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Biocatalytic Detoxification of Paralytic Shellfish Toxins
April L. Lukowski,†,‡ Nicholas Denomme,§,∥ Meagan E. Hinze,‡ Sherwood Hall,⊥ Lori L. Isom,§,#,∇
and Alison R. H. Narayan*,†,‡,○
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†
Program in Chemical Biology and ‡Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, United States
§
Department of Pharmacology and ∥Center for Consciousness Science, University of Michigan Medical School, Ann Arbor,
Michigan 48109, United States
⊥
United States Food and Drug Administration, College Park, Maryland 20740, United States
#
Department of Neurology, ∇Department of Molecular and Integrative Physiology, and ○Department of Chemistry, University of
Michigan, Ann Arbor, Michigan 48019, United States
S Supporting Information
*
ABSTRACT: Small molecules that bind to voltage-gated
sodium channels (VGSCs) are promising leads in the
treatment of numerous neurodegenerative diseases and pain.
Nature is a highly skilled medicinal chemist in this regard,
designing potent VGSC ligands capable of binding to and
blocking the channel, thereby offering compounds of potential
therapeutic interest. Paralytic shellfish toxins (PSTs),
produced by cyanobacteria and marine dinoflagellates, are
examples of these naturally occurring small molecule VGSC
blockers that can potentially be leveraged to solve human
health concerns. Unfortunately, the remarkable potency of
these natural products results in equally exceptional toxicity,
presenting a significant challenge for the therapeutic
application of these compounds. Identifying less potent analogs and convenient methods for accessing them therefore
provides an attractive approach to developing molecules with enhanced therapeutic potential. Fortunately, Nature has evolved
tools to modulate the toxicity of PSTs through selective hydroxylation, sulfation, and desulfation of the core scaffold. Here, we
demonstrate the function of enzymes encoded in cyanobacterial PST biosynthetic gene clusters that have evolved specifically for
the sulfation of highly functionalized PSTs, the substrate scope of these enzymes, and elucidate the biosynthetic route from
saxitoxin to monosulfated gonyautoxins and disulfated C-toxins. Finally, the binding affinities of the nonsulfated, monosulfated,
and disulfated products of these enzymatic reactions have been evaluated for VGSC binding affinity using mouse whole brain
membrane preparations to provide an assessment of relative toxicity. These data demonstrate the unique detoxification effect of
sulfotransferases in PST biosynthesis, providing a potential mechanism for the development of more attractive PST-derived
therapeutic analogs.
■
INTRODUCTION
Paralytic shellfish toxins (PSTs) are structurally complex small
molecules with nanomolar affinity for tetrodotoxin-sensitive
voltage-gated sodium channels (VGSCs).1,2 PSTs share a
common tricyclic, bisguanidinium ion-containing scaffold that
is differentially functionalized at the C11, C12, N1, and C13
positions (Figure 1).1,2 A subset of PSTs contain sulfate
groups, including gonyautoxins (GTX1−6, 6−11), C-toxins
C1−C4 (12−15), and M-toxins M1α (3), M1β (4), and M3
(5).2−4 Sulfated PSTs are often the most abundant species in
mixtures of toxins isolated from biological sources.5−7 While
risk of systemic toxicity has hindered clinical studies with
highly potent molecules from this class, such as saxitoxin (STX,
1) and neosaxitoxin (neoSTX, 2), PSTs bearing at least one
sulfate group, such as GTX5 (10), have greater potential as
safe pharmaceutical agents due to their reduced toxicity.8,9 For
example, gonyautoxins are being evaluated as agents for the
© 2019 American Chemical Society
treatment of pain and are currently in clinical trials for their
ability to reduce acute pain in small doses (i.e., 40−50 μg
injections). Importantly, these compounds elicit the desired
analgesic effect without the possible cardiotoxicity and abuse
potential observed with conventional local anesthetics.
Specifically, GTX5 (10) and mixtures of GTX2 (7) and
GTX3 (8) have been evaluated in clinical trials to treat chronic
tension headaches,10 muscle tension and spasms,11,12 and
postoperative pain associated with knee replacement surgery.13
Botulinum toxin proteins have been extensively developed for
these types of applications;14 however, small molecule agents
with similar capabilities possess significant advantages
compared to protein therapeutics, including increased stability,
Received: February 14, 2019
Accepted: March 28, 2019
Published: April 15, 2019
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Figure 1. Structures of select paralytic shellfish toxins and reported data on relative voltage-gated sodium channel affinity and toxicity for STX (1),
GTX2 (7), and GTX3 (8).
and disulfated PSTs (C-toxins 1−4) has not been elucidated
and the biocatalytic machinery has remained uncharacterized.
