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Structural characterization of neutral glycosphingolipids from Fusarium species
Biochimica et Biophysica Acta 1390 Ž1998. 186–196
Structural characterization of neutral glycosphingolipids from Fusarium species Rafael S. Duarte a , Carla R. Polycarpo a , Robin Wait b, Rudolf Hartmann c , Eliana Barreto Bergter a,) a
Instituto de Microbiologia, UniÕersidade Federal do Rio de Janeiro, 21 944 970-Cidade UniÕersitaria, ´ Rio de Janeiro, RJ, Brazil b Centre for Applied Microbiology and Research, Salisbury, SP4 0JG, UK c Institut fur ¨ Physiologische Chemie, UniÕersitat ¨ Bonn, Bonn, Germany Received 29 July 1997; revised 29 September 1997; accepted 3 October 1997
Abstract Glycosphingolipids were extracted from hyphae of Fusarium solani and from an unnamed Fusarium species, and were purified by silica and Iatrobead column chromatography. Their structures were determined by compositional analysis, nuclear magnetic resonance spectroscopy, gas chromatographyrmass spectrometry and by fast atom bombardment mass spectrometry of the native and peracetylated materials, which defined their sugar, long-chain base and fatty acid compositions. The locations of the double bonds in the bases were established by 2D NMR spectroscopy and by novel mass spectrometric approaches, including collisional activation of the protonated and lithium-cationized glycosphingolipids, and of the sphingadienene-derived fragment ion at mrz 276. From these results we propose that the structures of the glycosphingolipids from F. solani and Fusarium sp. are N-2X-hydroxyoctadecanoyl-1-O-b-D-glucopyranosyl-9-methyl-4,8-sphingadienine and N-2X-hydroxyoctadecenoyl-1-O-b-Dglucopyranosyl-9-methyl-4,8-sphingadienine, respectively. q 1998 Elsevier Science B.V. Keywords: Glycosphingolipid; Mass spectrometry, fast atom bombardment; NMR spectroscopy; Ž Fusarium.
1. Introduction Fusarium species are common hyaline soil saprophytes and plant pathogens which are the agents of serious diseases of a number of human food-plants, and pose a hazard to human health, both as opportunist pathogens Ž particularly in immunocompromised hosts. w1,2x, and through the production of mycotoxins w3x.
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Corresponding author. Fax: q55 021 560 8028; E-mail: [email protected]
Glycosphingolipids are abundant in fungi w4,5x, and these biologically active molecules have been implicated in cell–cell recognition, trans-membrane signalling, cellular growth and differentiation w6x. In pathogenic fungi glycosphingolipids may be immunogenic and relevant to virulence and pathogenesis, so elucidation of their structures can potentially offer new insights into the mechanism of host–pathogen interaction. The specific features of fungal lipid metabolism may also afford potential targets for improved antifungal agents. Although there is a certain amount of information available about the fatty acids and glycerolipids of
0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 1 7 9 - 3
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Fusarium w7x, little is known about the sphingolipid composition of the genus. In this paper we have performed the first detailed characterization of monohexosyl ceramides from F. solani and from an unnamed Fusarium isolate, and we demonstrate the value of collisional activation methods for analysis of sphingolipids from pathogenic fungi.
2. Material and methods 2.1. Microorganisms and growth conditions Fusarium solani and an unnamed Fusarium species were clinical isolates obtained from the Escola Paulista de Medicina, Sao ˜ Paulo, Brazil. Their identities were confirmed by conventional cultural techniques and by morphological examination. Both strains are available on request. The cells were grown at 308C with shaking in Sabouraud medium containing 10 grl peptone, 5 grl yeast extract and 20 grl glucose. After seven days the cells were harvested by filtration, washed three times with distilled water and stored at y208C. 2.2. Lipid extraction Lipids were isolated from intact hyphae of F. solani and Fusarium sp. by extractions at room tem perature with 10 sam ple volum es of chloroformrmethanol 2:1 Žvolrvol. followed by methanolrchloroform 2:1 Ž volrvol.. Evaporation of these combined extracts yielded a crude lipid mixture, which was subjected to mild alkaline methanolysis w8x and partitioned according to Folch et al. w9x. The upper phase was carefully removed and the lower phase was washed twice with Folch’s theoretical upper phase. Both phases were dried in vacuo. 2.3. Isolation and purification of neutral glycosphingolipids The lipids recovered from the lower layer of the Folch extract were purified by silica column chromatography, and neutral lipids, glycolipids and phospholipids were recovered by elution with chloroform, acetone and methanol, respectively.
