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Polysaccharide and glycolipid composition in Tritrichomonas foetus
Int. J. Biochem. Vol. 20, No. 3, pp. 329-335, 1988 Printed in Great Britain. All rights reserved
0020-711X/88 $3.00+0.00 Copyright © 1988 Pergamon Journals Ltd
POLYSACCHARIDE AND GLYCOLIPID COMPOSITION IN T R I T R I C H O M O N A S FOETUS BENEDITO PRADO DIAS FILHO l, CELUTA SALES ALVIANOI, WANDERLEY DE SOUZA2 and JAYME ANGLUSTER1'* qnstituto de Microbiologla, Universidade Federal do Rio de Janeiro, Ilha do Fundao, 21941, Rio de Janeiro, R. J., Brasil 2Instituto de Bioflsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fund~o, 21941, Rio de Janeiro, R. J., Brasil
(Received 22 April 1987) Abstract--1. The polysaccharide and glycolipid composition in Tritrichomonas foetus was studied by paper, thin-layer and gas-liquid chromatographic analysis. 2. The carbohydrate components of the polysaccharide were glucose (47%), galactose (34%) and mannose (19%). N-acetylneuraminic acid was the sialic acid derivative characterized in the flagellate whole cells. 3. The sialic acid density was estimated as 2.7 × 1 0 7 residues/cell. 4. The long-chain base dihydrosphingosine, the carbohydrates galactose (67%), glucose (21%) and mannose (12%) as well as the fatty acids myristic (48%) and palmitic (52%) acids were characterized as components of the total glycolipids of T. foetus. 5. Total glycolipids were fractionated: a galactocerebroside and a ganglioside were identified.
INTRODUCTION Cell surface carbohydrates are recognized constituents of antigenic determinants (Winzler, 1970), receptors for lectins, toxins (Nicolson, 1974), viruses (Schauer, 1985) and pathogenic microorganisms, which have been characterized in mammalian and other animal cells ( L e ~ e r and Svanborg-Edrn, 1981). Conversely, the immune surveillance system in mammals is able to recognize carbohydrate structures on foreign cells (Feizi, 1985) and pathogenic microorganisms (Leffler and Svanborg-Edrn, 1981). The carbohydrates of the cell membrane are present partly as glycolipids, which have been implicated as important mediators in cellular recognition and differentiation (Hakomori, 1981), tumorassociated markers and regulators of cell proliferation (Hakamori, 1984). The carbohydrate chain is the structure responsible for the glycolipid specific reactivity. In gangliosides, sialic acid units have been found to be important reactive groups (Hakamori, 1981; Schauer, 1982). Tritrichomonas foetus is a c o m m o n pathogenic protozoan of the urogenital tract of cattle, where it interacts mainly with epithelial cells (Honigberg, 1978). The few studies carried out on the cell surface of this flagellate include analyses of the surface charge (Silva-Filho et al., 1982; Silva-Filho and De Souza, 1986), detection of lectin receptors (Benchimol et al., 1981) and Ca:÷-binding sites (Benchimol et al., 1982b), as well as freeze-fracture observations (Benchimol et al., 1981, 1982a). N o information is available on isolated components of the cell membrane of T. foetus. A carbohydrate determination study showed that in this parasite glycogen is the prevalent intracellular storage polysaccharide (Manners and Rylei, 1955). A further study of the polysaccharides *To whom correspondence should be addressed.
and glycolipids, as well as the sialic acid derivatives of T. foetus was undertaken in the present work. MATERIALS AND METHODS
Microorganism Tritrichomonas foetus was isolated by Dr H. Guida (Embrapa, Rio de Janeiro, Brasil) from the urogenital tract of a bull in the State of Rio de Janeiro. The parasite has been maintained by weekly transfers at room temperture (23°C) in a medium with the following composition: 2.0% (w/v) trypticase (BBL), 1.0% (w/v) yeast extract, 0.5% (w/v) maltose, 0.1% (w/v) L-cysteine hydrochloride, 0.02% (w/v) ascorbic acid and 10% (v/v) of heat inactivated and filter-sterilized fresh bovine serum. For the experiments, cells were grown in 11 flasks containing 720 ml of medium. The inoculum consisted of about 4.5 x 106 cells/ml. The ceils, after being cultivated for 48 h at 37°C, were collected by centrifugation (1500g) at 4°C for 10min and were washed 3 times in cold phosphate-buffered saline (PBS) pH 7.2, 0.01 M. Carbohydrate composition of polysaccharides The polysaccharides were extracted from the washed cells with 6% KOH for 6 hr at 100°C. The alkaline extract was neutralized with glacial acetic acid, centrifuged and the supernatant precipitated with 3 volumes of ethanol. The precipitate was dissolved in water and lyophilized and the polysaccharides purified by column chromatography on Bio-Gel P-100 After hydrolysis with 3 N trifluoroacetic acid (which was removed by evaporation) for 3hr at 100°C the monosaccharide components of the polysaccharides were identified by paper chromatography with butanol-ethanolwater (3:2:1, v/v/v) as the solvent and ammoniacal silver nitrate and p-anisidine as the revealing reagents (Hough and Jones, 1962). The monosaccharides were quantified as their alditol acetates (Sawardeker et al., 1965) by gas-liquid chromatography (GLC) on a 3° neopentylglycol succinate (NPGS) of a stainless steel column (6 ft x 1/8 in.) with 100/200 gas chrom, at 210°C. An internal standard of allitol acetate was used.
