Characterization of the chemical diversity of glycosylated mycosporine-like amino acids in the terrestrial cyanobacterium Nostoc commune

Article abstract of DOI:10.1016/j.jphotobiol.2014.12.008

Mycosporine-like amino acids (MAAs) are UV-absorbing pigments, and structurally unique glycosylated MAAs are found in the terrestrial cyanobacterium Nostoc commune. In this study, we examined two genotypes of N. commune colonies with different water extract UV-absorption spectra. We found structurally distinct MAAs in each genotype. The water extract from genotype A showed a UV-absorbing spectrum with an absorption maximum at 335 nm. The extract contained the following compounds: 7-O-(β-arabinopyranosyl)-porphyra-334 (478 Da), pentose-bound shinorine (464 Da), hexose-bound porphyra-334 (508 Da) and porphyra-334 (346 Da). The water extract from genotype B showed a characteristic UV-absorbing spectrum with double absorption maxima at 312 and 340 nm. The extract contained hybrid MAAs (1050 Da and 880 Da) with two distinct chromophores of 3-aminocyclohexen-1-one and 1,3-diaminocyclohexen linked to 2-O-(β-xylopyranosyl)-β-galactopyranoside. A novel 273-Da MAA with an absorption maximum at 310 nm was also identified in genotype B. The MAA consisted of a 3-aminocyclohexen-1-one linked to a γ-aminobutyric acid chain. These MAAs had potent radical scavenging activities in vitro and the results confirmed that the MAAs have multiple roles as a UV protectant and an antioxidant relevant to anhydrobiosis in N. commune. The two genotypes of N. commune exclusively produced their own characteristic glycosylated MAAs, which supports that MAA composition could be a chemotaxonomic marker for the classification of N. commune.

Full text of DOI:10.1016/j.jphotobiol.2014.12.008

Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Characterization of the chemical diversity of glycosylated
mycosporine-like amino acids in the terrestrial cyanobacterium
Nostoc commune
Ehsan Nazifi a,1, Naoki Wada b, Tomoya Asano c,2, Takumi Nishiuchi c, Yoshiaki Iwamuro d,
Satoshi Chinaka d, Seiichi Matsugo b, Toshio Sakamoto a,b,
⇑
a Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan
b School of Natural System, College of Science and Engineering, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan
c Division of Functional Genomics, Advanced Science Research Center, Kanazawa University, Takara, Kanazawa 920-0934, Japan
d Forensic Science Laboratory, Ishikawa Prefectural Police Headquarters, 1-1 Kuratsuiki, Kanazawa 920-8553, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Mycosporine-like amino acids (MAAs) are UV-absorbing pigments, and structurally unique glycosylated
MAAs are found in the terrestrial cyanobacterium Nostoc commune. In this study, we examined two geno-
types of N. commune colonies with different water extract UV-absorption spectra. We found structurally
distinct MAAs in each genotype. The water extract from genotype A showed a UV-absorbing spectrum
with an absorption maximum at 335 nm. The extract contained the following compounds: 7-O-(b-arabi-
nopyranosyl)-porphyra-334 (478 Da), pentose-bound shinorine (464 Da), hexose-bound porphyra-334
(508 Da) and porphyra-334 (346 Da). The water extract from genotype B showed a characteristic UV-
absorbing spectrum with double absorption maxima at 312 and 340 nm. The extract contained hybrid
MAAs (1050 Da and 880 Da) with two distinct chromophores of 3-aminocyclohexen-1-one and 1,3-dia-
minocyclohexen linked to 2-O-(b-xylopyranosyl)-b-galactopyranoside. A novel 273-Da MAA with an
absorption maximum at 310 nm was also identified in genotype B. The MAA consisted of a 3-aminocyc-
Received 29 September 2014
Received in revised form 1 December 2014
Accepted 3 December 2014
Available online 13 December 2014
lohexen-1-one linked to a c-aminobutyric acid chain. These MAAs had potent radical scavenging activi-
ties in vitro and the results confirmed that the MAAs have multiple roles as a UV protectant and an
antioxidant relevant to anhydrobiosis in N. commune. The two genotypes of N. commune exclusively pro-
duced their own characteristic glycosylated MAAs, which supports that MAA composition could be a che-
motaxonomic marker for the classification of N. commune.
Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction
tion, N. commune cells rapidly recover respiration and photosyn-
thesis [5–8]. This phenomenon is termed anhydrobiosis [9–12].
The cyanobacterium Nostoc commune adapts to terrestrial envi-
ronmental conditions and has a cosmopolitan distribution on the
Earth [1]. In its natural habitats, N. commune forms visually con-
spicuous colonies that consist of extracellular matrix with filamen-
tous cells embedded inside [1,2]. N. commune colonies are
subjected to frequent cycles of desiccation and wetting. The desic-
cated colonies show little to no metabolic activity, yet they retain
the ability to grow for more than 100 years [3,4]. Upon rehydra-
N. commune is a prokaryotic model anhydrobiote that retains oxy-
genic photosynthetic capabilities in vegetative cells and does not
differentiate into akinetes (spores) [1,12]. In addition to extreme
desiccation tolerance, N. commune colonies are exposed to direct
solar radiation and can tolerate UV radiation stress [13,14]. The
desiccated N. commune has extreme longevity [3,4]; antioxidants
protect biomolecules from oxidation and are thought to be
involved in the mechanisms of anhydrobiosis in N. commune
[15]. N. commune has two types of UV-absorbing pigments which
are scytonemin and mycosporine-like amino acids (MAAs)
[14,16]. Both sunscreen pigments display radical scavenging activ-
ities [17–19]. Thus, these secondary metabolites are thought to
play multiple roles against environmental stresses such as UV radi-
ation and desiccation through their photoprotective and antioxida-
tive properties [15,20].
⇑
Corresponding author at: School of Natural System, College of Science and
Engineering, Kanazawa University, Kakuma, Kanazawa 920 1192, Japan. Tel.: +81 76
264 6227; fax: +81 76 264 6215.
E-mail address: tsakamot@staff.kanazawa-u.ac.jp (T. Sakamoto).
Present address: Department of Biology, Faculty of Basic Sciences, University of
1
Mazandaran, Babolsar, Iran.
2
Present address: Wakasa Seikatsu Co. Ltd., 22 Naginataboko-cho, Shijo-Karasuma,
Shimogyo-ku, Kyoto 600-8008, Japan.
http://dx.doi.org/10.1016/j.jphotobiol.2014.12.008
1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
155
Mycosporine-like amino acids (MAAs) are water-soluble pig-
ments that absorb UV radiation of 280–340 nm. There are structur-
ally distinct MAAs found in taxonomically diverse organisms [21–
27]. In cyanobacteria, MAAs protect the cells against solar radiation
[28,29]. The energy absorbed by MAAs is promptly dispersed into
the surroundings as heat [30,31]. Thus, MAAs do not function as
accessory pigments in photosynthesis [23]. In N. commune, a struc-
turally unique 1050-Da MAA with two distinct chromophores of 3-
aminocyclohexen-1-one and 1,3-diaminocyclohexen was found
[17]. There is also a pentose-bound porphyra-334 derivative
(478 Da) with an absorption maximum at 335 nm [17]. We
recently found the third type of N. commune colony with a water
extract that shows a characteristic UV-absorbing spectrum with
an absorption maximum at 325 nm. We identified novel glycosyl-
ated MAAs including two hexose-bound palythine–threonine
derivatives with a molecular mass of 612 Da in this particular type
of N. commune [19]. The glycosylated MAAs in N. commune are
thought to be localized in the extracellular matrix to allow interac-
tion with other constituents in its complex architecture [2,32,33].
These compounds have multiple functions including roles as UV
sunscreens and radical scavengers to protect the cells in terrestrial
environments [15].
[7,37]. In this study, another laboratory strain KU006 of N. com-
mune (genotype B) was isolated from the Kakuma Campus of
Kanazawa University (N 36.54388, E 136.70366) and purified by
streaking on agar plates. These N. commune cells were cultured at
25 °C under constant illumination from fluorescent lamps (15–
50
nitrogen source) containing 1.5% agar supplemented with a vita-
min mix at final concentrations of 1
g lꢀ1 biotin, 2 mg lꢀ1 thiamin
and 1
g lꢀ1 cyanocobalamin [38]. To remove fungus and bacterial
contaminations, 25
g mlꢀ1 of cycloheximide and 25 g mlꢀ1 of
l
mol mꢀ2 sꢀ1) on modified BG110 solid medium (without a
l
l
l
l
trimethoprim were added to the medium when necessary.
2.2. MAA standards
The known MAA standards including mycosporine–glycine,
palythine, shinorine, porphyra-334, and 13-O-(b-galactosyl)-por-
phyra-334 were kindly obtained from Dr. Ishihara, K. (National
Research Institute of Fisheries Science, Yokohama, Japan). These
compounds were used as authentic standards [39] for the compar-
ison of retention times on HPLC analysis.
2.3. Purification of MAAs from two types of N. commune colonies
In parallel with the studies of functional molecules in N. com-
mune [7,8,17–19,34–36], molecular taxonomical studies have
revealed that N. commune can be classified into four genotypes
based on differences in the 16S rRNA gene sequences. The sequence
differences are not great enough to be recognized as distinct species.
However, they are significantly different and allow the description
of genotype A to D [37]. These genotypes are difficult to distinguish
morphologically and there are no ecophysiological differences
allowing us to separate them. Recently, the N. commune that pro-
duces the 612-Da MAA was identified as genotype D, and the variety
of MAAs produced by genotype D N. commune were characterized
[19]. In our previous study [17], we examined the 478-Da MAA pro-
ducer and the 1050-Da MAA producer in N. commune. However, at
that time their genotypes had not been specified. In this study, the
genotypes were identified and various MAAs were purified from
the 478-Da MAA producer (genotype A) and the 1050-Da MAA pro-
ducer (genotype B). The chemical structures of the 478-Da MAA and
the 1050-Da MAA were reexamined to determine their sugar moie-
ties. The specific MAAs were found exclusively in their own types of
N. commune colonies. Additionally, the MAA compositions of the
laboratory culture strains of N. commune were examined to confirm
that each genotype produces its own characteristic MAAs in a geno-
type specific manner.
N. commune powder (35 g) was suspended in distilled water
(1200 ml). The MAAs were extracted by stirring at room tempera-
ture for 1 h. After centrifugation at 15,240g for 20 min at 4 °C, the
supernatant was vacuum-filtered with a Buchner sintered-glass fil-
ter funnel and then condensed to 300 ml with a rotary evaporator.
Ethanol was added to the filtrate to yield a final concentration of
70% (v/v) ethanol. The mixture was incubated at 4 °C for 1 h in
the dark to precipitate the 70% ethanol-insoluble materials. After
centrifugation at 15,240g for 20 min at 4 °C, the supernatant was
vacuum filtered with a Buchner sintered-glass filter funnel. The fil-
trate was evaporated and centrifuged at 21,500g for 10 min at 4 °C.
The supernatant was filtered through a 0.20-lm syringe filter
(Minisart RC 15, Sartorius Stedim, Göettingen, Germany) and
injected into a HPLC system with a Hitachi L-6200 pump that
was equipped with
a reverse phase column (IRICA C18,
20 ꢁ 250 mm, Shiseido Irica Technology, Kyoto, Japan). When we
fractionated the water extract of N. commune colonies with an
absorption maximum at 335 nm (genotype A), the mobile phase
was 0.2% acetic acid for the initial 135 min and 100% methanol
for the next 60 min. The flow rate was kept at 2 ml minꢀ1. For frac-
tionation of the water extract from N. commune colonies with
absorption maxima at 312 and 340 nm (genotype B) we used a
mobile phase that changed stepwise from 0.2% acetic acid during
the first 66 min to 5% (v/v) methanol with 0.2% (v/v) acetic acid
during the next 70 min. The mobile phase was 100% methanol dur-
ing the final 35 min. The flow rate was kept at 3 ml minꢀ1. The
absorbance at 330 nm (A330) was monitored with a Hitachi L-
4200 UV–VIS detector. The fractions with the MAAs were recov-
ered separately, condensed with a lyophilizer and injected into
an HPLC system equipped with a gel filtration column (TSKgel
G2500PW, TOSOH, Tokyo, Japan). The mobile phase was water at
a flow rate of 1 ml minꢀ1. The A330 was monitored with a Hitachi
L-4200 UV–VIS detector. The MAA fractions were recovered and
the final MAA products were lyophilized.
2. Material and methods
2.1. Microorganisms
Colonies of N. commune growing in the field were collected from
the Kakuma Campus of Kanazawa University (the genotype A: N
36.32715, E 136.42525 and N 36.32644, E 136.42537; the genotype
B: N 36.32816, E 136.42334), Ishikawa, Japan from April to Novem-
ber. Wet colonies naturally swelled after rain were harvested,
washed with tap water to remove soil, air-dried in the laboratory,
and stored at room temperature until used. The MAA was extracted
with water from N. commune powder and then the UV–VIS absorp-
tion spectrum was measured to identify the MAA type. The geno-
type was characterized by PCR direct sequencing of the 1.4-kb
DNA fragment containing the 16S rRNA gene as described previ-
ously [37]. The genotype A and genotype B samples of N. commune
were separately used for MAA purification and characterization.
The laboratory strain KU002 of N. commune (genotype A) has
been isolated and maintained at Kanazawa University since 2002
To examine the purity of the final MAA products and their
retention times on HPLC we performed HPLC analysis using a
reverse phase column (Inertsil ODS-3, 4.6 mm ꢁ 250 mm; GL Sci-
ences Inc., Tokyo, Japan). The mobile phase was selected according
to the targets. The methanol concentration in 0.2% (v/v) acetic acid
varied from 0% to 15% (v/v). The flow rate was at 1 ml minꢀ1. The
MAAs were detected by A330
.
To determine the extinction coefficients of the purified MAAs
the diluted solutions were prepared in water and the absorbance

