Isolation and identification of diadenosine 5′,5?-P 1,P4-tetraphosphate binding proteins using magnetic bio-panning

Article abstract of DOI:10.1016/j.bmcl.2011.09.070

We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap₄A)-'molecular hook' attached to magnetic beads enabling the isolation of Ap₄A-binding proteins from bacterial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is performed sequentially by mass spectrometry. The observation of positive controls suggests that these newly observed proteins are putative Ap₄A-binding partners, and we have expectations that others can be found with further technical improvements in our methods.

Full text of DOI:10.1016/j.bmcl.2011.09.070

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7175–7179
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Isolation and identification of diadenosine 50,5000-P1,P4-tetraphosphate
binding proteins using magnetic bio-panning
Wei Guo a,b, M. Ameruddin Azhar b,c, Yuhong Xu a, Michael Wright b,d, Ahmed Kamal c,
Andrew D. Miller b,d,
⇑
a Pharmacy School of Shanghai Jiao Tong University, Shanghai, China
b Imperial College Genetic Therapies Centre, Imperial College London, UK
c Organic-I Division, Indian Institute of Chemical Technology, Habsigida, Hyderabad, India
d Institute of Pharmaceutical Sciences, Franklin-Wilkins Building, King’s College London, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap4A)-
‘molecular hook’ attached to magnetic beads enabling the isolation of Ap4A-binding proteins from bacte-
rial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is
performed sequentially by mass spectrometry. The observation of positive controls suggests that these
newly observed proteins are putative Ap4A-binding partners, and we have expectations that others can
be found with further technical improvements in our methods.
Received 3 August 2011
Revised 16 September 2011
Accepted 19 September 2011
Available online 29 September 2011
Keywords:
Dinucleoside polyphosphate
Ap4A
Ó 2011 Elsevier Ltd. All rights reserved.
Bio-panning
Magnetic bead
Proteomics
Dinucleoside polyphosphates (NpnNs) are a family of ubiqui-
tous, naturally occurring molecules consisting of two nucleosides
bridged by 2–7 phosphate groups. Members of this family can be
extracted from a wide range of prokaryotic or eukaryotic cells or
tissues and have been suggested to possess a diverse range of
intracellular and extracellular biological roles.1,2 These include
activities in bacterial cell division,3 in the invasion of mammalian
cells,4 the regulation of apoptosis,5 plus roles in the cardio-vascular
system,6,7 neurotransmission,8,9 analgesia,10 and tissue protec-
tion.11 Most especially NpnNs may have a key role in cell stress
signalling and in management of the heat shock response.12 Final-
ly, two dinucleoside polyphosphates, (Diquafosol and Denufosol)
have even undergone clinical trials as treatments for dry eye and
cystic fibrosis.13
NpnN activities are potentially quite significant, there is now an
imperative to develop up-to-date methodologies to enhance our
understanding of the specific roles of Ap4A in biology in the first in-
stance with an eye thereafter to more general roles of the NpnN
family. As a first step towards this, we have developed new isola-
tion methods that were intended to enable the subsequent identi-
fication of novel, putative Ap4A binding proteins in diverse cellular
lysates by means of mass spectrometry.
