Secció Departamental de Microbiología, Facultat de Farmacia, Universitat de València, Vicent Andrés Estellés s/n, 46100 Burjassot, València, Spain1
Author for correspondence: Rafael Sentandreu. Tel: +34 96 386 42 99. Fax: +34 96 386 42 99. e-mail: rafael.sentandreu{at}uv.es
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ABSTRACT |
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Keywords: monoclonal antibodies, cell wall biosynthesis, lysis of yeast wall, N-glycosylated proteins, cell wall architecture
Abbreviations: Endo-H, N-acetylglucosaminidase H; GPI, glycosylphosphatidylinositol; IIF, indirect immunofluorescence
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INTRODUCTION |
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METHODS |
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Cell wall preparation.
Cells were collected by centrifugation (3000 g, 1015 min) and washed three times with chilled 0·001 M PMSF in 0·01 M Tris/HCl buffer, pH 7·2 (buffer A). Cell suspensions were broken by vortexing with glass beads [5 mg beads (mg cells)-1]; cell breakage was assessed by examining the preparations under a phase-contrast microscope, and complete breakage was obtained. The cell walls were sedimented (1200 g, 10 min) from the cell-free homogenate, washed four times with chilled buffer A, boiled for 10 min with 2% SDS in glass-distilled water and finally washed four more times with chilled 0·001 M PMSF in glass-distilled water. The supernatant resulting from isolation of the cell walls was further centrifuged at 40000 g for 40 min, the pellet (P40) being washed three times in buffer A and maintained at -20 °C.
Solubilization of wall proteins by digestion with hydrolytic enzymes.
Proteins were solubilized from purified walls (yeast or mycelium) by treatment with laminarinase, a 1,3-ß-glucanase (0·125 U ml-1 per 100150 µg purified freeze-dried cell walls in 0·010 M phosphate buffer, pH 6·3, containing 0·001 M sodium azide and 0·001 M PMSF in 0·1 M acetate buffer, pH 5·5, at 35 °C for 4 h) or chitinase (1 mg ml-1, specific activity 570 U g-1, 30 °C for 3 h). In both cases, suspensions were supplemented with pepstatin (0·7 µg ml-1) and leupeptin (0·5 µg ml-1). After treatment, the wall residue was recovered by centrifugation (1200 g, 15 min) and digested with the chitinase or with 1,3-ß-glucanase or zymolyase 20T (10 µg enzyme complex per 100150 µl purified cell walls in 0·001 M sodium azide and 0·01 M PMSF in 0·010 M Tris/HCl buffer, pH 7·4) at 28 °C for 2 h. The solubilized material was concentrated by freeze-drying. Beta-elimination of samples was carried out in 0·1 M NaOH at room temperature for 12 h. N-Acetylglucosaminidase H (Endo-H) digestion was performed as described previously (Pastor et al., 1984 ). Digestion with 1,6-ß-glucanase was carried out in 0·100 M sodium acetate, pH 5·5, at 37 °C for 20 h. The total sugar and protein contents in the lyophilized material were determined by the methods of Dubois et al. (1956)
and Lowry, respectively.
PAGE.
Proteins were separated by SDS-PAGE, performed basically as described by Laemmli (1970) . Polyacrylamide slab gradient gels (515%; ratio of acrylamide to bisacrylamide, 30:0·2) with 3·54% polyacrylamide stacking gel were used. Two volumes of the sample were mixed with one volume of electrophoresis sample buffer (35% glycerol, 14% 2-mercaptoethanol, 7% SDS, and 0·035% bromophenol blue as a tracking dye, in 0·5 M Tris/HCl, pH 6·8) and heated for 5 min in a boiling water bath prior to application onto the gels. Electrophoresis was performed at a constant current of 25 mA.
Western blot techniques.
After SDS-PAGE, the proteins were transferred to nitrocellulose paper by using an electroblotter (a Bio-Rad Trans-Blot cell with an LKB 2197 power supply) essentially as described by Burnette (1981) except that the transfer buffer used was 0·025 M Tris/0·192 M glycine, pH 8·3, plus 20% (v/v) methanol and the electrophoretic transfer was accomplished at 68 V cm-1 for 1517 h at 4 °C. Proteins were immunodetected according to the protocol described in the Bio-Rad Immun-Blot (GAR-HRP) assay kit, based on the procedure of Burnette (1981)
and Towbin et al. (1979)
, using mAb 3A10 at a final dilution of 1:6000 in 0·010 M Tris/HCl buffer (pH 7·4) containing 3% BSA as the blocking agent, 0·9% NaCl and 0·05% Tween 20. Diluted (1:10000) peroxidasegoat anti-mouse IgG (Bio-Rad) was used as the indicator antibody in the Western blot techniques.
Preparation of pustulan, 1,3-ß-glucan and colloidal chitin.