156
E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
at their absorption maxima were determined. The MAA dry weight
in 1 ml of solution was measured after lyophilization.
Na2SO4. The CH2Cl2 extract was analyzed by GC–MS (GC, Agilent
6890 series equipped with DB-5 ms column (0.25 mm i.d. ꢁ 30 cm;
MS, Agilent 5973 series). The temperature program for the GC and
MS conditions were set according to Hongbin et al. [42]. The acety-
lated sugar derivatives were identified according to their retention
time and the fragmentation pattern of the MS spectrum.
2.4. MS analysis
MALDI-TOF MS/MS analysis was performed at the Division of
Functional Genomics, Advanced Science Research Center, Kanaza-
wa University using a tandem mass spectrometer (4800 plus
MALDI TOF/TOF™ Analyzer; Applied Biosystems, Foster City, CA,
USA) with 2,5-dihydroxybenzoic acid (DHB) as a matrix. The sec-
ondary mass spectrum was recorded when applicable. The
expected resolution was less than 0.2 m/z. FAB MS analysis was
performed to determine the accurate mass and the predicted ele-
mental composition at the Research Institute for Instrumental
Analysis in Kanazawa University on a mass spectrometer (JMS-
SX102A) with glycerol as a matrix.
2.8. Measurement of antioxidant capacity
The radical scavenging activity was measured with 2,20-azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate
in a colorimetric assay [43]. The decolorization of ABTS radical cat-
ion at A734 was monitored spectrophotometrically for 1 h [17]. The
electron spin resonance (ESR) signals of the ABTS radical cation
were recorded with a free radical monitor (JES-FR30EX, JEOL,
Tokyo, Japan). Trolox (6-hydroxy-2,5,7,8-tetramethyl chroman-2-
carboxylic acid) and ascorbic acid were used as artificial and natu-
ral water-soluble antioxidant standards. Scytonemin is a hydro-
phobic UV-absorbing pigment from N. commune [18] and was
used for a comparison.
2.5. Spectroscopic methods
The UV–VIS spectra were recorded with a Hitachi U-2800 spec-
trophotometer. Fourier transformation infrared (FT-IR) spectra
were recorded with a Nicolet NEXUS 470 FT-IR by the KBr disk
method. The NMR spectra were recorded with a JEOL ECS400 spec-
trometer at the Research Institute for Instrumental Analysis in
Kanazawa University. 3-(Trimethylsilyl)-1-propanesulfonic acid-
d6 sodium salt (TMP) was used as an internal NMR standard.
2.9. Measurement of chlorophyll contents
Chlorophyll a (Chl a) was extracted with 100% methanol and the
concentrations were determined spectrophotometrically [44]. The
Chl a concentrations were calculated using the following equation
(A750 was subtracted to correct for light scattering):
g mlꢀ1Þꢂ ¼ ðA665 ꢀ A750Þ ꢁ 13:9
2.6. CE-MS analysis
½Chl að
l
Capillary electrophoresis mass spectrometry (CE-MS) analysis
was conducted using an Agilent Technologies CE System G1600
(Agilent Technologies, Waldbronn, Germany) coupled to an Agilent
Technologies 6410 triple quad mass spectrometer (Agilent Tech-
nologies, Wilmington, DE, USA) [40]. The analytes were separated
2.10. MAA composition analysis
The MAA composition in the laboratory cultures of N. commune
strain KU002 and KU006 was analyzed by HPLC. The colonies that
formed on agar plates were collected and desiccated using a lyoph-
ilizer (FDU-1200, EYELA, Tokyo, Japan). The dried materials (5–
50 mg) were suspended in 30% (v/v) methanol (1 ml) and the MAAs
were extracted at room temperature by mixing vigorously using a
tube mixer. After centrifugation at 21,500g for 5 min, the superna-
tant was collected in a new tube. This extraction step was repeated
three times and then the 30% methanol extract (approximately
3 ml) was lyophilized. The lyophilized powder was dissolved in
100% methanol (0.5 ml) and methanol insoluble materials were
removed by centrifugation at 21,500g for 5 min. Water was added
to the supernatant to yield 20% (v/v) methanol. The UV–VIS spec-
trum was recorded with a Hitachi U-2800 spectrophotometer to
determine the absorption maximum wavelength. Absorbance at
kmax (Amax) in a range of 300–400 nm was recorded and A400 was
subtracted to correct for light scattering when necessary. The total
MAA concentrations were calculated by Amax using an average
extinction coefficient of 120 l gꢀ1 cmꢀ1 [28]. The MAA containing
fraction was injected into a HPLC system with a Hitachi L-6000
pump that was equipped with a reverse phase column (Cholester,
4.6 ꢁ 150 mm, Nacalai Tesque, Kyoto Japan). The mobile phase was
10% (v/v) methanol with 0.1% (v/v) acetic acid. The flow rate was
0.6 ml minꢀ1. The A330 was monitored with a UV–VIS detector (IRI-
in an untreated fused-silica capillary of 50
lm ꢁ 85 cm (GL Sci-
ences, Tokyo, Japan) at a temperature of 25 °C. The pH of the back-
ground electrolyte (BGE) varied in the range of 3–11 by changing
the mix of formic acid and ammonium hydroxide. A sample solu-
tion was injected at 50 mbar for 10 s and then the BGE was injected
at 50 mbar for 3 s. After each analysis, the capillary was flushed
with BGE for 4 min. The power supply was operated in the con-
stant-voltage mode at +30 kV. The scanning or selected-ion-moni-
toring mode was used for the detection. The electrospray
ionization was conducted in the positive ion mode for all analytes.
The following instrument parameters were used for ionization: gas
temperature of 250 °C; N2 gas flow rate, 7 l minꢀ1, nebulizing N2
gas pressure 10 psi at electrophoresis and 0 psi at sample injection
and a capillary voltage of 4000 V. The sheath liquid was 10 mM
ammonium formate/methanol (50/50, v/v) and the flow rate was
maintained at 4 l .
l minꢀ1
2.7. Determination of sugars by GC–MS analysis
To determine the sugars in the glycosylated MAAs, the sugars
were released by acid hydrolysis. Following a reduction to alditol,
the acetylated derivatives were identified by GC–MS analysis [41].
CA
R873, Shiseido Irica Technology, Kyoto, Japan). The MAAs were
The purified glycosylated MAA (approximately 20
lg) was hydro-
identified according to their retention times and confirmed by co-
chromatography with the purified MAAs standards. The relative
amounts of total MAAs were estimated according to their peak
areas on the HPLC chromatograms.
lyzed by 5% H2SO4 aq. (100 L) at 75 °C for 8 h. After hydrolysis
l
the pH was adjusted to 11 with solid Na2CO3. Sodium borohydrate
solution (0.5 M in DMSO, 1 ml) was added and kept at 40 °C for
90 min and then incubated at 4 °C overnight to complete reduction.
Glacial acetic acid (99.9%, 100 ll), 1-methylimidazole (200 ll) and
acetic anhydride (1 ml) were added in this order at room tempera-
ture and kept at 40 °C for 10 min. After acetylation we added water
(2.5 ml) and the acetylated derivatives were extracted by CH2Cl2
(1 ml). An organic phase (lower layer) was recovered and dried with
2.11. Nucleotide sequence accession number
The nucleotide sequences of the 16S rRNA genes of N. commune
strain KU002 (genotype A) and N. commune strain KU006 (geno-

