Characterization of monoclonal antibody MEST-2 specific to glucosylceramide of fungi and plants

Marcos S. Toledo, Erika Suzuki, Steven B. Levery2, Anita H. Straus and Helio K. Takahashi1

Department of Biochemistry, Universidade Federal de São Paulo/Escola Paulista de Medicina, Rua Botucatu 862, São Paulo, SP, 04023–900, Brazil, and 2The Complex Carbohydrate Research Center, University of Georgia, 220 Riverbend Road, Athens, GA 30602, USA

Received on May 22, 2000; revised on September 21, 2000; accepted on September 25, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
An IgG2a monoclonal antibody anti-glucosylceramide was established and termed MEST-2. High performance thin layer chromatography immunostaining, and solid-phase radioimmunoassay showed that MEST-2 reacts with glucosylceramide from yeast and mycelium forms of Paracoccidioides brasiliensis, Histoplasma capsulatum, and Sporothrix schenckii; from hyphae of Aspergillus fumigatus; and from yeast forms of Candida albicans, Cryptococcus neoformans, Cryptococcus laurentii, and Cryptococcus albidus. Studies on the fine specificity of MEST-2 showed that it recognizes the ß-D-glucose residue, and that the 2-hydroxy group present in the fatty acid is an important auxiliary feature for the antibody binding. It was also demonstrated that phosphatidylcholine and ergosterol modulate MEST-2 reactivity to glucosylceramide, by solid-phase radioimmunoassay. Indirect immunofluorescence showed that MEST-2 reacts with the surface of yeast forms of P. brasiliensis, H. capsulatum and S. schenckii. Weak staining of mycelial forms of P. brasiliensis and hyphae of A. fumigatus was also observed. The availability of a monoclonal antibody specific to fungal glucosylceramide, and its potential use in analyzing biological roles attributed to glucosylceramide in fungi are discussed.

Key words: 2-hydroxy fatty acid/glucosylceramide/monoclonal antibody/Paracoccidioides brasiliensis/pathogenic fungi


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
As a part of a systematic study of fungal glycosphingolipids (GSLs), detailed characterizations of the neutral GSLs present in different pathogenic and nonpathogenic fungi were carried out. These studies showed that all fungi studied so far show as neutral GSLs only monohexosyl ceramide (CMH) (Toledo et al., 1995Go, 1999, 2000; Suzuki et al., 1997Go; Levery et al., l998Go, 2000).

Common features of fungal cerebrosides include a ceramide containing (4E,8E)-9-methyl-4,8-sphingadienine base and 2-hydroxy fatty acids, which may also be modified by (E)-{Delta}3 unsaturation (Mizushina et al., 1998Go). So far, the latter appears to be a modification unique to fungal cerebrosides.

Recently, we reported the detailed structural analysis of CMHs from yeast and mycelium forms of the thermally dimorphic mycopathogen, Paracoccidioides brasiliensis, and from hyphae of Aspergillus fumigatus. It demonstrated the presence of both saturated and (E)-{Delta}3 unsaturated 2-hydroxy fatty acids in their ceramide moieties (Toledo et al., 1999Go). Two strains of A. fumigatus were analyzed, and it was observed that they express both glucosylceramide (GlcCer) and galactosylceramide (GalCer). The differential synthesis of these two compounds seems to be related to the degree of {Delta}3 unsaturation in the 2-hydroxy fatty acids. In addition, a clear difference was also demonstrated in the amount of {Delta}3 unsaturation when comparing GlcCer from mycelium or yeast forms of P. brasiliensis (Toledo et al., 1999Go). The role of such chemical dimorphism in CMH in P. brasiliensis is still unknown, but may be related to signaling processes associated with morphological transitions in P. brasiliensis.