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The acetone fraction, containing the crude glycosphingolipids, was further purified on a silica gel column, which was sequentially eluted with chloroformrmethanol containing an increasing proportion of methanol Ž 95:5, 9:1 and 8:2 volrvol. and finally with 100% methanol. The fractions eluted with chloroformrmethanol Ž9:1 and 8:2 volrvol. were further purified by chromatography on Iatrobeads RS 2060 ŽMacherey & Nagel, Duren, Germany. , using the ¨ same elution system, yielding a purified glycosphingolipid fraction. Some of this material were peracetylated with pyridineracetic anhydride Ž1:1 volrvol; overnight at room temperature.. After removal of the reagents by evaporation under nitrogen, the peracetylated glycolipid was purified on a small Iatrobeads column Ž 0.5 = 5 cm. as previously described w4x. 2.4. High performance thin-layer chromatography (HPTLC) Native and peracetylated neutral glycolipids were analysed on HPTLC plates developed with chloroformrmethanolrwater Ž65:25:4 volrvol. and 1,2-dichloroethaneracetone Ž8:2 volrvol., respectively. The separated glycolipids were visualized with iodine vapour and by spraying with orcinolrsulphuric acid. 2.5. Sugar analysis Glycosphingolipids were hydrolyzed with 0.5 M sulphuric acid at 1008C for 18 h, and the resulting monosaccharides were characterized by paper chromatography w10x and quantified by gas chromatography Ž GC. as alditol acetate derivatives w11x using an OV-225 fused silica capillary column Ž 30 m = 0.25 mm i.d.. temperature programmed from 508C to 2208C at 508Crmin. 2.6. Characterization of fatty acids Fatty acid methyl esters were prepared by acid catalysed methanolysis using 1 ml of toluene: methanol Ž1:1 volrvol. containing 2.5% concentrated sulphuric acid Žovernight at 708C.. The samples were diluted with 0.5 ml of deionized water and extracted twice with hexane: chloroform Ž 4:1 volrvol.. The
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combined extracts were dried by vacuum centrifugation and trimethylsilylated by treatment with 100 ml of bis-Žtrimethylsilyl.trifluoracetamiderpyridine Ž1:1, volrvol; 30 min at 608C.. The reagent was removed by vacuum centrifugation, and the samples were dissolved in hexane for GCrMS analysis. 2.7. Characterization of long-chain bases Long-chain sphingosine bases were released from the purified glycosphingolipids by methanolysis Ž 1 M methanol–HCl made 10 M with respect to water. overnight at 808C w12x. The pH was adjusted to 11 with aqueous NaOH, and the methanolysate was extracted three times with two volumes of diethyl ether. The combined extracts were dried on a vacuum centrifuge and O-trimethylsilylated by treatment with bis-Ž trimethylsilyl. trifluoroacetamiderpyridine Ž 1:1, volrvol. for 30 min at 608C, and analysed by GC– MS.
an Ion Tech saddle-field atom gun supplied with high-purity xenon gas. Spectra of the underivatized glycolipids were recorded in both the positive and negative ion modes using either 3-nitrobenzyl alcohol, or a 1:1 Žvolrvol. mixture of glycerol and dithiothreitol–dithioerythritol Ž 5:1, wrw. as liquid matrices. Approximately 10 mg of each sample was loaded onto the FAB probe. The instrument was operated at an accelerating voltage of 4 kV and a resolution of 1000 Ž10% valley., and the magnet was scanned at 10 srdecade of mass over the range of 2000–200. Peracetyl derivatives Ž 1–5 mg. were analysed from 3-nitrobenzyl alcohol matrix. Cationization with lithium was achieved by mixing 1 ml of 0.05 M lithium iodide with the sample and the matrix on the target. Sugar- w13x and ceramide-derived w14,15x fragment ions are described using the nomenclature introduced by Domon and Costello. Collisional activation spectra were recorded by scanning the magnetic field Ž B . and the electrostatic
2.8. Gas chromatographyr mass spectrometry G as chrom atographyr m ass spectrom etry ŽGCrMS. was performed with a Kratos MS80 RFA spectrometer Ž Kratos, Manchester, UK. directly interfaced to a Carlo Erba 5160 chromatograph. Helium Ž0.7 mlrmin. was used as the carrier gas, and samples were introduced by splitless injection Žsplitless time 30 s. into a BPX-5 fused silica column Ž25 m = 0.2 mm; SGE, Milton Keynes, UK.. The injector and interface ovens were maintained at 2508C. 1 min after injection, the column oven temperature was programmed from 608C to 2008C at 408Crmin, then at a rate of 38Crmin to 2308C, with a final 88C ramp to 2658C. This temperature was maintained for 10 min. Electron ionization spectra were recorded at an ionization energy of 70 eV, trap current of 100 mA, and a source temperature of 2208C. Chemical ionization spectra were obtained using isobutane as the reagent gas with an emission current of 250 mA. The magnet was scanned at 0.6 srdecade over the range 550–40. 2.9. Fast atom bombardment–mass spectrometry Fast atom bombardment Ž FAB. spectra were obtained with a Kratos MS80 spectrometer, fitted with
Fig. 1. High performance thin-layer chromatogram of the glycosphingolipid from F. solani. Lane 1 shows the purified glycosphingolipid from F. solani, and lane 2 neutral glycosphingolipids from human erythrocytes and bovine brain ŽCMH s monohexosyl ceramide; CDH sdihexosyl ceramide; CTH s trihexosyl ceramide..
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analyser voltage Ž E ., while maintaining a constant ratio of B to E. The helium collision gas pressure was adjusted to give 50% attenuation of the ion beam.
2.10. Proton-NMR spectroscopy The peracetylated sample was dissolved in 0.5 ml CDCl 3. Spectra were recorded at 298 K on a Bruker AMX 500 spectrometer equipped with an X32 computer running standard Bruker software. For two-dimensional, homonuclear correlation spectrum Ž HHCOSY. the time-domain matrix comprised 2 K data points in t 2 and 256 data points in t 1. The matrix was zero-filled in t 1, multiplied in both dimensions by a sine squared function and Fourier transformed.
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3. Results 3.1. Monosaccharide identification Glycosphingolipids ŽGSL. extracted from F. solani and the Fusarium sp. were purified by column chromatography on silica gel and Iatrobeads. Thin-layer chromatography of the purified glycosphingolipids revealed in each case a single component with chromatographic mobility corresponding to a monohexosylceramide ŽCMH., which was the major neutral glycolipid in both F. solani and the Fusarium species ŽFig. 1.. The constituent monosaccharides were liberated by hydrolysis and identified by paper chromatography and GLC. The chemical shifts and coupling constants of the sugar protons of the peracetylated glycolipids
Fig. 2. Daughter ion spectrum of the Y0X ion at mrz 574 from the CMH of F. solani.
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were identical to those of an authentic sample of peracetylated glucosylceramide derived from Gaucher spleen. The 7.9 Hz coupling constant of H-1 in the CMH from both strains indicated that glucose residue was b-linked. 3.2. GC–MS analysis of the monohexosyl ceramide from F. solani GC–MS analysis of the methanolysed and trimethylsilylated CMH from F. solani revealed a single peak. Its EI mass spectrum showed a weak molecular ion at mrz 384, an ion at mrz 369 ŽM-15. and a base peak at mrz 325 ŽM-59.. This latter fragment, originating from facile cleavage between the carboxyl group and carbon 2, is characteristic of 2-hydroxy fatty acid methyl esters w16,17x, thus identifying the compound as 2-hydroxy-octadecenoic acid.