E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
157
type B) have been deposited in the GenBank/DDBJ/EMBL databases
with the following accession numbers: AB088375 (N. commune
strain KU002) and AB933330 (N. commune strain KU006).
(accession no. AB088375). The nucleotide sequence of the 16S
rRNA gene of N. commune that has a water extract with a charac-
teristic UV-absorbing spectrum containing the double absorption
maxima at 312 and 340 nm showed 100% identity to the sequence
of the genotype B1 of N. commune (accession no. AB094352). These
results suggest that N. commune colonies with a single absorption
maximum at 335 nm in the water extracts are genotype A and the
other N. commune colonies with double absorption maxima at 312
and 340 nm in the water extracts are genotype B.
3. Results
3.1. UV absorption spectra of water extracts and genotypes of N.
commune
The N. commune strain KU002 (genotype A) and N. commune
KU006 (genotype B) were cultured using laboratory conditions
and their MAA contents were characterized. The levels of chloro-
phyll and MAA were similar in both laboratory strains of N. com-
mune (Table 1), but the UV absorption spectra of these MAA
containing extracts were different (Fig. 1). The extract of N. com-
mune strain KU002 showed a single absorption maximum at
327 nm and the extract of N. commune strain KU006 showed a
characteristic spectrum with an absorption maximum at 312 nm
associated with a shoulder approximately 340 nm (Fig. 1). The N.
commune strain KU002 contained arabinose-bound porphyra-334
(478 Da) as a main component and its content was approximately
85% according to the estimation from the HPLC chromatogram
(Fig. 1A). Pentose-bound shinorine (464 Da) was also detected in
N. commune strain KU002 and its content was approximately 15%
(Fig. 1A). Conversely, the N. commune strain KU006 contained the
1050-Da MAA and its estimated content was approximately 80%
(Fig. 1B). The levels of MAAs detected in the two strains of N. com-
mune were approximately 0.01 to 0.03% of their dry weight
(Table 1). These data are in agreement with the previous reports
in field-isolated natural colonies of N. commune that showed the
amounts of MAAs are approximately 0.04% of their dry weight [36].
The genotypes of staring materials for MAA purification and
characterization were identified. The 1446-bp DNA fragment con-
taining the 16S rRNA gene was obtained by PCR using a genomic
DNA template from field-isolated natural colonies, of which the
water extract showed a UV-absorption spectrum with a single
absorption maximum at 335 nm. The nucleotide sequence showed
100% identity to the sequence of the genotype A1 of N. commune
Table 1
MAA contents in the laboratory cultured strains of Nostoc commune.
Strain
Chl aa
Total MAAb
(mg gꢀ1 [DW])
KU002 (genotype A)
KU006 (genotype B)
2.7 1.0 (n = 6)
2.2 0.3 (n = 6)
0.14 0.05c (n = 4)
0.25 0.08d (n = 4)
Data are presented as means SD.
a
Chlorophyll a (Chl a) was extracted with methanol and determined spectro-
photometrically [44].
Mycosporine-like amino acids (MAAs) were extracted with 30% methanol and
determined spectrophotometrically using an absorption coefficient of
120 l gꢀ1 cmꢀ1 [28].
Mixture of pentose-bound porphyra-334 (85%) and pentose-bound shinorine
(15%).
The 1050-Da MAA content was approximately 80%.
b
c
d
A
1
0.8
0.6
0.4
0.2
0
280
330
380
Wavelength, nm
B
0.8
0.6
0.4
0.2
0
280
330
380
Wavelength, nm
Fig. 1. Typical absorption spectra and HPLC chromatograms of the extracts of the laboratory cultures of Nostoc commune strain KU002 (A) and strain KU006 (B). The 30%
methanol extracts showed absorption maxima at 327 nm (A) and 312 nm (B), respectively. MAAs were analyzed by HPLC with a reverse-phase column (COSMOSIL Cholester,
4.6 mm ꢁ 150 mm, Nacalai Tesque) using 10% methanol and 0.1% acetic acid at a flow rate of 0.6 ml minꢀ1 as the mobile phase. MAAs were detected by the A330. N. commune
strain KU002 (A) contained arabinose-bound porphyra-334 (478 Da) and pentose-bound shinorine (464 Da). N. commune strain KU006 (B) contained the hybrid MAA
(1050 Da) with double absorption maxima at 312 and approximately 340 nm.

158
E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
These results suggest that the genetically distinct strains of N.
3.2. Variety of MAAs in genotype A
commune exclusively produce their own unique MAAs and that
the MAA production takes place under laboratory culture condi-
tions without supplemental UV irradiation. Stress conditions such
as UV irradiation and/or desiccation may enhance the MAA pro-
duction and this will be tested and reported in future studies.
The water extract from field-isolated natural colonies of geno-
type A showed a UV-absorbing spectrum with an absorption max-
imum at 335 nm. The extract from genotype A was examined by a
preparative HPLC system and four different MAAs were identified.
A
0
50
100
150
200
Retention time,min
B
C
D
E
0
9
18
0
10
20
0
10
20
0
10
20
30
40
min
min
min
min
λ
max= 333 nm
λ
max= 332 nm
F
G
0.5
0.5
0.2
0.2
-0.1
-0.1
250
350
450
250
350
450
Wavelength, nm
Wavelength, nm
λmax= 335 nm
λ
max= 334 nm
H
I
0.5
0.2
0.5
0.2
-0.1
-0.1
250
350
450
250
350
450
Wavelength, nm
Wavelength, nm
Fig. 2. HPLC chromatograms and absorption spectra of the purified MAAs from Nostoc commune colony with an absorption maximum at 335 nm (genotype A). (A) The crude
water-soluble extract was separated by an HPLC system equipped with a reverse phase column (IRICA C18, 20 ꢁ 250 mm, Shiseido Irica Technology) as described in Section 2.
The purified MAAs, 464-Da MAA (B), porphyra-334 (C), 508-Da MAA (D) and 478-Da MAA (E), were analyzed by an HPLC system equipped with a reverse phase column
(Inertsil ODS-3, 4.6 mm ꢁ 250 mm; GL Sciences Inc.) using 0.2% acetic acid at a flow rate of 1 ml minꢀ1 as the mobile phase and were detected by the A330. The purified 464-
Da MAA (F), porphyra-334 (G), 508-Da MAA (H) and 478-Da MAA (I) in water showed absorption maxima at 332 nm, 333 nm, 334 nm and 335 nm, respectively.

E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
159
The MAAs were the following: 478-Da MAA, 464-Da MAA, 508-Da
MAA, and porphyra-334 with a molecular mass of 346 Da (Fig. 2).
Each peak was collected and the purity was examined by an ana-
lytical HPLC system (Fig. 2B–E). The molecular masses were deter-
mined by MALDI-TOF–MS/MS analysis.
The 478-Da MAA with an absorption maximum at 335 nm
(Fig. 2I) was the main MAA from N. commune in genotype A and
accounted for approximately 62% of the total MAAs according to
the estimation from the HPLC chromatogram. The 478-Da MAA
has previously been characterized as a pentose-bound porphyra-
334 derivative [17]. The purified 478-Da MAA was acid hydrolyzed,
and the released pentose was determined by GC–MS analysis after
derivatization as described in Section 2. Penta-O-acetylcarbinol
(arabinose derivative) was detected (data shown in Supplement
Data). This finding indicates the presence of an arabinose moiety
in the 478-Da MAA. The structure was confirmed by the reexami-
nation of the NMR spectroscopic analysis (Table 2, Fig. S1). The cor-
relations in the HMBC spectra and vicinal coupling constant at the
anomer proton of 7.6 Hz confirm the presence of the b-form of
arabinose.
The HMBC correlation of the H10 proton with C7 carbon and vice
versa (Table 2, Fig. S1) indicates arabinose is linked at the C7 posi-
tion of porphyra-334. The 13C chemical shift at C50 carbon
appeared at 69.1 ppm, which showed a HMQC correlation with
two distinct proton signals at 3.66 and 3.92 ppm (Table 2). This
result indicates a pyranose-form arabinose because the methylene
protons at C50 are magnetically anisotropic. The long-range cou-
pling pattern determined by the HMBC measurements also sup-
ports the presence of arabinose in a pyranose form due to the
correlation between the lower field H50 proton and C10 carbon.
Fig. 3 shows the structure of 7-O-(b-arabinopyranosyl)-porphyra-
334 (478 Da) from the genotype A of N. commune.
The 464-Da MAA with an absorption maximum at 332 nm
(Fig. 2F) was the second major MAA in the genotype A and it rep-
resents approximately 15% of total MAAs. MALDI-TOF MS/MS anal-
ysis was performed on the parent molecular ion fragment with m/z
465 and a fragment with m/z 333. This is identical to the molecular
mass of shinorine [45,46] detected in the second MS (Table S1). The
Fig. 3. The structure of the 478-Da MAA, 7-O-(b-arabinopyranosyl)-porphyra-334,
isolated from the genotype A Nostoc commune. The structure of the sugar moiety of
pentose in the 478-Da MAA has been reexamined and updated from our previous
report [17].
neutral loss of 132 Da suggested the deletion of a pentose (C5H8O4)
from the fragment with m/z 465. Table S1 shows a summary of the
MALDI-TOF MS/MS analysis of the 464-Da MAA and strongly sug-
gests that the 464-Da MAA is
derivative.
a pentose-bound shinorine
Our prediction was confirmed by NMR spectroscopic analysis.
In the 13C NMR spectrum of the 464-Da MAA, the known chemical
shifts for shinorine [39,47] were observed in the 464-Da MAA
(Table S2). This finding suggests a shinorine scaffold in the 464-
Da MAA. Due to the contamination of oligosaccharides from the
extracellular polysaccharides it may be difficult to separate shino-
rine with our HPLC conditions. Therefore, it was difficult to use 2D
NMR analysis to determine the precise structure of the 464-Da
Table 2
Summary of the NMR analysis of the 478-Da MAA.
Position
1H (ppm)
Number of H
Multiplet
J-value (Hz)
13C (ppm)
HMBC correlation
1
2
3
161.6
128.4
163.2
35.9
4a
4b
5
2.83
2.94
1H
1H
d
d
ꢀ17.4
ꢀ17.4
C2, C3, C5, C6, C7
C2, C3, C5, C6
73.1
36.4
6a
6b
7a
7b
8
2.83
3.01
3.66
3.90
3.70
4.06
1H
1H
1H
1H
3H
1H
d
d
d
d
s
ꢀ17.4
ꢀ17.4
ꢀ10.2
ꢀ10.2
C1, C2, C4, C5, C7
C1, C2, C4, C5
C4, C5, C6, C10
C4, C5, C6, C10
C2
77.7
62.2
49.5
9
s
C3, C10
10
11
12
13
14
177.7
67.3
178.2
71.0
4.09
1H
d
4.2
C1, C12, C13, C14
4.31
1.26
1H
3H
dq
d
4.2, 6.0
6.0
22.2
C11, C13
10
20
30
40
4.37
3.61
3.68
3.95
3.66
3.92
1H
1H
1H
1H
1H
1H
d
7.2
7.2, 9.6
9.6, 3.6
3.6, 1.2, 2.4
1.2, ꢀ13.2
2.4, ꢀ13.2
106.4
73.5
75.0
71.1
69.1
C7
dd
dd
ddd
dd
dd
C10, C30
C30
C40
C10, C30, C40
a
50
b
50
NMR spectra were recorded with a JEOL ECA600 spectrometer in D2O as a solvent.
a
Higher field signals.
Lower field signals.
b

