Chemotypes significance of lichenized fungi by structural characterization of heteropolysaccharides from the genera Parmotrema and Rimelia

FEMS Microbiology Letters 246 (2005) 273–278 www.fems-microbiology.org Chemotypes significance of lichenized fungi by structural characterization of h...
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FEMS Microbiology Letters 246 (2005) 273–278 www.fems-microbiology.org

Chemotypes significance of lichenized fungi by structural characterization of heteropolysaccharides from the genera Parmotrema and Rimelia Elaine Rosechrer Carbonero a, Caroline Grassi Mellinger a, Sionara Eliasaro b, Philip Albert James Gorin a, Marcello Iacomini a,* a

Received 2 September 2004; accepted 14 April 2005 First published online 27 April 2005 Edited by G.M. Gadd

Abstract Galactoglucomannans were isolated from the lichenized fungi of the genus Parmotrema (Parmotrema austrosinense, Parmotrema delicatulum, Parmotrema mantiqueirense, Parmotrema schindlerii, and Parmotrema tinctorum and that of Rimelia (Rimelia cetrata and Rimelia reticulata) via successive hot alkaline extraction and precipitation with Fehling solution. The structure of each polysaccharide was investigated using 13C NMR and HSQC-DEPT spectroscopy, methylation analysis, and HPSEC-MALLS. The galactoglucomannans had a (1 ! 6)-linked main chain of a-Manp units, substituted preferentially at O-2 and O-4 by a-Galp and b-Galp nonreducing end-units, respectively. The C-1 region of the 13C NMR spectra of these heteropolysaccharides is typical of the lichen species, and is an additional tool in lichenized fungi classification.  2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Lichenized fungi; Parmeliaceae; Galactoglucomannan; Chemical structure;

1. Introduction The identification and classification of lichenized fungi was originally carried out on the basis of morphology. Since the 1860s, species differentiation was aided by the specific color reactions of their components [1,2], present at concentrations of 0.15% to 10%, or carotenoids [3,4]. The chemical analysis of compounds for taxonomic purposes was carried out by microcrystallization, chromatography, fluorescence and mass spectroscopy analysis [5]. Recently, advances in DNA technology *

Corresponding author. Tel.: +55 41 361 1655; fax: +55 41 266 2042. E-mail address: [email protected] (M. Iacomini).

13

C NMR

and fine chemical characterization of macromolecules served as a useful tools in the classification of lichens. The use of structurally different mannose-containing polysaccharides for the classification and identification of yeasts [6] led to the investigation of related polysaccharides isolated from ascomycetous lichens via Fehling precipitation. Their structure, as evidenced by chemical and 13C NMR studies, proved to be typical of the parent lichen and could thus be utilized in chemotyping studies [7–11]. In terms of macromolecules, the study of mannosecontaining polysaccharides as a taxonomic tool involves the structural diversity of the galactomannans from several lichenized fungi, and depends on their side-chain

0378-1097/$22.00  2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.019

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Departamento de Bioquı´mica e Biologia Molecular, Universidade Federal do Parana´, C.P. 19046, CEP 81531-990 Curitiba, PR, Brazil b Departamento de Botaˆnica, Universidade Federal do Parana´, C.P. 19031, CEP 81531-990 Curitiba, PR, Brazil

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2. Materials and methods

NaBH4 at 100 C for 3 h. The alkaline extract was neutralized with HOAc, dialyzed against tap water, and after 48 h was freeze dried. The crude fraction obtained from alkaline extraction was submitted to a freeze-thawing process, which furnished insoluble and soluble material, which were separated by centrifugation (15 min, 9000 rpm, 25 C). The soluble fraction was submitted to a second purification process using Fehling solution [17], resulting in a precipitate (Cu2+-ppt) and a soluble fraction (Cu2+-sup) which were separated by centrifugation under the above conditions. Each fraction was neutralized with HOAc, dialyzed against tap water and deionized with mixed ion exchange resins. 2.3. Monosaccharide composition Hydrolysis of the fractions were carried out with 1 M TFA at 100 C for 8 h and the hydrolyzates then evaporated to dryness, followed by successive reduction with NaBH4 and acetylation with Ac2O–pyridine (1:1 v/v; 2 ml) at room temperature for 12 h [18,19]. The resulting alditol acetates were analyzed by GCMS using a Varian model 3300 gas chromatograph linked to a Finnigan Ion-Trap, model 810 R-12 mass spectrometer, using a DB-225 capillary column (30 m · 0.25 mm i.d.), with helium as carrier gas. The analysis was carried out from 50–220 C at 40 C/ min maintaining the temperature constant to the end of analysis (18 min). The products were identified by their typical retention times and electron impact profiles.