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Assay of sialic acid Whole washed cells (109) were suspended in 2ml of distilled water dialysed for 5 hr at 4°C (four changes during this period). Subsequently, the suspension was acidified with formic acid to pH 2.0 and heated for 1 hr at 70°C followed by dialysis against 100 ml of distilled water (24 hr/4°C/two changes in the period). The cell suspension was then lyophilized and the residue hydrolysed in 2.0 ml of 0.1 M HCI for 1 hr at 80°C. After dialysis for 24 hr against 100 ml distilled water with two changes during this period, sialic acids in the combined dialysates of both hydrolytic steps were purified by ion-exchange chromatography as described (Kamerling et al., 1980). Sialic acids were assayed colorimetrically (Jourdian et al., 1971) and analysed by thin-layer chromatography on cellulose plates with the solvent mixture n-propanol-I M ammonia-water (6:2:1, v/v/v; Blix and Lindberg, 1960). Before use, the plates were activated in 0.1 M HC1 and dried under a current of air at room temperature for 30 min. Spots were located by spraying with a periodic acid-resorcinol reagent (Jourdian et al., 1971). Standards of Nacetylneuraminic and N-glycolyneuraminic acids (Sigma Chemical Co.) were used at 10 mg/ml. The nature of the sialic acids were confirmed by gas-liquid chromatography of the correspondent trimethylsilyl derivative on 3% OV 17/Gas Chrom 800-100 mesh at 210°C.
Extraction and purification of glycolipids Lipids were extracted from the washed protozoan cells at room temperature with each of the following solvent mixtures: chloroform-methanol, 2:1 and chloroform-methanol 1:2 (v/v). Both extracts were combined and evaporated to dryness, and a solvent mixture of chloroform-methanol water (1.0:2.0:1.4 v/v/v) was added to this dried residue in a funnel to isolate the crude glycolipids. The solvents were carefully mixed by turning the funnel up and down several times but shaking was omitted to prevent emulsification. When the two phases were distinctly separated (generally within 6 hr), the lower phase was removed and the upper phase set aside. To the lower was added methanol. After thorough shaking of the funnel, 0.01 M KC1 was then added slowly, and carefully mixed with the extract. The two upper phases were combined and evaporated to dryness after addition of isobutanol foaming. For the purification of the crude glycolipids fraction the residue of the upper phases was dissolved in chloroform-methanol-water (60:30:45, v/v/v) and left 24 hr at room temperature. The precipitate was removed by centrifugation. The glycolipid extract was evaporated and the residue dissolved in water and dialysed against running tap water for 48 hr and then against two changes of distilled water for 24 hr each. The dialyzed glycolipids were evaporated to dryness (Svennerholm and Fredman, 1980).
Carbohydrate composition of glycolipids Glycolipids were hydrolysed with 3 N trifluoroacetic acid tbr 3 hr at 100°C, after which the acid was removed by evaporation. The monosaccharide components of the glycolipids were detected by paper chromatography and their corresponding alditol acetates by GLC as described above (Albersheim et aL, 1967; Sawardeker et al., 1965).
Fatty acid composition of glycolipids Glycolipids (1.5 mg) were heated with 0.5 M methanolic HC1 (2 ml) at 80°C for 18 hr in a sealed tube. Methyl esters of fatty acids were extracted with three 2 ml portions of
hexane and then detected by gas-liquid chromatography as described elsewhere (Dias Filho et al., 1985).
Long chain composition of glycolipids Glycolipids were hydrolyzed in methanol--con. HC1water (82.0:6:9.4, v/v/v) at 70°C for 18 hr and the liberated fatty acids were removed by extraction with n-hexane. The lower phase was adjusted to pH 12, the long chain bases were extracted with diethyl ether and purified on a column packed with silica gel 60 (Karlsson, 1970). The column was eluted successively with chloroform-methanol (1:1, v/v), chloroform-methanol (1:4, v/v) and 100% methanol. The long chain bases were recovered with the last two solvents (Iwamori and Nagai, 1978) and then chromatographed on activated (105°C/1/2 hr) silica gel G plates using chloroform-methanol 2 N NH4OH (40:10:1 v/v/v) as solvent. Spots were located with ninhydrin spray (0.2 g ninhydrin in 95 ml n-butanol and 5 ml pyridine; Sambasivarao and McCluer, 1963). Standard of sphingosine and dihydrosphingosine (Sigma Chemical Co.) were used at 5 mg/ml.
