies Corporation, Cell Systems Division, 29851 Willow Creek Road, Eugene, OR 97402
Metabolic Antibody Labeling
Ac4ManNAz
Feed antibody expressing
cells azide sugars
ManNAz
(Sialic Acids)
N3- Gal
Gal-T(Y289L)
DIBO Alexa
Fluor® dye
N3
N3
GalT
1B
Alexa Fluor® 488
Signal
0 hr
CD45
SYPRO® Ruby
Protein Stain
0 hr
24 hr
1C
Alexa Fluor® 488
Signal
SYPRO® Ruby
Protein Stain
0 hr
24 hr
0 hr
24 hr
0 hr
24 hr
SYPRO® Ruby
Protein Stain
0 hr
24 hr
0 hr
24 hr
0 hr
24 h
Figure 1. Specific metabolic labeling of heavy chain can
be achieved in various cell lines
β-tubulin
24 hr
overnight at RT
Fluorophore-DIBO
+
-
+
5 hr
20 hr
-
-
+
Time
+
GalT
1 hr
-
+
2.5 hr
-
+
5 hr
-
20 hr
+
-
+
L
H
L
0
120
80
0
40
120
40
GalNAz (µM) only
80
0
0 ind
40
80
120
40
80
120
L
0
0 ind
40
80
120
40
80
120
0
25
50
75
0
25
50
75
0
25
50
75
0
25
50
75
L
0.2
0.1
0
0
25
50
75
GalNAz (µM) + ManNAz (µM)
0.2
0.1
0.0
0
40
80
120
GalNAz (µM) only
0h
24h
0.4
ManNAz
GalNAz
+
+ + ¯ + +
¯ ¯ + + ¯
¯ ¯ + ¯ ¯ +
¯ ¯ ¯
¯
¯ ¯ ¯ ¯ + + + ¯ ¯
Gal+Man
1 mM
0.08
0.06
0.04
- Gal-T
0.02
0.00
0.0
5.0
10.0
15.0
50 μM
2 mM
1 mM
0.1
H
0.0
0
40
80
120
GalNAz (µM) + ManNAz (µM)
SYPRO® Ruby
Protein Stain
on
on
lA
lA
F sti
F sti
ia
ia
+S F2 se e
+S F2 se e
al o a ig
al o a ig
G d G d
G d G d
β- En PN No
β- En PN No
L
DIBO-Alexa Fluor® 488 dye clicked to anti-TSH
Gal-T labeled antibody
0.6
0.5
0.4
0.3
0.2
0.1
0
50 μM
1 mM
2 mM
[UDP-GalNAz]
Figure 6. Click reaction with various DIBO dyes
Gal-T
®
BO
en
r®
r®
r®
DI
re
uo
uo
uo
Fl IBO
Fl O
Fl O G
A®
a
a DI B
a DI B o n BO
R
M
ex 7 D
ex 5
ex 8 eg DI
Al 64
Al 55
Al 48 Or
TA
- + - +
- +
- +
+
SYPRO® Ruby Protein Stain
-
+
-
+
-
+
-
+
-
+
Alk phos
H
H
L
DIBO-Dye Labeling:
Purified antibodies were labeled overnight at 25°C with 5 – 50 μM DIBO-Alexa
Fluor® dye, Oregon Green®, TAMRA™ or biotin conjugate. Antibodies were
desalted on Zeba™ 7K MW cutoff columns (Thermo Scientific) prior to functional
testing by Western blot.
Western Blotting:
Wortmanin-treated Jurkat cell extract (AKT, lane 1, 10 μg) or untreated Jurkat cell
extract (pAKT ser473; lane 2, 10 μg; lanes 3-10, two-fold dilutions) were separated
on 4-12% NuPAGE® Bis-Tris gels with MOPS buffer and blotted onto nitrocellulose.
Blots were incubated with the respective click-treated rabbit α-pAKT (ser 473)
antibody overnight. No click control and Alexa Fluor® 488 dye clicked α-pAKT
antibodies were indirectly detected with goat anti-rabbit (GAR) biotin and streptavidin
(SA) Qdot® 625 conjugate from the WesternDot 625™ kit (Invitrogen W10142).
Biotin clicked α-pAKT antibodies were directly detected via the click label with SAQdot® 625 conjugate. Blots were imaged on a FLA4000 imager (FujiFILM) using UV
epi illumination and a 605DF40 filter.
α-pAKT heavy chain metabolic labeling after enzyme digestion
Alexa Fluor® 647 Fluorescence
0.7
0.6
0.5
0.4
0.3
0.2
0.1
6000000
4000000
3000000
2000000
• Metabolic and enzymatic click chemistry labeling methods yield selective labeling
of antibody heavy chain glycans, which do not interfere with antigen binding.
