A desulfatation-oxidation cascade activates coumarin-based cross-linkers in the wound reaction of the giant unicellular alga dasycladus vermicularis

Article abstract of DOI:10.1002/anie.201100908

Plugged up: A rapid and efficient cascade of desulfatation, oxidation, and cross-linking mediates wound sealing in the giant-celled alga Dasycladus vermicularis (see picture). UV and fluorescence studies and mass spectrometry show the wound plug is a coumarin-based polymer. Copyright

Full text of DOI:10.1002/anie.201100908

DOI: 10.1002/anie.201100908
Phytochemistry
A Desulfatation–Oxidation Cascade Activates Coumarin-Based Cross-
Linkers in the Wound Reaction of the Giant Unicellular Alga
Dasycladus vermicularis**
Matthew Welling, Cliff Ross, and Georg Pohnert*
[
6,7]
Plants and algae protect themselves against environmental
threats by fast, wound-activated processes in which metabo-
lites that are stored within the tissue are transformed
hardening and browning process. The overall process leads
to the establishment of a rigid biopolymer (Figure 1). Herein
we provide evidence for the biochemical basis of this
biopolymerization event.
[
1]
enzymatically. For example, it was recognized early on
that plants and algae rely on the wound-activated action of
lipases and lipoxygenases to produce a structural variety of
[2]
oxylipins that can play important roles in chemical defense.
Wound-activated enzymatic reactions can not only be
employed to generate efficient chemical defenses against
herbivore attack or pathogenic invasion, but may also serve to
[
3]
mechanically protect the tissue or cells. Wound sealing is
particularly important for siphonous green macroalgae
because of their remarkable organization: they comprise a
single giant cell that can reach several meters in length. An
impressive example is the wound reaction of the siphonous
green alga Caulerpa taxifolia. Upon tissue disruption this alga
transforms the acetylated sesquiterpene caulerpenyne to
oxytoxin 2, a potent protein cross-linker that plays an integral
part in forming a protective polymer material that seals the
Figure 1. Wound-plug browning in the apical thallus of the unicellular
green algae D. vermicularis (left 2 h and right 24 h after wounding).
[
4]
wound. The rapid assimilation of cellular contents into an
insoluble wound plug prevents detrimental cytoplasmic loss
and limits the intrusion of extracellular components, which
could otherwise prove fatal. A number of other marine
siphonous-algal lineages also rely on activated cellular
metabolites for biopolymer formation, but little is known
about the underlying mechanisms. Herein we explore the
chemical basis of wound-plug formation in a member of the
order Dasycladales, Dasycladus vermicularis (Scropoli)
Krasser. This evolutionary ancient alga apparently lacks
caulerpenyne type molecules but is still capable of wound-
plug formation. Previous reports have described the chrono-
logical events involved in the wound-healing process in this
alga: an initial rapid gelling process followed by a delayed
We undertook chemical profiling of the metabolites found
in intact and wounded algae to monitor changes in secondary
metabolites potentially involved in biopolymerization. Sur-
prisingly, 3,6,7-trihydroxycoumarin (THyC) that has been
reported as the major secondary metabolite from D. vermic-
ularis was not detected in ultra performance liquid chro-
matography (UPLC)-MS chromatograms of MeOH extracts
of intact algae. Utilizing NMR spectroscopy, HR/MS, and MS/
MS the dominant metabolite in the algal extract was
[
5]
[
8]
determined
to
be
6,7-dihydroxycoumarin-3-sulfate
(DHyCS). The structure was confirmed by synthesis of an
authentic standard (see the Supporting Information). Pre-
vious studies on the secondary metabolites of D. vermicularis
have thus most likely been measuring DHyCS concentration
and not THyC as the UV-absorbance maximum falls at
[
*] Dr. M. Welling, Prof. Dr. G. Pohnert
Institute for Inorganic and Analytical Chemistry
Friedrich Schiller University Jena
[9,10]
approximately 345 nm for both compounds.
No other
Lessingstrasse 8, 07743 Jena (Germany)
E-mail: Georg.Pohnert@uni-jena.de
sulfated coumarins or higher substituted derivatives of THyC
were detected by monitoring the characteristic loss of Dm/z =
Prof. Dr. C. Ross
Department of Biology, University of North Florida
Jacksonville, FL 32224 (USA)
8
0 (SO ) within fragmentation patterns using UPLC-MS/MS
3
techniques. Interestingly, while DHyCS is stable at room
temperature the concentration of this compound in wounded
algal tissue rapidly diminishes as a function of time (Fig-
ure 2a–c). We reasoned that mechanical disruption of the cell
might result in the decompartmentalization of cellular
sulfatases which could transform DHyCS into THyC.