The long-chain bases were recovered from the methanolysed CMHs by ether extraction at alkaline pH. The mass spectra of their TMS derivatives contained a base peak at mrz 132, and weak ions at mrz 440 ŽM-15. and at mrz 350 ŽM-15-TMSOH.. Under chemical ionization conditions a protonated molecule was observed at mrz 456, which is consistent with a C 19 sphingadienine, though the spectra give no indication of the locations of the double bonds. 3.3. FAB MS analysis of the CMH from F. solani In the negative ion FAB spectrum of the native CMH from F. solani an abundant ion was observed at mrz 752.5, consistent with the deprotonated molecule of a monohexosyl ceramide containing hydroxyoctadecenoic acid and C 19 sphingadiene. A
Fig. 3. Helium collisional activation spectrum of the sphingadiene fragment at mrz 276 from the CMH of F. solani.
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fragment at mrz 590 represents loss of hexose via a Y-type process w13x. In the positive ion FAB spectrum the corresponding protonated molecule at mrz 754 was weak, though a more abundant wM q Hxq-18 ion was present at mrz 736. Loss of hexose from this wM q Hxq-18 species resulted in the base peak at mrz 574.8 ŽY0X .. Addition of 1 ml of 0.05 M NaI to the sample resulted in an abundant wM q Naxq ion at mrz 776. At the low mass end of the spectrum a characteristic peak at mrz 276, originating from the C 19 sphingadiene base was present w18x. Collisional activation of the Y0X ion at mrz 574 resulted in the spectrum shown in Fig. 2. Cleavage of the amide linkage w15,19x is responsible for the low mass ion at mrz 294 ŽWX . and its dehydration product at mrz 276 ŽWY . , which are diagnostic of a C 19 sphingadiene. The ion at mrz 394 Žand its associated dehydration product at mrz 376. is consistent with a 4,8-diene structure, in which cleavage
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of the doubly allylic C 6 –C 7 bond would be enhanced w18,20x. Further evidence for the location of the double bonds in the long-chain base was obtained by collisional activation of the sphingadiene fragment at mrz 276. Cleavages of the alkyl chain w21,22x indicated the presence of a 4,8 sphingadiene structure, with a methyl substituent on carbon 9 w18x Ž Fig. 3. . An identical daughter ion spectrum was obtained from collisional activation of mrz 276 in the FAB spectrum of the CMH from Aspergillus fumigatus, which has been shown by NMR spectroscopy to contain 9-methyl-4,8-sphingadiene w4x. Collisional activation of lithium cationized ceramides and GSL has been shown to provide more informative daughter ion spectra than those from protonated precursors w19,23,24x. Lithium cationization favours the production of high mass product ions formed by charge-site remote cleavages of the long-
Fig. 4. Helium collisional activation spectrum of the wM q Lixq Ž mrz 760. from the CMH of F. solani.
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Fig. 5. COSY spectrum of the CMH from F. solani.
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chain base and N-acyl chains. Typically the alkyl chains fragment by 1,4 elimination of C n H 2 nq2 , without double bond migration or other complex rearrangements w25x, which enables the location of structural features such as double bonds. The spectrum obtained by collisional activation of the lithium cationized CMH from F. solani Ž mrz 760. is shown in Fig. 4. Abundant fragments are observed at mrz 742 Žloss of water from wM q Lixq, and 598 Žfragment Y0 , attributable to loss of hexose. . The latter is accompanied by the corresponding 1,5 X 0 and Z 0 fragments at mrz 626 and 580, respectively. The prominent ion at mrz 480 originates by cleavage of the amide N–CO bond, which is particularly facile in ceramides which contain a-hydroxy acids, because of favoured transfer of the a-hydroxy hydrogen atom to the charge-retaining fragment w24x. The family of regularly spaced ions between mrz 744 and 536 originate from charge site remote fragmentations of the alkyl chains. Interpretation is somewhat complex, since cleavage of both the long-chain base and the N-acyl chain occurs. However, the prominent ion at mrz 578 supports a 4,8-sphingadiene structure, because cleavage in the doubly allylic position is favourable ŽFig. 4. . The mrz increment of 26 between mrz 562 and 536 is consistent with the double bond of 2-hydroxyoctadecenoic acid being in position 3 ŽFig. 4.. An identical collision spectrum was obtained from the lithium cationized CMH of A. fumigatus, which has been shown by NMR spectroscopy to be N-2X-hydroxyoctadecenoyl-1-O-hexopyranosyl9-methyl-4,8-sphingadiene w4x, which suggests that this material is identical to that from F. solani. These findings were confirmed by FAB MS of the peracetylated glycolipid. An wM q Naxq signal was observed at mrz 1028, indicating addition of six acetyl groups to the mass of the underivatized glycol-
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ipid, consistent with a hydroxy acid containing monohexosyl ceramide. Additional high mass signals were present at mrz 946 ŽM q H-HOAc. and mrz 886 ŽM q H-2HOAc. . An abundant ion at mrz 331 Ž B1 . was indicative of a terminal hexose; the fragment at mrz 229 was attributable to the loss of ketene plus acetic acid from mrz 331, further elimination of acetic acid giving mrz 169. The ceramide moiety was represented by peaks at mrz 658 ŽY0 q Naq., mrz 598 ŽY0-HOAc q Naq. and mrz 538 ŽY02HOAcq Naq.. The fragment at mrz 276 originates from the long-chain base, and has been discussed above. 3.4. NMR analysis of the CMH from F. solani The structure of the long-chain base was deduced from one- and two-dimensional NMR spectra Ž Fig. 5.. The HH-COSY spectrum enabled the connectivity of the long-chain base protons to be traced between C1 and C-11 and those of the fatty acid from C-2X to C-5X Žsee Fig. 6 for numbering of the carbon atoms. . The resonances at 5.30 ppm and 5.50 ppm, assigned to the long-chain base H-3 and the fatty acid H-2, respectively, are typical of protons directly bonded to O-acetylated carbons adjacent to olefinic bonds. The resonance at 5.39 ppm, assigned as H-4 of the long-chain base, exhibited a coupling constant Ž3JH,H . of 15.3 Hz to H-5, consistent with trans-double bonds. The 3,4 double bond of the fatty acid was likewise trans, because the coupling constant between H-3 Ž5.50 ppm. and H-4 was 14.3 Hz. 3.5. FAB MS analysis of CMH from the Fusarium sp. FAB MS analysis indicated that, in contrast to F. solani, the glycolipid from the Fusarium species
Fig. 6. Structure of the monohexosyl ceramide from F. solani. The numbers refer to the assigned resonances in the COSY spectrum ŽFig. 5..