Fractionation of glycolipids Glycolipids were separated by thin layer chromatography on activated (105°C/1/2hr) silica gel G plates using chloroform-methanol-2 N NH4OH (40:10:1) as solvent (Kochetkov et al., 1973). They were visualized by spraying with orcinol reagent (Skipski and Barclay, 1969). Standards of galactocerebroside and ganglioside from bovine brain (Sigma Chemical Co.) were used at 2 mg/ml. RESULTS
Carbohydrate composition o f polysaccharides Polysaccharides were isolated from T. foetus by alkaline K O H extraction. After total acid hydrolysis of the polysaccharides the c a r b o h y d r a t e c o m p o n e n t s identified by G L C were glucose (47%), galactose (34%) a n d m a n n o s e (19%). The presence of these m o n o s a c c h a r i d e s was confirmed by p a p e r chromatography.
Assay o f sialic acids Sialic acids, liberated from whole cells by acid hydrolysis, were evaluated by colorimetric determinations. A density o f 2.7 × 107 residues of sialic acid per cell was found. On thin-layer c h r o m a t o g r a p h y N-acetylneuramic acid was the only derivative o f sialic acid present in the hydrolysate from T. foetus cells (Fig. 1).
Carbohydrate, fatty acid and long-chain base composition o f glycolipids Glycolipids of T.foetus were isolated by extraction with c h l o r o f o r m - m e t h a n o l . The c a r b o h y d r a t e components, o b t a i n e d after total acid hydrolysis of the glycolipids, identified by G L C were glucose, galactose a n d mannose. A clear p r e d o m i n a n c e of galactose (67%) was observed (Table 1). The fatty acids composition of the glycolipids is also listed in Table 1. The two fatty acids detected by G L C C were myristic a n d palmitic acids. Sphingosine bases were released from the glycolipids sample by methanolysis. The thin-layer chro-
Table 1. Sugar and fatty acids composition (%) of glycolipids from T. foetus Sugar Fatty acids Mannose Galactose Glucose Myristic (C~4:0) Palmitic(C~:0) 12 67 21 48 52
#~ ~.~
~.~ ~.~
r~
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Fig. 3. Thin-layer chromatography of T. foetus total glyeolipids. (A) Mixture of gangliosides from bovine brain (from Sigma Chemical Co.); (B) total glyeolipids from T. foetus; (C) galaetoeerebroside from bovine brain (from Sigma Chemical Co.).
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Cell surface carbohydrates matography of the long-chain base is shown in Fig. 2. A single spot with the mobility of dihydrosphingosine was detected. Fractionation of glycolipids Thin-layer chromatography was employed for glycolipids separation. From the four main spots visualized, two of them were identified: one corresponding to a galactocerebroside and the other to a ganglioside, bovine brain standards taken as references (Fig. 3).
DISCUSSION Galactose, glucose and mannose were polysaccharide components detected in T. foetus. In general, this carbohydrate pattern resembled that observed in Trichomonas vaginalis (Warton and Honigberg, 1983) and in members of the Trypanosomatidae family such as species of Crithidia (Gottlieb, 1978; Esteves et al., 1982), Herpetomonas (Esteves et al., 1979; Lopes et al., 1983; Soares et al., 1984; Fiorini et al., 1985), Leishmania (Dwyer, 1977) and Trypanosoma (Dwyer and D'Alessandro, 1974; Pereira et al., 1980). Amino sugars including Nacetyl-D-glucosamine and N-acetyl-o-galactosamine were not found in the polysaccharide containing fraction. However, these components probably are part of surface glycoproteins since their presence is suggested by the reactivity of whole cells with specific lectins (Benchimol et al., 1981). The reactivity of T. foetus cells with the lectins concanavalin A and that of Geodia cydodonyum (Benchimol et al., 1981) indicates respectively that glucose and/or mannose and D-galactose were carbohydrate ligands exposed on the cell surface. However, glucose, at least in part is derived from glycogen which is a storage material in T. foetus and T. gallinae: in both parasites glycogen usually represents about 10-30% of the cell dry weight (Manners and Rylei, 1955). In contrast, with cytochemical studies, no indications were obtained that Trypanosomatidae posses reserve polysaccharides, so that all carbohydrate constituents are associated with cellular membranes, comprising the plasma membrane, the Golgi complex, endoplasmic reticulum and pinocytotic vesicles (Brooker, 1976; De Souza, 1976; De Souza et al., 1976; Esteves et al., 1979). The binding of colloidal iron and cationized ferritin particles to the parasite's surface, the high negative surface charge which was markedly reduced by neuraminidase treatment (Silva-Filho et al., 1982) and the binding of the lectin from Limulus polyphemus indicates that sialic acid is an important component exposed on the cell surface of T. foetus (Benchimol et al., 1981). Moreover, sialic acids comprise several derivatives of neuraminic acid includng N-acetyl, N-glycolyl and other substitutions (Blix et al., 1957). However, the present work shows, for the first time, that N-acetylneuraminic acid is the only derivative exposed on the plasma membrane of T. foetus. In the protozoa Crithidia deanei (Oda et al., 1984), Trypanosoma cruzi (Schauer et al., 1983) and the pathogenic fungi Sporothrix schenckii (Alviano et al., 1982) and Fonsecaea pedrosi (Souza et al., 1986), both the derivatives N-glycolyl and N-acetylneuraminic acids were detected.
