TY - JOUR
AU - Hazen, Kevin, C.
AB - Abstract Cell surface hydrophobicity of the opportunistic fungal pathogen Candida albicans has been linked to the level of cell wall protein glycosylation. Previous work demonstrated that outer chain mannosylation, rather than overall glycosylation, correlated with cell surface hydrophobicity. These studies further suggested that the phosphodiesterlinked, acid-labile β-1,2-mannan was the correlating element. The present work tests this hypothesis and extends the previous results. The composition of bulk mannan from hydrophobic and hydrophilic yeast cells, and the acid-labile mannan from both cell types are compared. Compositional analysis shows that the protein, hexose, and phosphorus content of bulk mannan is similar between the two phenotypes. Electrophoretic separation of acid-released and fluorophore-labeled mannan shows that the acid-labile oligomannosides from hydrophobic cells are longer and potentially in greater abundance than those from hydrophilic cells. These results suggest that regulation of a single step in cell wall protein outer chain mannosylation affects the cell surface ultrastructure and phenotype of C.albicans. β-1,2-oligomannose, Candida albicans, cell wall, glycosylation, hydrophobicity Introduction Cell surface hydrophobicity (CSH) is one of several characteristics of Candida albicans which play a role in the pathogenesis of this opportunistic fungal organism (Hazen, 1990; Cutler, 1991). Hazen and Hazen (1987a) developed an assay to determine CSH using hydrophobic microspheres and showed that CSH changed with growth phase and growth conditions (Hazen and Hazen, 1987b, 1988). Observations of the cell wall architecture provided a link between the CSH phenotype and structures within the cell wall. The current model of the cell wall describes it as having zones of enrichment of its various components: glucan, mannan, chitin, glycoproteins, etc. (Shepherd, 1987). The consensus of several cell wall composition studies is that the outermost layer is comprised of mannoproteins (reviewed in Cassone, 1989). Tokunaga et al. (1986), using freeze-fracture electron microscopy, showed that the outer region of the cell wall was comprised of fibrillar structures. They further concluded, using immunoelectronmicroscopy (with ferritinlabeled immunoglobulins raised against C.albicans cell wall polysaccharide) that these fibrils were the mannoproteins mentioned above. Subsequent freeze-fracture electron microscopy studies demonstrated that cells with different CSH status also exhibit differences in fibril structure and conformation (Hazen and Hazen, 1992). These differences in fibril structure also correspond to changes in CSH as a function of growth phase (Hazen and Hazen, 1993). From the results of these studies, it was concluded that CSH correlates with the status and structure of the mannoprotein surface fibrils. Additional studies supported this conclusion and demonstrated the importance of the glycan component in particular. When hydrophilic yeast cells were treated with tunicamycin, a global inhibitor of protein N-linked glycosylation, the cells became hydrophobic (Hazen and Hazen, 1992). Furthermore, the cell surface fibrils of tunicamycin-treated cells looked like those found on hydrophobic cells. However, the electrophoretic profiles of cell wall proteins enzymatically released from hydrophobic and hydrophilic cells are very similar (Hazen et al., 1990; Hazen and Hazen, 1992). Whelan et al. (1990) produced a set of C.albicans variants by selecting for decreased agglutination to antiserum raised against formalin-killed C.albicans (serotype A) blastoconidia. We have recently shown that the cell wall of one of these variants, which is hydrophobic under all growth conditions, contains cell surface fibrils that resemble those found on hydrophobic wildtype cells (Masuoka and Hazen, 1997). This previous work also examined mannan isolated from limited enzymatic digests of hydrophobic and hydrophilic wild-type cell walls. Compositional analysis of mannan extracted by this method unexpectedly showed no significant difference in the hexose content (Masuoka and Hazen, 1997). Two possible explanations arose for these results. First, the limited enzymatic digestion did not release sufficient material to allow an accurate assessment of mannan composition. Second, if the conformational alterations in the fibrillar mannoproteins, observed when wild-type cells undergo changes in CSH, are due to modifications in mannan composition, then these modifications are subtle. Such subtle differences would not be noticed if the majority of the glycan groups were unchanged. Table