2.1. Lichenized fungi (family, Parmeliaceae) Parmotrema austrosinense (Zahlbr.) Hale, Parmotrema delicatulum (Vain.) Hale, Parmotrema schindlerii Hale, Parmotrema mantiqueirense Hale, Parmotrema tinctorum (Nyl.) Hale, Rimelia cetrata (Ach.) Hale and Fletcher and Rimelia reticulata (Taylor) Hale and Fletcher were examined. Parmotrema spp. were collected in 1996, in Lapa, State of Parana´, Brazil, while Rimelia spp. are from Curitiba, State of Parana´, and have their vouchers (no. 33886, 33354, 33890, 33355, 28838, 38057, 38118, respectively) deposited in the UPCB (Herbarium name follows Holmgren et al. [16]).

LICHENIZED FUNGUS Cleaned, dried and powdered CHCl3: MeOH (2:1; v/v) at 60ºC, 3 h (x3)

MeOH: H2O (4:1; v/v) at 80ºC for 3 h (x3)

Extract of low molecular mass

Lichen residue II Aq. 2% KOH at 100ºC for 3 h (x3)

Alcaline extract

Lichen residue III

Freeze-Thawing Centrifugation

2.2. Isolation and purification of polysaccharides Lichenized fungus samples (P. austrosinense, 41 g; P. delicatulum, 32 g; P. schindlerii, 35 g; P. mantiqueirense, 43 g; P. tinctorum, 60 g; R. cetrata, 31 g; and R. reticulata, 26 g) were successively refluxed in CHCl3-MeOH (2:1 v/v; 300 ml) and 80% aqueous MeOH (300 ml), in order to extract low molecular components. The residual material was then extracted three times with 2% aq. KOH containing traces of

Lipid extract

Lichen residue I

Supernatant

Precipitate

Treatment with Fehling solution Centrifugation

Felhing supernatant

Fehling precipitate GALACTOGLUCOMANNAN

Fig. 1. Scheme of extraction and purification of the galactoglucomannans obtained from Parmotrema spp. and Rimelia spp.

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substituents on (1 ! 6)-linked a-D-mannopyranosyl main-chains [12]. These generally include monosubstituents at O-2 of a-D-Manp or a-D-Galp, at O-4 by b-Galp and sometimes with disubstitution occurring at O-2 and O-4 by a-D-Galp and b-D-Galp, or a-D-Manp and b-DGalp, respectively, although some of the main-chain units are frequently not substituted. Studies involving the taxonomy of lichenized fungi from Cladonia and Cladina species were carried out since classical taxonomy considered Cladina to be a subgenus of Cladonia, but thereafter lichenologists decided to be a distinct genus. Woranovicz-Barreira et al. [11] showed that galactoglucomannans are chemotypes which could be significant in aiding the taxonomy of Cladonia spp. and those of related genera. Ahti and Depriest [13] proposed, based on molecular phylogenetic results, that Cladina becomes a synonym of Cladonia. Subsequently, Carbonero et al. [14] studied the structures of polysaccharides of Cladina spp. and, based on their chemical characterization and when compared to those of Cladonia species, agreed with results obtained with DNA studies. Another suitable example of conflicting taxonomic data concerns the segregation of the genus Rimelia from the earlier Parmotrema, which was proposed by Hale and Fletcher [15]. Studies on the chemical elucidation of polysaccharides of species from these two genera are now reported as a taxonomic aid.

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a mixture of partially O-methylated alditol acetates, which was analyzed by GC-MS. The analysis was carried out from 50–215 C at 40 C/min maintaining the temperature constant to the end analysis (31 min), and the resulting partially O-methylated alditol acetates identified by their typical electron impact breakdown profiles and retention times [22,23].

Table 1 Yield of Fehling precipitates obtained from Parmotrema spp. and Rimelia spp. and their monosaccharide composition Lichenized fungus

Yield (%)a

Monosaccharide composition (%)b Man

Gal

Glc

Parmotrema austrosinense P. delicatulum P. mantiqueirense P. schindlerii P. tinctorum

6.7 3.8 5.6 3.2 5.4

50 49 50 50 51

44 44 43 43 42

5 6 6 7 6

Rimelia cetrata R. reticulata

3.4 5.2

53 52

40 40

7 8

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2.5. Determination of homogeneity and molar mass

a

Yields based on dry material. Alditol acetates obtained on successive hydrolysis, NaBH4 reduction, and acetylation, analyzed by GC-MS (DB-225 column). b

