Download Functional Assay for Botulinum Neurotoxin Type A

Functional Assay for Botulinum Neurotoxin Type A Utilizing the Neuronal Receptor Protein SV2c
Todd Christian1, Andreas Rummel2 and Nancy Shine1
1List
Biological Laboratories, Inc. Campbell, CA, USA.
2Institut
Presented at the
45th
für Toxikologie, Hanover, Germany
Annual Interagency Botulinum Research Coordinating Committee Meeting. September 2008 in Philadelphia, PA
Methods
Introduction
Multiple assays have been developed to detect botulinum toxin based solely on
antigenic properties and/or enzymatic activity. Now with the recent identification of
SV2c as the protein receptor for BTA1,2 new detection methods can be attempted
that combine multiple steps of the disease process of botulinum toxin. Capture of
the BTA with the SV2c receptor binding domain demonstrates the specific binding
which is then detected using a FRET peptide. Cleavage of this specific BTA
substrate indicates the specific endopeptidase activity of the BTA. By combining
these activities this assay mimics two of the three in vivo functions of the toxin.
Immobilized Protein A experiments: Immobilized Protein G resin (0.2ml ) was
washed and crosslinked to 50µg of Anti-GST antibody (Abcam 18183) using the
the Seize X Protein A Immunoprecipitation kit from Pierce. After crosslinking, the
resin was washed using the Gentle Ag/Ab binding and elution buffers from Pierce
(#2102). GST-human SV2c (125 µg) was added to the protein A resin with AntiGST antibodies and incubated overnight at 4ºC. The bound resin was washed
and 100µl was aliquoted into each spin column. Dilutions of BTA were added to
each column and incubated for 4 hours at room temperature. Unbound toxin was
washed away and SNAPtide® reaction buffer (50 mM HEPES, pH 8, 5 mM DTT, 1
mM ZnCl2, 0.2% Tween-20) and 20µM SNAPtide® (o-Abz/Dnp) #520 was added
to each column. The reactions were mixed at 37ºC for thirty minutes and then
overnight at room temperature. The reaction mixtures were centrifuged from the
columns and placed into a black 96 well plate. The resin was washed several
times with 50mM HEPES buffer and added to the wells. The plate was read using
a SPECTRAmax GEMINI XS fluorescence microplate reader (Molecular Devices).
The excitation and emission wavelengths were set to 321 nm and 418 nm,
respectively, for the o-Abz/Dnp-substrate
SV2c:MagneGST
ratio 0.6ug/ul
R² = 0.9999
SV2c:MagneGST
ratio 1.25ug/ul
R² = 0.9998
SV2c:MagneGST
ratio 2.5ug/ul
R² = 0.9992
120
SNAPtide reaction with 0.6ng BTA and a
30 min 37 degree reduction step
100
RFU
2000
80
60
1500
40
1000
20
0
500
2
4
6
17
Hours Incubated at Room Temperature
0
0
5
10
15
20
25
[BTA] ng
Figure 4: SV2c to MagneGST bead ratio. Several different ratios of SV2c to beads were
tested. The low ratio (0.6µg/µl) was 125µg SV2c:200µl beads. The middle ratio
(1.25µg/µl) was 250µg SV2c:200µl beads. The high ratio (2.5µg/µl) was 500µg SV2c:
200µl beads. At the lower concentrations of toxin the difference between the ratios was
quite small. The difference becomesnoticeable as the toxin concentration reaches 5 and
20ng.
Figure 7: Optimization of reaction time. The FRET peptide cleavage was monitored for a
series of time points in order to determine the optimal reaction time. Enzymatic activity with
0.6ng of BTA was clearly detected after 6 hours in the absence of a 30 minutes reduction
incubation at 37°C. When the 37°C reduction step was included, enzymatic activity was only
detected with 0.6ng of BTA after overnight incubation.