I Open in new tabDownload slide Characteristics of hydrophobic and hydrophobic cells and mannan isolated from each cell type Table I Open in new tabDownload slide Characteristics of hydrophobic and hydrophobic cells and mannan isolated from each cell type To examine potential changes in specific Candida mannan moieties, lectins and antibodies were used to probe western blots of cell wall glycoproteins (Masuoka and Hazen, 1997). Two antibodies in particular (mAb B6 and mAb B6.1, a gift of Prof. J.E.Cutler, Montana State University) provided interesting results. MAb B6 recognizes an epitope in the acid stable mannan and mAb B6.1 recognizes a β-1,2-mannotriose in the phosphodiester-linked, acid-labile region (Han et al., 1997). Western blots of cell wall proteins released by enzymatic digest suggested that B6 epitopes are more exposed on hydrophobic cells than on hydrophilic cells, and that B6.1 epitopes are present to a greater extent on hydrophilic cells than hydrophobic cells (Masuoka and Hazen, 1997). These results suggested a model in which the reduction of the phosphodiester-linked, β-1,2-oligomannosyl branches is sufficient to increase CSH. The present work was carried out to test this model. We have extended the previous studies to examine the composition of bulk mannan from hydrophobic and hydrophilic yeast cells. We have also examined the acidlabile mannan released from isolated bulk mannan by capillary zone electrophoresis (CZE) and fluorophore-assisted carbohydrate electrophoresis (FACE). Results Compositional analysis Mannan isolated from both hydrophilic and hydrophobic yeast cell walls was analyzed for protein, reducing hexose and phosphorus content (Table I). The percent dry weight of each component was not significantly different (α = 0.05) when comparing mannan from the two cell types. The extent of Alcian blue binding by hydrophilic and hydrophobic cells was determined as a measure of the phosphomannan content of the cell wall (Friis and Ottolenghi, 1970). Dye binding was assessed by visual inspection (1–4+) and by spectrophotometric determination as developed by Odani et al. (1997). Dye bound to both pellets was rated 4+ by visual inspection, which correlated with the results of the chemical assay for phosphorous above. However, the calculated amount of dye bound to hydrophobic cells was significantly less (α = 0.05, p = 0.005) than that bound to hydrophilic cells. Fig. 1 Open in new tabDownload slide FACE gel of standard β-1,4-oligomannosides (Man) and α-1,4-oligoglucosides (Glc). Degrees of polymerization are given to either side. Fig. 1 Open in new tabDownload slide FACE gel of standard β-1,4-oligomannosides (Man) and α-1,4-oligoglucosides (Glc). Degrees of polymerization are given to either side. Monosaccharide analysis Monosaccharide analysis of bulk mannan isolated from hydrophobic and hydrophilic cell walls indicated that the only neutral sugar present in the isolated mannan was mannose (not shown). No glucose was present, demonstrating the fidelity to the mannan preparations. The small amount of GlcNAc expected to be present (as part of the N-glycosylation core group) was not detectable. Analysis of the acid-labile phosphomannan FACE. Separations of standard oligosaccharides by FACE indicated that smaller groups have a higher mobility than larger groups (Figure 1 and Jackson, 1990). Further, in sideby- side electrophoretic runs of standard β-1,4-oligomannosides and β-1,4-oligoglucosides, groups having corresponding degrees of polymerization do not comigrate (with the exception of Man4 and Glc4). This suggests that the specific saccharide or anomeric configuration can affect oligosaccharide electrophoretic mobility. The acid-labile mannan from hydrophobic cells contains longer groups than that from hydrophilic cells (Figure 2). The signal from the longer groups, in both samples, is less intense than that from the shorter groups. This is probably due to lower abundance (for molar ratio figures, see Shibata et al., 1989). As was the case when the standard oligosaccharides were compared, sample bands did not tend to comigrate with standard oligomannoside bands. This supports the conclusion that the intersaccharide linkage affects oligosaccharide electrophoretic mobility. Because β-1,2-oligomannoside standards are not currently available, we have assigned each band to an individual oligomannoside with increasing degrees of polymerization. Fig. 2 Open in new tabDownload slide FACE gel of acid-labile mannan from hydrophilic (A) and hydrophobic (B) cell walls. Assigned degrees of polymerization are given to either side. Fig. 2 Open in new tabDownload slide FACE gel of acid-labile