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Fig. 7. Positive ion FAB spectrum of the peracetylated monohexosyl ceramides from the Fusarium species. The inset shows an expansion of the molecular ion region.
consisted of two components which differ in their fatty acid compositions. In the negative ion spectrum deprotonated molecules were observed at mrz 752 and 754, in an intensity ratio of about 0.8:1. Elimination of hexose results in the doublet of Y0 ions at mrz 592 and 590. The corresponding positive ion spectrum revealed wM q Naxq signals at mrz 776 and 778, and Y0X fragments at mrz 574 and 576. The low mass end of the spectrum was dominated by an abundant WY ion at mrz 276, and the corresponding WX signal at 294. The daughter ion spectrum obtained by collisional activation of the WY ion at mrz 276 was identical to those from the isobaric ions in F. solani and A. fumigatus. The positive ion FAB spectrum of the peracetylated sample is shown in Fig. 7. wM q Naxq ions were observed at mrz 1030.6 and 1028.6, and wM q Hx-60 fragments at mrz 948 and 946. The B 1 carbenium ion at mrz 331 is indicative of a terminal hexose, and this assignment is supported by the presence of the expected secondary
fragments at mrz 229, 169 and 109. Ceramide fragments were present as doublets at mrz 658 and 660 ŽY0 q Naq., and mrz 598 and 600 Ž Y0-HOAcq Naq., each doublet having a similar relative abundance ratio to the molecular ion clusters. The ions at mrz 886 and 538 were not assigned, but lacked the characteristic doublet structure of the fatty acyl substituted ions, and may be derived from a contaminant. These data thus suggest that the Fusarium species contains a mixture of two glucosylceramides, one of which is identical to that from F. solani, whereas the other differs in that it is acylated with 2-hydroxyoctadecanoic, instead of 2-hydroxyoctadecenoic acid.
4. Discussion Analysis by HPTLC, GC–MS, FAB–MS and proton NMR spectroscopy has shown that the major
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glycosphingolipid from F. solani is N-2X-hydroxyoctadecenoyl -1-O-b-D-glucopyranosyl -9-methyl-4,8sphingadienine, and that the other Fusarium species contains in addition N-2X-hydroxyoctadecanoyl-1-Ob-D -glucopyranosyl-9-methyl-4,8-sphingadienine. The main difference between the two species is thus the additional presence of ceramides containing 2-hydroxy hexadecanoic acid in the Fusarium sp. These results differ from those of Weiss et al. w26x who, on the basis of hydrolysis, reduction and periodate oxidation, reported that the monohexosylceramides of Fusarium lini contain predominantly C 18 and C 20 phytosphingosines rather than 9-methyl-4,8sphingadiene as detected in the present study. The possibility that phytosphingosine-containing glycolipids are present as minor components cannot, however, be excluded. The unusual long-chain base 9-methyl-4,8-sphingadiene was first described in cerebrosides from the plant pathogenic fungus Fusicoccum amygdali w27x, but appears to be widely distributed among fungi and has subsequently been isolated from Schizophyllum commune w28x, Aspergillus oryzae w29x and the edible fungi Clitocybe geotropa, C. nebularis w30x and Hypsizigus marmoreus w18x, though it has not been detected in the pathogenic species Sporothrix schenckii w5x and Paracoccidioides brasilensis w31x. A monoglucosylceramide with identical structure to that from F. solani has been detected in Aspergillus fumigatus and A. Õersicolor w4x. This finding is interesting because the clinical features of Fusarium and Aspergillus infections in humans are similar, as are the histopathological lesions that they induce. Moreover both exhibit similar septate and branched hyphae and are antigenically closely related w2x. The biological relevance of these molecules in fungi remains elusive. It is possible that their role is primarily structural, sphingolipids providing a more energetically efficient route to a surface glycocalyx than synthesis of a glycoprotein coat, a hypothesis initially proposed by Koscielak w32x in the context of the biological role of erythrocyte glycosphingolipids. The presence of glycosphingolipids as constituents of the cell membrane of Fusarium may modulate the response of the host to infection, since neutral glycosphingolipids isolated from the parasitic protozoan Trypanosoma cruzi are known to be antigenic and the
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ceramide moiety seems to be important for antibody recognition w33x. In higher eukaryotes ceramides are increasingly recognized as biologically active molecules which function as second messengers, mediating the effects of external stimuli on cell growth, differentiation and apoptosis w34x. It is possible that the glycosphingolipids of Fusarium species play a similar role in signal transduction and cell regulation, though this remains to be determined.
