©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure of a Novel Oligosaccharide-Mycosporine-Amino Acid Ultraviolet A/B Sunscreen Pigment from the Terrestrial Cyanobacterium Nostoc commune(*)

Günter A. Böhm (1)(§), Wolfgang Pfleiderer (1), Peter Böger (2), Siegfried Scherer (3)(¶)

From the (1) Fakultät für Chemie and the (2) Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, D-78434 Konstanz and the (3) Institut für Mikrobiologie, Weihenstephan, Technische Universität München, D-85350 Freising, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

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, NHphase), 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) NHat 25, 40, or 80 °C. Alternatively, 6 M HCl at 110 °C for 24 h under Natmosphere 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).


RESULTS

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 cmmgat 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 cmmg) 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 HO). 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).

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 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; MNa725 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.




DISCUSSION

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 cmmg. 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



FOOTNOTES

*
This work was supported by Deutsche Forschungsgemeinschaft Grant PF 36/62 and by the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry, Cambridge Centre for Molecular Recognition, Tennis Court Rd., Cambridge CB2 1QW, United Kingdom.

To whom correspondence should be addressed. Tel.: 8161-713516; Fax: 8161-714492; E-mail: 100424.2340@compuserve.com.

The abbreviations used are: HPLC, high pressure liquid chromatography; PD-MS, plasma desorption-mass spectrometry; E312, chromophore absorbing at 312 nm; E335, chromophore absorbing at 335 nm.


ACKNOWLEDGEMENTS

We thank M. Potts for helpful discussions and F. Garcia-Pichel for donation of reference samples.


REFERENCES
  1. Agrawal, P. K. (1992) Phytochemistry ( Oxf.) 31, 3307-3330 [CrossRef][Medline] [Order article via Infotrieve]
  2. Bock, K., Pedersen, C., and Pedersen, H. (1984) Adv. Carbohydr. Chem. Biochem. 42, 193-225
  3. Chioccara, F., Dela Gala, A., De Rosa, M., Novellino, E., and Prota, G. (1980) Bull. Soc. Chim. Belg. 89, 1101-1106
  4. Dill, K., Berman, E., and Pavia, A. A. (1985) Adv. Carbohydr. Chem. Biochem. 43, 1-49 [Medline] [Order article via Infotrieve]
  5. Foster, A. B., Horton, D., and Stacey, M. (1957) J. Chem. Soc. 81-86
  6. Fransson, L.-Å., Cöster, L., Nieduszynski, I. A., Phelps, C. F., and Sheehan, J. K. (1984) in Molecular Biophysics of the Extracellular Matrix (Arnott, S., Rees, D. A., and Morris, E. R., eds) pp. 95-118, Humana Press, Clifton, NJ
  7. Garcia-Pichel, F., and Castenholz, R. W. (1993) Appl. Environ. Microbiol. 59, 163-169 [Abstract]
  8. Garcia-Pichel, F., Wingard, C. E., and Castenholz, R. W. (1993) Appl. Environ. Microbiol. 59, 170-176 [Abstract]
  9. Gauch, R., Leuenberger, U., and Baumgartner, E. (1979) J. Chromatogr. 174, 195-200 [CrossRef]
  10. Gorin, P. A. J., and Mazurek, M. (1975) Can. J. Chem. 53, 1212-1223
  11. Hill, D. R., Hladun, S. L., Scherer, S., and Potts, M. (1994) J. Biol. Chem. 269, 7726-7734 [Abstract/Free Full Text]
  12. Ito, S., and Hirata, Y. (1977) Tetrahedron Lett. 2429-2430
  13. Karentz, D., McEuen, F. S., Land, M. C., and Dunlap, W. C. (1991) Mar. Biol. ( Berl.) 108, 157-166
  14. Plack, P. A., Fraser, N. W., Grant, P. T., Middleton, C., Mitchell, A. I., and Thomson, R. H. (1981) Biochem. J. 199, 741-747 [Medline] [Order article via Infotrieve]
  15. Post, A., and Larkum, A. W. D. (1993) Aquat. Bot. 45, 231-243
  16. Potts, M. (1994) Microbiol. Rev. 58, 755-805 [Abstract]
  17. Rebers, P. A., Wessman, G. E., and Robyt, J. F. (1986) Carbohydr. Res. 153, 132-135 [CrossRef]
  18. Scherer, S., and Potts, M. (1989) J. Biol. Chem. 264, 12546-12553 [Abstract/Free Full Text]
  19. Scherer, S., and Zhong, Z. P. (1991) Microb. Ecol. 22, 271-283
  20. Scherer, S., Chen, T. W., and Böger, P. (1988) Plant Physiol. 88, 1055-1057
  21. Shibata, K. (1969) Plant Cell Physiol. 10, 325-335
  22. Spiro, M. J., and Spiro, R. G. (1992) Anal. Biochem. 204, 152-157 [Medline] [Order article via Infotrieve]
  23. Takano, S., Uemura, D., and Hirata, Y. (1978) Tetrahedron Lett. 4909-4912
  24. Takano, S., Nakanishi, A., Uemura, D., and Hirata, Y. (1979) Chemistry Letters 419-420
  25. Wakabayashi, K., and Pigman, W. (1974) Carbohydr. Res. 35, 3-14 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.