A
900
SNAPtide reaction with
5ng BTA
800
700
SNAPtide reaction with
0.6 ng BTA
600
500
400
300
200
100
0
20
2500
10
5
µM SNAPtide® (o-Abz/Dnp) #520
2106
1941
2000
1733
Activity with 20ng BTA
during 25 degree binding
1705
1630
1600
1567 1557
B
800
SNAPtide (Mca/Dnp)
reaction with 5ng BTA
700
600
1500
1365
1364
Activity with 20ng BTA
during 37 degree binding
1000
The specific binding domain of SV2c for BTA has been shown to be the luminal
domain loop between transmembrane domains 7 and 8.1,2 A diagram of the
structure of SV2c is shown in Figure 1. We have purified the SV2c luminal
binding domain as a GST fusion and utilized it in our capture assays. There are
several strategies that we have attempted. We have immobilized the GST-SV2c
to anti-GST antibodies that are covalently attached to Protein A coated resin. We
have also bound the GST-SV2c to magnetic beads coated with glutathione
(MagneGST). See Figure 2 for an illustration of these two strategies.
SNAPtide reaction with 0.6ng BTA no
reduction step
140
RFU
With this assay we can examine two of the three steps of toxin activity, binding
and cleavage. In the future, we plan to establish this as a functional assay to
monitor binding and enzymatic activity of Botulinum Neurotoxin Type A in order to
demonstrate a direct correlation to the gold standard, the mouse bioassay.
160
2500
SNAPtide (Mca/Dnp)
reaction with 0.6ng BTA
500
RFU
Recent biochemical and molecular genetic studies have established synaptic
vesicle glycoprotein 2C (SV2c) as the protein receptor for Botulinum Neurotoxin
Type A (BTA). The luminal domain loop of SV2c, between transmembrane
domains 7 and 8, has been shown to be the location of BoNT/A binding
(Mahrhold et al FEBS Lett. 2006). Utilizing recombinant GST-SV2c, we have
shown through pull-down experiments that we can detect both the binding of BTA
to SV2c and enzymatic activity using the FRET peptide SNAPtide®. We describe
the reaction conditions and the detection limits using multiple FRET substrates.
180
3000
MagneGST experiments: MagneGST beads (200µl) were mixed with 125µg of
GST-SV2c and incubated for 1 hour with mixing in the presence of 1% BSA. The
MagneGST beads were washed and reconstituted in 1.2 ml of 50mM HEPES, pH
8 buffer. Fifty microliters of the beads were used in each reaction. Various
concentrations of Botulinum Neurotoxin Type A were added to each tube. Toxin
binding was 1-4 hours at room temperature with mixing. Unbound toxin was
washed away and 400µl of SNAPtide® reaction buffer (50 mM HEPES, pH 8, 5
mM DTT, 1 mM ZnCl2, 0.1% BSA) and 20µM SNAPtide® (o-abz/Dnp) #520 or
SNAPtide ® (Mca/Dnp) was added to each tube. The reaction tubes were mixed at
37ºC for thirty minutes and then overnight at room temperature, unless otherwise
noted. They were then placed on a magnetic stand and reaction mixture was
pipetted from the tubes. The beads were washed with 50mM HEPES buffer and
250µl of reaction and wash were placed into a single well of a black 96 well plate.
The plate was read using a SPECTRAmax GEMINI XS fluorescence microplate
reader (Molecular Devices). The excitation and emission wavelengths were set to
321 nm and 418 nm, respectively, for the o-Abz/Dnp substrate and 325nm /398nm
for the Mca/Dnp substrate.
RFU
Botulinum toxin progresses through a three step process leading to the
interruption of synaptic transmission. The first step is binding of toxin to neuronal
cells and incorporation of the toxin into an endosome; the second step is
translocation of the enzymatic light chain out of the endosome and into the
cytosol of the neuron; and the final step is cleavage of synaptosomal proteins to
prevent vesicle docking and neurotransmitter release.
RFU
Abstract
Figure 8: Test of different
substrate concentrations. Two
different FRET peptides were
tested at three different
concentrations using two
different BTA concentrations in
the MagneGST capture assay.
These assays had no 37°C
reduction step and were
overnight reactions. No
inhibitory affects were observed
using 20µM substrate.