Our work is informed by initial in vitro studies using enzymes
isolated from PST-producing dinoflagellate cultures performed
by Ishida and co-workers, which provided evidence for the
involvement of two sulfotransferases of unknown sequence in
gonyautoxin biosynthesis: one capable of N-sulfamation of the
C13 carbamate to afford GTX5 (10) from STX (1) and the
second conducting an O-sulfation to generate GTX2 (7) and
GTX3 (8) from a mixture of 11-α-hydroxySTX (16) and 11-βhydroxySTX (17).32,33 Because of enzyme instability, lack of
protein sequence information, and the general intractability of
manipulating dinoflagellate genomes, these enzymes were not
investigated further but provided the first piece of direct
evidence that sulfotransferases are critical to gonyautoxin
biosynthesis. Neilan and co-workers subsequently identified
the genes encoding the enzymes involved in gonyautoxin
biosynthesis in cyanobacteria and proposed that they are
located within the biosynthetic gene cluster for the parent
compound STX (1), naming them SxtN and SxtSUL,34−37 and
have specifically implicated SxtN from the cyanobacteria
Syctonema crispum as the catalyst for the conversion of STX
(1) to gonyautoxin 5 (GTX5, 10).36
To interrogate the process of PST sulfation in cyanobacteria
and elucidate the biosynthetic pathway toward the C-toxins
(12−15), we have biochemically characterized the enzymes
responsible for the installation of sulfo groups in vitro. Toward
this aim, we expressed and purified the O-sulfotransferase
SxtSUL from Microseira wollei and the N-sulfotransferase SxtN
from Aphanizomenon sp. NH-5 and assessed their activities on
a range of substrates. Using cascade reactions with SxtSUL,
SxtN, and the previously characterized Rieske oxygenase
GxtA,23 we have determined the cyanobacterial pathway
milder storage conditions, longer shelf lives, and a wider range
of dosing options.15,16 Synthetic routes for accessing saxitoxin
and gonyautoxins have been successfully developed,17−20
though they typically require lengthy, inefficient routes or
starting materials that are extracted from toxic shellfish.10,21
Elucidating a biosynthetic route for producing sulfated PST
derivatives stands to expedite production of these molecules
and will enable structural diversification for the discovery of
novel pharmaceutical agents and tools for fundamental
biological studies.22,23
The toxification and detoxification of PSTs to modulate
affinity for VGSCs have been employed by both prokaryotic24,25 and eukaryotic organisms.26,27 Typically, microorganisms responsible for the production of the sulfated
PSTs are consumed by shellfish, which subsequently
bioaccumulate the toxins and in some cases perform enzymatic
modifications that alter the molecules’ toxicity.26,27 For
example, it was recently reported that the marine scallop
Chlamys farreri enzymatically hydrolyzes the sulfate groups of
stored gonyautoxins to generate the more toxic parent
compounds STX (1) and neoSTX (2), weaponizing the
accumulated toxins for self-defense.28 C. farreri possess point
mutations in VGSC genes that reduce the affinity of PSTs for
the channels, enabling the sequestration of up to 40,000 μg
STX (1) per 100 g flesh, a quantity 500 times higher than the
safety limit issued by the United States Food and Drug
Administration and many other countries.28−30 This extraordinary adaptation has motivated our research into the
mechanisms of sulfotransferase-mediated modification of PSTs
by the microorganisms responsible for their production.
Despite the abundance of sulfated derivatives isolated from
natural sources,7,31 the order of events in the biosynthetic
route to singly sulfated derivatives (GTX1−6 and M-toxins)
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Figure 2. (A) Reaction of SxtSUL with a mixture of 11-α-hydroxySTX (16) and 11-β-hydroxySTX (17). (B) Observed epimerization of GTX3 (8)
to GTX2 (7) over time.