GlcCer is now recognized as an important compound related to fructification (Kawai and Ikeda, 1983, 1985; Kawai, 1989Go), membrane maintenance at low temperatures (Levery et al., l998Go), fungal dimorphism (San-Blas and San-Blas, 1985Go), mitogenesis (Marchell et al., 1998Go), or signal transduction (Patton and Lester, 1992Go; Boldin and Futerman, 2000). Thus, the availability of a monoclonal antibody (MAb) specifically directed to GlcCer containing 2-hydroxy fatty acids will be an effective tool leading to new approaches aiming to a more accurate understanding of the role and organizational pattern of GlcCer in the membrane/cell wall in different fungi.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Characterization of MAb MEST-2
Among a few hybridomas showing reactivity with GlcCer of P. brasiliensis, a clone secreting IgG2a monoclonal antibody was successfully established after repeated subcloning by limited dilution, and termed MEST-2. By high performance thin layer chromatography (HPTLC) immunostaining, it was verified that MEST-2 reacted with GlcCer from yeast and mycelium forms of P. brasiliensis, H. capsulatum, and S. schenckii; mycelia of Aspergillus fumigatus; yeast forms of Candida albicans, Cryptococcus neoformans, Cryptococcus laurentii, and Cryptococcus albidus. No reactivity was observed with GalCer of mycelium forms of A. fumigatus or yeast forms of S. schenckii (Figure 1). By solid-phase radioimmunoassay (RIA) the same results were observed, that is, MEST-2 was reactive with all GlcCer from the different fungi analyzed in this paper, whether isolated from yeast or mycelium forms, whereas no reactivity of MEST-2 was observed with GalCer of A. fumigatus and S. schenckii. Corroborating the data observed by HPTLC immunostaining, MEST-2 by solid-phase RIA did not react with GlcCer from Gaucher’s spleen or bovine buttermilk which present non-hydroxylated fatty acids, but reacted effectively with soybean GlcCer, comprised primarily (>95%) of ceramide containing 2-hydroxy fatty acids (Sullards et al., 2000Go) (Figures 1, 2). These results suggest that the 2-hydroxy group of the fatty acid (f.a.) is an important feature for the MEST-2 binding to GlcCer. The importance of 2-hydroxy fatty acids in the MEST-2 binding was further analyzed by solid-phase RIA using mixtures containing different molar proportion of non-hydroxylated f.a. glucosylceramide (Gaucher’s spleen) and 2-hydroxy f.a. glucosylceramide (P. brasiliensis), thus varying the levels of 2-hydroxy fatty acid GlcCer. As expected, it was observed a decrease of MEST-2 reactivity to glucosylceramide when the plates were adsorbed with a mixture 2-hydroxy f.a. glucosylceramide and non-hydroxylated f.a. glucosylceramide (1:1 and 1:9), a shift to right of the binding curve was observed. However, as shown in Figure 3B, if the binding curves are calculated considering only the amount of the 2-hydroxy fatty acid GlcCer present in each mixture the curves are exactly the same as the one with only 2-hydroxy fatty acid glucosylceramide alone. This experiment clearly demonstrate that: (1) glucosylceramide without 2-hydroxy fatty acid do not compete or enhance the MEST-2 binding to glucosylceramide presenting 2-hydroxy fatty acids and (2) a direct relationship between the MEST-2 reactivity and the amount of glucosylceramide presenting 2-hydroxy fatty acid.



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Fig. 1. HPTLC pattern and immunostaining with MEST-2 of CMH from different sources. Fungal Glc/GalCer were purified by a combination of chromatography in DEAE-Sephadex and silica gel 60, and preparative HPTLC in solvent B. About 3 µg of each purified CMH were applied on HPTLC plate and developed in solvent B. (A) Stained with orcinol/H2SO4 and (B) immunostaining with MEST-2. Lane 1, GlcCer from mycelium forms of P. brasiliensis; lane 2, GlcCer from yeast forms P. brasiliensis; lane 3, GalCer from hyphae of A. fumigatus; lane 4, GlcCer from hyphae of A. fumigatus ; lane 5, GlcCer from mycelium forms of A. niger; lane 6, GlcCer from mycelium forms of H. capsulatum; lane 7, GlcCer from yeast forms of H. capsulatum; lane 8, GlcCer from mycelium forms of S.schenckii; lane 9, GlcCer from yeast forms of S. schenckii; lane 10, GalCer from yeast forms of S. schenckii; lane 11, GlcCer from yeast forms of C. albicans; lane 12, GlcCer from yeast forms of C. neoformans; lane 13, GlcCer from yeast forms of C. laurentii; lane 14, GlcCer from yeast forms of C. albidus; lane 15, GlcCer from Gaucher’s spleen; lane 16, GlcCer (2-OH fatty acid) from soybean.