400
300
200
100
SNAPtide® FRET peptide
500
0
Magentic
20
Gluthione coated
Beads
(MagneGST )
GST-SV2c
0.5
Botulinum Toxin
1
2
5
4
Figure 5: Toxin exposure time at 25 and 37°C. The duration and temperature of the
toxin binding step does not significantly alter the amount of enzymatic activity detected.
Therefore, in an effort to shorten the assay time 1hour binding at room temperature was
chosen. Following the toxin binding, these assays were carried out using a 30 minute 37°C
reduction step followed by room temperature overnight incubation.
SNAPtide® FRET peptide
Protein A coated
Agarose bead
Protein A
Monoclonal
3
Hours
Type A
Our goal for this research is to establish an assay which demonstrates binding
and catalytic activity and that can compete with the mouse bioassay.
10
µM SNAPtide® (Mca/Dnp)
0
GST-SV2c
Anti-GST antibody
400
350
SNAPtide (Mca/Dnp)
R² = 0.9893
SNAPtide (o-Abz/Dnp)
R² = 0.9973
300
Botulinum Toxin
250
Figure 2: Capture Methods. Schematics showing the use
of SV2c in the binding and detection of Botulinum
Neurotoxin Type A.
SV2c
553
Lumen
RFU
Type A
579
454
200
150
800
0hrs @ 37 degrees
700
100
0.5hr @ 37 degree
600
1hr @ 37 degrees
603
50
800
727
700
Protein A:Ab:GST-SV2c R² = 0.9958
MagneGST:GST-SV2c
Cytosol
600
RFU
1
500
2hr @ 37 degrees
400
3hrs @ 37 degrees
0
0
1
2
3
4
5
6
[BTA] ng
R² = 0.9414
300
Mahrhold et. al. FEBS Letters 580(2006) 2011-2014
500
The binding domain for Botulinum Neurotoxin Type A has been shown to
be in the luminal loop, amino acids 454-579.
RFU
200
Figure 1: SV2C structure
400
100
300
0
5
200
100
Materials
SNAPtide®
substrate # 520 contains the FRET pair o-aminobenzoic acid/2,4
dinitrophenyl (o-Abz/Dnp) and SNAPtide® substrate Mca/Dnp contains (7methoxy-coumarin-4-yl)acetyl/2,4 dinitrophenyl as its FRET pair. Botulinum
Neurotoxin Type A (Product #130A) and the SNAPtide® substrates are products of
List Biological Laboratories. Plasmid encoding the binding domain of human
SV2c was provided by A. Rummel. MagneGST beads (V8611) were obtained
from Promega. Immobilized Protein A (#45215) was obtained from Pierce.
Monoclonal Anti-GST antibody was obtained from Abcam (ab18183).
0
0.00
1.00
2.00
3.00
4.00
5.00
6.00
[BTA] ng
Figure 3: Comparison of MagneGST and Protein A capture assays using
GST-SV2c. Both assays detect low amounts of Botulinum Neurotoxin. The
MagneGST assay is a simpler, more cost effective method and can achieve higher
signal to noise ratios.
[BTA] ng
0.6
Figure 9: Botulinum Neurotoxin Type A titration using both SNAPtide® (o-Abz/Dnp) #520
and SNAPtide® (Mca/Dnp). These assays were run with one hour toxin binding at room
temperature and a six hour room temperature cleavage reaction. Both substrates show a
linear response to the toxin concentrations. Three hundred picograms of BTA can be detected
with either peptide.
Figure 6: 37°C versus 25°C reduction step. The requirement of a 37°C incubation, prior
to room temperature overnight incubation of bound toxin with reaction buffer, was tested.
It was thought that incubation at 37°C would aid in the reduction of the toxin. However, as
the incubation at 37°C is extended, the loss of detectable activity is more apparent. This
effect is larger for 5ng than 0.6ng of BTA.
References:
1) Mahrold et al. FEBS Letters. 2006 April 3, 580(8):2011-4.
2) Dong et al. Science. 2006 April 28; 312(5773):592-6