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Sialic acid carries a negative charge on the cell surface, and may play a role in cell-to-cell recognition and receptor function (Schauer, 1982). Its biological role in T. foetus is still unclear. The few studies carried out on other microorganisms showed that sialic acid displays important functions such as the protective effect for S. schenkii against phagocytosis before an efficient immune response takes place (Oda et al., 1983); to difficult the uptake of bloodstream trypomastigotes by macrophages (Araujo-Jorge and De Souza, 1984) and to render trypomastigotes of T. cruzi unable to activate the alternative complement pathway (Kipnis et al., 1981). The glycolipids from T. foetus contains sphingosine base, two fatty acids, glucose, mannose and galactose. The sugar constituents of the glycolipids closely resembled those of T. foetus polysaccharides described above. The carbohydrate pattern of T. foetus glycolipids also shows a similarity with glycoconjugates of T. cruzi (Barreto-Bergter et al., 1985) and Leishmania (Palatnik, 1984). Similarities among structures of carbohydrate chains from mamalian glycoproteins and glycolipids have been also reported (Rauvala and Finne, 1979). T. foetus glycolipids only contain the normal saturated fatty acids myristic and palmitic acids which are components of total cell lipids (Dias Filho et al., 1985). This fatty acid profile is essentially different from that observed in glycolipids of extraneural tissue which are characterized by higher contents of the unsaturated fatty acids (Windeler and Feldman, 1970). Recently, in glycoconjugates of Trypanosoma mega a mixture of normal saturated and unsaturated ct-hydroxy fatty acid was detected (Vermelho et al., 1986). The sphingosine base composition of T. foetus glycolipids forming amide groups with fatty acids consist exclusively of dihydrosphingosine. In contrast, the glycolipid of T. mega contains sphingosine and traces of dihydrosphingosine (Vermelho et al., 1986). In Crithidia faseieulata a branched chain sphingolipid base, 19-methyl-C20 phitosphingosine, was previously noted (Carter et al., 1966). A galactocerebroside and a ganglioside were identified after glycolipid fractionation by thin-layer chromatography. It has been reported that in galactocerebrosides the galactosiiceramide linkage is of a Gal Bl-ceramide type (Hakamori, 1981). It can be stressed that in T. foetus glycolipids the content of galactose is high (about 67%). In addition, gangliosides are a family of glycolipids that contain sialic acids (Kohn et al., 1978). As referred, only Nacetylneuraminic acid was detected is T. foetus cells and therefore it is the sialic acid derivative constituent of the carbohydrate moiety of the gangliosides. In opposition, sialo-containing compounds from sea urchin Strongylocentrotus intermedius (Kochetkov et al., 1973) and bovine adrenal medula (Leeden et al., 1968) consist of a mixture of N-acetyl and Nglycolylneuraminic acids. Glycolipids have important biological functions such as cell recognition, control of cell growth, differentiation, etc. In addition, some glycolipids act as cell surface markers and important antigens (Hakomori, 1981,1984). Studies are in progress
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aiming to determine Trichomonas.
the role of sialic acid in
Acknowledgements--The authors thank Drs Hertha Meyer and Miklos Muller for reading the manuscript, Mr Luiz Rodrigues da Silva for dedicated technical assistance and Financiadora de Estudos e Projetos (FINEP) and Conselho Nacional de Desenvolvimento Cientifico e Tecnolrgico (CNPq) for financial support.