 


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Fig. 2. Binding specificity of monoclonal antibody MEST-2 to GlcCer from various sources. GlcCer, GalCer and CDH (first well 0.5 µg) were serially double diluted in ethanol and adsorbed on a 96-well plates. MEST-2 (100 µl) was added and incubated overnight at 4°C. The amount of antibody bound to GSLs was determined by incubation with rabbit anti-mouse IgG (2 h) and 105 c.p.m. of 125I-labeled protein A in 1% BSA. GlcCer from yeast (solid squares) and mycelium (open squares) forms of P. brasiliensis; (open triangles) GlcCer from soybean; (open circles) GlcCer from human Gaucher’s spleen; (inverted triangles) GlcCer from bovine buttermilk; (crossed squares) galactosylcerebrosides from bovine brain; (crossed circles) CDH and CTH from human erythrocytes.

 


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Fig. 3. MEST-2 reactivity to mixtures containing different molar proportion of non-hydroxylated fatty acid glucosylceramide and 2-hydroxy fatty acid glucosylceramide. About 25 µl of glucosylceramide solutions (0.32 mg/ml) containing different molar proportion of non-hydroxylated fatty acid glucosylceramide (Gaucher’s spleen) and 2-hydroxy fatty acid glucosylceramide (P. brasiliensis) were serially double diluted in ethanol and adsorbed on a 96-well plates. MEST-2 (100 µl) was added and incubated overnight at 4°C. The amount of antibody bound to GSLs was determined by incubation with rabbit anti-mouse IgG (2 h) and 125I-labeled protein A. (squares) 2-hydroxy f.a. glucosylceramide, (circles) mixture containing 2-OH f.a. GlcCer and non-hydroxylated f.a. GlcCer (1:1 molar ratio); (inverted triangles) mixture containing 2-OH f.a. GlcCer and non-hydroxylated f.a. GlcCer (1:9 molar ratio); (diamonds) non-hydroxylated f.a. GlcCer. (A) Horizontal axis expresses the amount of glucosylceramide adsorbed per well (non-hydroxy f.a. GlcCer plus 2-OH f.a. GlcCer); (B) horizontal axis expresses the concentration of only 2-OH f.a. GlcCer adsorbed per well.

 
Effect of temperature and lipids on MEST-2 binding
In order to determine whether other lipids could affect the conformation of glucosylceramides and therefore modulate the MEST-2 reactivity, a binding assay was also carried out at different temperatures using lipid mixtures containing different molar ratios of cerebrosides, phospholipids, and fungal sterols. As shown in Figure 4, by solid-phase RIA, MEST-2 reacts only with fungal and soybean glucosylceramide (Figure 4, A and B, respectively). Temperature did not alter the MEST-2 binding to GlcCer alone. On the other hand, it was observed an increase of MEST-2 reactivity to fungi GlcCer at 37°C in presence of phosphatidylcholine (Figure 4A). MEST-2 reactivity to fungi and soybean glucosylceramide increased to about 80% in the presence of phosphatidylcholine, at the same molar concentration of CMH, at 37°C. Conversely, presence of equimolar amount of ergosterol decreased the MEST-2 reactivity to fungi and soybean glucosylceramide to about 40% and 70%, respectively. No significant reactivity was detected with GlcCer from Gaucher’s spleen and with GalCer from Aspergillus fumigatus at different temperatures even in the presence of different molar ratios of phosphatidylcholine or mixtures containing ergosterol and phosphatidylcholine.



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Fig. 4. Binding specificity of monoclonal antibody MEST-2 to cerebrosides (CMH) immobilized in lipid mixed monolayer and effect of temperature; 25 µl of 32 µM solutions of phosphatidylcholine (PC), ergosterol (ERG) and phosphatidylcholine mixed to ergosterol (PC+ERG) were serially double diluted on a 96-well plates, in presence 25 µl of different cerebrosides (16 µM): (A) GlcCer from P. brasiliensis yeast forms; (B) GlcCer from soybean; (C) GlcCer from Gaucher’s spleen; and (D) GalCer from A. fumigatus. MEST-2 (l00 µl) was added to each well and incubated for 2 h at different temperatures, 4°C (open); 24°C (light shadow); and 37°C (heavy shadow). The amount of antibody bound to CMH was determined by incubation with rabbit anti-mouse IgG (2 h) and 125I-labeled protein A. (C) Control, well adsorbed only with CMH (0.4 nmol); 1, CMH (0.4 nmol) + lipids (0.1 nmol); 2, CMH (0.4 nmol) + lipids (0.2 nmol); 3, CMH (0.4 nmol) + lipids (0.4 nmol); and 4, CMH (0.4 nmol) + lipids (0.8 nmol).