Pustulan was prepared from the lichen Umbilicaria papullosa according to the method of Lindberg & McPherson (1954) , as modified by Reese et al. (1962)
. The lichen (100 g; Calbiochem) was pulverized and boiled for 15 min in 500 ml 0·165 M H2SO4. Thereafter, ethanol was added until the concentration reached 13% and then the mixture was centrifuged. The supernatant was collected and ethanol was added to reach 50%. After centrifugation, the white pellet was washed three times with acetone and dried at 37 °C.
Colloidal chitin was prepared as described by Pegg (1988) . Alkaline-insoluble glucan was prepared by extracting whole yeast with 2 M NaOH at 100 °C for 2 h. The insoluble residue (mainly 1,3-ß-glucan) was collected by centrifugation at 3000 g for 10 min and washed three times with glass-distilled water.
Concanavalin A affinity chromatography.
The zymolyase extract obtained from yeast or mycelial cells was dissolved in 40 mM Tris/HCl, pH 7·4, containing 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2 and 0·5 M NaCl (ConA buffer) and applied to a concanavalin A/Sepharose 4B (Pharmacia) column equilibrated with the same buffer. The column was washed with three bed volumes of ConA buffer, and the material bound by the concanavalin A was eluted with ConA buffer containing 0·5 M methyl -D-mannopyranoside. The eluted fractions were desalted by dialysis against distilled water and then lyophilized.
Production of mAbs.
For the production of mAbs, female BALB/c mice were inoculated intraperitoneally with 100 µl (50 mg protein) ConA-retained zymolyase-extracted material from C. albicans cell walls in PBS, emulsified with an equal volume of complete Freunds adjuvant. Two weeks later, the mice received a booster intraperitoneal injection with the same amount of antigen emulsified with incomplete Freunds adjuvant. One week after this immunization, the mice were tail-bled and the sera were tested for anti-antigen antibody titre by using an ELISA. Selected animals received two boosts with the same immunogen, and, 3 d after the last injection, the splenocytes from the immunized mice and P3-X63/Ag 8.653 murine myeloma cells were fused at a 5:1 ratio by using polyethylene glycol 1500 as the fusing agent. Cell fusion and selection of hybrids were performed as described by Galfre & Milstein (1981) . The fused cells were distributed in 96-well culture plates at an approximate density of 105 cells per well. Ten days after the fusion, the culture supernatants were screened for the presence of antibodies against the antigen (zymolyase-extracted material from C. albicans cell walls) by using an indirect ELISA. Positive hybridomas were cloned by limiting dilution in 96-well Nunclon plates containing feeder cells. Ten days later, supernatants from growing hybridomas were tested by using an ELISA, and hybridomas from positive wells were subcloned. The mAbs were purified from ascites by precipitation with 50% ammonium sulfate. The immunoglobulin class of antibodies was determined with a mouse monoclonal antibody isotyping kit (Sigma Immuno Chemicals).
ELISA.
These assays were performed as described by Voller et al. (1980) , but with the following modifications. Wells of Nunc-Immunoplate I (A/S Nunc) were coated with 25 µg (expressed as total sugar) antigen (the soluble material released from yeast cell walls by zymolyase digestion and retained by a concanavalin A affinity column) in 50 µl 0·030 M sodium carbonate buffer (pH 9·6) and incubated at 4 °C for 18 h. The plate was rinsed three times with Tris-buffered saline (TBS), 0·05% Tween 20 (TBST), then filled with 3% BSA in TBS and incubated for 2 h at 37 °C.
The mAbs from the ascitic fluid (see above) were diluted with PBSBT and added to the appropriate well, the plate being incubated at 37 °C for 1 h in a moist chamber. The wells were then rinsed three times with PBSBT, and 50 µl of a 1:100 dilution in BST of goat anti-mouse immunoglobulins conjugated to peroxidase (Sigma) was added to each well. After incubation for 1 h at 37 °C, the wells were rinsed with PBSBT. After the addition of substrate mixture containing o-phenylenediamine, the plate was incubated in the dark for 10 min and the reaction was stopped by the addition of 25 µl 3 M H2SO4 to each well. The colour intensity was determined at 492 nm with an automated plate reader (Easy Reader EAR 400; Institut Pasteur Laboratories).
Indirect immunofluorescence for detection of cell-surface antigens.