160
E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
MAA. We then performed CE-MS analysis on the 464-Da MAA to
rule out the possibility that the oligosaccharides might be cova-
lently bound to a shinorine scaffold. A single peak with m/z 465
was detected on the electropherogram and this confirmed its
molecular mass of 464 Da (data not shown). In the 464-Da MAA,
pentose is likely bound to the C7 position of shinorine via an O-gly-
coside bond similar to the 478-Da MAA as an arabinose-bound por-
phyra-334 derivative. There was a chemical shift at the C7 position
that was downfield shifted to 77.7 ppm (Table S2).
The 508-Da MAA with an absorption maximum at 334 nm
(Fig. 2H) was also purified and its estimated amount was only 3%
of total MAAs. MALDI-TOF MS/MS analysis was performed on the
parent molecular ion fragment with m/z 509 and a fragment with
m/z 347, which is identical to the molecular mass of porphyra-334
[45,46] that was detected in the second MS (Table S3). The neutral
loss of 162 Da suggested the deletion of a hexose (C6H10O5) from
the fragment with m/z 509. Table S3 shows a summary of the
MALDI-TOF MS/MS analysis for the 508-Da MAA. These fragmenta-
tion patterns were similar to those from the MS analyses of known
MAAs [48]. These data suggest that the 508-Da MAA is a hexose-
bound porphyra-334 derivative. The NMR analysis of the 508-Da
MAA from genotype A could not be performed due to the limitation
of the sample purified. Therefore, we compared the retention times
of the known hexose-bound porphyra-334 isoforms using an ana-
lytical HPLC. The data showed different retention times (Fig. S2)
and suggested the different chemical structures were due to either
variety of hexose or a different binding position of hexose to por-
phyra-334. In the 508-Da MAA from the genotype A of N. commune,
hexose is likely to be bound to the C13 position of porphyra-334
via an O-glycoside bond. However, this tentative structure must
be examined in a future study.
The 347-Da MAA with an absorption maximum at 333 nm
(Fig. 2G) was purified and its estimated amount was 1% of total
MAAs. Its kmax, molecular mass and retention time on the HPLC
chromatogram were identical to those of porphyra-334. This find-
ing indicated that the 347-Da MAA is porphyra-334 from N.
commune.
In genotype A of N. commune we found 7-O-(b-arabinopyrano-
syl)-porphyra-334 (478 Da), pentose-bound shinorine (464 Da),
hexose-bound porphyra-334 (508 Da) and porphyra-334
(346 Da). Fig. 3 shows their chemical structures and one plausible
biosynthetic pathway. It has been reported that 4-deoxygadusol is
a common precursor for the biosynthesis of MAAs [20,25,49]. The
4-deoxygadusol could conjugate with a nitrogen substituent (e.g.,
amino acids) to generate MAAs [20,26,27]. The gene product of
Ava-3856 from Anabaena variabilis can convert 4-deoxygadusol
and glycine into mycosporine–glycine [49]. The condensation of
mycosporine–glycine with an amino acid would be a common
reaction in the generation of amino acid bi-substituted MAAs such
as shinorine and porphyra-334 because most contain a glycine res-
idue [20,49].
In our predicted pathway for the 478-Da MAA producer of the
genotype A (Fig. 4), porphyra-334 and shinorine could be gener-
ated via the addition of threonine and serine, respectively, to the
mycosporine–glycine intermediate. The following glycosylation of
porphyra-334 and shinorine could produce the 478-Da MAA, the
508-Da MAA and the 464-Da MAA. The glycosylation of the core
ring of mycosporine–glycine may occur and there is no evidence
Fig. 4. A plausible biosynthetic pathway for the glycosylated MAAs in the genotype A of Nostoc commune. The addition of arabinose to porphyra-334 produces 7-O-(b-
arabinopyranosyl)-porphyra-334 (478 Da) as a main MAA. A hexose-bound porphyra-334 (508 Da) is also produced by the glycosylation of prophyra-334. Shinorine can be
produced via conjugation of mycosporine–glycine and serine and the following glycosylation produces a pentose-bound shinorine (464 Da) as a minor pathway. The
structures of the sugar moieties in the pentose-bound shinorine (464 Da) and the hexose-bound porphyra-334 (508 Da) have not yet determined.

E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
161
to rule out this possibility. In the proposed biosynthetic pathway,
the glycosylation used to produce the pentose-bound porphyra-
334 and shinorine derivatives is a unique process in genotype A
of N. commune. Additional molecular genetic studies to identify
the genes involved in the glycosylation will elucidate the biosyn-
thesis of the glycosylated MAAs unique in the terrestrial cyanobac-
terium N. commune.
The 1050-Da MAA has a characteristic absorption maximum at
312 nm and an associated shoulder approximately 340 nm
(Fig. 5G) and was a main MAA in genotype B of N. commune. The
1050-Da MAA accounted for approximately 43% of the total MAAs
according to the estimation from the HPLC chromatogram. The
1050-Da MAA has previously been characterized and its unique
structure consists of two distinct chromophores of 3-aminocycloh-
exen-1-one and 1,3-diaminocyclohexen and two pentose and hex-
ose sugars [17]. The structure was reexamined using the purified
1050-Da MAA as described below in the text.
3.3. MAAs in genotype B
The 880-Da MAA has an absorption maximum at 331 nm and is
associated with a shoulder at 312 nm (Fig. 5E). The 273-Da MAA
has an absorption maximum at 310 nm (Fig. 5F). Both MAAs were
found and purified from genotype B of N. commune. The contents of
these MAAs varied in samples and the estimated amounts were
approximately 16% (880-Da MAA) and 8% (273-Da MAA), respec-
tively. Because there are no MAAs with a molecular mass of
The water extract from genotype B of N. commune showed a
characteristic UV-absorbing spectrum with double absorption
maxima at 312 and 340 nm. The extract was examined by a pre-
parative HPLC system and three different MAAs including 1050-
Da MAA, 880-Da MAA and 273-Da MAA were identified (Fig. 5).
Each peak was collected and the purity was examined by an ana-
lytical HPLC (Fig. 5B–D). The molecular masses were determined
by MALDI-TOF–MS/MS analysis.
A
0
40
80
120
160
Retention time,min
B
C
D
40
40
0
20
min
0
20
min
40
0
20
min
λmax= 331 nm
λmax= 310 nm
λmax= 312 nm
E
F
G
0.5
0.2
0.5
0.2
0.5
0.2
-0.1
-0.1
-0.1
250
350
450
250
350
450
250
350
450
Wavelength, nm
Wavelength, nm
Wavelength, nm
Fig. 5. HPLC chromatograms and absorption spectra of the purified MAAs from Nostoc commune colony with absorption maxima at 312 and 340 nm (genotype B). (A) The
crude water-soluble extract was separated by an HPLC system equipped with a reverse phase column (IRICA C18, 20 ꢁ 250 mm, Shiseido Irica Technology) as described in
Materials and Methods. The purified 880-Da MAA was analyzed by an HPLC system equipped with a reverse phase column (Inertsil ODS-3, 4.6 mm ꢁ 250 mm; GL Sciences
Inc.) using 0.2% acetic acid at a flow rate of 1 ml minꢀ1 as the mobile phase and were detected by the A330 (B). The 273-Da MAA (C) and 1050-Da MAA (D) were analyzed by
the same HPLC system but 5% methanol with 0.2% acetic acid was used as the mobile phase. The purified 880-Da MAA (E), 273-Da MAA (F) and 1050-Da MAA (G) in water
showed absorption maxima at 331 nm, 310 nm and 312 nm, respectively.