 
Inhibition assays of antibody binding using methyl-glycosides
The importance of the sugar moiety in the reactivity of MEST-2 with GlcCer was confirmed by an inhibition assay using different methyl-glycosides. The assay was carried out by solid-phase RIA on 96-well plates coated with highly purified fungi GlcCer, using as inhibitors several methyl-{alpha}- and ß-D-glycosides (glucopyranoside, galactopyranoside, and mannopyranoside) in concentrations ranging from 293 nM to 75 mM. Only methyl-ß-D-glucoside at a concentration of 37.5 mM, was able to inhibit about 85% of the binding of MEST-2 to GlcCer isolated from the different fungi (Figure 5).



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Fig. 5. Inhibition of monoclonal antibody MEST-2 binding to GlcCer from different sources by methyl-glycosides. 96-well plates were adsorbed with different fungal CMH: (A) GlcCer from yeast forms of P. brasiliensis; (B) GlcCer from mycelium forms of P. brasiliensis; (C) GlcCer from yeast forms of S. schenckii; (D) GlcCer from yeast forms of H. capsulatum; (E) GlcCer from C. albicans and (F) GlcCer from A. fumigatus. Different methyl-glycosides (first well 150 mM) were serially double diluted with PBS and preincubated with MEST-2. The inhibition assay was carried out as described in Materials and methods. The effects of the methyl-glycosides are expressed as percentages of inhibition of MEST-2 binding to the GlcCer. Solid squares, methyl-ß-D-glucopyranoside; open squares, methyl-{alpha}-D-glucopyranoside; inverted triangles, methyl-ß-D-galactopyranoside; open circles, methyl-{alpha}-D-galactopyranoside; and diamonds, methyl-{alpha}-D-mannopyranoside.

 
Indirect immunofluorescence with MEST-2
As shown in Figure 6, indirect immunofluorescence studies with MEST-2 showed clear staining of the surface of yeast forms of P. brasiliensis and H. capsulatum. Weak staining of the surface of the mycelial forms of P. brasiliensis and H. capsulatum and of hyphae of A. fumigatus was also observed. In A. fumigatus, it a remarkable difference was detected in the fluorescence pattern with MEST-2 between hyphae and the conidiophore, that is, hyphae presented only a weak fluorescence whereas the conidial heads were highly reactive with MEST-2. Also, as expected, yeast forms of S. schenckii gave strong immunofluorescence with MEST-2, whereas mycelium forms reacted only weakly with the antibody (data not shown). On the other hand, yeast forms of C. albicans, C. albidus, C. laurentii, and C. neoformans showed no immunofluorescence with MEST-2, although GlcCer isolated from these fungi was reactive with MEST-2 by HPTLC immunostaining or solid-phase RIA.



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Fig. 6. Indirect immunofluorescence of different fungi with MAb MEST-2. (A) and (B) yeast forms of P. brasiliensis; (C) and (D), mycelium forms of P. brasiliensis; (E) and (F) mycelium forms of A. fumigatus; (G) and (H) yeast forms of H. capsulatum; (I) and (J) mycelium forms of H. capsulatum. (A, C, E, G, and I), fluorescence. (B, D, F, H, and J), phase contrast.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
An increasing number of studies has pointed to possible roles for cerebrosides in biological processes of various organisms. For example, physiological activities described for GlcCer include stimulation of mitogenesis in mammalian cells (Marchell et al., 1998Go) and induction of fruiting body formation in fungi such as Schizophyllum commune and Coprinus cinereus (Kawai and Ikeda, 1983, 1985; Kawai, 1989Go; Mizushina et al., 1998Go). A MAb directed to GlcCer containing 2-hydroxy fatty acid represents a relevant reagent for immunochemical and biological studies of GlcCer in different fungi.