2.4. Methylation analysis Each sample (5 mg) was per-O-methylated according to the method of Ciucanu and Kerek [20], using powdered NaOH in Me2SO–MeI. The per-O-methylated derivatives were hydrolyzed with 50% v/v sulfuric acid (1 h, 0 C), followed by dilution to 5.5% v/v (5 h, 100 C), neutralization (BaCO3) and filtration [21]. The resulting mixture of O-methylaldoses was reduced with NaBH4 or NaBD4 and acetylated as cited above to give

Table 2 Partially O-methylated alditol acetates obtained from methylated galactoglucomannans O-Me-alditol acetatesa

2,3,4,6-Me4Man 2,3,4,6-Me4Glc 2,3,5,6-Me4Gal 2,3,4,6-Me4Gal 2,4,6-Me3Glc 2,4,6-Me3Man 2,4,6-Me3Gal 2,3,6-Me3Man 3,4,6-Me3Gal 2,3,4-Me3Man 2,3,4-Me3Gal 2,6-Me2Man 4,6-Me2Gal 3,6-Me2Gal 2,3-Me2Man 3,4-Me2Man 2,4-Me2Man 2,3-Me2Gal 2-MeMan 3-MeMan Man

Molar %

b,c

Pa

Pd

Pm

Ps

Pt

Rc

Rr

1.1 1.6 – 40.0 3.3 0.4 0.4 0.2 – 22.0 1.1 0.2 0.6 0.5 1.9 7.2 0.2 0.4 0.4 18.1 0.4

0.9 1.4 0.6 40.3 3.6 – 0.3 0.1 – 21.4 0.5 0.2 0.2 0.5 4.4 7.8 0.2 0.1 0.1 16.4 0.2

0.9 3.1 0.5 39.6 2.5 0.4 – – 1.7 15.7 0.7 0.3 0.3 0.9 3.8 6.1 0.1 0.2 0.8 19.3 0.7

1.3 2.4 0.7 39.7 3.8 0.3 0.3 0.4 – 19.7 0.8 0.5 0.4 0.4 3.6 6.6 – – 0.3 18.4 0.4

1.3 1.7 0.2 39.2 3.2 – 0.5 0.9 – 20.9 1.2 0.3 0.4 0.7 2.4 7.4 0.4 0.4 0.3 18.3 0.3

1.0 3.8 0.3 38.4 4.1 – 0.2 – 1.3 17.9 0.6 0.3 0.1 0.3 6.2 7.6 – 0.3 0.2 17.1 0.3

0.9 4.1 0.7 38.1 3.9 – 0.5 – 1.7 14.1 1.1 0.2 0.3 1.0 6.5 8.7 – 0.4 0.3 16.8 0.7

a O-Me-alditol acetates obtained by methylation analysis, followed by successive hydrolysis, reduction and acetylation, analyzed by GC-MS (column DB-225). b % of peak area relative to total peak area. c The symbols are: Pa, P. austrosinense; Pd, P. delicatulum; Pm, P. mantiqueirense; Ps, P. schindlerii; Pt, P. tinctorum; Rc, R. cetrata; Rr, R. reticulata).

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The elution profiles of fractions were determined by high performance size-exclusion chromatography (HPSEC), using a WATERS 510 HPLC pump at 0.6 ml/min with four gel permeation columns in series with exclusion sizes of 7 · 106, 4 · 105, 8 · 104, and 5 · 103 Da, using a refraction index (RI) detector. The eluent was 0.1 mol/l aq. NaNO3 containing 200 ppm aq. NaN3. Samples, previously filtered through a membrane (0.22 lm; Millipore), were injected (250 ll loop) at 2 mg/ ml. The specific refractive index increment (dn/dc) was determined, with the samples being dissolved in 50 mM NaNO3 and five increasing concentrations, ranging from 0.2 to 1.0 mg/ml, were used to determine the slope of the increment. Results were processed in software provided by the manufacturer (Wyatt Technologies).

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2.6. Nuclear magnetic resonance spectroscopy

3. Results and discussion

NMR spectra were obtained using a 400 MHz Bruker model DRX Avance spectrometer with a 5 mm inverse probe. 13C NMR (100.6 MHz) and HSQC1DEPT analyses were performed at 50 or 30 C, with samples being dissolved in D2O, the OH groups being exchanged with D2O followed by freeze-drying. Chemical shifts of samples are expressed in ppm (d) relative to acetone at d 30.20 and 2.22 for 13C and 1H signals, respectively.

Samples of seven species of lichenized fungi from Parmotrema and Rimelia genus were submitted to purification procedures, according to Fig. 1, and after the treatment with Fehling solution supernatant and precipitated fractions were obtained. Table 1 shows all Fehling precipitated fractions to contain mannose, galactose and glucose as monosaccharide components. The average obtained from all the fractions was 51% Man, 42% Gal and 7% of Glc, and it is important to observe that

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Fig. 2. 13C NMR spectra of heteropolysaccharide from Parmotrema austrosinense (a), P. delicatulum (b), P. mantiqueirense (c), P. schindlerii (d), P. tinctorum (e), Rimelia cetratum (f), and R. reticulata (g).

