From the
Water-soluble UV-A/B-absorbing pigments are secreted by cells of
the cosmopolitan terrestrial cyanobacterium Nostoc commune.
The pigments constitute a complex mixture of monomers with molecular
masses of up to 1801 Da. Two different chromophores with absorption
maxima at 312 and 335 nm are linked to different amino acids and to
oligosaccharides consisting of galactose, glucose, xylose, glucuronic
acid, and glucosamine. The 335 nm chromophore is a
1,3-diaminocyclohexen derivative, while the chromophore with an
absorption maximum at 312 nm is most likely a 3-aminocyclohexen-1-one
derivative. These UV-inducible substances are the first mycosporines to
be described covalently linked to oligosaccharides. The pigments are
located in the extracellular glycan sheath of Nostoc colonies,
where they form complexes of extremely high molecular mass that are
attached noncovalently to the glycan sheath. Pigments occur in
concentrations that permit the cells to attenuate a significant part of
incident UV-B radiation.
Microorganisms possess a variety of UV-protecting mechanisms,
some of which ( e.g. the SOS response) have been elucidated in
great detail. Additionally, cells are equipped with radical quenchers
and antioxidants that provide protection by scavenging harmful radicals
or oxygen species. A variety of cells, including cyanobacteria, produce
UV-absorbing substances that are thought to serve as sunscreen
molecules (Garcia-Pichel and Castenholz, 1993; Karentz et al.,
1991; Post and Larkum, 1993; Scherer et al., 1988). The
occurrence of colorless UV-B-absorbing substances in cyanobacteria has
been known for at least 25 years (Shibata, 1969). More recently, the
induction of such compounds by UV-B was demonstrated (Garcia-Pichel
et al., 1993; Scherer et al. 1988). However, the
structure of these cyanobacterial secondary products is not known.
The terrestrial cyanobacterium Nostoc commune is well
adapted to live under extraordinary environmental conditions. Besides
withstanding extreme water stress (for review, see Potts (1994)), this
organism can tolerate high levels of UV radiation. In this paper, the
structure of a chromophore of a UV-B-absorbing pigment that is
synthesized upon UV radiation is reported.
After hydrolysis in the presence
of 2
M trifluoroacetic acid, the anomeric protons of both
glucose and galactose were detected in the NMR spectra of hydrolysis
products of the pigment. Analysis of the saccharides by TLC and HPLC
yielded glucose, galactose, and glucosamine. The product (named t38) of
the trifluoroacetic acid hydrolysis was purified by HPLC and subjected
to a second hydrolysis under more vigorous conditions (6
M HCl, 6 h, 100 °C). Thereafter, galactose, xylose, and
glucuronic acid were detected by HPLC, which was confirmed by
We thank M. Potts for helpful discussions and F.
Garcia-Pichel for donation of reference samples.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Extraction and Isolation of the Pigment
750-g
fresh, wet colonies of N. commune var. Vaucher, collected
after rains in South Germany during Spring 1992, were extracted in 2.5
liters of methanol:water (3:7) for 1.5 h at 40 °C. After filtration
and reduction of the volume to 0.1, the green filtrate was further
purified by protein precipitation with chloroform. Additional proteins
were removed by DE52 anion-exchange chromatography, and the
flow-through fraction was eluted 2-fold from a gel exclusion
chromatography column (Sephadex G-25; Pharmacia, Uppsala, Sweden).
After concentration by freeze-drying, fractions were separated by
reversed-phase HPLC(
)
(5-µm RP-18 column, 125
4 mm; E. Merck AG, Darmstadt, Germany) with a linear gradient
from 100% water to 5% acetonitrile in water (v/v) in 30 min. The use of
other packing materials (RP-4, RP-8, NH
phase), buffers,
ion-pairing reagents, and 0.1% (w/v) acetic acid failed to achieve
better resolutions. For determination of the apparent molecular mass,
the pigment was eluted from a Superose 12 HR 10/30 column (Pharmacia)
calibrated with aminobenzoic acid ethyl ester derivatives of
maltooligosaccharides.
Chemical Hydrolysis
Pigments were treated with 2
M trifluoroacetic acid at 100 °C for 3 h or with 6
M HCl at 100 °C for 6 h. The solution was neutralized with solid
NaHCO. Hydrolyses were monitored after 1, 2, 3, 12, and 24
h by reversed-phase HPLC using the following gradient (RP-18 column):
0-5% acetonitrile in water from 0 to 30 min and 5-100%
acetonitrile from 30 to 60 min. This gradient was also used for
preparative separation of hydrolysis mixtures.