The fine specificity of MEST-2 was assessed by inhibition assays using five different methyl-glycosides, and only ß-D-Glc was able to inhibit, by about 85%, MEST-2 binding to GlcCer of P. brasiliensis, H. capsulatum, S. schenckii, A. fumigatus, and C. albicans. However, the striking lack of reactivity of MEST-2 with Gaucher’s spleen GlcCer led us to analyze other structural features involved in the recognition of fungal GlcCer by this antibody. Structures such as the 9-methyl group and {Delta}8 unsaturation of the sphingosine are likely to be embedded in the cell membrane, and they presumably do not have any influence in the interaction between MEST-2 and fungal GlcCer. Also, the {Delta}3 unsaturation of C-18 2-hydroxy fatty acid, described in fungal cerebrosides, seems not to be relevant to MEST-2 recognition of fungal GlcCer, since no difference was observed in MEST-2 reactivity with GlcCer isolated from yeast or mycelium forms of P. brasiliensis, which present 15% and 55% of {Delta}3 unsaturation, respectively (Toledo et al., 1999Go). One likely key feature for the MEST-2 reactivity with fungal and plant GlcCer appeared to be the 2-hydroxy group of the fatty acid, which is absent in Gaucher’s GlcCer (Koerner et al., 1979Go) and in commercial buttermilk GlcCer (Matreya, Inc.), but present in GlcCer of fungi and soybean. The glucocerebroside isolated from Gaucher’s spleen has been analyzed a number of times previously, and found to have ceramides consisting of (d18:1) 4-sphingenine N-acylated exclusively with non-hydroxy fatty acid. A positive electrospray ionization-mass spectrometry (+ESI-MS) profile of Gaucher’s spleen GlcCer confirmed these data, 2-hydroxy fatty N-acylation, if present, would constitute less than 1% (data not shown). The GlcCer from buttermilk used in this study, also do not present 2-hydroxy fatty acid, according to data from the manufacturer (Matreya Inc.). In contrast, by +ESI-MS, the ceramide present in the soybean GlcCer standard employed for this study is >95% N-2'-hydroxyhexadecanoyl-(4E,8E/Z)-sphinga-4,8-dienine (data not shown), essentially as described by Shibuya et al. (1990)Go and Sullards et al. (2000)Go. Detailed mass spectrometric characterization of all fungal cerebrosides used in this study, as their Na+ and/or Li+ adducts, by both +ESI-MS and +ESI-MS/collision induced decomposition-mass spectrometry (CID-MS), have been or will be described elsewhere (Levery et al., l998Go, 2000; Toledo et al., 1999Go, and unpublished observations). These studies confirm that the major ceramides in all cases consist of (>95%) N-2'-hydroxyalkanoyl- or N-2'-hydroxy-(E)-3-alkenoyl- (or both) -(4E,8E)-9-methyl-sphinga-4,8-dienine. The possibility of the 2-hydroxy group being the primary epitope recognized by MEST-2 was discarded by the fact that this antibody does not react with GalCer of A.fumigatus which presents 2-hydroxy fatty acids (Toledo et al., 1999Go). Therefore, the minimum epitope required for optimum binding of MEST-2 with GlcCer would comprise primarily the ß-D-Glc residue and a secondary site represented by the 2-hydroxy group of the fatty acid, which present a direct relationship to MEST-2 binding capacity, as determined by solid-phase RIA using different proportions of glucosylceramides containing hydroxy and non-hydroxy fatty acids.