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Silva-Filho F. C., Elias C. A. and De Souza W. (1982) The surface charge of Tritrichomonasfoetus. J. ProtozooL 29, 551-555. Silva-Filho F. C. and De Souza W. (1986) Effect of colchicine, vinblastine, and cytochalasin B on cell surface anionic sites of Tritrichomonas foetus. J. ProtozooL 33, 6-10. Skipski V. P. and Barclay M. (1969) Thin-layer chromatography of lipids. Meth Enzymol. 14, 530-598. Soares R. M. A., Alviano C. S., Silva-Filho F. C., Esteves M. J. G., Angluster J. and De Souza W. (1984) Effect of dimetilsulfoxide on the cell surface of Herpetomonas samuelpessoai. J. submicrosc. Cytol. 16, 735-739. Souza E. T., Silva-Filho F. C., De Souza W., Alviano C. S., Angluster J. and Travassos C. R. (1986) Indentification of sialic acids on the cell surface of hyphae and conidia of the human pathogen Fonsecaea pedrosio. J. med. vet. MycoL 24, 145-153. Svennerholm L. and Fredman P. (1980) A procedure for the quantitative isolation of brain gangliosides. Biochim. biophys. Acta 617, 97-109. Vermelho A. B., Hogge L. and Barreto-Bergter E. (1986) Isolation and characterization of a neutral glycosphingolipid from the epimastigote form of Trypanosoma mega. J. Protozool. 33, 208-213. Warton A. and Honigberg B. M. (1983) Analysis of surface saccharides in Trichomonas vaginalis strains with various pathogenicity levels by fluorescein-conjugated plant lectins. Z. Parasitenk. 69, 149-159. Windeler A. S. and Feldman G. L. (1970) The isolation and partial structural characterization of some ocular gangliosides. Biochim. biophys. Acta 202, 361-366. Winzler R. J. (1970) Carbohydrates in cell surfaces. Int. Rev. CytoL 29, 77-125.
0020-711X/88 $3.00+0.00 Copyright © 1988 Pergamon Journals Ltd
POLYSACCHARIDE AND GLYCOLIPID COMPOSITION IN T R I T R I C H O M O N A S FOETUS BENEDITO PRADO DIAS FILHO l, CELUTA SALES ALVIANOI, WANDERLEY DE SOUZA2 and JAYME ANGLUSTER1'* qnstituto de Microbiologla, Universidade Federal do Rio de Janeiro, Ilha do Fundao, 21941, Rio de Janeiro, R. J., Brasil 2Instituto de Bioflsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fund~o, 21941, Rio de Janeiro, R. J., Brasil
(Received 22 April 1987) Abstract--1. The polysaccharide and glycolipid composition in Tritrichomonas foetus was studied by paper, thin-layer and gas-liquid chromatographic analysis. 2. The carbohydrate components of the polysaccharide were glucose (47%), galactose (34%) and mannose (19%). N-acetylneuraminic acid was the sialic acid derivative characterized in the flagellate whole cells. 3. The sialic acid density was estimated as 2.7 × 1 0 7 residues/cell. 4. The long-chain base dihydrosphingosine, the carbohydrates galactose (67%), glucose (21%) and mannose (12%) as well as the fatty acids myristic (48%) and palmitic (52%) acids were characterized as components of the total glycolipids of T. foetus. 5. Total glycolipids were fractionated: a galactocerebroside and a ganglioside were identified.
INTRODUCTION Cell surface carbohydrates are recognized constituents of antigenic determinants (Winzler, 1970), receptors for lectins, toxins (Nicolson, 1974), viruses (Schauer, 1985) and pathogenic microorganisms, which have been characterized in mammalian and other animal cells ( L e ~ e r and Svanborg-Edrn, 1981). Conversely, the immune surveillance system in mammals is able to recognize carbohydrate structures on foreign cells (Feizi, 1985) and pathogenic microorganisms (Leffler and Svanborg-Edrn, 1981). The carbohydrates of the cell membrane are present partly as glycolipids, which have been implicated as important mediators in cellular recognition and differentiation (Hakomori, 1981), tumorassociated markers and regulators of cell proliferation (Hakamori, 1984). The carbohydrate chain is the structure responsible for the glycolipid specific reactivity. In gangliosides, sialic acid units have been found to be important reactive groups (Hakamori, 1981; Schauer, 1982). Tritrichomonas foetus is a c o m m o n pathogenic protozoan of the urogenital tract of cattle, where it interacts mainly with epithelial cells (Honigberg, 1978). The few studies carried out on the cell surface of this flagellate include analyses of the surface charge (Silva-Filho et al., 1982; Silva-Filho and De Souza, 1986), detection of lectin receptors (Benchimol et al., 1981) and Ca:÷-binding sites (Benchimol et al., 1982b), as well as freeze-fracture observations (Benchimol et al., 1981, 1982a). N o information is available on isolated components of the cell membrane of T. foetus. A carbohydrate determination study showed that in this parasite glycogen is the prevalent intracellular storage polysaccharide (Manners and Rylei, 1955). A further study of the polysaccharides *To whom correspondence should be addressed.