162
E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
880 Da nor 273 Da reported in the literature their chemical struc-
tures were further characterized in the present study.
The mass spectra analyses and 13C NMR analysis indicated that
the ornithine moiety connected two 3-aminocyclohexen-1-one
and a 1,3-diaminocyclohexen chromophores to construct the scaf-
fold of the 1050-Da MAA. In HMBC analysis, the C1 and C3 atom in
the central imine-type ring showed a correlation with methylene
protons (H22 and H9) at C22 and C9 carbon, respectively (Table 3,
3.3.1. 1050-Da hybrid MAA
The 1050-Da MAA is a unique hybrid MAA with double absorp-
tion maxima at 312 and 340 nm and is consistent with the pres-
Fig. S3). Furthermore, the
at 179.1 and 179.2 ppm (C13 and C26) and showed a correlation
with the -proton (H12, H25) instead of H9 and H22 (Table 3,
Fig. S3). These results suggest the d-amino group in ornithine is
linked to the central ring of 1,3-diaminocyclohexen and the
a-carboxyl group in ornithine appeared
ence
of
two
3-aminocyclohexen-1-one
and
a
1,3-
diaminocyclohexen chromophores [17]. In the previous report,
we proposed the tentative structure of the 1050-Da MAA but its
details, especially the structure of the disaccharide moiety, was
not characterized [17].
a
a-
Table 3
Summary of the NMR analysis of the 1050-Da MAA.
Position
1H (ppm)
Number of H
Multiplet
J-value (Hz)
13C (ppm)
HMBC correlation
1
2
162.0
128.4
162.3
36.28
3
4a
2.76⁄⁄
3.02
1H
1H
d
d
ꢀ15.0
ꢀ17.4
C2, C3, C5, C6
C1, C2, C4, C5
4b
5
73.5
36.33
6a
2.81⁄⁄
3.06
3.70
3.91
3.65
3.41
1.68
1.98
4.26
1H
1H
1H
1H
3H
2H
2H
2H
1H
d
d
d
d
ꢀ17.4
ꢀ17.4
10.2
6b
7a
77.8
7b
10.8
8
9
s
62.4
45.2
29.3
C2
C3
m
m
m
m
10
11
12
13
14
15
16
17a
17b
18
19a
19b
20
21
22
23
24
25
26
27
28
29
30a
30b
31
32a
32b
33
34
32.2à
61.0àà
179.1ààà
162.4
132.9
187.1⁄
45.6
C11, C13
2.34
1H
1H
d
d
ꢀ16.8
ꢀ17.4
C15, C16, C18, C19, C20
C14, C15
2.65⁄⁄⁄⁄
75.1
35.7
2.78⁄⁄⁄
2.85
3.53
3.55
3.41
1.68
1.98
4.26
1H
1H
2H
3H
2H
2H
2H
1H
d
d
s
s
m
m
m
m
ꢀ16.2
ꢀ17.4
70.6
62.2
45.2
29.3
C17, C18, C19
C15
C1
32.4à
61.2àà
179.2ààà
162.5
132.9
187.2⁄
45.7
C24, C26
2.34
1H
1H
d
d
ꢀ16.8
ꢀ16.8
C28, C29, C31, C32, C33
C27, C28
2.66⁄⁄⁄⁄
75.1
35.7
2.79⁄⁄⁄
2.85
3.53
1H
1H
2H
3H
d
d
s
ꢀ16.8
ꢀ17.4
70.6
62.2
C30, C31, C32
C28
3.55
s
10
20
30
40
50
60
4.50
3.69
3.82
3.93
3.68
3.77
1H
1H
1H
1H
1H
2H
d
–
–
–
–
–
7.2
–
–
–
–
105.0
83.1
75.7
71.6
78.1
63.9
C7
C10, C100
C20, C30
C60
–
100
200
300
400
4.61
3.25
3.41
3.72
3.25
3.78
1H
1H
1H
1H
1H
1H
d
–
–
–
–
–
8.4
–
–
–
–
107.6
76.8
78.5
72.1
68.4
C20
C100
C200, C400
a
500
C300
b
500
–
NMR spectra were⁄r⁄ec⁄o⁄⁄rded with a JEOL ECA600 spectrometer in D2O as a solvent.
à
àà ààà
⁄
, ,
,
,
,
,
,
,
,
⁄⁄⁄⁄: Exchangeable signals.
– Not analyzed due to the overlapping signals.
a
Higher field signals.
Lower field signals.
b

E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
163
amino group is linked to the exterior ring of 3-aminocyclohexen-1-
one.
The sugar composition of the 1050-Da MAA was analyzed as
described in Materials and Methods. The two monosaccharide
derivatives hexa-O-acetylgalactitol (galactose derivative) and
penta-O-acetylxylitol (xylose derivative) were detected (data
shown in Supplement Data). These results indicate the presence
of galactose and xylose moieties in the 1050-Da MAA. The coupling
scheme and the details of the disaccharide structure were further
characterized by the reexamination of the NMR spectroscopic
analysis.
Chemical shift values of the anomer carbon of galactose
(105.0 ppm) and xylose (107.6 ppm) in the 1050-Da MAA (Table 3)
were similar to 1-O-methyl-b-
and 1-O-methyl-b- -xylopyranoside (105.1 ppm) rather than 1-O-
methyl- -galactopyranoside (100.1 ppm) and 1-O-methyl-
D-galactopyranoside (104.5 ppm)
D
a
-D
a-D-
xylopyranoside (100.6 ppm), respectively. Furthermore, the vicinal
coupling constants of the anomeric proton of galactose and xylose
were 7.2 and 8.4 Hz, respectively (Table 3) and are much larger
Fig. 6. The structure of the 1050-Da hybrid MAA isolated from the genotype B
Nostoc commune. The structure of disaccharide moiety has been reexamined and
updated, thus it is different from our previous report [17].
than that of a typical a-form. These results indicate that both steric
structures at anomer carbons are b form. The 13C chemical shift at
C500 carbon in the xylose residue appeared at 68.4 ppm and showed
a HMQC correlation with two distinct proton signals at 3.25 and
3.78 ppm (Table 3). The data indicate a pyranose-form xylose
because the methylene protons at C500 are magnetically
anisotropic.
acid scaffold, which may also be produced via glycosylation of a
mycosporine–ornithine molecule (Fig. 7). The glycosylation may
occur after assembling the core structure consisting of three rings.
There is no evidence to specify when it is glycosylated and where
the glycosylation reactions take place. Assuming that the glycosyl-
ation occurs at the initial step to produce a glycosylated 4-deoxyg-
adusol scaffold, two mycosporine–ornithine molecules can
successively be conjugated via imination to produce the final prod-
uct of the 1050-Da MAA. In this alternative pathway, the glycosyl-
ated 4-deoxygadusol molecule will be a core ring to link with two
mycosporine–ornithine molecules at their d-amino groups. The
enzymes and their responsible genes for the predicted biosynthetic
pathway of the 1050-Da MAA in N. commune are unknown. Thus,
further molecular genetic studies are required to elucidate their
identity.
In the 13C NMR spectrum, the characteristic signal appeared at
83.1 ppm and we were able to assign it to the C20 carbon in galact-
ose (Table 3). According to the HMQC analysis, this typical peak at
83.1 ppm showed a correlation with the proton at 3.69 ppm and
with both anomer carbons in the HMBC analysis. This result indi-
cates that the carbon at 83.1 ppm must be present between two
anomer carbons (C10 and C100). The correlation signal between the
anomer carbon of galactose (C10) and the methylene proton at C7
in the central imine-type ring was observed (Table 3). The data
suggest the galactose binds to the central ring at the anomer posi-
tion and xylose links the C20 carbon of galactose with the chemical
shift at 83.1 ppm (Fig. S3). The assignment of the signal appearing
at 83.1 ppm is a key to identifying the disaccharide structure of the
1050-Da MAA. We further examined the data to confirm the cou-
pling scheme of the disaccharide.
3.3.2. 880-Da MAA
An MAA with an absorption maximum at 331 nm associated
with a shoulder at 312 nm (Fig. 5E) was purified (Fig. 5B). Its
molecular mass of 880 Da and it has a double absorption maxima
at 312 and 331 nm, which suggests it is a hybrid structure consist-
ing of both 3-aminocyclohexen-1-one and 1,3-diaminocyclohexen
chromophores. The absorption coefficient of the 880-Da MAA was
56.65 l gꢀ1 cmꢀ1 at 331 nm in water. The calculated molar absorp-
The DEPT 90 spectrum showed the carbon at 83.1 ppm was
assigned to the methine (CH) carbon (data shown in Supplement
Data), indicating that xylose does not bind at the C60 position.
The 13C chemical shifts of the galactose moiety in the 1050-Da
MAA were compared to those of galactose derivatives and we
tion coefficient at 331 nm was 4.98 ꢁ 104 Mꢀ1 cmꢀ1
.
found the 1,2-O-dimethyl-b-
galactopyranose moiety in 1-O-methyl-2-O-fucopyranosyl-b-
D
-galactopyranoside and the b-
D
-
-
The IR spectrum of the 880-Da MAA was compared to the 1050-
Da MAA (Table S5). Similar IR absorption peaks suggest that the
880-Da MAA has a similar structure to the 1050-Da MAA. The
absorption peak at 1548 cmꢀ1 in the 880-Da MAA indicated the
presence of the conjugated imine is a characteristic structure of
MAA chromophore and this corresponds to the 1541 cmꢀ1 peak
in the 1050-Da MAA (Table S5). The absorption peaks in the range
of 1200–1300 cmꢀ1 and the characteristic absorption peak at
3374 cmꢀ1 in the 880-Da MAA indicated the structure contains
sugar(s) and several hydroxyl functional groups, respectively
(Table S5).
D
galactopyranoside (Table S4). The results suggest the b-xylopyra-
nose link is located at the C20 position of the galactose moiety.
Fig. 6 shows a plausible structure of the 1050-Da MAA. The pro-
posed chemical structure of the 1050-Da MAA, especially the
disaccharide moiety, has been updated based upon the reexamina-
tion of NMR data. We proposed that xylose binds to the C20 posi-
tion of galactose as a pyranose form (Fig. 6). In our previous
report we have assumed the pentose (furanose form) binds to hex-
ose at the C6’ position in the 1050-Da MAA [17].
Fig. 7 shows a plausible biosynthetic pathway for the 1050-Da
MAA in the genotype B of N. commune. Mycosporine–ornithine is
an assumed intermediate used to produce the 1050-Da MAA. It
can be generated via the addition of ornithine to 4-deoxygadusol,
which is a common precursor in MAA biosynthesis (Fig. 7). The
symmetrical structure consists of three mycosporine rings and
two ornithine molecules and can be generated via two consecutive
conjugation reactions. During this reaction two mycosporine–orni-
thine molecules are added to a glycosylated mycosporine-amino-
The fragmentation pattern of the 880-Da MAA in the MALDI-
TOF MS/MS analysis (Table S6) was similar to the 1050-Da MAA.
The difference was the fragments resulted from the 880-Da MAA
were 170 Da less than those from the 1050-Da MAA [17]. This
molecular mass of 170 Da is identical to 4-deoxygadusol, which
is a precursor for the biosynthesis of MAA. These results suggest
that the hydrolysis of the 1050-Da MAA to remove a cyclohexe-
none chromophore (170 Da) could produce the 880-Da MAA
(Fig. S4). Thus, the double absorption maxima of 1050-Da MAA

164
E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
Fig. 7. A plausible biosynthetic pathway for the 1050-Da MAA in the genotype B of Nostoc commune. R1 represents an amino acid, e.g., ornithine. R2 represents the glycosyl
group and 2-O-(b-xylopyranosyl)-b-galactopyranoside is bound at this position of the final product of the 1050-Da MAA.