E.R. Carbonero et al. / FEMS Microbiology Letters 246 (2005) 273–278

Fig. 3. HSQC-DEPT of heteropolysaccharide from Parmotrema austrosinense in D2O at 30 C (chemical shifts are expressed as d, ppm).

(3.74) from the non reducing ends of Galp, Glcp, and Manp units. According to the present data, we can conclude that the galactoglucomannans showed much similarity between the two distinct genera Parmotrema and Rimelia, but with minor differences, typical of the species. The galactoglucomannans have main chains of (1 ! 6)linked a-D-mannopyranosyl residues, that are mainly unsubstituted and disubstituted at O-2 and O-4 with a-Galp and b-Galp side-chains, respectively. These results agree with previous data on other species of these genera [9,27], and show the chemical method based on polysaccharides to be useful as an additional tool in lichenized fungi classification.

Acknowledgements The authors thank the Brazilian agencies, Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), and Fundac¸a˜o Arauca´ria for financial assistance, and Dr. G. Torri, from the Istituto di Ricerche Chimiche e Biochimiche ‘‘G. Ronzoni’’, Milan, Italy, for preparation of the HSQC-DEPT spectrum.

References [1] Purvis, W. (2000) Lichens 122 p. Craft Print, Singapore. [2] Aghoramurth, K., Sarma, K.G. and Seshadri, T.R. (1961) Chemical investigation of Indian lichens. J. Sci. Ind. Res. 20, 166–168.

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no species showed a significant variation of monosaccharide composition when compared to the average data. All Fehling precipitate fractions showed homogeneous elution profiles when analyzed by HPSECMALLS, and as all the elution profiles were similar, the averaged specific refractive index increment was dn/dc = 0.148. The samples had Mw of 53.7 kDa for P. austrosinense, 68.2 kDa for P. delicatulum, 62.7 kDa for P. schindlerii, 64.5 kDa for P. mantiqueirense, 39.4 kDa for P. tinctorum, 38.5 kDa for R. cetrata and 59.7 kDa for R. reticulata. Methylation analysis of Fehling precipitate fractions (Table 2) showed highly branched structures based on resulting partially O-methylated alditol acetates (GCMS) with high proportion of non-reducing ends of Galp, besides small percentages of Manp, Galf and Glcp. They also showed 2,3,4-Me3Man, 2,3-Me2Man, 3,4-Me2Man, and 3-MeMan, corresponding to the main chains formed by a-Manp-(1 ! 6) units, which were nonsubstituted, substituted at O-2, O-4, and disubstituted at O-2,4. Small amounts of Manp fully substituted units were also observed. Substitutions at O-2, O-3, and O-6; disubstitutions at O-2,3; O-2,4; and O-4,6 were observed for Galp units. Glcp was 3-O-substituted, besides its nonreducing end-units. The 13C NMR spectra of the galactoglucomannan (Fig. 2) contained major signals in common, but there were minor differences typical of the species. In general, we have found that such 13C NMR spectra correspond to the lichen species [10,11,14,24], to the extent that have been used for classification and identification [9–11]. The 13C NMR spectra of all species (Fig. 2), contained C-1 signals that indicated predominant branched structures with nonreducing end-units of b-D-Galp(1 ! 4)-a-D-Manp (d 104.6), a-D-Galp-(1 ! 2)-a-DManp (d 102.8) [12,25], along with 6-O-(d 101.6) and 2,6-di-O-and 2,4,6-tri-O-substituted (d 99.8) units of aD-Manp from the polysaccharide core [12,26]. The signal at d 80.8 arose from 2-O-substituted a-D-Manp units [27]. The HSQC spectrum of the P. austrosinense galactoglucomannan (Fig. 3) defined its a-and b-glycosidic configurations: the nonreducing end-units of Galp that had a b-configuration by virtue of a high-field H-1 signal at 4.42 (C-1 d 104.6), and an a-configuration due to a low field H-1 signal at d 5.17 (102.8). The low-field H-1 signals in d 4.98 (101.6) and 5.23 (99.8) indicated that the units of Manp had the a-configuration. Its HSQC-DEPT spectrum showed inverted signals in d 67.9 and d 67.3 suggesting a substituted CH2 group, probably from C-6 of a-Manp units, data in agreement with the methylation analysis that gave mainly 3-Me and 2,3,4-Me3Man derivatives. The non substituted C6Õs appeared at d 62.4 (3.92), d 62.7 (3.78) and d 62.9

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