mannan from hydrophilic (A) and hydrophobic (B) cell walls. Assigned degrees of polymerization are given to either side. CZE. Acid-labile mannan oligosaccharides, released from bulk mannan and labeled with ANTS were also separated by CZE (Figure 3). The CZE results concur with the FACE results: the smaller molecules elute first; the signal decreases as the retention times (and oligosaccharide length) increase; and mannan from hydrophobic cells have longer groups or a greater amount of the longer groups than mannan from hydrophilic cells. Also analogous to the FACE separations, the retention times for standard oligomannoside peaks do not (except for mannose) correspond with sample peaks. This was determined by spiking sample runs with each standard oligomannoside in turn. The difference in retention times, or electrophoretic mobility in the case of FACE gels, for the samples and the standards is not due to problems with reproducibility because both the standards and the sample peaks were reliably reproduced from run to run. We conclude that the lack of correspondence in retention times is due to differences in the particular intersaccharide linkages. Another possibility is that changes in retention are due to branching. However, since the presence of branching in the acid-labile groups has not been reported, the former explanation is more likely. Discussion Our previous results regarding the relationship between cell wall protein mannosylation and CSH suggested a model in which the abundance of phosphomannan itself was inversely related to the extent of CSH (Masuoka and Hazen, 1997). This model was based on comparisons of wild-type cells to variant cells which were hydrophobic under all growth conditions. The present studies were designed to extend those results and to examine more closely the acid-labile mannan from hydrophilic and hydrophobic wild-type cells. Compositional analysis, carried out on bulk mannan rather than the enzymatically released material studied in the previous work (Masuoka and Hazen, 1997), showed no significant difference between the two cell types. The reason for the discrepancy in the phosphorus determination results is unclear. Certainly visual inspection of a stained pellet of cells is a much cruder method of measurement, but the difference between Alcian blue binding and the colorimetric phosphorus assay is harder to explain. It may result from differences in the moiety being measured (i.e., phosphorus associated with phosphomannan alone vs. that plus any phosphorus associated with polypeptides), or it may simply be a reflection of the variance in the two assay data sets. Fig. 3 Open in new tabDownload slide CZE: representative electropherograms of the acid-labile mannan from hydrophilic (top) and hydrophobic (bottom) cells. Fig. 3 Open in new tabDownload slide CZE: representative electropherograms of the acid-labile mannan from hydrophilic (top) and hydrophobic (bottom) cells. These results, taken together, suggest that there is no observable difference in the composition of bulk mannan from hydrophobic and hydrophilic cells. These assays do not, however, take into account aspects of structure, only of content. It is possible, for example, that a decrease in the acid-labile component is compensated for by an increase in the acid-stable portion. Furthermore, the similarity observed in the compositional analysis, monosaccharide analysis and Alcian blue staining results does not explain the dramatic difference between hydrophobic and hydrophilic cells such as those seen when comparing the fibrils in freeze-fracture electron micrographs (Hazen and Hazen, 1992). Analysis of the acid-labile β-1,2-mannan suggests that the differences in this group between hydrophobic and hydrophilic cells play a significant role in hydrophobic surface status. Acid release of the phosphomannan provides a convenient de facto separation of the two groups following labeling with ANTS. The phosphodiester link is broken such that the phosphorus stays with the main glycan group (Okubo et al., 1981). This exposes a reducing end on the acid-labile group to which ANTS can attach. Conversely, there is no reducing end available on the acid-stable mannan (acid does not break the Glc-NAc-Asn bond) so labeling of these groups does not occur. Thus, when the oligosaccharides are separated and visualized via the ANTS label, only the acid-labile groups can be seen. When the acid-labile phosphomannan is separated from bulk mannan in this way and separated into its component oligosaccharides, there is a clear difference between hydrophobic and hydrophilic cells. The hydrophobic cells contain longer acidlabile groups