E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
165
at 312 nm associated with a shoulder approximately 340 nm
(Fig. 4G) changes to the absorption maximum at 331 nm associ-
ated with a shoulder at 312 nm for the 880-Da MAA (Fig. 5E).
The MALDI-TOF MS/MS analysis of the parent molecular ion
fragment with m/z 881 indicated the presence of a hexose and a
pentose in its structure similar to the 1050-Da MAA (Table S6).
There are also galactose and xylose moieties in the 880-Da MAA
that were confirmed by sugar composition analysis (data shown
in Supplement Data).
MAA symmetric molecule produces asymmetric molecules like
the 880-Da MAA. It is unclear whether this hydrolysis of the
1050-Da MAA is enzymatic or an artificial event. In either case, this
is the first report of the 880-Da MAA with both 3-aminocyclohex-
en-1-one and 1,3-diaminocyclohexen chromophores.
3.3.3. Mycosporine-GABA
The 273-Da MAA was purified from genotype B of N. commune
(Fig. 5C) and its chemical structure was characterized. The UV
absorption spectrum of the purified 273-Da MAA showed a single
absorption peak at 310 nm (Fig. 5F) and its absorption coefficient
was 106.05 l gꢀ1 cmꢀ1 at 310 nm in water. An accurate molecular
mass was determined by FAB MS to predict the elemental compo-
sition. A molecular ion fragment with m/z 274.1295 was detected
The 13C NMR spectrum of the 880-Da MAA was similar to the
1050-Da MAA (Table S7). However, the signal number of the
880-Da MAA was greater than the 1050-Da MAA. This result was
inconsistent with the assumption that the 880-Da MAA is a break
down compound of the 1050-Da MAA. In the spectrum of the 880-
Da MAA there are multiple signals we were able to assign to the
ornithine moiety (C11, C24 and C12, C25) and they appeared close
together (Table S7). This finding suggests the purified 880-Da MAA
may be a mixture of isomers whose structures are different at the
ornithine moiety.
and its predicted molecular formula was
C12H20NO6 within
1 ppm error. The calculated molar absorption coefficient at
310 nm was 2.89 ꢁ 104 Mꢀ1 cmꢀ1
.
The IR spectrum of the 273-Da MAA was compared with known
MAAs methyl ester of mycosporine–glycine [50] and porphyra-334
[51] (Table S8). Similar IR absorption peaks were observed and this
result suggests that the 273-Da MAA has a similar structure to
these MAAs.
Mycosporine–glycine is known to be hydrolyzed to form 4-
deoxygadusol and glycine [50]. Both peripheral carbonyl-type
rings in the 1050-Da MAA are thought to be susceptible to hydro-
lysis. If one peripheral carbonyl-type ring is removed from the
1050-Da MAA by hydrolysis (Fig. S4) then the symmetric molecule
changes to asymmetric molecules with a chiral center at C5 carbon
in the central ring of the 880-Da MAA (Fig. S5). Three substituent
groups on the central imine-type ring of the 880-Da MAA, labeled
by X, Y and Z, have chiral centers individually (Fig. S5). To test this
assumption of the 880-Da MAA, CE-MS analysis was performed on
the purified 880-Da MAA sample. A symmetrical single peak of the
880-Da MAA was detected on the electropherogram when ana-
lyzed at 6pH 7 and this result confirmed its purity. At pH 11, the
peak derived from the 880-Da MAA was split on the electrophero-
gram and suggests the mixture of isomers with the identical m/z
881. The 880-Da MAA was purified using an octadecylsilyl (ODS)
column and a symmetrical single peak of the purified 880-Da
MAA was recorded (Fig. 5B), which suggests these isomers are dif-
ficult to separate on a regular ODS column. Thus, HPLC analysis
was performed using another type of a reverse phase column with
the cholesteryl-bonded silica gel (COSMOSIL Chorester, Nacalai).
Consistent with the result of CE-MS analysis, two peaks with sim-
ilar signal intensities were detected when the purified 880-Da
MAA was analyzed using the HPLC system with the cholesteryl-
bonded silica gel column (Fig. S6B). These results demonstrate that
the purified 880-Da MAA contains a pair of diastereomer in equal
amounts and confirm the idea that hydrolysis of the 1050-Da
MALDI-TOF MS/MS analysis was performed on the molecular
ion fragment with m/z 274 and fragments with m/z 186, m/z 137
and m/z 87 were detected (Table S9). The fragment with m/z 137
could be a divalent ion of the 273-Da MAA. The fragment with
m/z 87 was thought to be a c-aminobutyric acid chain. The frag-
ment with m/z 186 corresponded to the remaining 3-aminocycloh-
exen-1-one scaffold. This fragmentation pattern of the elimination
of an amino-acid side chain was common in the results of the MS
analysis of the known MAAs [45,48,52].
Table 4 shows the summary of 13C and 1H NMR analysis of the
273-Da MAA. Signals with identical chemical shifts to mycospo-
rine–glycine [50] were observed in the 273-Da MAA, indicating
the presence of the oxocarbonyl-type chromophore (C1, 2, 3, 4, 5,
6, 7, 8). In addition, signals with identical chemical shifts to
nobutyric acid (C9, 10, 11, 12) were observed. These data indicate
the -aminobutyric acid is a side chain linked to 3-aminocyclohex-
c-ami-
c
en-1-one chromophore (Fig. S7). These results are consistent with
the MS analysis of the 273-Da MAA. This scaffold was named as
mycosporine-GABA. This is the first report of the unique mycospo-
rine-GABA structure, which is not listed in the previously reported
MAAs [21–27].
c-aminobutyric acid can be generated via decar-
boxylation of glutamic acid and the addition of
c
-aminobutyric
acid to 4-deoxygadusol may produce mycosporine-GABA. The
identification of the mycosporine-GABA in the genotype B of N.
Table 4
Summary of the NMR analysis of the 273-Da MAA.
Position
1H (ppm)
Number of H
Multiplet
J-value (Hz)
13C (ppm)
COSY correlation
HMBC correlation
1
2
3
185.7
131.1
159.8
33.7
4a
4b
5
2.74
2.86
1H
1H
d
d
17.4
17.4
H4b
H4a
C3
73.1
44.0
6a
6b
7
2.36
2.64
3.51
3.55
3.39
1.90
2.44
1H
1H
2H
3H
2H
2H
2H
d
d
s
s
t
17.4
17.4
H6b
H6a
C1, C5
68.7
60.0
43.0
26.2
32.1
178.9
C4, C5, C6
C2
8
9
10
11
12
6.7
6.7, 7.1
7.1
H10
C3
tt
t
H9, H11
H10
C9, C11, C12
C9, C12
NMR spectra were recorded with a JEOL ECS400 spectrometer in 30% methanol-d4 in D2O as a solvent.
a
Higher field signals.
Lower field signals.
b

166
E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
commune provides one new molecule to the characterized MAAs.
However, the total amount was a small portion of all MAAs
(approximately 8% or less).
imately 310 nm (Fig. 5E–G). The results indicate the presence of 3-
aminocyclohexen-1-one chromophore in their structures [27]. The
double absorption maxima of the purified 1050-Da MAA (Fig. 5G)
and the purified 880-Da MAA (Fig. 5E) are caused by 1,3-diamino-
cyclohexen and 3-aminocyclohexen-1-one chromophores in their
structures (Fig. 6, Fig. S5). These chromophores expand their UV-
absorbing range covering UV-A/B wavelengths. The hybrid-type
MAAs exclusively occur in genotype B of N. commune and have
not been reported in any other organisms. Ecophysiological roles
specific for the hybrid MAAs are unknown and microbial ecological
features for the genotype B of N. commune remain to be studied.
We have proposed the biosynthetic pathway of the 1050-Da hybrid
MAA (Fig. 7) and this will be examined to identify the genes and
enzymes involved by molecular biological and biochemical
approaches in future studies.
The different MAA contents in these two types of N. commune
suggest there is molecular diversity of MAAs in N. commune and
they are genetically different to produce their type-specific MAAs.
Consistent with this idea, it has been reported that the genotype D
of N. commune type-specifically produces the 612-Da MAA, which
is a glycosylated palythine–threonine derivative [19]. Four major
genotypes of N. commune have been reported and their 16S rRNA
sequences show >96% identity and they are recognized as the same
species [37]. Thus, the diversity of these MAAs in N. commune are
unexpectedly high. We have characterized a variety of MAAs in
the genotype A and B (this study) and in genotype D [19]. The vari-
ety of the MAA contents in the genotype C of N. commune remains
to be characterized. In order to generalize the relation between the
genotypes and the MAA types, further studies on MAA contents
and their biosynthetic pathways among the different genotypes
of N. commune are ongoing and will be reported in future studies.
3.4. Radical scavenging activity in MAAs from N. commune
Table 5 shows the radical scavenging activity detected in the
purified 880-Da MAA and 273-Da MAA. We compared the activi-
ties in the previously reported MAAs from N. commune. Both
showed ABTS radical scavenging activities when we monitored
the decolorization of ABTS radical. During the time course experi-
ments, the decolorization of ABTS radical increased as incubation
times increased from 10 min to 2 h. These findings suggest the
MAAs are slow-acting radical scavengers and may be similar to
the other known MAAs from N. commune. The molecule used as a
standard, Trolox, is known to be a fast-acting scavenger and the
reaction is completed within 10 min. Based on these results, the
incubation time was fixed for 1 h in the assay as described by Mat-
sui et al. [17]. We directly monitored the decrease of ABTS radical
cation by ESR and found higher activities than those measured by
the colorimetry assay (Table 5). The IC50 values by the colorimetry
assay maybe underestimated because of changes of A734 not due to
the reaction, e.g. the increase of light scattering during the incuba-
tion for 1 h. These results indicate that the 880-Da MAA and 273-
Da MAA are strong radical scavengers in vitro and are consistent
with previous reports of MAAs from N. commune [17,19].
4. Discussion
4.1. Chemical diversity of MAAs in N. commune
Two types of N. commune with water extracts that showed dif-
ferent UV-absorbing spectra were examined and several distinct
MAAs were identified (Figs. 1, 2 and 5). One type of N. commune
showed a single absorption maximum at 335 nm in the water
extract. This genotype was identified as genotype A and produced
7-O-(b-arabinopyranosyl)-porphyra-334 (478 Da), pentose-bound
shinorine (464 Da), hexose-bound porphyra-334 (508 Da) and por-
phyra-334 (346 Da) (Fig. 4). All of these compounds have absorp-
tion maxima approximately 330 nm (Fig. 2F–I), indicating the
presence of 1,3-diaminocyclohexen chromophore conjugated with
two nitrogen substituent via C1 and 3 in their structures [27]. The
other N. commune colonies had double absorption maxima at 312
and 340 nm in the water extract and were identified as genotype
B. This genotype produced the 1050-Da, 880-Da and 273-Da MAAs
(Fig. 6, Figs. S4 and S7), which all have absorption maxima approx-
4.2. MAAs as potent antioxidative compounds
UV radiation causes oxidative damages in cyanobacterial cells
including lipid peroxide formation, DNA strand breaks, and chloro-
phyll bleaching concomitant with deactivation of photosynthesis
and growth inhibition [53]. MAAs are suggested to have a protec-
tive role against UV-induced oxidative stress in algae [54] and cya-
nobacteria [55]. It has been reported that oxocarbonyl-type MAAs
such as mycosporine–glycine [56–58] and mycosporine–taurine
[55] show radical scavenging activities in vitro. The 273-Da MAA
(mycosporine-GABA) is
a non-glycosylated oxocarbonyl-type
MAA consisting of a cyclohexenone chromophore with a high
activity (Table 5). Conversely, porphyra-334 has photoprotective
properties but not antioxidant functions [30,56]. This suggests that
imine-type MAAs are not antioxidative compounds. We confirmed
that no radical scavenging activity was detected in porphyra-334
using our assay system (data not shown) that routinely accesses
the activities in the glycosylated MAAs.
In N. commune, we have demonstrated that the glycosylated
MAAs show radical scavenging activities in vitro and suggested that
the glycosylation of porphyra-334, shinorine and palythine–threo-
nine provide the antioxidant function of these imine-type MAAs
(Table 5). The hybrid-type MAAs such as the 1050-Da MAA and
the 880-Da MAA have potent radical scavenging activities in vitro
(Table 5). The presence of the cyclohexenone chromophore in the
hybrid-type MAAs suggests their radical scavenging activities.
However, it remains to be elucidated whether the glycosylation
enhances the activities. The energy absorbed by MAAs is dispersed
as heat [30,31] without generating reactive oxygen species (ROS).
Thus, it will be interesting to determine whether UV radiation
affects the radical scavenging activity of these glycosylated MAAs
in N. commune. Future in vitro studies will answer this question
and provide further evidence of the multifunctional abilities of
these compounds.
Table 5
Radical scavenging activity of MAAs.a
Assay
Colorimetryb
ESRc
References
IC50 (mM)
Ascorbic acidd
0.28
0.25
0.57
0.60
9.5
58
16
0.51
1.04
0.16
0.17
0.036
0.11
0.18
29
0.25
0.08
0.05
[This study]
[This study]
[18]
[This study]
[17]
[19]
[19]
[This study]
[17]
Troloxd
Scytonemine
273-Da MAA (kmax, 310 nm)
478-Da MAA (kmax, 335 nm)
508-Da MAA (kmax, 334 nm)
612-Da MAA (kmax, 322 nm)
880-Da MAA (kmax, 331 nm)
1050-Da MAA (kmax, 312 nm)
IC50 (50% inhibitory concentration) values are shown.
a
Radical scavenging activity was measured with ABTS radical cation as the
organic radical source.
Decolorization of ABTS radical was monitored with a spectrophotometer for 1 h.
ESR signals were monitored with a free radical monitor (JEOL JES-FR30EX).
Ascorbic acid and Trolox were used as standards.
b
c
d
e
A hydrophobic UV-absorbing pigment (544 Da) isolated from N. commune.