The immunofluorescence assay was carried out basically as described by Sundstrom & Kenny (1985) . Organisms were washed twice in PBS, fixed for 1020 min with 0·5% formalin in PBS, and stored in this suspension until used. The cell concentration was adjusted to 106 cells ml-1 by the addition of PBS, and drops from this suspension were placed on the wells of Microslides (BioMérieux), then allowed to air-dry. Drops (10 µl) from different antiserum dilutions in PBS [dilutions of ascitic fluid in the case of mAb (alternatively, undiluted culture supernatants were also used), or dilutions of the yeast-adsorbed polyclonal antibodies against cell walls from mycelium] were dropped over the antigen. Microslides were placed in a moist chamber at 37 °C for 30 min and washed in two changes of PBS for a total of 15 min. FITC-conjugated goat anti-rabbit or anti-mouse IgG (10 µl; Ortho-Diagnostics Systems) was added to the slides, which were incubated again at 37 °C for 30 min in a moist chamber, washed with PBS as described above, rinsed with glass-distilled water, and mounted in PBS containing 90% glycerol. The indirect immunofluorescence (IIF) of insoluble polysaccharides was investigated in a similar way, but omitting the fixation step. The cells (or polysaccharides) were examined under a Zeiss Photomicroscope III equipped for epifluorescence (UV filter no. 487702; excitation line 365/366 nm). The fluorescence was dependent upon the reaction of the cells with the mAb, since no fluorescent cells were observed if they were incubated only with the second antibody.
Miscellaneous.
Reagents used for gel electrophoresis and blotting were from Bio-Rad. The SDS molecular mass markers were from Sigma and Pharmacia. Culture media compounds and complete and incomplete Freunds adjuvants were purchased from Difco. Laminarinase (EC 3 . 2 . 1 . 6), chitinase (EC 1 . 2 . 3 . 14) from Serratia marcescens, peptastin, antipain and the antiprotease cocktail were from Sigma. Unless specifically indicated otherwise, all other chemicals were from Sigma. The 1,6-ß-glucanase from Trichoderma harzianum was a gift from T. Benitez of the University of Seville (De la Cruz et al., 1995 ).
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RESULTS |
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Further experiments were carried out with mAb 3A10. To determine whether mAb 3A10 reacts with a sugar or a protein epitope, the antibody was reacted against insoluble preparations of pustulan (1,6-ß-glucan), colloidal chitin and alkali-insoluble wall polysaccharide (mainly 1,3-ß-glucan). IIF was negative with each of these three preparations, demonstrating that the mAb did not recognize these polysaccharides (IFF not included) and, as a consequence, mAb 3A10 probably recognized a mannoprotein antigen(s). The three polysaccharides (1,6-ß-glucan, 1,3-ß-glucan and chitin) are stable under the mild alkali conditions used in the ß-elimination.
IIF was also used to determine whether the 3A10 antigen was located on the surface of cells. Exponentially growing yeast and mycelial cells were collected and reacted with the mAb. Yeast cells showed no fluorescence (Fig. 1a, panel 2) whereas mycelia were stained (but not the original parent yeast cells) (Fig. 1b
, panel 2). This result suggested that the epitope recognized by mAb 3A10 was absent or differently located in yeast and mycelial cell walls. The latter possibility was confirmed when we found that yeast cells extracted with hot SDS were stained with mAb 3A10 (Fig. 1c
, panel 2) and the mycelia appeared brighter (Fig. 1b
, d
, panel 2).
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DISCUSSION |
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As expected of a secreted antigen, material reacting with mAb 3A10 was found in a mixed membrane preparation containing membranous fragments belonging to the secretory pathway (Fig. 2). Two bands with apparent molecular masses of 70 and 80 kDa, whose mobilities were not affected by treatment with Endo-H (an enzyme that releases N-linked mannose residues), 1,3-ß-glucanase or 1,6-ß-glucanase, were detected in these particles. Treatment with alkali (ß-elimination) destroyed the reacting material. It is known that under these conditions the sugar residues linked to threonine or serine in a mannoprotein (O-linkages) are released and the protein degraded. This behaviour suggests that the antigen molecules do not contain 1,3-ß-glucan, 1,6-ß-glucan or N-linked mannan moieties, but contain only O-glycosidically linked mannose residues. The detection of two bands with apparent molecular masses of 70 and 80 kDa might also indicate that the smaller one is a degradation product, or that the larger one carries an additional repeat (if it is a protein with internal repeats; Kapteyn et al., 2000
). As the reactivities of the two bands are similar in all preparations, the second hypothesis would appear to be the correct one. The two bands were also solubilized from the walls by SDS treatment (Fig. 2
), suggesting that non-covalent linkages retained them. SDS-PAGE demonstrated that the material extracted with the detergent contained, in addition to the two bands of 70 and 80 kDa, polydispersed high-molecular-mass material. The electrophoretic mobilities of the two first bands were not modified by Endo-H treatment, which, interestingly, increased the mobility of the third band. The three bands disappeared after ß-elimination, but their mobilities did not change when laminarinase or 1,6-ß-glucanase treatments were used. Therefore, it may be concluded that the polydispersed band (in reality two bands; Figs 4