Our approach to isolation was based on the use of superpara-
magnetic magnetic particles. Such particles have been used in a
wide range of applications, including molecular imaging16 and
magnetic thermotherapy.17 Larger ‘beads’ have also been used as
a solid matrix for the separation of proteins, peptides, DNA/RNA
and small-molecule drugs18 and even for targeted delivery.19–21
Earlier in the 1990s, streptavidin-coated magnetic iron oxide beads
were used to enrich intracellular binding partners of proteins or
peptides for characterisation from whole-cell lysates.22,23 Here
we report on how this concept can be adapted further for the mag-
netic bio-panning, isolation and characterisation of new Ap4A
binding proteins. Central to our approach was the preparation of
a synthetic, biotin-conjugated Ap4A-‘molecular fish-hook’ with
the capacity to bind to bona fide Ap4A binding proteins on the
one hand and simultaneously to streptavidin-coated superpara-
magnetic particles on the other hand. It is our expectation that spe-
cific protein interactions with Ap4A are most likely by recognition
Diadenosine tetraphosphate (Ap4A) is one of the most studied
members of the NpnN family. Even so, the in vivo biology of
Ap4A remains poorly understood, and only a very limited numbers
of protein binding-partners are known.13–15 Given that known
Abbreviations:
Ap4A,
diadenosine
tetraphosphate;
ATP,
adenosine
50-triphosphate; ESI-MS, electrospray ionisation mass spectroscopy; IMP, inosine
50-monophosphate; IMPDH, IMP dehydrogenase; NAD, nicotinamide adenine
dinucleotide; NpnN, dinucleoside polyphosphate.
⇑
Corresponding author. Tel.: +44 (0) 7879 635 513.
E-mail address: andrew.d.miller@kcl.ac.uk (A.D. Miller).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.070

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W. Guo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7175–7179
O
P
NH2
O
O
N
O
O
O
O
O
NH2
N
O
N
O
O
O
P
P
P
P
P
O
O
O
O
N
O
O
O
O
N
O
N
O
O
xs NaIO4 (aq)
rt, 15 min
N
N
O
1.
2.
HO
OH
O
O
O
O
O
P
NH2
N
O
N
O
O
P
O
O
P
O
N
O
O
S
O
S
N
3.
NH2
HN
O
N
4.
HN
NH
H
HN
H
NH
H
HN
+
H
O
water/DMSO
rt, 1.5 h
H
N
H
N
O
O
OH
HO
i. NaBH4 (aq), rt, 15 min
ii. ATP, LysU mixture, 37oC, 2 h
O
P
O
NH2
N
O
O
O
O
O
N
O
N
P
P
P
O
N
N
O
O
O
O
O
N
N
O
O
S
N
N
NH2
5.
O
HN
NH
H
HN
H
O
H
N
O
Scheme 1. Tandom chemical/enzymatic synthesis of biotin-LC-Ap4A.
of the adenines or the polyphosphate chain. Our conjugate (5) was
designed via modification of the ribose to reduce the risk of bind-
ing interference. Given the attachment to a relatively large mag-
netic bead, we also chose a long linker to reduce steric hindrance.
We have previously reported on the use of tandem chemical/
enzymatic synthetic procedures for the production of fluorescent
Ap4A analogues for use in affinity binding studies.24 In particular,
the ability of LysU – a heat inducible lysyl-tRNA synthetase from
Escherichia coli (E. coli) – to produce a variety of Ap4A-like ana-
logues from mononucleotides has proved to be very valuable.
Forming phosphate–phosphate bonds via chemical synthetic ap-
proaches is often difficult or hard to control (with resulting purifi-
cation problems), although there have been some interesting
recent developments.25 LysU-catalysed biosynthesis is more lim-
ited in term of acceptable substrates but is fast and highly specific
– often giving 90–95% yields with optimised conditions.
A number of alternate synthetic routes were investigated but
the simplest was a one-pot biotinylation involving pre-oxidation
of adenosine triphosphate (ATP) (1) with sodium periodate to yield
dial (2) that was conjugated in situ to biotin-LC-hydrazide (3)
(Sigma–Aldrich) (see Scheme 1).26 The resulting biotin-LC-ATP
(4) was then coupled enzymatically to a second ATP, giving bio-
tin-LC-Ap4A (5) in a reaction that could be easily monitored by
ion-exchange HPLC.24 The overall yield was 30% – that was subop-
timal due to only partial biotinylation in step II. However, the
advantage of this one-pot process was found to be the ease of puri-
fication (any excess Ap4A produced is degraded to Ap3A during the
long LysU incubation,27 allowing product (5) to elute from purifica-
tion column in especially pure form.
