Development of an Affinity-Based Proteomic Strategy for the Elucidation of Proanthocyanidin Biosynthesis

Full text of DOI:10.1002/cbic.201100044

DOI: 10.1002/cbic.201100044
Development of an Affinity-Based Proteomic Strategy for the Elucidation of
Proanthocyanidin Biosynthesis
Cꢀline Chalumeau,[a] Denis Deffieux,*[a] Stꢀphane Chaignepain,[b] and Stꢀphane Quideau*[a]
Proanthocyanidins (PAs), also known as condensed tannins, are
oligo/polymeric chains of flavan-3-ols.[1] These polyphenolic fla-
vonoid secondary metabolites are ubiquitously found in plants
for which they essentially assume protective functions against
microbial pathogens and herbivores.[2] Also found in grapes,
PAs are an important factor in the gustative quality of wines.
They are biosynthetically produced through the flavonoid
pathway, one of the best-studied biochemical pathways in
plants.[3,4] During the last decade, advances in genetic tech-
niques and recombinant expression technologies have allowed
isolation, from different sources, of most of the structural
genes encoding the enzymes involved in flavonoid biosynthe-
sis.[5] However, the last steps leading to the formation of PAs
remain unclear and are still under debate (Scheme 1).[6–8] Leu-
coanthocyanidins (i.e., (2R,3S,4S)-flavan-2,3-trans-3,4-cis-diols)
are presumed to be the common precursors of both PAs and
anthocyani(di)ns. Results from in vitro studies have shown that
leucoanthocyanidin dioxygenase (LDOX, also referred to as
ANS—anthocyanidin synthase), a 2-oxoglutarate-dependent
enzyme, is involved in the conversion of colorless leucoantho-
cyanidins into unstable colored anthocyanidins.[9–11] These fla-
vylium ions are then stabilized as anthocyanins through the
action of an anthocyanidin 3-glucosyltransferase (3-GT).[11,12]
Two additional enzymes catalyze the production of flavan-3-ols
from leucoanthocyanidins. A leucoanthocyanidin reductase
(LAR) directly converts leucoanthocyanidins into (2R,3S)-trans-
flavan-3-ols such as (+)-catechin (1),[13] whereas (2R,3R)-cis-
Scheme 1. Putative last steps of the biosynthesis of proanthocyanidins and
anthocyani(di)ns.
flavan-3-ols such as (À)-epicatechin (2) indirectly derive from
leucoanthocyanidins through the action of an anthocyanidin
reductase (ANR) on anthocyanidins (Scheme 1).[14] PAs could
then result from the oligomerization of flavan-3-ols such as 1
and 2, and/or their flavan-3,4-diol precursor(s), but the exact
nature of the starter and extension units, as well as whether or
not a dedicated enzyme (i.e., a “proanthocyanidin synthase”) is
involved in the coupling process, remains unknown.[6–8]
of this oligomerization process. The recent availability of the
grapevine genome sequence[15] has boosted efforts towards
the proteomic analysis of tissues from various parts of the
plant.[16] However, despite outstanding progress in electropho-
retic and chromatographic techniques coupled to mass spec-
trometry-based proteomics, the quantitative resolution and
identification of all proteins in a given proteome extracted
from a plant tissue entail serious drawbacks. Proteins extracted
from recalcitrant plant materials contain a high abundance of
interfering compounds such as secondary metabolites, and
thus efficient extraction is necessary in order to allow well-re-
solved two-dimensional polyacrylamide gel electrophoresis (2-
DE).[17] Moreover, the complexity of the resulting extracted pro-
teome (more than 700 proteins can be found just in grape
skin[18]) and the wide dynamic range of protein concentrations
often impede quantitative determination by 2-DE or a shotgun
strategy.[19,20] Therefore, proteomic analysis of grapevine tissues
have so far been essentially limited to the qualitative identifica-
tion of changes in protein expression during plant develop-
ment by performing comparative 2-DE analyses.[17,18]
In this context, the discovery of any novel enzyme involved
in PA biosynthesis by proteomic analysis would certainly
supply valuable information regarding the mechanistic nature
[a] C. Chalumeau, Dr. D. Deffieux, Prof. Dr. S. Quideau
Universitꢀ de Bordeaux, Institut des Sciences Molꢀculaires (UMR-CNRS 5255)
Institut Europꢀen de Chimie et Biologie (IECB)
2 rue Robert Escarpit, 33607 Pessac Cedex (France)
Fax: (+33)540-00-22-15
E-mail: d.deffieux@iecb.u-bordeaux.fr
s.quideau@iecb.u-bordeaux.fr
[b] Dr. S. Chaignepain
Universitꢀ de Bordeaux
Pꢁle Protꢀomique, Centre de Gꢀnomique Fonctionnelle
146 rue Lꢀo Saignat, 33076 Bordeaux (France)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100044.