Glycoside Analysis
TLC analysis of the hydrolyzed
samples was performed according to Gauch et al. (1979) and
Rebers and Wessman (1986) for amino sugars. HPLC analysis of the
hydrolyzed samples was as described (Spiro and Spiro, 1992). Glucose,
galactose, mannose, rhamnose, fucose, xylose, ribose, arabinose,
glucosamine, galactosamine, glucuronic acid, and galacturonic acid were
used as reference substances.
Amino Acid Analysis
Samples were hydrolyzed in 1%
(w/v) NHor 12.5% (w/v) NH
at 25, 40, or 80
°C. Alternatively, 6
M HCl at 110 °C for 24 h under
N
atmosphere with 0.1% (w/v) phenol was used. Amino acids
were analyzed as o-phthalaldehyde derivatives on a 244-mm
Supersphere column (E. Merck AG) using a Merck-Hitachi F1000
fluorescence detector.
Spectroscopic Methods
UV spectra were recorded
with a Perkin-Elmer Lambda 5 UV-visible spectrometer. NMR spectra were
recorded on an AC 250-MHz (Bruker, Rheinstetten, Germany) or a JNM-GX
400-MHz (Jeol, Tokyo) spectrometer in DO as solvent. The
signals of the deuterated solvents were used as references. PD-MS
spectra were recorded on a Bio Ion 20K spectrometer. 10-20 µg
of the sample were loaded on a nitrocellulose-coated Mylar target. Fast
atom bombardment-MS spectra were recorded on a Finnigan MAT
312/AMD-5000 spectrometer. Matrices were glycerin, 6
M HCl
(1:1) or glycerol/thioglycerol/acetic acid (1:7:2).
Extraction and Purification
Starting from
desiccated material, 10-15% of the total amount of pigment can be
extracted in water at 25 °C. Wet colonies do not release any
pigments under these conditions. The isolation procedure described
under ``Experimental Procedures'' yielded 7 mg of pigment/g
of dry material with an absorption coefficient of 17 cmmg
at 312 nm. Fig. 1 A shows the
absorption spectrum of the pigment extract after 2-fold Sephadex column
chromatography. This is by no means a pure preparation. It consists of
a variety of compounds, each containing both chromophores in various
ratios, which is indicated by the fractionation of the eluate by
reversed-phase HPLC (Fig. 1 B). Every peak in the HPLC
chromatogram absorbs at 312 and 335 nm and still is a mixture of
compounds as shown by PD-MS (data not shown).
Figure 1:
A, UV spectrum of the pigment in
HO (
= 17 cm
mg
)
after 2-fold gel exclusion chromatography with Sephadex G-25. The
quotient 260/312 nm was used as a gauge of the progress of the
purification. B, HPLC of the pigment with detection at 330 nm
(for conditions, see ``Experimental Procedures''). No peaks
missing the chromophores were detected at 220 nm. Use of buffers or
ion-pairing reagents did not result in a better resolution. The reason
for the broad peaks is presumably the complex mixture of closely
related compounds and the formation of complexes in the aqueous eluent.
The seven fractions indicated were taken and examined by PD-MS. Each
fraction was a mixture of several compounds. Probably the complexes are
destroyed during the desorption process in the spectrometer. Note that
all fractions show the same UV spectrum. C, PD-MS of the
pigment (15 µg in H
O). From the ion at m/ z 1646, a series of peaks with
m = 162 can be seen,
indicating a series of homologous oligohexoses (no fragmentation is
normally found with PD-MS). Below m/ z 600, no
reliable data can be obtained with this technique. The peak at
m/ z 1802 is probably the one with the highest mass as
similar results are found with gel exclusion
chromatography.
Molecular Size
Gel exclusion chromatography on
Superose 12 with distilled water as eluent gave two peaks containing
the pigment. The first one represented 85% of the absorption at 280 nm
and showed an extremely high apparent molecular mass, eluting in the
exclusion volume (2,000,000 Da). The smaller peak (15% of total
absorption) was at an apparent molecular mass of 3200 Da. 0.2
M NaCl as eluent resulted in a single sharp peak with an apparent
molecular mass of 3200 Da. The use of the chaotroph sodium
trichloroacetate (1
M, pH 7.0) as eluent resolved a single
peak with an apparent molecular mass of 2000 Da. A plasma desorption
mass spectrum (Fig. 1 C) of the pigment in distilled
water indicated a maximum molecular mass of 1801 Da. PD-MS normally
yields no fragmentation ions with oligosaccharides. Therefore, it can
be assumed that the largest pigment molecule has indeed a mass of 1801
Da. Starting from a molecular mass of 1646 Da, there are several peaks
with a distance of
m = 162, which would correlate with a
homologous series of oligohexoses.