toward GTXs, M-toxins, and C-toxins. Beyond in vitro
characterization of C-toxin biosynthesis, we aim to demonstrate the impact of sulfation of PSTs on binding affinity to
VGSCs present in mouse brain extracts. PST toxicities have
been evaluated for decades using a myriad of methods
including in vivo mouse toxicity assays, mouse or rat whole
brain homogenate binding assays, and electrophysiological
recordings with squid axon model systems or VGSCs in
heterologous expression systems such as human embryonic
kidney (HEK) or Chinese hamster ovary (CHO) cells.38−48
For example, studies with heterologously expressed VGSCs
indicate that GTX2 (7) and GTX3 (8) are comparable in
toxicity to STX (1),43 while squid axon studies demonstrate
that GTX2 (7) and GTX3 (8) are significantly lower in
toxicity relative to STX (1, Figure 1).45 While there are many
ways to assess toxicity, it is accepted that binding affinity of
PSTs to VGSCs directly correlates with toxicity.42,49 We aim to
understand the structure activity relationships associated with
PST sulfation using the reliable [3H]-STX competition binding
assay with mouse whole brain membrane preparations
containing VGSCs in a physiologically relevant environment
in order to directly compare the binding affinities of
monosulfated, disulfated, and nonsulfated PSTs.39
(6) and GTX2 (7), and GTX3 (8) and GTX4 (9),
respectively. Initially, we found soluble expression and
subsequent purification of SxtSUL to be challenging. We
determined that coexpression of GroEL and GroES chaperone
proteins with SxtSUL in a standard BL21(DE3) expression cell
line was necessary to obtain sufficient quantities of soluble
protein (see Supporting Information).50 BLAST analysis
indicated the presence of a conserved 3′-phosphoadenosine5′-phosphosulfate (PAPS) binding site; thus, PAPS was
employed as the sulfate donor in these reactions.51 In analytical
assays with a mixture 11-α-hydroxySTX (16) and 11-βhydroxySTX (17), SxtSUL preferentially converted 11-βhydroxySTX (17) to GTX3 (8, Figure 2A). However, a
small amount of GTX2 (7) was observed in the reaction
mixture, which could arise through two distinct pathways,
either direct sulfation of 11-α-hydroxySTX (16) or from
epimerization of GTX3 (8) to GTX2 (7), a phenomenon
reported in the total synthesis of GTX2 (7) and GTX3 (8) by
Du Bois and co-workers.18
To determine the operative route to GTX2 (7), we
conducted a cascade reaction with SxtSUL and the recently
characterized Rieske oxygenase, GxtA, responsible for the
stereoselective conversion of STX (1) to 11-β-hydroxySTX
(17).23 This approach allowed for exclusive analysis of the
epimerization pathway by generating 11-β-hydroxySTX (17)
in situ. At early time points, we observed production of only
GTX3 (8), which epimerized to GTX2 (7) over time. After 8
days of incubation at 30 °C, a 3:1 mixture of GTX3 (8) and
GTX2 (7) was present (Figure 2B). Precipitation was also
evident based on the decrease in ion counts over time.
Additionally, the epimerization of 11-β-hydroxySTX (17) to
11-α-hydroxySTX (16) was not observed (Supporting
Information Figure S7). The formation of GTX2 (7) and
GTX3 (8) from a single hydroxylated precursor, 11-βhydroxySTX (17), is also supported by (1) the absence of
genes for GxtA homologues that would be capable of
■
RESULTS AND DISCUSSION
Characterization of SxtSUL as an O-Sulfotransferase.