Magnetic bio-banning of the lysate with Ap4A-labelled and control
beads was then carried out according to Figure 1.30 All washes,
eluted fractions and the beads themselves were analysed by means
of SDS–PAGE electrophoresis.
Interesting results from this initial phase were then repeated
and after Coomassie Brilliant Blue staining of the gels, appropriate
bands were excised, trypsinised, then subject to analysis by mass
spectrometry.31 Proteins identified from the lysate (using the Esch-
erichia_coli_08 protein bank on the NCBL website) were found to
include the important positive control GroEL protein, a previously
identified and well known Ap4A-binding protein.12–14 The most
dominant putative new Ap4A binding protein identified was ino-
sine 50-monophosphate (IMP) dehydrogenase (IMPDH) but others
were also identified (see Supplementary data, Table 1).
LysU (another positive control) was not observed in our screen
but this is not surprising since the bacterial cultures were grown
at 37 °C, normal growth temperature, and the gene for LysU is only
expressed under heat shock conditions. Otherwise, we were also
unable to observe several other known E. coli Ap4A-binding proteins.
However, these are either membrane bound proteins (not investi-
gated here) or else active Ap4A-hydrolases which would be able to
consume the biotin-LC-Ap4A ‘molecular fish-hook’ enzymatically
during the magnetic bio-panning process after binding. Accordingly,
studies are now underway to develop Ap4A-‘molecular fish-hooks’
that are resistant to hydrolysis but retain binding efficacy.
Our magnetic molecular bio-panning approach was also ex-
tended to the investigation of murine brain lysates32 using the
same procedures as above (see Fig. 2). In this case, several hsp70
family proteins were identified (Hspa8, Hspa5 and Hspa1b). While
these have not been previously reported to bind Ap4A, the prokary-
otic hsp70 family equivalent ‘DnaK’ has.14 This suggests that these
hsp70 proteins may also be considered an encouraging positive
control. Otherwise, as with the E. coli lysate, several other novel
and interesting putative Ap4A-binding proteins were identified
(see Supplementary data, Table 2).
Attachment of the biotin-LC-Ap4A ‘molecular fish-hook’ (5) to
streptavidin-linked magnetic beads (M-270 Dynabeads™, Invitro-
gen) was carried out with stirring at room temperature, followed
by a series of wash steps.28 Gratifyingly, positive control LysU (in
50 mM Tris–HCl, 100 mM NaCl, 10 mM MgCl2 pH 8.0) was found
to bind to the resulting Ap4A-labelled beads but not to unlabelled
(control) beads. Following this confirmation, initial studies were
then carried out with an E. coli lysate prepared as described.29
Although our studies are still at an early stage, we have still suc-
ceeded in identifying several new, potentially interesting putative

W. Guo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7175–7179
7177
wash
mix
A
B
separate
mix
incubate
wash
separate
add
Ap A
4
separate
mix
incubate
Streptavidin beads
Magnet
Biotin-C10-Ap A
Cell/tissue lysate
4
Figure 1. Flow-diagram of target screening of Ap4A from cell or tissue lysate using a magnetic bio-panning technique. Biotin-conjugated Ap4A-‘molecular fish-hooks’ (biotin-
LC-Ap4A, 5) (shown in red) are mixed with streptavidin-associated superparamagnetic particles (grey spheres): unbound material is removed by wash and separation (A); (B)
lysate is added for the binding of putative Ap4A-binding proteins (green spheres); unbound material is removed by wash and separation: Ap4A-binding proteins (green
spheres) are steadily eluted and separated from the particles after Ap4A and other wash steps (see Fig. 2).