Acknowledgements This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientıfico e Tec´ Ž . nologico CNPq , Financiadora de Estudos e Projetos ´ ŽFINEP., Conselho de Ensino e Pesquisa da UFRJ ŽCEPG., Fundac¸ao ˜ de Amparo a` Pesquisa do Estado do Rio de Janeiro ŽFAPERJ. and Programa de ŽPRONEX. . Nucleos de Excelencia ´ ˆ
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Structural characterization of neutral glycosphingolipids from Fusarium species Rafael S. Duarte a , Carla R. Polycarpo a , Robin Wait b, Rudolf Hartmann c , Eliana Barreto Bergter a,) a
Instituto de Microbiologia, UniÕersidade Federal do Rio de Janeiro, 21 944 970-Cidade UniÕersitaria, ´ Rio de Janeiro, RJ, Brazil b Centre for Applied Microbiology and Research, Salisbury, SP4 0JG, UK c Institut fur ¨ Physiologische Chemie, UniÕersitat ¨ Bonn, Bonn, Germany Received 29 July 1997; revised 29 September 1997; accepted 3 October 1997
Abstract Glycosphingolipids were extracted from hyphae of Fusarium solani and from an unnamed Fusarium species, and were purified by silica and Iatrobead column chromatography. Their structures were determined by compositional analysis, nuclear magnetic resonance spectroscopy, gas chromatographyrmass spectrometry and by fast atom bombardment mass spectrometry of the native and peracetylated materials, which defined their sugar, long-chain base and fatty acid compositions. The locations of the double bonds in the bases were established by 2D NMR spectroscopy and by novel mass spectrometric approaches, including collisional activation of the protonated and lithium-cationized glycosphingolipids, and of the sphingadienene-derived fragment ion at mrz 276. From these results we propose that the structures of the glycosphingolipids from F. solani and Fusarium sp. are N-2X-hydroxyoctadecanoyl-1-O-b-D-glucopyranosyl-9-methyl-4,8-sphingadienine and N-2X-hydroxyoctadecenoyl-1-O-b-Dglucopyranosyl-9-methyl-4,8-sphingadienine, respectively. q 1998 Elsevier Science B.V. Keywords: Glycosphingolipid; Mass spectrometry, fast atom bombardment; NMR spectroscopy; Ž Fusarium.
1. Introduction Fusarium species are common hyaline soil saprophytes and plant pathogens which are the agents of serious diseases of a number of human food-plants, and pose a hazard to human health, both as opportunist pathogens Ž particularly in immunocompromised hosts. w1,2x, and through the production of mycotoxins w3x.
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Corresponding author. Fax: q55 021 560 8028; E-mail: [email protected]
Glycosphingolipids are abundant in fungi w4,5x, and these biologically active molecules have been implicated in cell–cell recognition, trans-membrane signalling, cellular growth and differentiation w6x. In pathogenic fungi glycosphingolipids may be immunogenic and relevant to virulence and pathogenesis, so elucidation of their structures can potentially offer new insights into the mechanism of host–pathogen interaction. The specific features of fungal lipid metabolism may also afford potential targets for improved antifungal agents. Although there is a certain amount of information available about the fatty acids and glycerolipids of
0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 1 7 9 - 3
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Fusarium w7x, little is known about the sphingolipid composition of the genus. In this paper we have performed the first detailed characterization of monohexosyl ceramides from F. solani and from an unnamed Fusarium isolate, and we demonstrate the value of collisional activation methods for analysis of sphingolipids from pathogenic fungi.
2. Material and methods 2.1. Microorganisms and growth conditions Fusarium solani and an unnamed Fusarium species were clinical isolates obtained from the Escola Paulista de Medicina, Sao ˜ Paulo, Brazil. Their identities were confirmed by conventional cultural techniques and by morphological examination. Both strains are available on request. The cells were grown at 308C with shaking in Sabouraud medium containing 10 grl peptone, 5 grl yeast extract and 20 grl glucose. After seven days the cells were harvested by filtration, washed three times with distilled water and stored at y208C. 2.2. Lipid extraction Lipids were isolated from intact hyphae of F. solani and Fusarium sp. by extractions at room tem perature with 10 sam ple volum es of chloroformrmethanol 2:1 Žvolrvol. followed by methanolrchloroform 2:1 Ž volrvol.. Evaporation of these combined extracts yielded a crude lipid mixture, which was subjected to mild alkaline methanolysis w8x and partitioned according to Folch et al. w9x. The upper phase was carefully removed and the lower phase was washed twice with Folch’s theoretical upper phase. Both phases were dried in vacuo. 2.3. Isolation and purification of neutral glycosphingolipids The lipids recovered from the lower layer of the Folch extract were purified by silica column chromatography, and neutral lipids, glycolipids and phospholipids were recovered by elution with chloroform, acetone and methanol, respectively.
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The acetone fraction, containing the crude glycosphingolipids, was further purified on a silica gel column, which was sequentially eluted with chloroformrmethanol containing an increasing proportion of methanol Ž 95:5, 9:1 and 8:2 volrvol. and finally with 100% methanol. The fractions eluted with chloroformrmethanol Ž9:1 and 8:2 volrvol. were further purified by chromatography on Iatrobeads RS 2060 ŽMacherey & Nagel, Duren, Germany. , using the ¨ same elution system, yielding a purified glycosphingolipid fraction. Some of this material were peracetylated with pyridineracetic anhydride Ž1:1 volrvol; overnight at room temperature.. After removal of the reagents by evaporation under nitrogen, the peracetylated glycolipid was purified on a small Iatrobeads column Ž 0.5 = 5 cm. as previously described w4x. 2.4. High performance thin-layer chromatography (HPTLC) Native and peracetylated neutral glycolipids were analysed on HPTLC plates developed with chloroformrmethanolrwater Ž65:25:4 volrvol. and 1,2-dichloroethaneracetone Ž8:2 volrvol., respectively. The separated glycolipids were visualized with iodine vapour and by spraying with orcinolrsulphuric acid. 2.5. Sugar analysis Glycosphingolipids were hydrolyzed with 0.5 M sulphuric acid at 1008C for 18 h, and the resulting monosaccharides were characterized by paper chromatography w10x and quantified by gas chromatography Ž GC. as alditol acetate derivatives w11x using an OV-225 fused silica capillary column Ž 30 m = 0.25 mm i.d.. temperature programmed from 508C to 2208C at 508Crmin. 2.6. Characterization of fatty acids Fatty acid methyl esters were prepared by acid catalysed methanolysis using 1 ml of toluene: methanol Ž1:1 volrvol. containing 2.5% concentrated sulphuric acid Žovernight at 708C.. The samples were diluted with 0.5 ml of deionized water and extracted twice with hexane: chloroform Ž 4:1 volrvol.. The
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combined extracts were dried by vacuum centrifugation and trimethylsilylated by treatment with 100 ml of bis-Žtrimethylsilyl.trifluoracetamiderpyridine Ž1:1, volrvol; 30 min at 608C.. The reagent was removed by vacuum centrifugation, and the samples were dissolved in hexane for GCrMS analysis. 2.7. Characterization of long-chain bases Long-chain sphingosine bases were released from the purified glycosphingolipids by methanolysis Ž 1 M methanol–HCl made 10 M with respect to water. overnight at 808C w12x. The pH was adjusted to 11 with aqueous NaOH, and the methanolysate was extracted three times with two volumes of diethyl ether. The combined extracts were dried on a vacuum centrifuge and O-trimethylsilylated by treatment with bis-Ž trimethylsilyl. trifluoroacetamiderpyridine Ž 1:1, volrvol. for 30 min at 608C, and analysed by GC– MS.