1000000
0
G
GalNAz
Conclusions
5000000
β-
+
Si
al
A
GalNAz
+ ManNaz
7000000
al
PN
G
as
PN e F
+S Gas
ia e
l
F
+S PN A
ia Ga
lA s
+ eF
βG
a
No l
di
g
Si
al
PN
A
+S Gas
ia e
lA F
g
di
di
No
g
Si
al
A
0.0
L
Effect of glycosidic degestion on Gal-Tgenerated click labeling of α-pAKT heavy chain
0.8
No
Metabolic Labeling:
HEK293, CHO or mouse hybridoma cells were cultured with 40 – 120 μM azido
sugar (GalNAz and/or ManNAz) for 3 days. Antibodies were isolated from the cell
culture supernatant with Protein A resin.
Alexa Fluor® 488/SYPRO® Ruby Signal
L
Enzymatic Labeling:
Antibodies (20 – 40 μg) were incubated overnight at ambient temperature in 50 – 120
μL of non-phosphate buffer (e.g., 25 mM HEPES pH 6.8, 50 mM NaCl, 10 mM
MnCl2) containing 0.05 – 2 mM UDP-GalNAz, 3 – 20 μM β-Gal-T1 enzyme and 0 –
0.05 U/μL CIP alkaline phosphatase. Glycosidase treatment with β-(1-4)
galactosidase, Sialidase A, Endo F2 or PNGase F were performed simultaneously
with the Gal-T reaction by adding 1 μL of the desired enzyme stock solution to the
reaction at the beginning of the incubation period. Excess UDP-GalNAz was
removed by Protein A resin or dialysis prior to click conjugation reactions.
20.0
Time (hr)
SYPRO® Ruby Protein Stain
2 mM
0.2
H
Materials and Methods
50 μM
0.3
Alexa Fluor®
647 Signal
SYPRO® Ruby Protein Stain
ManNAz
GalNAz
Gal+Man
Sialidase A
+ + ¯ + + ¯ + +
β-galactosidase ¯ ¯
+ ¯ ¯ + ¯ ¯
¯
PNGase F ¯ ¯ ¯
+
¯ ¯ ¯ ¯ + + + ¯ ¯
[UDP-GalNAz]
tr
o
hr l
17
hr
C
on
t
3 ro l
hr
17
hr
C
on
t
3 ro l
hr
17
hr
0.5
C
on
t
3 ro l
hr
17
hr
C
on
t
3 ro l
hr
17
hr
C
on
t
3 ro l
hr
17
hr
Antibody from CHO cells
0.6
C
on
0h
24 h
0.3
0.10
Figure 5. Dependence of the Gal-T reaction on UDP-GalNAz
concentration
3
0.4
+ Gal-T
0.12
C
on
0.3
β-tubulin
CD45
Antibody from HEK 293F cells
0.5
Alexa Fluor® 488/SYPRO® Ruby Signal
Alexa Fluor® 488/SYPRO® Ruby Signal
0.4
0.14
GalNAz (µM) + ManNAz (µM)
Alexa Fluor® 488 Signal
Antibodies from mouse hybridoma
0.5
DIBO-Alexa Fluor® 647 dye clicked to anti-TSH
Gal-T labeled antibody
H
H
GalNAz (µM) + ManNAz (µM)
Alexa Fluor® 488 Signal
Stable triazole conjugate
1 hr 2.5 hr
-
H
Figure 2. Metabolic and enzymatic click labeling is selective
for specific antibody glycan subclasses
Azide-modified molecule
Labeled Antibody
SYPRO® Ruby Protein Stain
Alexa Fluor® 647 Signal
Time
Alexa Fluor® 488
Signal
Gal
Reaction ON
Antibody-Azide
Figure 4. Time-course for DIBO-Alexa Fluor® dye conjugation
of Gal-T labeled antibodies
React ON
N3
β-tubulin CD45
DIBO Alexa
Fluor® dye
UDP-GalNAz
3-4 days
N3
Alexa Fluor® 647/SYPRO® Ruby Signal
OAc
O
OAc
NH
O
Alexa Fluor® 488/SYPRO® Ruby Signal
AcO
Harvest Abs
Alexa Fluor® 488/SYPRO® Ruby Signal
Click chemistry based antibody labeling scheme
N3
OAc
O
OAc
GalNAz
(Core GalNAc)
1A
Here we describe click chemistry-mediated antibody labeling methods
that result in site-specific labeling of antibody heavy chain glycans using
mild conditions. The methods involve the metabolic or enzymatic
incorporation of azide-modified sugars into antibody heavy chain glycans
away from the antibody binding domain. The metabolic labeling approach
involves feeding antibody-expressing cultured cells tetraacetylated azido
modified sugars that specifically incorporate into heavy chain N- or Olinked glycans1. The in vitro enzymatic labeling approach involves the use
of a modified β-Gal-T1 enzyme that specifically labels terminal Nacetylglucosamine (GlcNAc) residues on N-linked antibody sugars with
azide-modified N-acetylgalactosamine azide (GalNAz)2,3. The azidelabeled antibodies are purified from the cell supernatants or labeling
reactions using standard techniques and modified using mild, copper-free,
click reaction conditions4.