The immunofluorescence profile of mycelium forms of P. brasiliensis with MEST-2 suggested that the organization of GlcCer or cell wall is different from that observed in yeast forms. Similarly, in hyphae of A. fumigatus, MEST-2 reacted almost exclusively with the head of the conidiophore. In general, the immunofluorescence pattern of the various yeast forms with MEST-2 studied in this work is consistent with a model where GlcCer is distributed evenly throughout the fungi surface in yeast forms, and accessible to MEST-2, whereas in mycelium forms the presentation of GlcCer is not favorable to interact with this antibody. Since both forms of dimorphic fungi present CMH in similar amounts, as previously reported (Toledo et al., 1995Go, 1999, 2000), the strong reactivity of MEST-2 with CMH of yeast forms possibly is associated with the cell wall organization and morphological transition of mycelium to yeast forms (San-Blas, 1985Go). The results obtained by solid-phase RIA using mixtures of GlcCer of different sources, ergosterol and phosphatidylcholine, indicate that the antibody binding to glucosylceramide containing 2-hydroxy fatty acids is favored by a specific conformation induced by phosphatidylcholine or a mixture of phosphatidylcholine and ergosterol. On the other hand, the reactivity of MEST-2 to fungi and soybean GlcCer decreased significantly in the presence of ergosterol alone. It should be noted that different temperatures (4°C to 37°C) did not alter the specificity of MEST-2. These results are consistent with the notion that the surface phospholipids and fungal sterols may modulate the MEST-2 reactivity/accessibility, but they do not alter the MEST-2 specificity.

MAbs MEST-1 (an IgG3 monoclonal antibody directed to terminal residues of galactofuranose linked in ß1->3 or ß1->6; see Suzuki et al., 1997Go) and MEST-2 should be effective reagents in studies for the distribution of glycoinositolphosphoryl ceramides and GlcCer in the plasma membrane/cell wall of yeast and mycelium forms of different fungi by electron microscopy, thus providing more accurate information regarding GSLs expression in the processes of growth, morphological transition, and infectivity.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Fungal isolates and growth conditions
Paracoccidioides brasiliensis strain Pb18 was provided by Dr. C. Fava-Netto, Sao Paulo, SP, Brazil; strain B339 was provided by Dr. A. Restrepo-Moreno, Medellin, Colombia. Yeast and mycelium forms of the P. brasiliensis were grown at 37°C and 23°C, respectively, in PGY (peptone 5 g/l, glucose 15 g/l, yeast extract 5 g/l) using 2.5 l Fernbach flasks in a shaker at 100 r.p.m.. Aspergillus fumigatus (ATCC strain 9197) mycelia were grown in malt extract (20 g/l) at 25°C. Histoplasma capsulatum strain 496 from human pulmonary lesion and Sporothrix schenckii strain 65 from Human foot cutaneous lesion, were kindly provided by Dr. O. Gompertz, Sao Paulo, SP, Brazil. Yeast and mycelium forms of both fungi were grown in brain heart infusion (BHI) (37 g/l) at 37°C and 25°C, respectively. The yeast form of Candida albicans, strain ATCC 10231 was grown in modified PGY supplemented with malt extract (peptone 5 g/l, glucose 10 g/l, yeast extract 3 g/l, and malt extract 3 g/l) at 28°C. Yeast forms of Cryptococcus neoformans, strain 512 VFSB; Cryptococcus laurentii, strain 40043; and Cryptococcus albidus, strain 40077 were grown in PGY at 25°C. After 1 week both yeast and mycelium forms of the various fungi were inactivated with 0.1% of thimerosal, and after an additional 48 h the fungi were collected by filtration on Whatman no. 1 filter paper, except for yeast forms of S. schenkii and H. capsulatum, which were harvested by centrifugation at 5000 r.p.m. for 20 min.