than hydrophilic cells. This was observed using both CZE and FACE (Figures 2, 3). Because the acid-labile mannan is a minor component of the overall N-linked glycan (Shibata et al., 1989), it may be that this difference was not observable against the background of total reducing sugar. It is also possible, as mentioned above, that compensating changes in the acid-stable mannan in hydrophilic cells occurs. The present results show that the previous model, based on mannan enzymatically released from wild-type and variant cell walls (Masuoka and Hazen, 1997), was too simplistic and needed to be radically changed. Rather than longer groups being on hydrophilic cells, the longer groups are found on hydrophobic cells. These results seemed to conflict with our earlier experiments with the B6 and B6.1 monoclonal antibodies (see Introduction). However, reexamination of the B6.1 epitope characterization (Han et al., 1997) indicated that the two sets of results were consistent. B6.1 interacts only with a β-1,2-mannotriose. Oligomannosides of length greater or lesser than three did not inhibit binding of the antibody to phosphomannan. Thus, the presence of longer acid-labile mannan on hydrophobic cells would lead to the observed decrease in B6.1 signal on Western blots (Masuoka and Hazen, 1997). The difference in acid-labile mannan between hydrophobic and hydrophilic cells raises the question of how this change in phosphomannan is involved in CSH. CPK and computer modeling (Bohne et al., 1998) indicate that β-1,2-oligomannose groups form a tight helix with one face which is predominantly hydrogens. This structure suggests an inflexible helix and one which might provide interfibril interactions that lead to the fibril folding and aggregation seen in freeze-fracture electron micrographs (Hazen and Hazen, 1992). We are now in the process of cloning the enzymes involved in C.albicans phosphomannan synthesis to test the effect of their activity on CSH status. Studies are also underway that will determine any differences in the acid stable mannan between hydrophobic and hydrophilic cells. However, the results presented here suggest that regulation of a single step in cell wall protein glycosylation, namely the synthesis or elongation of the acid-labile β-1,2-oligomannosides, is sufficient for regulation of C.albicans CSH status. Materials and methods Cell culture Candida albicans strain A9 (serotype B) was provided by Prof. Richard Calderone at Georgetown University. Cultures were maintained as frozen stocks. Yeast cells from frozen stocks were subcultured three times at 24 h intervals in yeast nitrogen base (Difco, Detroit, MI) buffered with 0.055 M sodium phosphate (pH 7.2) and supplemented with 2% (w/v) glucose. Prior to use in experiments, the cells were harvested by centrifugation and washed three times with cold, sterile, distilled water. Growth temperature was used as a convenient, in vitro method for controlling CSH. Cell surface hydrophobicity was measured by the hydrophobic microsphere assay developed by Hazen and Hazen (1987a). Wild-type cells grown at 23°C are hydrophobic at stationary phase (26 h incubation period). Those grown at 37°C are hydrophilic at stationary phase (Hazen and Hazen, 1987a,b). Biochemical assays Protein concentration was determined by the bicinchoninic acid assay (BCA, Pierce Chemical) (Smith et al., 1985). Bovine serum albumin was used as the standard protein to generate a standard curve. Hexose content was determined by the phenol-sulfuric acid method of Dubois et al. (1956). Sample absorbances were compared to a mannose standard curve. Phosphorus was determined by the method of Chen et al. (1956). A standard curve was prepared with monobasic potassium phosphate from a 2 mg/ml stock. Alcian blue binding Alcian blue binding to yeast cells was measured using a method based on that of Odani et al. (1997). A standard curve was created by making a serial dilution of 0.1% (w/v) Alcian blue 8GX (Sigma) in 0.02 N HCl, measuring the absorbance at 600 nm and plotting OD600 vs. µg of Alcian blue. A sample taken from a stationary phase yeast culture was diluted to give an OD600 of approximately 1.0. Five milliliters of this dilution were transferred to a fresh 12 × 75 mm borosilicate tube and the cells pelleted in a Serofuge. The pelleted cells were washed once with 2 ml of 0.02 N HCl, suspended in a staining solution of 0.005% (w/v) Alcian blue in 0.02 N HCl (for a total of 100 µg dye in the staining solution), and allowed to stand at room temperature for 10 min. The tube was then centrifuged for 3 min and the supernatant fluid removed and rapidly filtered through a 0.45 µm cellulose acetate