an Ion Tech saddle-field atom gun supplied with high-purity xenon gas. Spectra of the underivatized glycolipids were recorded in both the positive and negative ion modes using either 3-nitrobenzyl alcohol, or a 1:1 Žvolrvol. mixture of glycerol and dithiothreitol–dithioerythritol Ž 5:1, wrw. as liquid matrices. Approximately 10 mg of each sample was loaded onto the FAB probe. The instrument was operated at an accelerating voltage of 4 kV and a resolution of 1000 Ž10% valley., and the magnet was scanned at 10 srdecade of mass over the range of 2000–200. Peracetyl derivatives Ž 1–5 mg. were analysed from 3-nitrobenzyl alcohol matrix. Cationization with lithium was achieved by mixing 1 ml of 0.05 M lithium iodide with the sample and the matrix on the target. Sugar- w13x and ceramide-derived w14,15x fragment ions are described using the nomenclature introduced by Domon and Costello. Collisional activation spectra were recorded by scanning the magnetic field Ž B . and the electrostatic
2.8. Gas chromatographyr mass spectrometry G as chrom atographyr m ass spectrom etry ŽGCrMS. was performed with a Kratos MS80 RFA spectrometer Ž Kratos, Manchester, UK. directly interfaced to a Carlo Erba 5160 chromatograph. Helium Ž0.7 mlrmin. was used as the carrier gas, and samples were introduced by splitless injection Žsplitless time 30 s. into a BPX-5 fused silica column Ž25 m = 0.2 mm; SGE, Milton Keynes, UK.. The injector and interface ovens were maintained at 2508C. 1 min after injection, the column oven temperature was programmed from 608C to 2008C at 408Crmin, then at a rate of 38Crmin to 2308C, with a final 88C ramp to 2658C. This temperature was maintained for 10 min. Electron ionization spectra were recorded at an ionization energy of 70 eV, trap current of 100 mA, and a source temperature of 2208C. Chemical ionization spectra were obtained using isobutane as the reagent gas with an emission current of 250 mA. The magnet was scanned at 0.6 srdecade over the range 550–40. 2.9. Fast atom bombardment–mass spectrometry Fast atom bombardment Ž FAB. spectra were obtained with a Kratos MS80 spectrometer, fitted with
Fig. 1. High performance thin-layer chromatogram of the glycosphingolipid from F. solani. Lane 1 shows the purified glycosphingolipid from F. solani, and lane 2 neutral glycosphingolipids from human erythrocytes and bovine brain ŽCMH s monohexosyl ceramide; CDH sdihexosyl ceramide; CTH s trihexosyl ceramide..
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analyser voltage Ž E ., while maintaining a constant ratio of B to E. The helium collision gas pressure was adjusted to give 50% attenuation of the ion beam.
2.10. Proton-NMR spectroscopy The peracetylated sample was dissolved in 0.5 ml CDCl 3. Spectra were recorded at 298 K on a Bruker AMX 500 spectrometer equipped with an X32 computer running standard Bruker software. For two-dimensional, homonuclear correlation spectrum Ž HHCOSY. the time-domain matrix comprised 2 K data points in t 2 and 256 data points in t 1. The matrix was zero-filled in t 1, multiplied in both dimensions by a sine squared function and Fourier transformed.
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3. Results 3.1. Monosaccharide identification Glycosphingolipids ŽGSL. extracted from F. solani and the Fusarium sp. were purified by column chromatography on silica gel and Iatrobeads. Thin-layer chromatography of the purified glycosphingolipids revealed in each case a single component with chromatographic mobility corresponding to a monohexosylceramide ŽCMH., which was the major neutral glycolipid in both F. solani and the Fusarium species ŽFig. 1.. The constituent monosaccharides were liberated by hydrolysis and identified by paper chromatography and GLC. The chemical shifts and coupling constants of the sugar protons of the peracetylated glycolipids
Fig. 2. Daughter ion spectrum of the Y0X ion at mrz 574 from the CMH of F. solani.
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were identical to those of an authentic sample of peracetylated glucosylceramide derived from Gaucher spleen. The 7.9 Hz coupling constant of H-1 in the CMH from both strains indicated that glucose residue was b-linked. 3.2. GC–MS analysis of the monohexosyl ceramide from F. solani GC–MS analysis of the methanolysed and trimethylsilylated CMH from F. solani revealed a single peak. Its EI mass spectrum showed a weak molecular ion at mrz 384, an ion at mrz 369 ŽM-15. and a base peak at mrz 325 ŽM-59.. This latter fragment, originating from facile cleavage between the carboxyl group and carbon 2, is characteristic of 2-hydroxy fatty acid methyl esters w16,17x, thus identifying the compound as 2-hydroxy-octadecenoic acid.