Extraction and purification of glycosphingolipids (GSLs)
GSLs were extracted by homogenizing yeast or mycelium forms (~30 g) in an Omni-mixer (Sorvall Inc. Wilmington, DE), three times with 200 ml of isopropanol/hexane/water (IHW, 55:20:25, v/v/v, upper phase discarded), and twice with 200 ml of chloroform/methanol (CM, 2:1, v/v). The five extracts were pooled, dried on rotary evaporator, dialyzed against water, and lyophilized. The CMHs used in this study were purified by a methodology avoiding acetylation/Florisil chromatography/deacetylation (Saito and Hakomori, 1971Go), in order to allow immunochemical analysis of fungal CMHs as close as possible to their native composition and state. Neutral and acidic GSLs were separated in a DEAE-Sephadex A-25 column as described by Yu and Ledeen (1972)Go. The neutral GSL fraction was further purified from other contaminants by Folch’s partitioning (Folch-Pi et al., 1951Go), and chromatography on silica gel 60 using a step-wise gradient of chloroform/methanol from 9:1 to 1:1 (v/v) (Sweeley, 1969Go). Fractions containing CMHs, were assessed by HPTLC on silica gel 60 plates (E. Merck, Darmstadt, Germany) using solvent A: chloroform/methanol/CaCl2 0.02% (60:40:9; v/v/v), and stained with orcinol/H2SO4. For preparative-scale HPTLC separated GSL bands were visualized under UV after spraying with primulin 0.01% in 80% aqueous acetone (Straus et al., 1993Go). Pure GlcCer was obtained from CMH fraction (separated from GalCer and other contaminants) by using solvent B: chloroform/methanol/NH4OH/NH4Cl 0.83% (50:36.8:6:7.2; v/v/v/v). GSLs were isolated from silica gel scraped from the plates by repeated sonication in IHW, as described previously (Straus et al., 1997Go). Galactocerebrosides were purchased from Sigma (St. Louis, MO). Ergosterol, GlcCer from Human Gaucher’s spleen, soybean GlcCer, bovine buttermilk GlcCer from Matreya, Inc. (Pleasant Gap, PA).

Production of hybridomas
About 300 ng of GlcCer purified from mycelium forms of P. brasiliensis was dissolved in 1.5 ml of distilled water and mixed with 1.2 mg of acid-treated, heat-inactivated Salmonella minnesota. Aliquots (100 µl) of this suspension containing 20 µg of the antigen were used to immunize 6-week-old BALB/c mice, by i.v. route, through the caudal vein once a week, over 4 weeks. After a rest period of 30 days, the immune response was boosted with 200 µl of the immunogenic complex. Three days later, the mice were sacrificed and their spleen removed. The lymphocytes were fused with NS-1 myeloma cells and placed in 96-well plates. Hybrids secreting immunoglobulins reacting with GlcCer were detected by solid-phase RIA. Only clones showing strong reactivity with GlcCer of mycelium forms of P.brasiliensis were cloned by limited dilution as described previously (Takahashi et al., 1988Go; Straus et al., 1992Go).

Binding assay
Various GSLs were adsorbed on 96-well plates (Falcon Microtest III flexible assay plates, Oxnard, CA). Solutions (25 µl/well, 300 ng/first well) in ethanol of different GSLs were serially diluted, dried at 37°C and the wells blocked with 1% bovine serum albumin (BSA) in 0.01 M phosphate-buffered saline (PBS), pH 7.2 (200 µl) for 2 h, and sequentially incubated with MAb MEST-2 (100 µl) overnight at 4°C, rabbit anti-mouse IgG (50 µl) for 2 h, and with 50 µl of 125I-labeled protein A in PBS with 1% of BSA (about 105 c.p.m./well) for 1 h. The amount of MAb MEST-2 bound to GlcCer was determined by measuring the radioactivity in each well in a gamma counter (Suzuki et al., 1997Go).

MEST-2 reactivity to mixtures containing different molar proportion of non-hydroxylated fatty acid glucosylceramide (Gaucher’s spleen) and 2-hydroxy fatty acid glucosylceramide (P. brasiliensis) was carried out as described before, where 25 µl of glucosylceramide from Gaucher or P. brasiliensis (0.32 mg/ml) or mixtures containing 2-hydroxy f.a. glucosylceramide (0.16 mg/ml) and non-hydroxylated f.a. glucosylceramide (0.16 mg/ml), or 2-hydroxy f.a. glucosylceramide (0.032mg/ml) and non-hydroxylated f.a. glucosylceramide (0.288 mg/ml) were serially double diluted in ethanol and adsorbed on a 96-well plates.

MEST-2 binding assay to immobilized CMH in a mixed lipids monolayer
Twenty-five microliters of solutions of phosphatidylcholine, ergosterol, and a mixture of phosphatidylcholine with ergosterol in ethanol (32 µM/first well) were serially diluted on 96-well plates. Immediately, 25 µl of a CMH solution at 16 µM were added to each well and mixed. The plates were dried at 37°C and the wells blocked with 1% BSA in PBS (200 µl) for 2 h, and incubated with MAb MEST-2 (100 µl) for 2 h at 4°C, 25°C, and 37°C. The amount of antibody bound to CMH was determined as described in Binding assay.