syringe filter (Corning). The OD600 of the filtered supernatant fluid was measured. The amount of dye (x, µg) in the supernatant fluid was determined using the standard curve generated above and correcting for the amount of dye typically bound to the filter membrane. The Alcian blue staining level was arrived at by normalizing the amount of dye bound to the cells (100 − x) to cell OD. Preparation of mannan Cells are grown to stationary phase (26 h, third transfer). A typical culture volume of 1 l was inoculated with 5 ml from the second transfer. Cells are harvested by centrifugation at 2300 × g for 15 min, washed once with 200 ml of sterile distilled water. Washed pellets are suspended in an excess of acetone. The supernatant acetone is removed and the cells are dried over Drierite and under vacuum. A 11 culture typically yields 2.5–3.0 g of this dried material. Initial extraction of glycan from the dried cells is based on the method of Peat et al. (1961). The dried cells are rehydrated in 60 ml of distilled water in a 500 ml polycarbonate centrifuge bottle. The resulting suspension is then autoclaved for 90 min. After cooling to room temperature, the bottle is centrifuged at 2300 × g for 15 min. The supernatant fluid is transferred to a 250 ml beaker. The water soluble glycan (glucan and mannan) is precipitated by adding 1.8 g sodium acetate and 180 ml (3 volumes) of absolute ethanol and allowing the beaker to stand at room temperature for 1 h. The mixture is transferred to a 500 ml polycarbonate centrifuge bottle and centrifuged at 2300 × g for 15 min. The pellet is dissolved in 20 ml of distilled water and dialyzed against distilled water overnight at 4°C. Following dialysis, mannan is isolated using a strategy based on that used by Lloyd (1970) and Okubo et al. (1981). Fraction designations are those used by Hamada et al. (1981). Fraction B contains the majority of the mannose. Fraction C is the glucan fraction. Basic fractionation Following dialysis, the crude extract is transferred to 100 ml beakers. To the dialyzed solution is added 1/2 V0 of an 8% (w/v) cetyltrimethylammonium bromide (CTAB, Calbiochem, La Jolla, CA) solution, slowly and with mixing. When making the stock solution, gentle warming is required to completely dissolve the CTAB powder. Further, it is usually necessary to prewarm the stock solution (e.g., in a 37°C water bath) to redissolve precipitated material before each use. The resulting extract/CTAB mixture is allowed to stand at room temperature for 2 h after which it is centrifuged at 3000 × g for 10 min. The precipitate = fraction A containing remaining cellular debris. The supernatant fluid is transferred to a 100 ml beaker and V0 of a 1% (w/v) boric acid solution is added, slowly and with mixing. The pH of the solution is adjusted to 8.8 with 2.0 N NaOH and allowed to stand at room temperature for 1 h. The mixture is again centrifuged at 3000 × g for 10 min. The precipitate = fraction B (mannan). The supernatant fluid is removed and reserved. The pellet is washed with 1/2 V0 of 0.5% (w/v) sodium acetate, pH 8.8, which may be added to the supernatant fluid above. This supernatant fluid = fraction C (glucan) which can be precipitated by adding 3 volumes of absolute ethanol. Purification of fraction B The washed pellet (fraction B) is dissolved in 1/2 V0 of 2% (v/v) acetic acid (aqueous). To this solution is added, slowly and with stirring, 1–1/2 V0 of 0.67% (w/v) sodium acetate in ethanol (1 g/150 ml). The resulting mixture is allowed to stand at room temperature for 1 h after which it is centrifuged at 3000 × g for 10 min. The pellet is washed with V0 of 2% (v/v) acetic acid (ethanolic) and then dissolved in 1/2 V0 of dH2O. The pH is adjusted to 7.0 with 0.1 N NaOH. The solution is then dialyzed against dH2O overnight at room temperature. Following dialysis, the retentate is poured into 3 volumes of 1% (w/v) sodium acetate (ethanol) and the resulting mixture centrifuged at 3000 × g for 10 min. The final pellet is dissolved in 4 ml of water and lyophilized. Monosaccharide analysis Monosaccharide analysis of isolated bulk mannan was carried out using a commercially available kit (Bio-Rad). This kit contained the reagents and standards necessary for glycan hydrolysis (three conditions for release of neutral sugars, amine sugars, and sialic acids); labeling of the monosaccharides with 2-aminoacridone (AMAC); and separation by gel electrophoresis. Acid treatment Phosphomannan groups were released from bulk mannan by mild acid hydrolysis (Okubo et al., 1981). Lyophilized mannan was dissolved in 10 mM HCl (10 mg/ml). A 1 ml aliquot was transferred to a 1.5 ml microcentrifuge tube