and glycolipids, as well as the sialic acid derivatives of T. foetus was undertaken in the present work. MATERIALS AND METHODS
Microorganism Tritrichomonas foetus was isolated by Dr H. Guida (Embrapa, Rio de Janeiro, Brasil) from the urogenital tract of a bull in the State of Rio de Janeiro. The parasite has been maintained by weekly transfers at room temperture (23°C) in a medium with the following composition: 2.0% (w/v) trypticase (BBL), 1.0% (w/v) yeast extract, 0.5% (w/v) maltose, 0.1% (w/v) L-cysteine hydrochloride, 0.02% (w/v) ascorbic acid and 10% (v/v) of heat inactivated and filter-sterilized fresh bovine serum. For the experiments, cells were grown in 11 flasks containing 720 ml of medium. The inoculum consisted of about 4.5 x 106 cells/ml. The ceils, after being cultivated for 48 h at 37°C, were collected by centrifugation (1500g) at 4°C for 10min and were washed 3 times in cold phosphate-buffered saline (PBS) pH 7.2, 0.01 M. Carbohydrate composition of polysaccharides The polysaccharides were extracted from the washed cells with 6% KOH for 6 hr at 100°C. The alkaline extract was neutralized with glacial acetic acid, centrifuged and the supernatant precipitated with 3 volumes of ethanol. The precipitate was dissolved in water and lyophilized and the polysaccharides purified by column chromatography on Bio-Gel P-100 After hydrolysis with 3 N trifluoroacetic acid (which was removed by evaporation) for 3hr at 100°C the monosaccharide components of the polysaccharides were identified by paper chromatography with butanol-ethanolwater (3:2:1, v/v/v) as the solvent and ammoniacal silver nitrate and p-anisidine as the revealing reagents (Hough and Jones, 1962). The monosaccharides were quantified as their alditol acetates (Sawardeker et al., 1965) by gas-liquid chromatography (GLC) on a 3° neopentylglycol succinate (NPGS) of a stainless steel column (6 ft x 1/8 in.) with 100/200 gas chrom, at 210°C. An internal standard of allitol acetate was used.
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Assay of sialic acid Whole washed cells (109) were suspended in 2ml of distilled water dialysed for 5 hr at 4°C (four changes during this period). Subsequently, the suspension was acidified with formic acid to pH 2.0 and heated for 1 hr at 70°C followed by dialysis against 100 ml of distilled water (24 hr/4°C/two changes in the period). The cell suspension was then lyophilized and the residue hydrolysed in 2.0 ml of 0.1 M HCI for 1 hr at 80°C. After dialysis for 24 hr against 100 ml distilled water with two changes during this period, sialic acids in the combined dialysates of both hydrolytic steps were purified by ion-exchange chromatography as described (Kamerling et al., 1980). Sialic acids were assayed colorimetrically (Jourdian et al., 1971) and analysed by thin-layer chromatography on cellulose plates with the solvent mixture n-propanol-I M ammonia-water (6:2:1, v/v/v; Blix and Lindberg, 1960). Before use, the plates were activated in 0.1 M HC1 and dried under a current of air at room temperature for 30 min. Spots were located by spraying with a periodic acid-resorcinol reagent (Jourdian et al., 1971). Standards of Nacetylneuraminic and N-glycolyneuraminic acids (Sigma Chemical Co.) were used at 10 mg/ml. The nature of the sialic acids were confirmed by gas-liquid chromatography of the correspondent trimethylsilyl derivative on 3% OV 17/Gas Chrom 800-100 mesh at 210°C.
Extraction and purification of glycolipids Lipids were extracted from the washed protozoan cells at room temperature with each of the following solvent mixtures: chloroform-methanol, 2:1 and chloroform-methanol 1:2 (v/v). Both extracts were combined and evaporated to dryness, and a solvent mixture of chloroform-methanol water (1.0:2.0:1.4 v/v/v) was added to this dried residue in a funnel to isolate the crude glycolipids. The solvents were carefully mixed by turning the funnel up and down several times but shaking was omitted to prevent emulsification. When the two phases were distinctly separated (generally within 6 hr), the lower phase was removed and the upper phase set aside. To the lower was added methanol. After thorough shaking of the funnel, 0.01 M KC1 was then added slowly, and carefully mixed with the extract. The two upper phases were combined and evaporated to dryness after addition of isobutanol foaming. For the purification of the crude glycolipids fraction the residue of the upper phases was dissolved in chloroform-methanol-water (60:30:45, v/v/v) and left 24 hr at room temperature. The precipitate was removed by centrifugation. The glycolipid extract was evaporated and the residue dissolved in water and dialysed against running tap water for 48 hr and then against two changes of distilled water for 24 hr each. The dialyzed glycolipids were evaporated to dryness (Svennerholm and Fredman, 1980).
Carbohydrate composition of glycolipids Glycolipids were hydrolysed with 3 N trifluoroacetic acid tbr 3 hr at 100°C, after which the acid was removed by evaporation. The monosaccharide components of the glycolipids were detected by paper chromatography and their corresponding alditol acetates by GLC as described above (Albersheim et aL, 1967; Sawardeker et al., 1965).
Fatty acid composition of glycolipids Glycolipids (1.5 mg) were heated with 0.5 M methanolic HC1 (2 ml) at 80°C for 18 hr in a sealed tube. Methyl esters of fatty acids were extracted with three 2 ml portions of
hexane and then detected by gas-liquid chromatography as described elsewhere (Dias Filho et al., 1985).