[
**] We thank the Volkswagen Stiftung for generous support within the
framework of a Lichtenberg Professorship.
Supporting information for this article (a detailed experimental
section mentioning sample collection, extraction procedures, and
all experiments as well as the spectroscopic data of DHyCS) is
available on the WWW under http://dx.doi.org/10.1002/anie.
Hydrolysis of sulfate esters by sulfatase enzymes regulates
the activity of a broad range of biomolecules and thus controls
201100908.
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Figure 2. a)–c) UPLC-ESI-MS ion trace of DHyCS (m/z 273) in algal extracts at 0, 2, and 20 min after injury, respectively. d) THyC mediated a-
lactalbumin aggregation under oxidative conditions monitored by light-scattering assays at 350 nm (* 20 mm THyC, * 40 mm THyC, ! 80 mm
THyC). e) SDS PAGE (10%), lane 1: protein ladder, lane 2 : Boiled D. vermicularis cells exhibiting protein content of intact cells, lane 3:
D. vermicularis cells boiled 4 h after wounding. The arrows indicate polymer in the loading pocket (top) and proteins in intact algae.
important functions including hormone regulation and signal-
trapped by glutathione (GSH), cysteine, and dithiothreitol.
Metabolites resulting from the transformation with one or
two GSH, or with the other thiol-containing nucleophiles,
could be detected and characterized by UV spectroscopy,
HR/MS and MS/MS analysis (Figure 3, see the Supporting
Information). The ability of the oxidized THyC to form
covalent bonds to amino groups was evaluated by using the
probe Bodipy FL STP ester, which has an available reactive
amine group. In the presence of oxidants, the percentage of
free reactive amino groups decreased as the THyC concen-
tration increased (see the Supporting Information). These
data provide evidence that ubiquitous thiol- and amine-
bearing functionalities in algal tissue (e.g. proteins), could
readily react under ambient conditions with oxidized THyC.
Moreover, when D. vermicularis cells were wounded in the
presence of GSH, THyC/GSH adducts were detected (Sup-
porting Information). This observation suggests that condi-
tions necessary for the quinone chemistry to take place are
met in vivo. The transformations involved most likely proceed
by a radical mechanism, which is also supported by the fact
that reactive oxygen species, generated in the presence of a
lipoxygenase and its substrate linolenic acid, also lead to
THyC oxidation (see the Supporting Information). Since the
sulfated DHyCS is not transformed by H O or the peroxidase
[
11]
ing pathways. Sulfatase activity could indeed be detected in
an in-vitro assay using the model substrate 4-methylumbelli-
ferone sulfate which was rapidly hydrolyzed by wounded
algae (see the Supporting Information). Whereas the product
4
-methylumbelliferone could be readily detected, THyC was
only found in trace amounts during and after the wound
reaction. We therefore concluded that THyC might be further
transformed in the wounded algae. Because hydroxycoumar-
[
12]
ins can be easily oxidized, we reasoned that reactive oxygen
species, which were previously detected in wounded D. ver-
[7]
micularis, could serve as an oxidant for THyC. If the kinetics
[7]
of H O production and DHyCS transformation are com-
2
2
pared it becomes clear that the oxidant is only detected after
the complete transformation of DHyCS (see the Supporting
Information). In model reactions it could be demonstrated
that H O only slowly oxidized THyC. This oxidation was
2
2
considerably accelerated in the presence of horseradish
peroxidase (HRP) and was complete after 4 min. The HRP
was utilized to mimic the presence of oxidase activity in
[7]
wounded algae (see the Supporting Information). The
observations are in accordance with a mechanism in which
H O is produced continuously after wounding and acts as
2
2
substrate for the enzymatic oxidation of THyC. The oxidant
would then only be
2
2
detected once the sub-
strate THyC is quantita-
tively transformed.
To assess the ability
for THyC to serve as a
cross-linking agent under
oxidizing
conditions,
model reactions using
this compound, H O ,
2
2
HRP,
and
selected
amino- and sulfhydryl-
bearing model com-
pounds were employed.