The long-chain bases were recovered from the methanolysed CMHs by ether extraction at alkaline pH. The mass spectra of their TMS derivatives contained a base peak at mrz 132, and weak ions at mrz 440 ŽM-15. and at mrz 350 ŽM-15-TMSOH.. Under chemical ionization conditions a protonated molecule was observed at mrz 456, which is consistent with a C 19 sphingadienine, though the spectra give no indication of the locations of the double bonds. 3.3. FAB MS analysis of the CMH from F. solani In the negative ion FAB spectrum of the native CMH from F. solani an abundant ion was observed at mrz 752.5, consistent with the deprotonated molecule of a monohexosyl ceramide containing hydroxyoctadecenoic acid and C 19 sphingadiene. A
Fig. 3. Helium collisional activation spectrum of the sphingadiene fragment at mrz 276 from the CMH of F. solani.
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fragment at mrz 590 represents loss of hexose via a Y-type process w13x. In the positive ion FAB spectrum the corresponding protonated molecule at mrz 754 was weak, though a more abundant wM q Hxq-18 ion was present at mrz 736. Loss of hexose from this wM q Hxq-18 species resulted in the base peak at mrz 574.8 ŽY0X .. Addition of 1 ml of 0.05 M NaI to the sample resulted in an abundant wM q Naxq ion at mrz 776. At the low mass end of the spectrum a characteristic peak at mrz 276, originating from the C 19 sphingadiene base was present w18x. Collisional activation of the Y0X ion at mrz 574 resulted in the spectrum shown in Fig. 2. Cleavage of the amide linkage w15,19x is responsible for the low mass ion at mrz 294 ŽWX . and its dehydration product at mrz 276 ŽWY . , which are diagnostic of a C 19 sphingadiene. The ion at mrz 394 Žand its associated dehydration product at mrz 376. is consistent with a 4,8-diene structure, in which cleavage
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of the doubly allylic C 6 –C 7 bond would be enhanced w18,20x. Further evidence for the location of the double bonds in the long-chain base was obtained by collisional activation of the sphingadiene fragment at mrz 276. Cleavages of the alkyl chain w21,22x indicated the presence of a 4,8 sphingadiene structure, with a methyl substituent on carbon 9 w18x Ž Fig. 3. . An identical daughter ion spectrum was obtained from collisional activation of mrz 276 in the FAB spectrum of the CMH from Aspergillus fumigatus, which has been shown by NMR spectroscopy to contain 9-methyl-4,8-sphingadiene w4x. Collisional activation of lithium cationized ceramides and GSL has been shown to provide more informative daughter ion spectra than those from protonated precursors w19,23,24x. Lithium cationization favours the production of high mass product ions formed by charge-site remote cleavages of the long-
Fig. 4. Helium collisional activation spectrum of the wM q Lixq Ž mrz 760. from the CMH of F. solani.
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Fig. 5. COSY spectrum of the CMH from F. solani.
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chain base and N-acyl chains. Typically the alkyl chains fragment by 1,4 elimination of C n H 2 nq2 , without double bond migration or other complex rearrangements w25x, which enables the location of structural features such as double bonds. The spectrum obtained by collisional activation of the lithium cationized CMH from F. solani Ž mrz 760. is shown in Fig. 4. Abundant fragments are observed at mrz 742 Žloss of water from wM q Lixq, and 598 Žfragment Y0 , attributable to loss of hexose. . The latter is accompanied by the corresponding 1,5 X 0 and Z 0 fragments at mrz 626 and 580, respectively. The prominent ion at mrz 480 originates by cleavage of the amide N–CO bond, which is particularly facile in ceramides which contain a-hydroxy acids, because of favoured transfer of the a-hydroxy hydrogen atom to the charge-retaining fragment w24x. The family of regularly spaced ions between mrz 744 and 536 originate from charge site remote fragmentations of the alkyl chains. Interpretation is somewhat complex, since cleavage of both the long-chain base and the N-acyl chain occurs. However, the prominent ion at mrz 578 supports a 4,8-sphingadiene structure, because cleavage in the doubly allylic position is favourable ŽFig. 4. . The mrz increment of 26 between mrz 562 and 536 is consistent with the double bond of 2-hydroxyoctadecenoic acid being in position 3 ŽFig. 4.. An identical collision spectrum was obtained from the lithium cationized CMH of A. fumigatus, which has been shown by NMR spectroscopy to be N-2X-hydroxyoctadecenoyl-1-O-hexopyranosyl9-methyl-4,8-sphingadiene w4x, which suggests that this material is identical to that from F. solani. These findings were confirmed by FAB MS of the peracetylated glycolipid. An wM q Naxq signal was observed at mrz 1028, indicating addition of six acetyl groups to the mass of the underivatized glycol-
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ipid, consistent with a hydroxy acid containing monohexosyl ceramide. Additional high mass signals were present at mrz 946 ŽM q H-HOAc. and mrz 886 ŽM q H-2HOAc. . An abundant ion at mrz 331 Ž B1 . was indicative of a terminal hexose; the fragment at mrz 229 was attributable to the loss of ketene plus acetic acid from mrz 331, further elimination of acetic acid giving mrz 169. The ceramide moiety was represented by peaks at mrz 658 ŽY0 q Naq., mrz 598 ŽY0-HOAc q Naq. and mrz 538 ŽY02HOAcq Naq.. The fragment at mrz 276 originates from the long-chain base, and has been discussed above. 3.4. NMR analysis of the CMH from F. solani The structure of the long-chain base was deduced from one- and two-dimensional NMR spectra Ž Fig. 5.. The HH-COSY spectrum enabled the connectivity of the long-chain base protons to be traced between C1 and C-11 and those of the fatty acid from C-2X to C-5X Žsee Fig. 6 for numbering of the carbon atoms. . The resonances at 5.30 ppm and 5.50 ppm, assigned to the long-chain base H-3 and the fatty acid H-2, respectively, are typical of protons directly bonded to O-acetylated carbons adjacent to olefinic bonds. The resonance at 5.39 ppm, assigned as H-4 of the long-chain base, exhibited a coupling constant Ž3JH,H . of 15.3 Hz to H-5, consistent with trans-double bonds. The 3,4 double bond of the fatty acid was likewise trans, because the coupling constant between H-3 Ž5.50 ppm. and H-4 was 14.3 Hz. 3.5. FAB MS analysis of CMH from the Fusarium sp. FAB MS analysis indicated that, in contrast to F. solani, the glycolipid from the Fusarium species
Fig. 6. Structure of the monohexosyl ceramide from F. solani. The numbers refer to the assigned resonances in the COSY spectrum ŽFig. 5..