, 5
and 6
) is N-glycosylated in addition to being O-glycosylated. Although we initially considered the possibility that the high-molecular-mass highly polydispersed antigen(s) was a totally distinct cell wall protein, we rejected this hypothesis, as the antigen would also have been detected at the level of the mixed membrane preparation. It is therefore possible that the two O-glycosylated antigens found in the mixed membrane preparation are the precursors of the N-glycosylated, high-molecular-mass mannoproteins, given that they are the only antigens found in the mixed membrane preparation and are not covalently linked to the wall (SDS-extractable). The possibility of high-molecular-mass mannan residues being transferred to asparagine residues of the antigen molecules, giving rise to N-glycosidic linkages, is unlikely since this process occurs only intracellularly. The alternative possibility, whereby the antigens of 70 and 80 kDa are transferred to an N-glycosylated mannoprotein(s), seems more likely. We have no information about the nature of the putative N-glycosylated mannan linked to the antigens, about how this molecular complex(es) might be formed, or about the enzymic activity (or activities) that catalyses this reaction(s). A transglutaminase activity was detected in the cell walls of C. albicans, S. cerevisiae and Yarrowia lipolytica (Ruiz-Herrera et al., 1995
). This enzyme (EC 2 . 3 . 2 . 13) catalyses the formation of inter-peptidic cross-links between the carboxamide group of a glutamine residue in one protein and the
-amino group of a lysine residue of another peptide. Participation of an N-glycosylated protein in the incorporation of an O-glycoslyated protein has been found in the case of a mycelial specific protein (Elorza et al., 1989
) and with Ywp1 of Y. lipolytica (Ramón et al., 1999
). In this case, the protein was also retained in the wall by disulfide bridges to lattice proteins, as in S. cerevisiae (Cappellaro et al., 1998
).
Three bands were also detected in the materials released by laminarinase (a 1,3-ß-glucanase) and four in those released by chitinase. This result was expected, as in C. albicans 90% of the GPI proteins are attached via 1,6-ß-glucan to 1,3-ß-glucan and the remaining ones are attached through 1,6-ß-glucan to chitin (Kapteyn et al., 2000 ). Similarly in S. cerevisiae, 12% of cell wall proteins are attached directly to chitin through a 1,6-ß-glucan moiety (Kapteyn et al., 1997
), and Cwp1p is found as a 1,6-ß-glucanase-extractable and non-extractable, chitin-linked fraction (Van der Vaart et al., 1996
; Kapteyn et al., 1997
). Although the mobility of the high-molecular-mass band was different, the behaviour of the three antigens towards additional digestions or ß-elimination was similar to that found in the material released by SDS. Therefore, it is hypothesized that part of each antigen molecule is covalently linked to a high-molecular-mass N-glycosylated molecule, and, in successive steps, associates with the wall structure by forming covalent bonds with either 1,3-ß-glucan or chitin. The observation that part of the antigen remaining in the wall after extraction with either 1,3-ß-glucanase or chitinase was released by further digestion with chitinase or 1,3-ß-glucanase suggests that parts of the antigens are cross-linked to both 1,3-ß-glucan and chitin. These results are consistent with the idea that, after the formation of covalent linkages between the antigens and the 1,3-ß-glucan or chitin, these molecules bound to the 1,3-ß-glucan are cross-linked to chitin, and that those initially bound to chitin are now linked to 1,3-ß-glucan. If that were the case, formation of these new linkages would lead to the transformation of the cell wall from an elastic structure to a rigid one. In S. cerevisiae, in agreement with our observations, nascent wall GPI proteins carrying 1,6-ß-glucan moieties are retained initially at the level of the plasma membrane (Kapteyn et al., 2000
), and later are transferred to a 1,3-ß-glucan molecule that finally appears cross-linked to chitin (Kollár et al., 1997
). Enzymic activities that participate in the formation of these linkages are the Gas1p of S. cerevisiae (Ram et al., 1998
) and Phr1p and Phr2p of C. albicans (Popolo & Vai, 1998
; Fonzi, 1999
). The expression of Phr1p and Phr2p is regulated by ambient pH; these enzymes process 1,3-ß-glucans by making acceptor sites available for the attachment of mannoproteins carrying 1,6-ß-glucans. In null PHR1 and PHR2 mutants, and at the non-permissible pH, there is an increase in the amounts of proteins in the spent media, and mannoproteins cross-linked to chitin are detected. We are currently trying to determine the fate of the 3A10 antigens in these mutants.
In conclusion, the results reported in this paper indicate that some mannoproteins in C. albicans are incorporated into the wall architecture directly, or after interacting with an N-glycosylated protein(s), by binding to 1,3-ß-glucan or chitin probably via 1,6-ß-glucan (Kapteyn et al., 2000 ). In a second step, they are cross-linked to the alternative polysaccharide (chitin or 1,3-ß-glucan).
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ACKNOWLEDGEMENTS |
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Received 29 November 2000;
revised 28 February 2001;
accepted 27 March 2001.
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