Long chain composition of glycolipids Glycolipids were hydrolyzed in methanol--con. HC1water (82.0:6:9.4, v/v/v) at 70°C for 18 hr and the liberated fatty acids were removed by extraction with n-hexane. The lower phase was adjusted to pH 12, the long chain bases were extracted with diethyl ether and purified on a column packed with silica gel 60 (Karlsson, 1970). The column was eluted successively with chloroform-methanol (1:1, v/v), chloroform-methanol (1:4, v/v) and 100% methanol. The long chain bases were recovered with the last two solvents (Iwamori and Nagai, 1978) and then chromatographed on activated (105°C/1/2 hr) silica gel G plates using chloroform-methanol 2 N NH4OH (40:10:1 v/v/v) as solvent. Spots were located with ninhydrin spray (0.2 g ninhydrin in 95 ml n-butanol and 5 ml pyridine; Sambasivarao and McCluer, 1963). Standard of sphingosine and dihydrosphingosine (Sigma Chemical Co.) were used at 5 mg/ml.
Fractionation of glycolipids Glycolipids were separated by thin layer chromatography on activated (105°C/1/2hr) silica gel G plates using chloroform-methanol-2 N NH4OH (40:10:1) as solvent (Kochetkov et al., 1973). They were visualized by spraying with orcinol reagent (Skipski and Barclay, 1969). Standards of galactocerebroside and ganglioside from bovine brain (Sigma Chemical Co.) were used at 2 mg/ml. RESULTS
Carbohydrate composition o f polysaccharides Polysaccharides were isolated from T. foetus by alkaline K O H extraction. After total acid hydrolysis of the polysaccharides the c a r b o h y d r a t e c o m p o n e n t s identified by G L C were glucose (47%), galactose (34%) a n d m a n n o s e (19%). The presence of these m o n o s a c c h a r i d e s was confirmed by p a p e r chromatography.
Assay o f sialic acids Sialic acids, liberated from whole cells by acid hydrolysis, were evaluated by colorimetric determinations. A density o f 2.7 × 107 residues of sialic acid per cell was found. On thin-layer c h r o m a t o g r a p h y N-acetylneuramic acid was the only derivative o f sialic acid present in the hydrolysate from T. foetus cells (Fig. 1).
Carbohydrate, fatty acid and long-chain base composition o f glycolipids Glycolipids of T.foetus were isolated by extraction with c h l o r o f o r m - m e t h a n o l . The c a r b o h y d r a t e components, o b t a i n e d after total acid hydrolysis of the glycolipids, identified by G L C were glucose, galactose a n d mannose. A clear p r e d o m i n a n c e of galactose (67%) was observed (Table 1). The fatty acids composition of the glycolipids is also listed in Table 1. The two fatty acids detected by G L C C were myristic a n d palmitic acids. Sphingosine bases were released from the glycolipids sample by methanolysis. The thin-layer chro-
Table 1. Sugar and fatty acids composition (%) of glycolipids from T. foetus Sugar Fatty acids Mannose Galactose Glucose Myristic (C~4:0) Palmitic(C~:0) 12 67 21 48 52
#~ ~.~
~.~ ~.~
r~
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Fig. 3. Thin-layer chromatography of T. foetus total glyeolipids. (A) Mixture of gangliosides from bovine brain (from Sigma Chemical Co.); (B) total glyeolipids from T. foetus; (C) galaetoeerebroside from bovine brain (from Sigma Chemical Co.).
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Cell surface carbohydrates matography of the long-chain base is shown in Fig. 2. A single spot with the mobility of dihydrosphingosine was detected. Fractionation of glycolipids Thin-layer chromatography was employed for glycolipids separation. From the four main spots visualized, two of them were identified: one corresponding to a galactocerebroside and the other to a ganglioside, bovine brain standards taken as references (Fig. 3).