Oligosaccharide Composition
Even after
reversed-phase HPLC, the NMR spectra of the complex pigment mixture
were far too complex for an interpretation. However, NMR spectra
clearly demonstrated the presence of saccharide structures (data not
shown). No cleavage of the pigment by applying 11 glycosidases
separately as well as in combinations was obtained. Control experiments
showed that the pigment mixture did not inhibit the enzymes used in
these digestions (data not shown).
C NMR analysis of the hydrolysis product, t38
().
Amino Acid Analysis
After hydrolysis of the
pigment in 1 or 12.5% NHat different temperatures, no
amino acids could be detected. When hydrolysis was performed in 6
M HCl for 24 h, the following amino acids were found in amounts
equivalent to one-tenth of the molar amount of pigment (assuming an
average molecular mass of the pigment of 1200 Da): Asx, Glx, Ser, Gly,
Thr, and Ala.
Properties of the Chromophores
The most
conspicuous feature of the two chromophores, besides the absorption
maxima, is their complementary stability against acids and bases. The
actions of acids or bases are irreversible processes; no isosbestic
points are found. E312 is destroyed at pH 2 at 25 °C within
minutes. E335 is stable for hours in 2
M trifluoroacetic acid
at 100 °C, but is destroyed at pH values higher than 12. Chemical
hydrolysis in the presence of 2
M trifluoroacetic acid at 100
°C for 3 h produced a definitively less polar product on
reversed-phase HPLC (t38) (Fig. 2). This fragment had only one
absorption maximum at 335 nm. Apparently, the 312 nm chromophore was
destroyed. After preparative HPLC purification, subsequent HPLC
analysis showed a single peak in the chromatogram, designated t38.
Nevertheless, fast atom bombardment-mass spectrometry revealed that
this fraction still is a mixture of several compounds (data not shown).
The highest molecular mass was 703 Da (MH;
MNa
725 was also found). The
H NMR data
showed the presence of methoxy, hydroxymethyl, and cyclic methylene
protons.
C NMR data of the chromophore based on hydrolysis
of HPLC-purified t38 are listed in .
Figure 2:
Contour plot of the HPLC chromatogram of
the pigment after hydrolysis with 2
M trifluoroacetic acid for
3 h at 100 °C. The peak with a retention time of 38 min (t38)
containing the E335 chromophore is the hydrolysis product and was
characterized by spectroscopic methods.
Structure of the Chromophores
The NMR data of
the hydrolysis fragment t38 (see Fig. 2) were compared with those
published in the literature for the 334 nm mycosporine isolated from
the eucaryotic red alga Porphyra tenera (Takano et
al., 1979). All NMR signals for the
1,3-diamino-2-methoxycyclohexen core are found in the H and
C NMR spectra of t38, which contains E335 ().
A characteristic feature of the Nostoc pigments is the
complementary sensitivity of the two chromophores to acids and bases,
which is also a feature of mycosporines (Takano et al., 1978;
Chioccara et al., 1980). The E312 chromophore has UV
spectroscopic and hydrolysis properties identical to those of
mycosporine-Gly (Ito and Hirata, 1977), a 3-aminocyclohexene-1-one
derivative with only 1 amino acid that is readily hydrolyzed by hot
water. The product formed by hydrolysis is an unstable 1,3-diketone
with an absorption maximum at 268 nm in acidic solution (Ito and
Hirata, 1977; Plack et al., 1981). Hydrolysis of the
Nostoc pigment E312 showed a similar transient absorption.
Therefore, we suggest tentatively that the chromophore core of E312
might also be a mycosporine, although we do not have NMR data available
to support this assertion.
Saccharide and Amino Acid Content of the
Pigment
The complex pigment mixture contains glucose, galactose,
glucosamine, glucuronic acid, and xylose in unknown molarities. It may
be significant that a water stress protein of N. commune (Scherer and Potts, 1989) is secreted and is associated with the
UV-absorbing pigments and a xylanxylanohydrolase activity (Hill et
al., 1994). Usually, oligosaccharides are hydrolyzed in 2
M HCl at 100 °C. E335 is stable under these conditions, and only
part of the saccharides are liberated from the pigment. Based on
C NMR data, unhydrolyzable C-glycosides as
structural elements could be excluded: signals between 85 and 100 ppm
( cf.