To interrogate the biosynthetic route to highly functionalized
PSTs, we sought to identify the chemical functions and
substrate scopes of SxtSUL and SxtN as well as the sequence of
enzymatic reactions that lead to the various gonyautoxins and
C-toxins. SxtSUL is proposed to be an O-sulfotransferase and
the only characterized O-sulfated PST derivatives possess
sulfate groups at the C11 position, as observed in GTX1−
GTX4 (6−9) and C1−C4 (12−15). Thus, we sought to assess
the activity of SxtSUL using a mixture of 11-α-hydroxySTX
(16) and 11-β-hydroxySTX (17), putative precursors to GTX1
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than 1 mM were observed with α-STOH (20) and β-STOH
(19) (Supporting Information Figure S5). Additionally, steadystate kinetic analysis of PAPS using constant saturating STX
(1) revealed a KM of 6.2 ± 1.4 μM, a value consistent with
other characterized PAPS-dependent sulfotransferases (Figure
3).52
In Vitro Enzyme Cascades. To determine the order of
events leading to toxins C1 (12) and C2 (13), we conducted
cascade reactions with SxtN, GxtA, and SxtSUL using STX (1)
as a substrate. As SxtN was only capable of reacting with
substrates at or below the STX (1) oxidation state, we
hypothesized that SxtN must react with STX (1) before GxtA
hydroxylation and SxtSUL installation of the C11 sulfate
group. In reactions containing only SxtN and GxtA, the
emergence of a new product with the exact mass of GTX2 (7)
and GTX3 (8), m/z = 396, and higher polarity than GTX5
(10) was observed by hydrophilic interaction liquid
chromatography (HILIC)-MS (Figure 4B). By MS/MS
analysis, this new product corresponded to hydroxylated
GTX5, a natural product toxin previously isolated and
named M1, consisting of epimers M1α (3) and M1β (4).4,53
The retention time and MS/MS fragmentation pattern differed
from GTX3 (8) with the enhanced fragmentation peak at 316
suggested the cleavage of the sulfocarbamate with a remaining
C11 hydroxylated tricycle (Supporting Information Figure
S10). This fragmentation pattern was also observed in the
reported MS/MS spectrum of M1 epimers.4 Furthermore, the
addition of SxtSUL to a reaction containing SxtN and GxtA
resulted in the formation of GTX3 (8) and toxin C2 (13,
Figure 4C), confirming the identity of the M1 epimer
produced by SxtN and GxtA to be M1β (4). The identity of
toxin C2 (13) was verified using an authentic standard by LCMS (Figure 4C). Collectively, these experiments represent the
first direct characterization of the biosynthetic enzymes and
relevant biosynthetic intermediates necessary for the generation of M-toxins the disulfated C-toxins from STX (1) in
cyanobacteria, demonstrating an imperative order of events.
Affinity for Voltage-Gated Sodium Channels. Previous
in vitro work using a high-throughput 96-well [3H]-STX
binding assay developed by Usup and co-workers reported the
monosulfated PSTs to have a reduced affinity compared to
STX (1) and neoSTX (2) for the tetrodotoxin-sensitive
VGSCs present in rat brain preparations.42 In vivo work using
the mouse toxicity assay also indicated the sulfated PSTs have
reduced toxicity compared to STX (1) and neoSTX (2).54 To
pharmacologically characterize the therapeutic potential of the
PSTs explored in this study and assess relative toxicity,
competitive [3H]-STX binding assays using mouse whole brain
membrane preparations were performed.39
Competitive binding curves for the unlabeled toxins against
5 nM [3H]-STX are shown in Figure 5. The relative Ki values
for each unlabeled toxin are shown in Figure 5. Ki values
ranged from 0.875 to 625.2 nM for STX (1) and a mixture of
toxins C1 (12) and C2 (13), respectively. The competitive
binding curves for GTX2 (7) and GTX3 (8), 11-αhydroxySTX (16) and 11-β-hydroxySTX (17), GTX5 (10),
and toxins C1 (12) and C2 (13) were shifted to the right of
unlabeled STX (1), indicating a lower affinity than STX (1) for
the VGSCs present in the mouse whole-brain preparations.
The rank order of binding affinity according to Ki values was
STX (1) > GTX2/3 (7, 8) > 11-α/β-hydroxySTX (16, 17) >
GTX5 (10) > C1/C2 (12, 13). At 10 μM, each of the
unlabeled toxins displaced more than 92% of the [3H]-STX
producing 11-α-hydroxySTX (16), (2) the absence of genes
for SxtSUL homologues to react with 11-α-hydroxySTX (16),
and (3) the absence of 11-α-hydroxySTX (16) in natural
product extracts. These data indicate that only one Osulfotransferase, SxtSUL, is required to generate GTX1−
GTX4 (6−9) from 11-β-hydroxylated precursors. The enzyme
required for N1 hydroxylation and the order of events in the
biosynthesis of GTX1 (6) and GTX4 (9) are not known.