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We thus surmised that analysis of the complex grapevine
proteome might benefit from a preliminary protein sorting by
affinity chromatography (AC). Such a strategy based on pre-
fractionation of proteins prior to mass spectrometry-based pro-
teomics has already been used with success on various pro-
teomes,[21–25] but not from grapevine. Thus, we describe herein
the development of an AC tool designed for investigating PA
biosynthesis by using immobilized flavanoid probes and in-
tended for the proteomic analysis of grapevine tissues.
ical grafting. The amino-PEGA resin, a copolymer of poly(ethyl-
ene glycol) and polyacrylamide with terminal amino
groups,[27,28] meets these criteria, and has previously been used
with success in biochemical assays.[29–31] We therefore selected
the commercially available PEGA1900 resin, which has a loading
capacity of 0.2 mmolgÀ1 and a molecular weight cut-off
around 70 kDa, hence affording good permeability towards
macromolecules and, in this case, towards the 40 kDa LDOX
protein. Next, the site of attachment of the flavanoid probes to
a spacer unit was chosen so as to minimize the impact of any
structural modification of the native flavanols upon the molec-
ular-recognition specificity of the enzyme. Since the primary
function of LDOX appears to be a stereospecific hydroxylation
at C-3 of the central pyran C ring,[32–35] we initially opted to in-
troduce a spacer unit on the distal catecholic B ring. We settled
on the use of a three-carbon spacer by taking advantage of
the 2-(methoxycarbonyl)ethylidene (Mocdene) group recently
developed by Vilarrasa and co-workers for the protection of
1,2-diols and catechols.[36,37] This cyclic acetal unit was chemo-
selectively installed onto (+)-1 by treating it with methyl pro-
The two flavanols (+)-catechin (1) and (À)-epicatechin (2),
both presumably engaged in the oligomerization process lead-
ing to PAs, were selected as potential affinity-based probes.
From the point of view of molecular recognition, these two
flavan-3-ol probes should potentially target any unknown
enzyme, as well as the three known enzymes (LDOX, ANR, and
LAR) of the last steps in flavonoid biosynthesis. To evaluate the
suitability of our AC tool, we selected LDOX as a model
enzyme. Recent in vitro experiments carried out by Matern
and co-workers[26] have shown that LDOX is capable of reacting
not only with (2R,3S,4S)-flavan-3,4-diols (leucoanthocyanidins),
but also with the flavan-3-ol (+)-1, which also harbors a
(2R,3S)-trans-configured C ring, to generate the flavan-3-one
C4-C4 dimer 3 (Scheme 2). However, neither its enantiomer
pynoate in the presence of
a slight excess of DMAP
(Scheme 3). Unfortunately, we failed to saponify the resulting
methyl ester 1a to its corresponding carboxylic acid 1c, re-
quired for amidation with the amino groups of the PEGA resin.
Treatment of 1a with LiOH (1 equiv) led to partial cleavage of
the Mocdene acetal. Thus, we instead used benzyl propynoate
to generate under Vilarrasa’s conditions the benzyl ester ana-
logues 1b and 2b from (+)-1 and (À)-2, respectively. Standard
hydrogenolysis of these benzyl esters furnished the carboxylic
acids 1c and 2c in good yields, ready for attachment to the
resin (Scheme 3).