DISCUSSION Galactose, glucose and mannose were polysaccharide components detected in T. foetus. In general, this carbohydrate pattern resembled that observed in Trichomonas vaginalis (Warton and Honigberg, 1983) and in members of the Trypanosomatidae family such as species of Crithidia (Gottlieb, 1978; Esteves et al., 1982), Herpetomonas (Esteves et al., 1979; Lopes et al., 1983; Soares et al., 1984; Fiorini et al., 1985), Leishmania (Dwyer, 1977) and Trypanosoma (Dwyer and D'Alessandro, 1974; Pereira et al., 1980). Amino sugars including Nacetyl-D-glucosamine and N-acetyl-o-galactosamine were not found in the polysaccharide containing fraction. However, these components probably are part of surface glycoproteins since their presence is suggested by the reactivity of whole cells with specific lectins (Benchimol et al., 1981). The reactivity of T. foetus cells with the lectins concanavalin A and that of Geodia cydodonyum (Benchimol et al., 1981) indicates respectively that glucose and/or mannose and D-galactose were carbohydrate ligands exposed on the cell surface. However, glucose, at least in part is derived from glycogen which is a storage material in T. foetus and T. gallinae: in both parasites glycogen usually represents about 10-30% of the cell dry weight (Manners and Rylei, 1955). In contrast, with cytochemical studies, no indications were obtained that Trypanosomatidae posses reserve polysaccharides, so that all carbohydrate constituents are associated with cellular membranes, comprising the plasma membrane, the Golgi complex, endoplasmic reticulum and pinocytotic vesicles (Brooker, 1976; De Souza, 1976; De Souza et al., 1976; Esteves et al., 1979). The binding of colloidal iron and cationized ferritin particles to the parasite's surface, the high negative surface charge which was markedly reduced by neuraminidase treatment (Silva-Filho et al., 1982) and the binding of the lectin from Limulus polyphemus indicates that sialic acid is an important component exposed on the cell surface of T. foetus (Benchimol et al., 1981). Moreover, sialic acids comprise several derivatives of neuraminic acid includng N-acetyl, N-glycolyl and other substitutions (Blix et al., 1957). However, the present work shows, for the first time, that N-acetylneuraminic acid is the only derivative exposed on the plasma membrane of T. foetus. In the protozoa Crithidia deanei (Oda et al., 1984), Trypanosoma cruzi (Schauer et al., 1983) and the pathogenic fungi Sporothrix schenckii (Alviano et al., 1982) and Fonsecaea pedrosi (Souza et al., 1986), both the derivatives N-glycolyl and N-acetylneuraminic acids were detected.
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Sialic acid carries a negative charge on the cell surface, and may play a role in cell-to-cell recognition and receptor function (Schauer, 1982). Its biological role in T. foetus is still unclear. The few studies carried out on other microorganisms showed that sialic acid displays important functions such as the protective effect for S. schenkii against phagocytosis before an efficient immune response takes place (Oda et al., 1983); to difficult the uptake of bloodstream trypomastigotes by macrophages (Araujo-Jorge and De Souza, 1984) and to render trypomastigotes of T. cruzi unable to activate the alternative complement pathway (Kipnis et al., 1981). The glycolipids from T. foetus contains sphingosine base, two fatty acids, glucose, mannose and galactose. The sugar constituents of the glycolipids closely resembled those of T. foetus polysaccharides described above. The carbohydrate pattern of T. foetus glycolipids also shows a similarity with glycoconjugates of T. cruzi (Barreto-Bergter et al., 1985) and Leishmania (Palatnik, 1984). Similarities among structures of carbohydrate chains from mamalian glycoproteins and glycolipids have been also reported (Rauvala and Finne, 1979). T. foetus glycolipids only contain the normal saturated fatty acids myristic and palmitic acids which are components of total cell lipids (Dias Filho et al., 1985). This fatty acid profile is essentially different from that observed in glycolipids of extraneural tissue which are characterized by higher contents of the unsaturated fatty acids (Windeler and Feldman, 1970). Recently, in glycoconjugates of Trypanosoma mega a mixture of normal saturated and unsaturated ct-hydroxy fatty acid was detected (Vermelho et al., 1986). The sphingosine base composition of T. foetus glycolipids forming amide groups with fatty acids consist exclusively of dihydrosphingosine. In contrast, the glycolipid of T. mega contains sphingosine and traces of dihydrosphingosine (Vermelho et al., 1986). In Crithidia faseieulata a branched chain sphingolipid base, 19-methyl-C20 phitosphingosine, was previously noted (Carter et al., 1966). A galactocerebroside and a ganglioside were identified after glycolipid fractionation by thin-layer chromatography. It has been reported that in galactocerebrosides the galactosiiceramide linkage is of a Gal Bl-ceramide type (Hakamori, 1981). It can be stressed that in T. foetus glycolipids the content of galactose is high (about 67%). In addition, gangliosides are a family of glycolipids that contain sialic acids (Kohn et al., 1978). As referred, only Nacetylneuraminic acid was detected is T. foetus cells and therefore it is the sialic acid derivative constituent of the carbohydrate moiety of the gangliosides. In opposition, sialo-containing compounds from sea urchin Strongylocentrotus intermedius (Kochetkov et al., 1973) and bovine adrenal medula (Leeden et al., 1968) consist of a mixture of N-acetyl and Nglycolylneuraminic acids. Glycolipids have important biological functions such as cell recognition, control of cell growth, differentiation, etc. In addition, some glycolipids act as cell surface markers and important antigens (Hakomori, 1981,1984). Studies are in progress
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aiming to determine Trichomonas.
the role of sialic acid in
Acknowledgements--The authors thank Drs Hertha Meyer and Miklos Muller for reading the manuscript, Mr Luiz Rodrigues da Silva for dedicated technical assistance and Financiadora de Estudos e Projetos (FINEP) and Conselho Nacional de Desenvolvimento Cientifico e Tecnolrgico (CNPq) for financial support.
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