), typical for C-glycosides, are
missing. For the same reason, N-glycosides can be ruled out
(Dill et al., 1985). Some other unusually stable glycosidic
bonds are known that contain an amino function near the glycosidic bond
(Foster et al., 1957). Protonation of nitrogen prevents the
attack of a proton at the glycosidic oxygen atom and thereby
hydrolysis. As mycosporines are substituted with amino acids or amino
alcohols, such structures are probably the cause for the unusual
stability of the glycosidic bonds in the Nostoc pigment. This
is consistent with our finding that glycosidases are unable to liberate
saccharides from the pigment. Amino acids can be liberated from most of
the mycosporines by very dilute base (Takano et al., 1978) or
even hot water (mycosporine-Gly) (Ito and Hirata, 1977). We could not
detect any amino acids after such hydrolysis procedures. As acetals,
glycosides are stable under basic conditions. Therefore, if the amino
acid is linked to an oligosaccharide, this oligosaccharide-amino acid
conjugate cannot be detected by amino acid analysis.
O-Glycosides of Ser and Thr give
-eliminination under
basic conditions, resulting in
-aminoacrylic acid derivatives
(Wakabayashi and Pigman, 1974), which also cannot be detected by normal
amino acid analysis. However, after treatment with 6
M HCl at
110 °C, we found the amino acids Asx, Glx, Ser, Thr, Gly, and Ala.
Ala and Asx have not yet been reported as components of mycosporine
amino acids.
Proposed Structure and Native Size of the E335
Pigment
Based on the data presented, we suggest the structure of
E335 that is depicted in Fig. 3. The chromophore of the pigment
is a mycosporine with 2 amino acid residues. Saccharides such as
galactose, xylose, and glucuronic acid are coupled to these amino acid
residues, forming a stable ``inner shell'' of saccharides
that is only hydrolyzable under vigorous conditions. An ``outer
shell'' of glucose, galactose, and glucosamine is attached that is
easily hydrolyzable. Based on PD-MS, a molecular mass of 1801 Da for
the the largest pigment molecule was found, which corresponds well with
our results from gel exclusion chromatography. One mycosporine
chromophore with Thr and Ser as substituents plus two inner shell
saccharides (Gal) attached would have a molecular mass of 701 Da. The
largest pigment molecule with a molecular mass of 1801 Da could
therefore be composed of up to eight oligosaccharides. Most probably,
the UV-B-absorbing pigments in the glycan sheath of N. commune are a complex mixture of molecules of different sizes and amino
acid and saccharide composition. Among the mycosporines described in
this paper are some with an unusually high molecular mass and the first
ones to be described that have oligosaccharides attached.
Figure 3:
A hypothetical structure of the pigment
consisting of the chromophore E335, the amino acids serine and
threonine, and the two saccharide shells, R (galactose,
xylose, and glucuronic acid) and R
(galactose, glucose, and
glucosamine). The inner shell, R
, is more stable toward
hydrolysis than the outer shell,
R
.
Our data
show that the pigments form large complexes in aqueous solutions that
are held together by noncovalent interactions (compare with data of
Fransson et al. (1984)). The extraordinary high molecular mass
complexes observed during gel exclusion chromatography may well be due
to the ability of the pigment to associate tightly with the
extracellular polysaccharide sheath of Nostoc by noncovalent
interaction with the glycan sheath, preventing its loss during drying
and rewetting cycles of the colonies occurring daily in some habitats
(Scherer and Zhong, 1991).
Sunscreen Function of Pigment Complexes
There is
clear evidence that the Nostoc pigments serve as a sunscreen.
First, the pigments are inducible by UV-B (Scherer et al.,
1988). Second, the UV-absorbing pigment is located extracellularly in
the glycan sheath of the colony; during extraction of the material
under mild conditions, no intracellular compounds were detected in the
extract, which is indicated by the complete lack of phycobiliproteins
in the aqueous extracts (see also Hill et al. (1994)). Third,
the actual contribution of the mycosporines to UV-B absorption is
significant. On the average, wet N. commune colonies collected
in the field are 0.5 mm thick and contain 40 µg of UV-B-absorbing
pigment/cm. The pigment has an average
of 17 cm
mg
. If the pigments are distributed
homogeneously throughout the glycan sheath, an average sunscreen factor
of 0.7 can be calculated (compare with data of Garcia-Pichel and
Castenholz (1993)). This means that two out of three photons will be
absorbed by the UV-absorbing pigment.
Table: C NMR chemical shifts
(solvent D
O,
in ppm
relative to
Me
SO-d
,
39.50 ppm) for the carbon atoms of the saccharide part of t38
(containing E335) correlated with
Me
SO-d
literature data from Gorin and Mazurek (1975), Bock et al.
(1984), Dill et al. (1985), and Agrawal (1992)
Table: C NMR chemical shifts
(solvent D
O,
in ppm
relative to
Me
SO-d
,
39.50 ppm) for the 335 nm chromophore compared with literature data
from Takano et al. (1979) for the mycosporine core
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.