which was then sealed closed with a caplock (PGC Scientific, Gaithersburg, MD). Samples were heated in a boiling water bath for 60 min. (Okubo et al., 1981). After boiling, the samples were cooled to room temperature, neutralized with 1 N NaOH and dried in a centrifugal vacuum evaporator. Labeling of oligosaccharides by ANTS Labeling of oligosaccharides was carried out using the method of Jackson (Jackson, 1990; Jackson and Williams, 1991). A stock 3 M solution of ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid, Molecular Probes, Eugene, OR) was prepared by dissolving the ANTS in acetic acid:water (3:17 v/v). The solution was heated in a 65°C water bath and vortex mixed periodically until the crystals completely dissolved (Jackson, 1994). Aliquots of this ANTS stock solution were stored at −80°C until use. A stock solution of sodium cyanoborohydride (1 M in DMSO) was prepared fresh. To the dried samples were added 200 µl of the ANTS stock solution followed by 200 µl of the sodium cyanoborohydride stock solution. Samples were then incubated overnight at 37°C. α-1,4-oligomannosides (Man2-Man6, Vector Labs, Covington, LA) and maltooligosaccharides (Sigma) were also labeled with ANTS and used as standards. Capillary zone electrophoresis (CZE) CZE was carried out using a BioFocus 2000 instrument (Bio- Rad, Hercules, CA). The capillary was bare fused silica (polyimide outer coating), 75 Ém ID and 20 cm length, (CElect, Supelco, Bellefonte, PA). The CZE running buffer was 50 mM phosphate:triethylamine pH 2.6 (Chiesa and Horváth, 1993). Labeled samples were diluted 1:20 in buffer before electrophoretic runs. Prior to each set of runs on a given day, a preparation method was run consisting of: water (90 s), 0.1 N NaOH (2 ×180 s), water (180 s), buffer (180 s). Injection of sample was electrokinetic (5 kV for 5 s). Electrophoresis was carried out at 9 kV constant. Detection of ANTS labeled oligosaccharides was done at 223 nm (Oefner and Chiesa, 1994). In between sample runs the capillary was washed: 30 s with water and 60 s with buffer. Fluorophore-assisted carbohydrate electrophoresis (FACE) Large-format FACE gels were prepared using a method based on that of Jackson (1990). In the separations described here, 20–30% T gradient gels were used rather than straight 30% gels. Precast gels, available commercially (Bio-Rad), were used as an alternative. Labeled samples were diluted in sample buffer (62.5 mM Tris, pH 6.8, 20% (v/v) glycerol) prior to loading into the wells. Tracking dye (sample buffer containing bromophenol blue, xylene cyanole FF, and Thorin) was added into an unused well. Electrophoresis running buffer was 25 mM Tris, 192 mM glycine, pH 8.5. Large format gels were run at 4°C and 200 V constant. Precast gels were run at 15 mA constant (according to the manufacturer's suggestions) in an electrophoresis apparatus cooled by Peltier cooling (Glyko, Inc., Novato, CA). Runs were stopped when the front-running dye was approximately 1 cm from the bottom of the gel. Following electrophoresis, the gels were imaged using either a GlycoDoc imager (Bio-Rad) for precast gels, or an Alpha Imager 2000 (Alpha Innotech, San Leandro, CA) for large-format gels. Acknowledgments This work was supported in part by PHS grants F32AI09428 and RO1AI31048. Abbreviations AMAC 2-aminoacridone ANTS 8-aminonaphthalene-1,3,6-trisulfonic acid CTAB cetyltrimethylammonium bromide CZE capillary zone electrophoresis DMSO dimethylsulfoxide FACE fluorophore-assisted carbohydrate electrophoresis OD optical density V0 sample starting volume References Bohne A. , Lang E. , von der Lieth C. . W3-SWEET: carbohydrate modeling by internet , J. Mol. Model. , 1998 , vol. 4 (pg. 33 - 43 ) Google Scholar Crossref Search ADS WorldCat Cassone A. . Cell wall of Candida albicans: its function and its impact on the host , Curr. Top. Med. Mycol. , 1989 , vol. 3 (pg. 248 - 314 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Chen P.S. Jr. , Toribara T.Y. , Warner H. . Microdetermination of phosphorus , Anal. Chem. , 1956 , vol. 28 (pg. 1756 - 1758 ) Google Scholar Crossref Search ADS WorldCat Chiesa C. , Horváth C. . Capillary zone electrophoresis of malto-oligosaccharides derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid , J. 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TI - Differences in the acid-labile component of Candida albicans mannan from hydrophobic and hydrophilic yeast cells
JO - Glycobiology
DO - 10.1093/glycob/9.11.1281
DA - 1999-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/differences-in-the-acid-labile-component-of-candida-albicans-mannan-SKEvT4eDDS
SP - 1281
EP - 1286
VL - 9
IS - 11
DP - DeepDyve
ER -