Substrate Scope of SxtN. Analysis of in vitro reactions
indicated that SxtSUL performed only a single sulfation, thus a
second sulfotransferase is required to introduce the sulfate
onto the nitrogen atom appended to the carbamate group of
the PST scaffold to afford disulfated C-toxins (12-15). Neilan
and co-workers recently demonstrated that SxtN from
Syctonema crispum can install the sulfo group onto the
carbamate of STX (1), generating GTX5 (10).36 However,
the ability of SxtN to act on a broad panel of PSTs or to
perform O-sulfations on 11-α-hydroxySTX (16) or 11-βhydroxySTX (17) to generate GTX2 (7) or GTX3 (8) has not
been assessed. Our initial experiments with heterologously
expressed SxtN were conducted in vitro with STX (1),
neoSTX (2), a mixture of 11-α-hydroxySTX (16) and 11-βhydroxySTX (17), GTX2 (7) and GTX3 (8), dideoxysaxitoxin
(ddSTX, 18), α-saxitoxinol (α-STOH, 20), and β-saxitoxinol
(β-STOH, 19). While reaction with neoSTX (2) and GTX2
(7) and GTX3 (8) did not result in product formation, STX
(1) was converted to a single product, GTX5 (10)
(Supporting Information Figure S3). Additionally, 11-αhydroxySTX (16) and 11-β-hydroxySTX (17) were not
observably sulfamated by SxtN, definitively demonstrating
that SxtN exclusively acts as a N-sulfotransferase. Interestingly,
reactions with substrates at a lower oxidation level than STX
(1) such as ddSTX (18), α-STOH (20), and β-STOH (19)
resulted in the formation of sulfonated products in each case
(Figure 3). Relative percent conversions were assessed for each
of these transformations by LC-MS and suggest that STX (1)
is the preferred substrate of SxtN (Supporting Information
Figure S3). Steady-state kinetic analysis of SxtN with STX (1)
revealed a kcat/KM of 0.44 M s−1 (Figure 3). KM values greater
Figure 3. SxtN reactions with non-native substrates and steady-state
kinetics with PAPS and STX (1).
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Figure 4. Cascade reactions with SxtN, GxtA, and SxtSUL. (A) Scheme of cascade reactions en route to natural products boxed in gray. (B) LC-MS
traces of one and two enzyme reactions compared to product standards. (C) LC-MS traces of SxtN + GxtA + SxtSUL reaction compared to C1
(12) and C2 (13) product standard.
negatively charged functional groups in the design of safer,
lower toxicity PST analogs as therapeutics.
Conclusions. In summary, we have elucidated the
biosynthetic steps necessary to achieve GTX2 (7), GTX3
(8), M1β (4), and C2 (13) from STX (1) using individual and
cascade reactions with the sulfotransferases SxtN and SxtSUL
and oxygenase GxtA. We have also directly demonstrated the
detoxification effect of further modifications to the STX (1)
scaffold by sulfation using competitive [3H]-STX displacement
assays in mouse whole brain. This work sheds light on the
remarkable biological processes regulating PST toxicity in
cyanobacteria. The characterized enzymes herein have the
potential to become powerful biocatalytic tools for producing
sulfated PSTs, empowering the study of VGSC biology and the
development of therapeutics for VGSC disorders.
present, except the mixture of C1 (12) and C2 (13), which
only reached ∼80% total displacement of [3H]-STX. The
curves for GTX2/3 (7, 8) and 11-α/β-hydroxySTX (16, 17)
clustered together, signifying similar VGSC affinity. However,
the Ki of GTX5 (10) was roughly 2-fold higher than these
toxins and the Ki measured for the mixture of C1 (12) and C2
(13) was over 10-fold higher, indicating a more significant role
in VGSC binding affinity for the negatively charged sulfate
appended to the nitrogen of the carbamate than the C11
position.
In light of recent structural studies demonstrating binding
interactions of STX (1) with the pore of the insect VGSC,
NavPaS, it is now known that the amine of the carbamate of
STX (1) forms a key hydrogen bond with the side chain of
Gln1062.55 Similar H-bond donating residues are not reported
within the vicinity of the C11 position. These observations
correlate with the results of a study by Du Bois and co-workers
assessing different functional groups at the carbamate position
of STX (1), where the addition of steric bulk did not reduce
the molecule’s VGSC potency unless the group was negatively
charged.56 Thus, the reduction in affinity observed with
sulfocarbamate-containing PSTs compared to C11-sulfated
PSTs in our study is corroborated by structural information.