Ap4A-binding partners. From the E. coli list, IMPDH (also known as
guanosine 50-monophosphate oxidoreductase) is responsible for
catalysing the rate-limiting step of de novo guanosine 50-triphos-
phate biosynthesis and the nicotinamide adenine dinucleotide
(NAD)-dependent reduction of IMP into xanthosine 50-monophos-
phate.33 Indeed, several adenine dinucleotides and dinucleotide-
like compounds, for example, NAD, thiazole-4-carboxamide- and
selenazole-4-carboxamide-adenine dinucleotides,34 and myco-
phenolic adenine dinucleotide,35 are reported to act as potent
inhibitors of IMPDH function. Given the structural similarities,
Ap4A could certainly bind into the IMP/NAD pocket of IMPDH
and exhibit putative biological effects. These putative effects will
need to be investigated by means of biological and biophysical
studies.12
kD
- 200
- 116
- 97.2
- 66.4
- 44.3
The chaperone/peptidyl-prolyl cis-trans isomerase SurA is
known to promote protein folding by increasing the rate of prolyl
residue cis-trans isomerisation. Once again, the reason why this
protein might be an Ap4A binding protein will require some
detailed study. Given the apparent modulatory behaviour of Ap4A
seen in most recent studies,9,12 there may be a very significant role
for Ap4A-binding to SurA protein given that inactivation of SurA
typically results in a reduction of E. coli growth rate, viability and
increased susceptibility to certain antibiotics.36 On another tack,
our discovery that a LysR family transcriptional regulator is a
1A
1C
2A
2C
3A
3C
4A
4C
Figure 2. Silver stained SDS-PAGE of putative protein-binding partners to biotin-
LC-Ap4A from murine brain lysate. Columns labelled with ‘A’ are from Ap4A-bearing
beads, ‘C’ are negative controls. Samples are from: 1, third buffer wash; 2, non-
specific NaCl elution; 3, specific Ap4A elution; 4, remaining beads.

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W. Guo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7175–7179
7. Bobbert, P.; Schluter, H.; Schultheiss, H. P.; Reusch, H. P. Biochem. Pharmacol.
2008, 75, 1966.
8. Gomez-Villafuertes, R.; Pintor, J.; Gualix, J.; Miras-Portugal, M. T. J. Pharmacol.
Exp. Ther. 2004, 308, 1148.
putative Ap4A-binding protein is also very interesting given that
the LysRS isozyme – LysU – appears to be the primary source of
Ap4A biosynthesis in E. coli.
9. Melnik, S.; Wright, M.; Tanner, J. A.; Tsintsadze, T.; Tsintsadze, V.; Miller, A. D.;
Lozovaya, N. J. Pharmacol. Exp. Ther. 2006, 318, 579.
10. Giraldez, L.; Diaz-Hernandez, M.; Gomez-Villafuertes, R.; Pintor, J.; Castro, E.;
Miras-Portugal, M. T. J. Neurosci. Res. 2001, 64, 174.
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I.; Chen, S.; Thiemermann, C. J. Neurosci. 2003, 23, 7958.
12. Tanner, J. A.; Wright, M.; Christie, E. M.; Preuss, M. K.; Miller, A. D. Biochemistry
2006, 45, 3095.
Turning to the murine brain, carbamoyl-phosphate synthase 1
(Cps1) is identified here as a putative Ap4A-binding protein. This
is also intriguing. Cps1 is involved in the urea cycle, where the en-
zyme plays an important role in removing excess ammonia from
the cell. The known ligands of Cps1 include ATP and other nucleo-
tides.37 Indeed Cps1 requires ATP to transform excess ammonia
into carbamoyl phosphate, and Ap5A that has been found previ-
ously to act as a CpsI inhibitor.38 Could Ap4A have similar proper-
ties and act as a modulator of urea cycle to prevent the over
accumulation of extracellular ammonia that might otherwise
cause neurological damage problems? Once more detailed biolog-
ical and biophysical studies are now required. Finally, the identifi-
cation of three Hsp70 family proteins (namely Hspa8, Hspa5 and
Hspa1b) as putative Ap4A binding proteins also appears very sig-
nificant. Previous studies have concluded that Ap4A-binding to
molecular chaperone proteins is associated with the modulation
of cellular stress responses and recovery from stress injury.13,39
Therefore, Ap4A binding with these Hsp70 family proteins could in-
deed conceivably follow the same pattern.