OAc
O
HN
AcO
AcO
Enzymatic Antibody labeling
Ac4GalNAz
tr
ol
3
hr
17
h
C
on r
tr
ol
3
hr
17
C hr
on
tr
ol
3
hr
17
hr
Introduction
Standard antibody labeling techniques can sometimes lead to decreased,
or even complete loss of, binding affinity due to disruption of the antigen
binding domain. This is often due to the modification of lysine residues in
the antigen binding domain with amine-reactive compounds that results in
blocking antibody-antigen interaction. Monoclonal antibodies can be
particularly susceptible to active-site blockage as the single amino acid
sequence may contain vulnerable lysine residues at or near the antigen
binding domain resulting in crippled or inactivated antibodies. Another
common labeling method involves the partial reduction and labeling of
cysteine residues in the antibody hinge region. In addition to potentially
blocking the antigen binding domain, the method typically results in a
significant fraction of fully or partially dissociated antibody fragments
depending upon the extent of reduction which can vary widely between
specific antibodies. Another chemical labeling method involves the
modification of vicinal alcohols on the antibody sugars using periodate
oxidation followed by reductive amination. This method requires multiple
steps, uses relatively harsh reaction conditions, and the labeling efficiency
tends to be antibody dependent.
β-Gal+
SialA
Endo
F2
No
PNGase
digestion
F
ManNaz
Figure 3. Click labeling does not affect binding affinity
A. GalNAz + ManNAz Labeled Antibodies
W lysate dilutions
Ctl
α-pAKT (no click control) Æ GAR-biotin Æ SA-Qdot® 625
• Metabolic labeling results in the modification of both N- and O-linked sugars on
antibody heavy chains.
• Metabolic and enzymatic labeling methods result in the differential modification
of glycan subtypes that is dependent on the expression cell type.
• DIBO copper-free click chemistry labeling reagents offer a gentle, selective and
easy method for labeling azide-tagged macromolecules.
α-pAKT (Alexa Fluor® 488 dye clicked) Æ GAR-biotin Æ SA-Qdot® 625
α-pAKT (biotin clicked) Æ SA-Qdot® 625
B. Gal-T Labeled Antibodies
W lysate dilutions
Ctl
α-pAKT Gal-T (biotin clicked) Æ SA-Qdot® 625
α-pAKT (EndoF2) Gal-T (biotin clicked) Æ SA-Qdot® 625
AKT is constitutively phosphorylated in Jurkat cells and phosphorylation was suppressed with Wortmannin treatment in control
cells (W Ctl). A) Blots of lysate dilutions were prepared and indirect detection was performed with untreated pAKT antibody
(top) or with metabolically labeled pAKT antibody clicked with Alexa Fluor® 488 dye (middle). Direct detection was performed
with metabolically labeled pAKT antibody clicked with biotin alkyne (bottom). B) Direct detection was performed with
enzymatically labeled pAKT antibody clicked with biotin alkyne with (top) or without (bottom) Endo F2 glycosidase digestion,
which cleaves N-linked high mannose or biantennary oligosaccharides leaving a GlcNAc residue remaining on the
asparagine.
References
1. Dube DH and Bertozzi CR. Curr Opin Chem Biol. 2003;7(5):616-625.
2. Khidekel N, Arndt S, Lamarre-Vincent N, Lippert A, Poulin-Kirstien KG, Ramakrishnan B, Qasba PK, Hsieh-Wilson LC.
J Am Chem Soc 2003;125(52):16162.
3. Boeggeman E, Ramakrishnan B, Pasek M, Manzoni M, Puri A, Loomis KH, Waybright TJ and Qasba PK. Bioconjugate
Chem. 2009;20:1228.
4. Ning X, Guo J, Wolfert MA, and Boons GJ. Angew Chem In Ed. 2008;47(12):2253.
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
© 2011 Life Technologies Corporation. All rights reserved.
The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.
Zeba is a trademark of Thermo Scientific.

Download Report