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Fig. 7. Positive ion FAB spectrum of the peracetylated monohexosyl ceramides from the Fusarium species. The inset shows an expansion of the molecular ion region.
consisted of two components which differ in their fatty acid compositions. In the negative ion spectrum deprotonated molecules were observed at mrz 752 and 754, in an intensity ratio of about 0.8:1. Elimination of hexose results in the doublet of Y0 ions at mrz 592 and 590. The corresponding positive ion spectrum revealed wM q Naxq signals at mrz 776 and 778, and Y0X fragments at mrz 574 and 576. The low mass end of the spectrum was dominated by an abundant WY ion at mrz 276, and the corresponding WX signal at 294. The daughter ion spectrum obtained by collisional activation of the WY ion at mrz 276 was identical to those from the isobaric ions in F. solani and A. fumigatus. The positive ion FAB spectrum of the peracetylated sample is shown in Fig. 7. wM q Naxq ions were observed at mrz 1030.6 and 1028.6, and wM q Hx-60 fragments at mrz 948 and 946. The B 1 carbenium ion at mrz 331 is indicative of a terminal hexose, and this assignment is supported by the presence of the expected secondary
fragments at mrz 229, 169 and 109. Ceramide fragments were present as doublets at mrz 658 and 660 ŽY0 q Naq., and mrz 598 and 600 Ž Y0-HOAcq Naq., each doublet having a similar relative abundance ratio to the molecular ion clusters. The ions at mrz 886 and 538 were not assigned, but lacked the characteristic doublet structure of the fatty acyl substituted ions, and may be derived from a contaminant. These data thus suggest that the Fusarium species contains a mixture of two glucosylceramides, one of which is identical to that from F. solani, whereas the other differs in that it is acylated with 2-hydroxyoctadecanoic, instead of 2-hydroxyoctadecenoic acid.
4. Discussion Analysis by HPTLC, GC–MS, FAB–MS and proton NMR spectroscopy has shown that the major
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glycosphingolipid from F. solani is N-2X-hydroxyoctadecenoyl -1-O-b-D-glucopyranosyl -9-methyl-4,8sphingadienine, and that the other Fusarium species contains in addition N-2X-hydroxyoctadecanoyl-1-Ob-D -glucopyranosyl-9-methyl-4,8-sphingadienine. The main difference between the two species is thus the additional presence of ceramides containing 2-hydroxy hexadecanoic acid in the Fusarium sp. These results differ from those of Weiss et al. w26x who, on the basis of hydrolysis, reduction and periodate oxidation, reported that the monohexosylceramides of Fusarium lini contain predominantly C 18 and C 20 phytosphingosines rather than 9-methyl-4,8sphingadiene as detected in the present study. The possibility that phytosphingosine-containing glycolipids are present as minor components cannot, however, be excluded. The unusual long-chain base 9-methyl-4,8-sphingadiene was first described in cerebrosides from the plant pathogenic fungus Fusicoccum amygdali w27x, but appears to be widely distributed among fungi and has subsequently been isolated from Schizophyllum commune w28x, Aspergillus oryzae w29x and the edible fungi Clitocybe geotropa, C. nebularis w30x and Hypsizigus marmoreus w18x, though it has not been detected in the pathogenic species Sporothrix schenckii w5x and Paracoccidioides brasilensis w31x. A monoglucosylceramide with identical structure to that from F. solani has been detected in Aspergillus fumigatus and A. Õersicolor w4x. This finding is interesting because the clinical features of Fusarium and Aspergillus infections in humans are similar, as are the histopathological lesions that they induce. Moreover both exhibit similar septate and branched hyphae and are antigenically closely related w2x. The biological relevance of these molecules in fungi remains elusive. It is possible that their role is primarily structural, sphingolipids providing a more energetically efficient route to a surface glycocalyx than synthesis of a glycoprotein coat, a hypothesis initially proposed by Koscielak w32x in the context of the biological role of erythrocyte glycosphingolipids. The presence of glycosphingolipids as constituents of the cell membrane of Fusarium may modulate the response of the host to infection, since neutral glycosphingolipids isolated from the parasitic protozoan Trypanosoma cruzi are known to be antigenic and the
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ceramide moiety seems to be important for antibody recognition w33x. In higher eukaryotes ceramides are increasingly recognized as biologically active molecules which function as second messengers, mediating the effects of external stimuli on cell growth, differentiation and apoptosis w34x. It is possible that the glycosphingolipids of Fusarium species play a similar role in signal transduction and cell regulation, though this remains to be determined.
Acknowledgements This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientıfico e Tec´ Ž . nologico CNPq , Financiadora de Estudos e Projetos ´ ŽFINEP., Conselho de Ensino e Pesquisa da UFRJ ŽCEPG., Fundac¸ao ˜ de Amparo a` Pesquisa do Estado do Rio de Janeiro ŽFAPERJ. and Programa de ŽPRONEX. . Nucleos de Excelencia ´ ˆ
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