However, preliminary assays on the activity of the LDOX
enzyme by using the B-ring-modified catechin derivatives 1a–c
in solution turned out negative. An explanation of these disap-
pointing observations could be found in Schofield’s co-crystal
structure of the LDOX enzyme from Arabidopsis thaliana with
dihydroquercetin (DHQ) as a substrate analogue,[30] in which
the two DHQ B-ring hydroxyl groups are engaged in hydrogen
bonding with the Tyr142 residue of the LDOX active site.
Therefore, the suppression of the two catechin B-ring hydroxyl
groups resulting from the installation of the spacer unit in 1a–
c might have been be detrimental to the recognition (and
transformation) of such derivatives by the LDOX enzyme.
The alternative, to install a spacer unit onto the flavanol
A ring, was thus implemented. Because Schofield’s structure
also indicates that the DHQ A-ring hydroxyl group at C-7 is hy-
drogen-bonded to the side-chain carboxylate group of the
LDOX Glu306 residue,[32] we set out to preserve this phenolic
hydroxyl group and to exploit the prominent nucleophilic
character of the A-ring C-8 center to forge a functionalized C–
C-linked tether at that position. This was achieved by a regio-
selective Vilsmeier–Haack formylation of perbenzylated (+)-1
and (À)-2 (Scheme 3).[38] The aldehydes 1d and 2d were both
obtained in about 30% over three steps and were then sub-
mitted to Horner–Wadsworth–Emmons (HWE) reaction condi-
tions[39] with triethyl or benzyl diethyl phosphonoacetate. The
resulting four enoates were finally submitted to hydrogenolysis
Scheme 2. LDOX assays with (+)-catechin (1) and (À)-epicatechin (2).
(À)-1 nor its epimer (À)-2 is processed by the enzyme.[26]
Therefore, LDOX constitutes an appropriate model enzyme for
this proof-of-concept study, since it should be competent only
for binding with solid-supported (+)-1, but not with (À)-2.
We used an N-terminal His6-tagged recombinant LDOX from
Vitis vinifera cabernet sauvignon, which was partially purified
by using Ni-chelating affinity chromatography. The activity of
this tagged enzyme was checked by using (+)-1 and (À)-2 as
substrates in the presence of the requisite co-factors. LC-MS
analyses of these reaction mixtures confirmed the aforemen-
tioned observations made by Matern and co-workers who
used a recombinant LDOX from Gerbera hybrida[26] (Scheme 2
and see the Supporting Information for details).
With this active LDOX enzyme to hand, we then focused our
attention on finding an adequate solid support on which to
attach our two flavonoid probes by using an appropriate
spacer. An ideal resin has good permeability towards the LDOX
protein and is compatible with both the aqueous media used
for biochemical assays and the organic solvents used for chem-
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echin (1) and 2 grafted onto the
same resin but through their cat-
echolic B rings could constitute
negative controls that would
allow us to discriminate nonspe-
cific interactions with LDOX
(vide infra). Hence, the B-ring-
tethered carboxylic acid deriva-
tives 1c and 2c (see Scheme 3)
were grafted onto PEGA beads
using 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide
(EDCI)
and 1-hydroxy-1H-benzotriazole
(HOBt) as condensation reagents
in DMF at room temperature
(Scheme 5). The effectiveness of
the reactions was confirmed by
a
2,4,6-trinitrobenzenesulfonic
acid (TNBS) test that indicated
no appreciable amount of re-
maining free amino groups in
the case of (epi)catechin-grafted
resins 1i and 2i. However, in the
case of the grafting of the A-
ring-tethered carboxylic acid de-
rivatives 1h and 2h under the
same conditions, the coupling
reactions turned out to be quasi-
ineffective, mainly leading to an
Scheme 3. Reagents and conditions: a) DMAP, CH3CN/MeOH (R=Me) or CH3CN/tBuOH (R=Bn), 3 h, RT; b) H2, Pd/
C, EtOH, 3 h, RT; c) BnBr, K2CO3, DMF, 48 h, RT; d) BnBr, NaH, 6 h, DMF, 08C; e) POCl3, DMF, 4 h, 758C, then 15 h at
RT; f) NaH, DMF, 15 h (R=Et) or 24 h (R=Bn), RT; g) H2, Pd/C, THF/MeOH/CH2Cl2, 24 h, RT.
to mediate the cleavage of their benzyl protecting groups, as
well as the hydrogenation of their olefinic bond; this furnished
the two ethyl propanoate-tethered flavanols 1g and 2g, and
their carboxylic acids 1h and 2h in good yields (Scheme 3).