E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
167
[2] R.F. Helm, M. Potts, Extracellular Matrix (ECM), in: B.A. Whitton (Ed.), Ecology
of Cyanobacteria II, Kluwer Academic Publishers, Dordrecht, Netherlands,
2012, pp. 461–480.
[3] C.B. Lipman, The successful revival of Nostoc commune from a herbarium
specimen eighty-seven years old, Bull. Torr. Bot. Club 68 (1941) 664–666.
[4] R.E. Cameron, Species of Nostoc vaucher occurring in the Sonoran Desert in
Arizona, Trans. Am. Microsc. Soc. 81 (1962) 379–384.
[5] S. Scherer, A. Ernst, T.-W. Chen, P. Böger, Rewetting of drought-resistant blue-
green algae: time course of water uptake and reappearance of respiration,
photosynthesis, and nitrogen fixation, Oecologia 62 (1984) 418–423.
[6] K. Satoh, M. Hirai, J. Nishio, T. Yamaji, Y. Kashino, H. Koike, Recovery of
4.3. Glycosylated MAAs and anhydrobiosis
We have isolated and characterized 10 MAAs from N. commune
[17,19] (this study) and 8 are glycosylated. Moreover, all of the
main MAA in each genotype such as 7-O-(b-arabinopyranosyl)-
porphyra-334 (478 Da) in the genotype A, the 1050-Da hybrid
MAA in the genotype B and two hexose-bound palythine–threo-
nine (612 Da) in the genotype D are glycosylated. The finding sug-
gests that glycosylation could be requisite for the interaction of
these MAAs within the extracellular matrix and their functions in
N. commune. The glycosylation may stabilize these MAAs. How-
ever, this possibility must be demonstrated in future studies. The
known glycosylated MAAs mycosporine–glutaminol–glucoside
and mycosporine–glutamicol–glucoside have been reported in
rock-inhabiting microcolonial fungi [59] and the terrestrial cyano-
bacteria from rock surfaces [60]. These results suggest an ecologi-
cal role of glycosylated MAAs in adaptation to terrestrial
environments. Using these glycosylated MAAs N. commune may
have acquired the capabilities of surviving terrestrial environments
and sustaining viability in a desiccated state. However, biochemi-
cal mechanisms for the protection of cells by these glycosylated
MAAs remain to be elucidated.
Trehalose is a nonreducing disaccharide and is known to be an
anti-stress molecule that protects biological membranes and pro-
teins under various stressed conditions [61]. In N. commune, treha-
lose accumulation occurs in response to water loss during
desiccation [8]. The extracellular polysaccharides are main compo-
nents of the extracellular matrix in N. commune [34] and play a
major role in protecting cells during desiccation [7]. Glycosylated
MAAs may function together with trehalose and the extracellular
polysaccharides to protect cells from damage under stressed con-
ditions. The crystal structure of palythine has demonstrated that
there are twelve types of hydrogen bonds between one palythine
molecule and three water molecules [62], indicating that various
types of hydrogen bonds are formed not only intra- but also
inter-molecularly to form a three dimensional network. The hydro-
gen bond network structure with water molecules is expected to
contribute to the vitrification of cells in a desiccated condition
[15]. In N. commune, MAAs are thought to be localized in the extra-
cellular matrix and bound to extracellular polysaccharides [2] and
an extracellular matrix protein called water stress protein (WspA)
by physical interaction [32,33]. The complex structures of the
extracellular matrix consisted of polysaccharides, proteins, sugars
and MAAs may synergistically function in vitrifying to protect cells
in a desiccated condition [15]. Further studies on the glycosylated
MAAs in N. commune will be needed to address their functions in
the mechanisms of anhydrobiosis.
photosynthetic systems during rewetting is quite rapid in
a terrestrial
cyanobacterium, Nostoc commune, Plant Cell Physiol. 43 (2002) 170–176.
[7] Y. Tamaru, Y. Takani, T. Yoshida, T. Sakamoto, Crucial role of extracellular
polysaccharides in desiccation and freezing tolerance in the terrestrial
cyanobacterium Nostoc commune, Appl. Environ. Microbiol. 71 (2005) 7327–
7333.
[8] T. Sakamoto, T. Yoshida, H. Arima, Y. Hatanaka, Y. Takani, Y. Tamaru,
Accumulation of trehalose in response to desiccation and salt stress in the
terrestrial cyanobacterium Nostoc commune, Phycol. Res. 57 (2009) 66–73.
[9] M. Potts, Desiccation tolerance: a simple process?, Trend Microbiol. 9 (2001)
553–559.
[10] J.S. Clegg, Cryptobiosis – a peculiar state of biological organization, Comp.
Biochem. Physiol. Part B 128 (2001) 613–624.
[11] L.M. Crowe, Lessons from nature: the role of sugars in anhydrobiosis, Comp.
Biochem. Physiol. Part A 131 (2002) 505–513.
[12] D. Billi, M. Potts, Life and death of dried prokaryotes, Res. Microbiol. 153
(2002) 7–12.
[13] R.W. Castenholz, F. Garcia-Pichel, Cyanobacterial responses to UV-radiation,
in: B.A. Whitton, M. Potts (Eds.), The Ecology of Cyanobacteria, Kluwer
Academic Publishers, Dordrecht, Netherlands, 2000, pp. 591–611.
[14] R.W. Castenholz, F. Garcia-Pichel, Cyanobacterial responses to UV radiation, in:
B.A. Whitton (Ed.), Ecology of Cyanobacteria II, Kluwer Academic Publishers,
Dordrecht, Netherlands, 2012, pp. 481–499.
[15] N. Wada, T. Sakamoto, S. Matsugo, Multiple roles of photosynthetic and
sunscreen pigments in cyanobacteria focusing on the oxidative stress,
Metabolites 3 (2013) 463–483.
[16] P.N. Leão, N. Engene, A. Antunes, W.H. Gerwick, V. Vasconcelos, The chemical
ecology of cyanobacteria, Nat. Prod. Rep. 29 (2012) 372–391.
[17] K. Matsui, E. Nazifi, S. Kunita, N. Wada, S. Matsugo, T. Sakamoto, Novel
glycosylated mycosporine-like amino acids with radical scavenging activity
from the cyanobacterium Nostoc commune, J. Photochem. Photobiol. B. 105
(2011) 81–89.
[18] K. Matsui, E. Nazifi, Y. Hirai, N. Wada, S. Matsugo, T. Sakamoto, The
cyanobacterial UV-absorbing pigment scytonemin displays radical-
scavenging activity, J. Gen. Appl. Microbiol. 58 (2012) 137–144.
[19] E. Nazifi, N. Wada, M. Yamaba, T. Asano, T. Nishiuchi, S. Matsugo, T. Sakamoto,
Glycosylated porphyra-334 and palythine–threonine from the terrestrial
cyanobacterium Nostoc commune, Mar. Drugs 11 (2013) 3124–3154.
[20] Q. Gao, F. Garcia-Pichel, Microbial ultraviolet sunscreens, Nat. Rev. 9 (2011)
791–802.
[21] W.M. Bandaranayake, Mycosporines: are they nature’s sunscreens?, Natural
Product Reports 1998 (1998) 159–171
[22] C.S. Cockell, J. Knowland, Ultraviolet radiation screening compounds, Biol. Rev.
74 (1999) 311–345.
[23] A. Oren, N. Gunde-Cimerman, Mycosporines and mycosporine-like amino
acids: UV protectants or multipurpose secondary metabolites?, FEMS
Microbiol Lett. 269 (2007) 1–10.
[24] R.P. Rastogi, R.P. Sinha, Biotechnological and industrial significance of
cyanobacterial secondary metabolites, Biotechnol. Adv. 27 (2009) 521–539.
[25] J.M. Shick, W.C. Dunlap, Mycosporine-like amino acids and related gadusols:
Biosynthesis, accumulation, and UV-protective functions in aquatic organisms,
Annu. Rev. Physiol. 64 (2002) 223–262.
[26] S.P. Singh, D.-P. Häder, R.P. Sinha, Cyanobacteria and ultraviolet radiation
(UVR) stress: Mitigation strategies, Ageing Res. Rev. 9 (2010) 79–90.
[27] J.I. Carreto, M.O. Carignan, Mycosporine-like amino acids: relevant secondary
metabolites. chemical and ecological aspects, Mar. Drugs 9 (2011) 387–446.
[28] F. Garcia-Pichel, R.W. Castenholz, Occurrence of UV-absorbing, mycosporine-
like compounds among cyanobacterial isolates and an estimate of their
screening capacity, Appl. Environ. Microbiol. 59 (1993) 163–169.
[29] F. Garcia-Pichel, C.E. Wingard, R.W. Castenholz, Evidence regarding the UV
Acknowledgments
We thank Prof. K. Takahashi (Kanazawa Univ.) for generous
assistance with IR analysis and Prof. K. Tanaka (Tokyo Institute of
Technology) for suggestion to use antibiotics for the culture. This
work was supported by HABA Laboratories, Inc. (research grant).
sunscreen role of
a mycosporine-like compound in the cyanobacterium
Gloeocapsa sp, Appl. Environ. Microbiol. 59 (1993) 170–176.
[30] F.R. Conde, M.S. Churio, C.M. Previtali, The photoprotector mechanism of
mycosporine-like amino acids. Excited-state properties and photostability of
porphyra-334 in aqueous solution, J. Photochem. Photobiol. B. 56 (2000) 139–
144.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
[31] F.R. Conde, M.S. Churio, C.M. Previtali, The deactivation pathways of the
excited-states of the mycosporine-like amino acids shinorine and porphyra-
334 in aqueous solution, Photochem. Photobiol. Sci. 3 (2004) 960–967.
[32] D.J. Wright, S.C. Smith, V. Joardar, S. Scherer, J. Jervis, A. Warren, R.F. Helm, M.
Potts, UV irradiation and desiccation modulate the three-dimensional
extracellular matrix of Nostoc commune (cyanobacteria), J. Biol. Chem. 280
(2005) 40271–40281.
j.jphotobiol.2014.12.008.
References
[1] M. Potts, Nostoc, in: B.A. Whitton, M. Potts (Eds.), The Ecology of
Cyanobacteria, Kluwer Academic Publishers, Dordrecht, Netherlands, 2000,
pp. 465–504.
[33] D.R. Hill, S.L. Hladun, S. Scherer, M. Potts, Water stress proteins of Nostoc
commune (cyanobacteria) are secreted with UV-A/B-absorbing pigments and