These data suggest the importance of considering bulky,
■
METHODS
Protein Expression and Purification. GxtA and VanB were
expressed and purified as described previously.23 A pMCSG7
expression vector containing sxtSUL was transformed into the E.
coli BL21(DE3) containing the pGro7 plasmid for the coexpression of
GroEL and GroES chaperones, and a pMCSG7 vector containing
sxtN was transformed into BL21(DE3) only. All proteins were
purified using a GE Healthcare Ä KTA Pure FPLC with 5 mL of
HisTrap, 5 mL of MBPTrap, or HiPrep 16/60 Sephacryl S-200 high945
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Synthesis and Standard Preparation. Product standards of
ddSTX (18), α-STOH (20), β-STOH (19), STX (1), neoSTX (2),
11-α-hydroxySTX (16), 11-β-hydroxySTX (17), GTX2 (7), and
GTX3 (8) were prepared as previously described.23 GTX5 (10), C1
(12), and C2 (13) were authentic standards purchased from National
Research Council Canada.
■
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschembio.9b00123.
Supplemental figures and methods (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: arhardin@umich.edu.
ORCID
Nicholas Denomme: 0000-0002-7631-0385
Alison R. H. Narayan: 0000-0001-8290-0077
Notes
Figure 5. Concentration response curves showing specific binding of
PSTs to mouse whole-brain membrane samples in competition with 5
nM [3H]-saxitoxin. Ki values listed are determined from concentration
response curves.
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This research was supported by funds from the University of
Michigan Life Sciences Institute and the National Institute of
General Medical Science R35GM124880 (to A.R.H.N.) and
the National Institute of Neurological Disorders and Stroke
R37NS076752 (to L.L.I.). We thank D. Ellinwood for
assistance with synthesis and J. Iñiguez-Lluhi ́ for helpful
discussions.
resolution columns (GE Healthcare) using conditions described in
detail in the Supporting Information Appendix. Purified proteins were
frozen in liquid nitrogen and stored at −80 °C. Proteins were
discarded after one freeze−thaw cycle.
Enzymatic Reactions and Analysis. All reactions were
performed on analytical scale in 50 μL volumes prepared in 1.5 mL
plastic centrifuge tubes. For reactions containing individual enzymes,
5 μM SxtSUL or SxtN was combined with 200 μM of substrate (STX
(1) for SxtN and a mixture of 11-α-hydroxySTX (16) and 11-βhydroxySTX (17) for SxtSUL), 100 μM PAPS, and 50 mM HEPES
pH 7.0 buffer. Reactions were incubated at RT (22 °C) for 2 h
without agitation. Cascade reactions containing multiple enzymes
were performed by combining 20 μM SxtSUL, 20 μM SxtN, 5 μM
GxtA, 5 μM VanB, 200 μM STX (1), 200 μM PAPS, 500 μM NADH,
100 μM Fe(NH4)2(SO4)2, and 50 mM TrisHCl pH 7.0 buffer.
Reactions were incubated at RT overnight (∼10 h) without agitation.
All reactions were quenched by the addition of 3× volume HPLCgrade acetonitrile (150 μL) and centrifuged at 12,000×g for 20 min.
For LC-MS analysis, 100 μL of the supernatant was diluted with a
mixture of 50% v/v acetonitrile and 50% v/v sterile-filtered ddH2O
containing 1% v/v formic acid and 6 μg mL−1 15N-arginine as an
internal standard. All separations were performed using an Acquity
UPLC BEH Amide 1.7 μm, 2.1 × 100 mm hydrophobic interaction
liquid chromatography (HILIC) column from Waters with an
isocratic mobile phase of 18% water with 0.1% formic acid and
82% of 95% acetonitrile, 5% water and 0.1% formic acid (v/v) at 0.4
mL min−1.
Mouse Whole Brain Binding Assays. Whole brain membranes
were prepared from adult (postnatal day (P) 30−150) wildtype
C57Bl/6J mice as described previously.39 Equilibrium [3H]-STX
binding in the presence or absence of PST was measured at 4 °C for
at least 1 h using a vacuum filtration assay with a saturating
concentration (5 nM) C11 labeled [3H]-STX (20 Ci mmol−1,
American Radiolabeled Chemicals Inc.). A stock of each unlabeled
toxin was diluted in binding buffer to give a final concentration of
10 000, 1000, 100, 10, 1, 0.1, and 0.01 nM. A 7-point concentration−
response curve was generated for each unlabeled PST in competition
with 5 nM [3H]-STX. Two independent experiments were conducted
for each unlabeled PST, and in each experiment each sample was
tested in duplicate. The average DPM reading of duplicate values was
used for analysis.
■
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