13. Gendaszewska-Darmach, E.; Kucharska, M. Purinergic Signal. 2011, 7, 193.
14. Johnstone, D. B.; Farr, S. B. EMBO J. 1991, 10, 3897.
15. Fuge, E. K.; Farr, S. B. J. Bacteriol. 1993, 175, 2321.
16. Bjornerud, A.; Johansson, L. NMR Biomed. 2004, 17, 465.
17. Hilger, I.; Hergt, R.; Kaiser, W. A. IEE Proc. Nanobiotechnol. 2005, 152, 33.
18. Salaklang, J.; Steitz, B.; Finka, A.; O’Neil, C. P.; Moniatte, M.; van der Vlies, A. J.;
Giorgio, T. D.; Hofmann, H.; Hubbell, J. A.; Petri-Fink, A. Angew. Chem., Int. Ed.
2008, 47, 7857.
19. Li, W.; Ma, N.; Ong, L. L.; Kaminski, A.; Skrabal, C.; Ugurlucan, M.; Lorenz, P.;
Gatzen, H. H.; Lutzow, K.; Lendlein, A.; Putzer, B. M.; Li, R. K.; Steinhoff, G. J.
Gene Med. 2008, 10, 897.
20. Neuberger, T.; Schöpf, B.; Hofmann, H.; Hofmann, M.; von Rechenberg, B. J.
Mag. Mag. Mat. 2005, 293, 483.
21. Arruebo, M.; Fernández-Pacheco, R.; Ibarra, M. R.; Santamaría, J. Nano Today
2007, 2, 22.
22. Kausch, A. P.; Owen, T. P., Jr.; Narayanswami, S.; Bruce, B. D. Biotechniques 1999,
26, 336.
23. Luers, G. H.; Hartig, R.; Mohr, H.; Hausmann, M.; Fahimi, H. D.; Cremer, C.;
Volkl, A. Electrophoresis 1998, 19, 1205.
In conclusion, we report that a first proof of concept study has
been concluded for the identification of putative Ap4A binding pro-
teins from two different biological sources by a magnetic bio-pan-
ning procedure. Given the structural similarities between Ap4A and
adenine mononucleotides (e.g. ADP/ATP) there will always be con-
cern as to the specificity of the identified partners. The use of a high
[Ap4A] elution wash should increase the probability of isolating
specific binders however this study must be followed up with
other biophysical, NMR or probe-based studies before they could
be definitively declared as such.40 On balance, however, the meth-
od appears to work. We predict that other interesting binding pro-
tein partners will be identified as our methodology is refined and
as the search is extended to include other NpnN family members
and other lysate sources. The results will then guide future inves-
tigations into the role of Ap4A and other NpnN family members in
biology and medicine.