168
E. Nazifi et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 154–168
associate with 1,4-b-D-xylanxylanohydrolase activity, J. Biol. Chem. 269
(1994) 7726–7734.
[48] K. Whitehead, J.I. Hedges, Electrospray ionization tandem mass spectrometric
and electron impact mass spectrometric characterization of mycosporine-like
amino acids, Rapid Commun. Mass Spectrom. 17 (2003) 2133–2138.
[49] E.P. Balskus, C.T. Walsh, The genetic and molecular basis for sunscreen
biosynthesis in cyanobacteria, Science 329 (2010) 1653–1656.
[50] S. Ito, Y. Hirata, Isolation and structure of mycosporine from the zoanthid
Palythoa tuberculosa, Tetrahedron Lett. 18 (1977) 2429–2430.
[51] A. Torres, C.D. Enk, M. Hochberg, M. Srebnik, Porphyra-334, a potential natural
source for UVA protective sunscreens, Photochem. Photobiol. Sci. 5 (2006)
432–435.
[52] K.H.M. Cardozo, V.M. Carvalho, E. Pinto, P. Colepicolo, Fragmentation of
mycosporine-like amino acids by hydrogen/deuterium exchange and
electrospray ionisation tandem mass spectrometry, Rapid Commun. Mass
Spectrom. 20 (2006) 253–258.
[53] Y.Y. He, M. Klisch, D.-P. Häder, Adaptation of cyanobacteria to UV-B stress
correlated with oxidative stress and oxidative damage, Photochem. Photobiol.
76 (2002) 188–196.
[54] T.-M. Lee, C.-T. Shiu, Implications of mycosporine-like amino acid and
antioxidant defenses in UV-B radiation tolerance for the algae species
Ptercladiella capillacea and Gelidium amansii, Mar. Environ. Res. 67 (2009) 8–16.
[55] L. Zhang, L. Li, Q. Wu, Protective effects of mycosporine-like amino acids of
Synechocystis sp. PCC 6803 and their partial characterization, J. Photochem.
Photobiol. B. 86 (2007) 240–245.
[56] W.C. Dunlap, Y. Yamamoto, Small-molecule antioxidants in marine organisms:
antioxidant activity of mycosporine-glycine, Comp. Biochem. Physiol. B 112
(1995) 105–114.
[34] F.M. Morsy, S. Kuzuha, Y. Takani, T. Sakamoto, Novel thermostable
glycosidases in the extracellular matrix of the terrestrial cyanobacterium
Nostoc commune, J. Gen. Appl. Microbiol. 54 (2008) 243–252.
[35] T. Yoshida, T. Sakamoto, Water-stress induced trehalose accumulation and
control of trehalase in the cyanobacterium Nostoc punctiforme IAM M-15, J.
Gen. Appl. Microbiol. 55 (2009) 135–145.
[36] T. Sakamoto, K. Kumihashi, S. Kunita, T. Masaura, K. Inoue-Sakamoto, M.
Yamaguchi, The extracellular-matrix-retaining cyanobacterium Nostoc
verrucosum accumulates trehalose, but is sensitive to desiccation, FEMS
Microbiol. Ecol. 77 (2011) 385–394.
[37] H. Arima, N. Horiguchi, S. Takaichi, R. Kofuji, K. Ishida, K. Wada, T. Sakamoto,
Molecular genetic and chemotaxonomic characterization of the terrestrial
cyanobacterium Nostoc commune and its neighboring species, FEMS Microbiol.
Ecol. 79 (2012) 35–45.
[38] R.W. Castenholz, Culturing methods for cyanobacteria, Methods Enzymol. 167
(1988) 68–93.
[39] C. Oyamada, M. Kaneiwa, K. Ebitani, M. Murata, K. Ishihara, Mycosporine-like
amino acids extracted from scallop (Patinopecten yessoensis) ovaries: UV
protection and growth stimulation activities on human cells, Mar. Biotechnol.
10 (2008) 141–150.
[40] Y. Iwamuro, R. Iio-Ishimura, S. Chinaka, N. Takayama, K. Hayakawa, Analysis of
11-nor-
D
9-tetrahydrocannabinol-9-carboxylic acid and its glucuronide in
urine by capillary electrophoresis/mass spectrometry, Biomed. Chromatogr.
26 (2012) 1452–1456.
[41] A.B. Blakeney, P.J. Harris, R.J. Henry, B.A. Stone, A simple and rapid preparation
of alditol acetates for monosaccharide analysis, Carbohydr. Res. 113 (1983)
291–299.
[57] H.-J. Suh, H.-W. Lee, J. Jung, Mycosporine glycine protects biological systems
against photodynamic damage by quenching singlet oxygen with
efficiency, Photochem. Photobiol. 78 (2003) 109–113.
a high
[42] Z. Hongbin, X. Zhiyu, Y. Yang, W. Rong, L. Ping, S. Bo, Determination of alditols
in wine by gas chromatography-mass spectrometry after acetate
derivatization, Chin. J. Chromatogr. 31 (2013) 786–790.
[43] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans,
Antioxidant activity applying an improved ABTS radical cation decolorization
assay, Free Radical Biol. Med. 26 (1999) 1231–1237.
[44] N. Tandeau de Marsac, J. Houmard, Complementary chromatic adaptation:
physiological conditions and action spectra, Methods Enzymol. 167 (1988)
318–328.
[45] K. Whitehead, D. Karentz, J.I. Hedges, Mycosporine-like amino acids (MAAs) in
phytoplankton, a herbivorous pteropod (Limacina helicina), and its pteropod
predator (Clione antarctica) in McMurdo Bay, Antarctica, Mar. Biol. 139 (2001)
1013–1019.
[58] I. Yakovleva, R. Bhagooli, A. Takemura, M. Hidaka, Differential susceptibility to
oxidative stress of two scleractinian corals: antioxidant functioning of
mycosporine-glycine, Comp. Biochem. Physiol. Part B 139 (2004) 721–730.
[59] M. Volkmann, K. Whitehead, H. Rütters, J. Rullkötter, A.A. Gorbushina,
Mycosporine–glutamicol–glucoside:
a
natural UV-absorbing secondary
metabolite of rock-inhabiting microcolonial fungi, Rapid Commun. Mass
Spectrom. 17 (2003) 897–902.
[60] M. Volkmann, A.A. Gorbushina, A broadly applicable method for extraction and
characterization of mycosporines and mycosporine-like amino acids of
terrestrial, marine and freshwater origin, FEMS Microbiol. Lett. 255 (2006)
286–295.
[61] A.D. Elbein, Y.T. Pan, I. Pastuszak, D. Carroll, New insights on trehalose: a
multifunctional molecule, Glycobiology 13 (2003) 17R–27R.
[62] A. Furusaki, T. Matsumoto, I. Tsujino, I. Sekikawa, The crystal and molecular
structure of palythine trihydrate, Bull. Chem. Soc. Jpn. 53 (1980) 319–323.
[46] M. Yoshiki, K. Tsuge, Y. Tsuruta, T. Yoshimura, K. Koganemaru, T. Sumi, T.
Matsui, K. Matsumoto, Production of new antioxidant compound from
mycosporine-like amino acid, porphyra-334 by heat treatment, Food Chem.
113 (2009) 1127–1132.
[47] I. Tsujino, K. Yabe, I. Sekikawa, Isolation and structure of a new amino acid,
shinorine, from the red alga Chondrus yendoi Yamada et Mikami, Bot. Mar. 23
(1980) 65–68.