With this new set of three-carbon-tethered flavanols to
hand, we first verified that they were competent substrates for
the LDOX enzyme. The ethyl propanoate-bearing (+)-catechin
derivative 1g was the only compound transformed by the
enzyme. A new compound exhibiting a pseudomolecular ion
peak [M+H]+ at m/z 775 was detected by ESI-MS analysis of
the enzymatic reaction mixture. By analogy with the known
C4–C4 catechin dimer 3 (see Scheme 2), this compound is ten-
tatively assigned to the C4–C4 bis-ketonic dimer 4 of 1g
(Scheme 4, see the Supporting Information for details). The
fact that the (+)-catechin derivative 1h remained intact upon
exposure to LDOX could be due to its highly polar carboxylic
acid function, which might interfere with the enzyme recogni-
tion process. The absence of enzymatic activity on the corre-
sponding (À)-epicatechin derivatives 2g and a fortiori 2h was
of course expected, since (À)-epicatechin itself is not a compe-
tent substrate for LDOX (vide supra).[26]
Scheme 4. LDOX assays with tethered (+)-catechin 1g, 1h and (À)-epicate-
chin 2g and 2h.
intramolecular lactonization into the flavonoid derivatives 1j
and 2j in 69 and 51% yield, respectively. Microwave heating of
the reaction mixture led to a better level of grafting, but was
still incomplete, as evidenced by the TNBS test. In order to cir-
cumvent this problem, the lactones 1j and 2j isolated from
the initial EDCI/HOBt-mediated reactions were then heated in
DMF at 508C for 10 h under microwave assistance. Under
these conditions, ring-opening of the two lactones occurred ef-
ficiently upon nucleophilic attack by the PEGA amino groups
and afforded the desired (epi)catechin-grafted resins 1k and
2k (Scheme 5). The four grafted resins 1i/k and 2i/k were fur-
The main conclusion drawn from these preliminary LDOX ac-
tivity assays was that grafting (+)-catechin (1) to the PEGA
resin terminal amino groups through a three-carbon acyl
spacer installed at the catechin A-ring C-8 locus appeared to
be a viable approach for our intended AC design. (À)-Epicate-
chin (2) similarly grafted onto the resin, as well as both (+)-cat-
1
ther analyzed by H high resolution magic angle spinning (HR-
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the LDOX protein and these resin materials (lanes 6 and 7). In
gratifying contrast, LDOX was successfully bound to the resin
grafted with (+)-catechin through its A ring (i.e., 1k), as shown
by the observation of a protein band at a molecular weight
close to 40 kDa (lane 9).
This result confirmed that the (+)-catechin-derived resin-
supported flavanoid probe 1k could be used to capture LDOX
in a competitive manner. The unexpected but weaker electro-
phoretic band observed when using the resin grafted with (À)-
epicatechin through its A ring (i.e., 2k, see lane 8) can be at-
tributed to a specific but lower binding affinity of the epicate-
chin unit with LDOX. In fact, one can conceivably argue that
the LDOX enzyme recognizes both catechin epimers 1 and 2,
but that the stereochemistry at the C-ring C-3 locus is the de-
termining factor in enabling the enzymatic reaction to pro-
ceed. To complement this proof-of-concept approach to the
proteomic analysis of flavonoid enzymes, the two SDS-PAGE
protein bands at ~40 kDa (lanes 8 and 9) were in-gel digested.