Under these conditions
THyC is rapidly oxidized
Figure 3. ESI-MS/MS of a) a THyC–glutathion adduct and b) a THyC–(glutathione) adduct with indicative
2
and the product can be fragments.
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Scheme 1. Process by which THyC is transformed in to a potent protein cross-linker. Stage 1: Sulfate ester hydrolysis by sulfatase upon cellular
decompartmentalization. Stage 2: Radicals formed from THyC oxidation react with amino acid side chains. Stage 3: Further oxidation and reaction
with amino acid side chains leads to cross-linked products increasing the stability of the wound plug.
treatment (see the Supporting Information) it can be assumed
that the radical attack preferentially occurs at the C3-OH
group by formation of a mesomeric radical which adds a
nucleophile to form first addition products (Figure 3, see the
Supporting Information). These can then undergo further
transformation to higher functionalized THyC derivatives
fluorescence investigations of the wound plug show that the
THyC fluorescence and the characteristic UV absorption are
still observed (see the Supporting Information). Nevertheless
MS-analysis, which is more sensitive than UV spectroscopy,
does not indicate any THyC or low molecular weight
derivatives of this metabolite. This result is in accordance
with the presence of a coumarine-based polymer in the plugs.
Cross-linking based on the oxidation of hydroxylated aro-
matic metabolites is a common process in wounded plant
tissue and also the basis for insect cuticle sclerotization. In
these cases substrates and amino acids are usually in separate
(
Figure 3, Scheme 1). Alternatively, the reactions could
follow the oxidation/addition pathways involving quinones
which have been described for the thiol addition to dopamine
and the protein cross-linking in privet trees under oxidative
[
13,14]
conditions.
[
16]
To provide additional evidence for the involvement of
THyC in an oxidative biopolymerization process, the metab-
olite was incubated with HRP and H O in the presence of the
compartments to prevent uncontrolled reactions.
The
process observed in D. vermicularis follows a novel biopoly-
merization regulation principle based on desulfatation of
stable storage metabolites. Sulfatation is often found in nature
as protection for reactive metabolites. Thus sulfatation and
desulfatation processes are involved in the regulation of
2
2
test protein a-lactalbumin. 30–40 min after the start of
incubation the mixture turned brown and a precipitate was
clearly visible. This reaction was also observed with other
randomly selected test proteins, indicating an unspecific
activity. Acid hydrolysis and automated amino acid analysis
revealed that this precipitate was rich in a-lactalbumin.
Although solution browning occurred even in the absence of a
test protein, precipitation occurred only upon addition of a-
lactalbumin. A light-scattering assay was used to quantify the
degree of polymerization when oxidized THyC was combined
[
11]
important cellular activities. Sulfatation of THyC at the C3
position might prevent oxidation and minimize autotoxicity
during regular growth. It is of note that wound-plug formation
based on protein cross-linking in siphonous green algae can
be mediated by fundamentally different processes. While the
siphonous Caulerpales rely on a terpenoid acetate that is
transformed by esterases to form a highly active protein cross-
[
15]
[4]
with a-lactalbumin. Three concentrations of THyC were
incubated with a-lactalbumin, HRP, and H O and polymer-
linker, D. vermicularis accomplishes protein cross-linking in
a fundamentally different manner. A further screening of
siphonous algae is required for an in depth understanding of
the evolution of wound-repair mechanisms which are essen-
tial for such giant-celled organisms.
2
2
ization was monitored for 80 min (Figure 2d). 20 mm of THyC
was capable of promoting protein polymerization and the
amount of aggregation increased with 40 mm and 80 mm of
THyC. Control experiments showed no turbidity when THyC
was omitted from the reaction mixture thus indicating its
essential involvement in aggregation. Cellular DHyCS con-
centrations above 100 mm can be estimated based on photo-
Received: February 4, 2011
Revised: May 23, 2011
Published online: June 30, 2011
[
10]
metric quantifications. According to our data this concen-
tration would clearly be sufficient for protein polymerization.
To verify if the observed protein aggregation is also found in
Dasycladus cells, we used sodium dodecyl sulfate polyacryl-
amide gel electrophoresis (SDS-PAGE) to monitor the
distribution of protein content before and after wounding.
Protein analysis of ground cells revealed a band in the loading
Keywords: coumarins · enzyme catalysis · polymers ·
protein cross-linking · siphonous algae
.
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