24. Wright, M.; Tanner, J. A.; Miller, A. D. Anal. Biochem. 2003, 316, 135.
25. Mohamady, S.; Taylor, S. D. J. Org. Chem. 2011, 76, 6344.
26. Carried out as follows: (i) 5 mM aqueous ATP ((1) retention time 3.93 min) was
stirred with excess sodium periodate to give ribose-oxidised dialdehyde-ATP
((2) 4.06 min); (ii) The pH of the mixture was adjusted to 4.5 and a concentrated
solution of biotin-LC-hydrazide (3) in DMSO was slowly added over 30 min, with
vigorous stirring. HPLC analysis after a further hour showed the presence of
biotin-LC-ATP (4) at 3.69 min; (iii) the periodate was quenched with sodium
borohydride (3.87 min) and the Tris-HCl added to 50 mM, pH 8.0; (iv) finally,
L-
lysine (to 2 mM), MgCl2 (10 mM), ZnCl2 (160 M), LysU (10 M), inorganic
l
l
pyrophosphatase (Sigma–Aldrich; 6 U/mL) was added in sequence and the
mixture warmed to 37 °C. Aqueous ATP (to 6 mM) was then added in four
portions over the period of 1 h and resulting mixture left for a further hour to give
biotin-LC-Ap4A (6) as the majority product (4.24 min); (v) purification was easily
carried out by SOURCE-15Q/TEAB ion exchange HPLC as previously described24
and combined product fractions lyophilised to a highly hydroscopic, white
crystalline solid, before storage at ꢀ20 °C. Major side products from this reaction
included Ap3A, ADP and 4. Compound 5 was identified by ESI-MS: [MꢀH]ꢀ
1185.7 m/z, expected (C36H52N15O21P4Sꢀ) 1186.2 m/z and 31P NMR (202 MHz,
D2O, 21500x) d ꢀ11.63 (2P, m, J
22.2, Pa,d), ꢀ23.16 (1P, d, J 18.2, Pc),
a
b/d
c
cd
ꢀ23.28 (1P, d, Jb 24.2, Pb), purity confirmed as >95% by HPLC.
a
Acknowledgments
27. Wright, M.; Boonyalai, N.; Tanner, J. A.; Hindley, A. D.; Miller, A. D. FEBS J. 2006,
273, 3534.
28. Beads and 5 were stirred in coupling buffer (5 mM Tris–HCl, pH 7.5, 0.5 mM
EDTA, 1 M NaCl) at rt for 30 min, before settling of the beads with the aid of a
neodymium ceramic magnet, removal of the supernatant and washing (3ꢁ)
with coupling buffer. Beads were then resuspension in phosphate buffer
(20 mM sodium phosphate, pH 8.0) before being stored at 4 °C (<3 days).
29. E. coli BL21 (DE3), pLysS cells were grown at 37 °C in LB medium supplemented
This study was partially funded by the Natural Science Founda-
tion of China (No. 30825045). W.G. wishes to thank the Chinese
Scholarship Council and A.A. the British Council for partial Ph.D.
studentship funding. We also thank Key Lab of Proteomics,
Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai, China
to provide mass spectrometry analysis.
The mice were housed in the specific pathogen-free Animal
Centre of Shanghai Jiao Tong University and all the experimental
operations were done according to the ethic review from The Ani-
mal Care & Welfare Committee of that institute.
with carbenicillin (50 lg/ml) until OD600 = 1.5, then harvested by
centrifugation. Cell pellets were lysed by probe sonication in lysis buffer
(50 mM Tris–HCl, 2 mM BME, 0.1% Triton X-100, 0.6 mM benzamidine, 3 mg
DNAase, 20 mM MgCl2, protease inhibitor tablet; pH 8.0). After addition of
streptomycin sulphate, the lysate was centrifuged at 12,000g to remove cell
debris and precipitated nucleotides. It was then fractionated with 20% and 60%
ammonium sulfate, with the precipitated protein fractions being redissolved in
HEPES buffer (50 mM, 100 mM NaCl, protease inhibitor tablet, pH 8.0) before
storage with 30% glycerol, in small portions at ꢀ20 °C.
30. As follows: (i) 30
(50 mM Tris–HCl, 100 mM NaCl, 10 mM MgCl2, 10 mM KCl, pH 8.0); (ii)
incubated at 4 °C for 40 min with 30 L of each lysate fraction in 200 L Tris–
lL of beads were washed (3ꢁ) with 200 lL Tris–HCl buffer
Supplementary data
l
l
HCl buffer; (iii) beads washed (3ꢁ) with Tris–HCl; (iv) initial elution for 10 min
(4 °C) in Tris–HCl with NaCl concentration increased to 1 M; (v) competition
elution with 4 mM Ap4A in Tris–HCl buffer.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.070.
31. LTQ, Thermo Finnigan, positive charge testing mode, micro spray injecting way,
170 °C capillary temperature, 0.15 mm and 15 cm column, 470-1800 DAL
scanning scope.
References and notes
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normal saline. The brains were rapidly removed and put in ice-cold RIPA lysis
buffer (25 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium
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