The resulting peptide fragments were then extracted from the
gel and analyzed by LC-MS/MS. Peptide sequences were then
identified through a proteomic database search from which
we could verify a positive identification of the LDOX enzyme
from Vitis labrusca x Vitis vinifera with a score of 28% (Support-
ing Information).
Scheme 5. Grafting of PEGA resin with flavanols through their A or B rings.
Reagents and conditions: a) EDCI, HOBt, DMF, 5 h (20 h for 1h and 2h), RT;
b) microwave 50 W, DMF, 10 h, 508C.
MAS) NMR spectroscopy; this confirmed the attachment of the
flavanol units onto the PEGA resin (Supporting Information).[40]
Finally, we performed an SDS-PAGE analysis in order to eval-
uate the capacity of these grafted resins to act as affinity matri-
ces for our recombinant LDOX enzyme (Figure 1). This partially
In summary, we have designed an affinity chromatography
tool based on specific flavanol–protein interactions for the de-
velopment of a proteomic approach to the identification of un-
known (and known) proteins involved in the last steps of the
proanthocyanidin biosynthesis. Here, two of their putative
monomeric precursors, (+)-catechin and (À)-epicatechin, were
covalently linked through their A rings to a PEGA resin. These
grafted resins constitute suitable materials for batch affinity
purifications of flavonoid enzymes. A proof of concept for this
approach was established by using a recombinant LDOX pro-
tein from Vitis vinifera as an enzyme model system. This tool
should help proteomic efforts aimed at elucidating the last
steps of flavonoid biosynthesis, while facilitating the extraction
and purification of specific proteins from plant tissues. We
shall next exploit this coupled affinity chromatography–proteo-
mic analysis methodology to investigate protein materials
from grape tissues at different growth and maturity stages
with the aim of unveiling the role of enzyme(s) in proantho-
cyanidin biosynthesis.
Figure 1. Affinity-based SDS-PAGE analysis of recombinant LDOX by using
(epi)catechin-grafted resins 1i, 1k and 2k. Lane M: protein markers; lane 1:
partially purified LDOX; lane 2: first column wash from PEGA resin; lane 3:
first column wash from resin 1i; lane 4: first column wash from resin 2k;
lane 5: first column wash from resin 1k; lane 6: fraction after elution with
free PEGA resin; lane 7: fraction after elution with resin 1i; lane 8: fraction
after elution with resin 2k; lane 9: fraction after elution with resin 1k.
Acknowledgements
purified LDOX enzyme was thus added to suspensions of the
resin-supported flavanol 1i, 1k or 2k, or the ungrafted PEGA
resin control. After 4 h of incubation with the four resins,[41]
each resin was washed twice with a phosphate buffer solution
(0.2m, pH 6.0), in order to remove unbound LDOX (Figure 1,
lanes 2 to 5). After the second wash, no significant amount of
LDOX was detected in the column washings by SDS-PAGE anal-
ysis (not shown). Next, after elution with a denaturing Laemmli
buffer solution (lanes 6 to 9), no protein was detected in frac-
tions eluted from the resin grafted with (+)-catechin through
its B ring (i.e., 1i) or from the ungrafted resin. These negative
controls confirmed the absence of any interaction between
We thank the Conseil Interprofessionnel du Vin de Bordeaux
(CIVB) for their generous financial support, including Cꢀline Cha-
lumeau’s research assistantship. We also thank Dr. Bernard Gal-
lois (UMR-CNRS 5248) for the vector containing the gene for the
Vitis vinifera LDOX, Dr. Claudine Trossat (INRA-UMR A 1287), Dr.
Sꢀbastien Fribourg (IECB, INSERM U869) and Sabrina Rousseau
(IECB, UMS CNRS-Universitꢀ de Bordeaux 3033) for their help and
advice in LDOX expression and purification, and Axelle Grelard
(UMR-CNRS 5248) and Cꢀcile Courreges (IECB, UMS CNRS-Univer-
sitꢀ de Bordeaux 3033) for their help in performing HR-MAS NMR
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Received: January 18, 2011
Published online on April 20, 2011
ChemBioChem 2011, 12, 1193 – 1197
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
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