Methyl-glycosides
Methyl-{alpha}- and ß-D-galactopyranoside, methyl-{alpha}- and ß-D-glucopyranoside and methyl-{alpha}-D-mannopyranoside were purchased from Sigma.

Inhibition of antibody binding by different methyl glycosides
Initially, 75 µl of a 150 mM solution of different methyl glycosides were serially diluted with PBS in a 96-well plate. Each methyl glycoside solution was incubated with 75 µl of MEST-2 at room temperature (Straus et al., 1993Go). After 2 h, aliquots of 100 µl were taken and incubated overnight at 4°C in 96-well plates pre-coated with GlcCer (40 ng/well) essentially as described under Binding assay.

High performance thin layer chromatography (HPTLC) immunostaining
CMHs purified from different fungi were separated by HPTLC, and the immunostaining of the plates was performed by the procedure of Magnani et al. (1980)Go. GSLs (3 µg) were separated in solvent A or B, after development, the plates were dried soaked in 0.5% polymethacrylate in hexane, dried, and blocked for 2 h with 1% of BSA in PBS. Plates were then incubated with MAb MEST-2 overnight followed by sequential incubations with rabbit anti-mouse IgG and 125I-labeled protein A (2 x 107 c.p.m./50 ml of BSA/PBS).

Indirect immunofluorescence
Fungi were fixed with 1% formaldehyde in PBS for 10 min. Cells were washed and resuspended in 1 ml of PBS, and 20 µl of the solution was added to a coverslip pretreated with polylysine 0.1% during 1 h. Air-dried preparations were soaked for 1 h in PBS containing 5% of BSA, and incubated subsequently with culture supernatant of MEST-2 MAb (2 h), biotin-conjugated goat anti-mouse IgG (1 h), and avidin-conjugated fluorescein (1 h). After each incubation the coverslips were washed five times with PBS. The coverslips were examined with an epifluorescence microscope (Straus et al., 1993Go). Control experiments for each fungi were carried out in the presence of an irrelevant monoclonal antibody (BM-8, IgG2a isotype), and no fluorescence was observed.

Electrospray ionization mass spectrometry (ESI-MS) and tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS)
Mass spectrometric analysis was performed on a Sciex API-III (Concord, Ontario, Canada) tandem quadrupole instrument equipped with a standard IonSpray source; samples were introduced into the source by direct infusion of dilute solutions in 100% methanol. Molecular ion profiles of gluco- and galactocerebroside samples used in this study were obtained by positive ion mode ESI-MS, either as their Na+ adducts, under conditions described by Toledo et al. (1999, 2000), and/or as their Li+ adducts as described by Levery et al. (2000)Go. Analysis of individual unit-mass resolved cerebroside components by positive ion mode ESI-MS/CID-MS was carried out on the Li+ molecular ion adducts as described by Levery et al. (2000)Go.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by FAPESP, CNPq, and PRONEX (Brazil; M.S.T., E.S., A.H.S., and H.K.T.); a Glycoscience Research Award from Neose Technologies, Inc. (S.B.L.); and the National Institutes of Health Resource Center for Biomedical Complex Carbohydrates (NIH #5 P41 RR05351, S.B.L.).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
BSA, bovine serum albumin; BHI, brain heart infusion; CID-MS, tandem collision-induced dissociation mass spectrometry; CM, chloroform/methanol; CMH, monohexosyl ceramide; ERG, ergosterol; +ESI-MS, positive electrospray ionization mass spectrometry; f.a., fatty acid; GalCer, galactosylceramide; GlcCer, glucosylceramide; GSL, glycosphingolipid; HPTLC, high performance thin layer chromatography; IHW, isopropanol/hexane/water; MAb monoclonal antibody; PBS, 0.01 M phosphate-buffered saline; PC, phosphatidylcholine; PGY, peptone, glucose, yeast extract; RIA, radioimmunoassay.


    Footnotes
 
1 To whom all correspondence should be addressed at Universidade Federal de São Paulo/Escola Paulista de Medicina, Department of Biochemistry, Rua Botucatu, 862, Ed. J.L. Prado, Sao Paulo, SP, 04023–900, Brazil Back


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 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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