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Eukaryotic Cell, August 2004, p. 955-965, Vol. 3, No. 4
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.4.955-965.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Proteomic Analysis of Candida albicans Cell Walls Reveals Covalently Bound Carbohydrate-Active Enzymes and Adhesins

Piet W. J. de Groot,1,2* Albert D. de Boer,1 Jeff Cunningham,2 Henk L. Dekker,2 Luitzen de Jong,2 Klaas J. Hellingwerf,1 Chris de Koster,2 and Frans M. Klis1

Laboratories for Microbiology,1 Biomacromolecular Mass Spectrometry, Swammerdam Institute for Life Sciences, University of Amsterdam, 1018 WV Amsterdam, The Netherlands2

Received 5 May 2004/ Accepted 18 June 2004


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ABSTRACT
 
Covalently linked cell wall proteins (CWPs) of the dimorphic fungus Candida albicans are implicated in virulence. We have carried out a comprehensive proteomic analysis of the covalently linked CWPs in exponential-phase yeast cells. Proteins were liberated from sodium dodecyl sulfate (SDS)-extracted cell walls and analyzed using immunological and advanced protein sequencing (liquid chromatography-tandem mass spectrometry [LC/MS/MS]) methods. HF-pyridine and NaOH were used to chemically release glycosylphosphatidylinositol-dependent proteins (GPI proteins) and mild alkali-sensitive proteins, respectively. In addition, to release both classes of CWPs simultaneously, cell walls were digested enzymatically with a recombinant ß-1,3-glucanase. Using LC/MS/MS, we identified 14 proteins, of which only 1 protein, Cht2p, has been previously identified in cell wall extracts by using protein sequencing methods. The 14 identified CWPs include 12 GPI proteins and 2 mild alkali-sensitive proteins. Nonsecretory proteins were absent in our cell wall preparations. The proteins identified included several functional categories: (i) five CWPs are predicted carbohydrate-active enzymes (Cht2p, Crh11p, Pga4p, Phr1p, and Scw1p); (ii) Als1p and Als4p are believed to be adhesion proteins. In addition, Pga24p shows similarity to the flocculins of baker's yeast. (iii) Sod4p/Pga2p is a putative superoxide dismutase and is possibly involved in counteracting host defense reactions. The precise roles of the other CWPs (Ecm33.3p, Pir1p, Pga29p, Rbt5p, and Ssr1p) are unknown. These results indicate that a substantial number of the covalently linked CWPs of C. albicans are actively involved in cell wall remodeling and expansion and in host-pathogen interactions.


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INTRODUCTION
 
The cell wall of the human pathogenic yeast Candida albicans is an essential organelle that helps to withstand turgor pressure and determines the shape of the cell (46). It has adhesive properties and is instrumental in virulence, biofilm formation, and possibly also protection against host defense reactions (23, 27, 31, 35, 59, 70). The basic organization of the cell wall of C. albicans is similar to that of Saccharomyces cerevisiae and has a bilayered structure (39, 62). The inner part, consisting mainly of ß-1,3-glucan molecules, is a skeletal layer that is fortified by hydrogen bonds and extended with covalently bound ß-1,6-glucan and chitin chains. The outer part, which determines cell surface properties, consists mainly of mannoproteins which are covalently bound to ß-1,6- or ß-1,3-glucan (17, 45). In addition, some secretory proteins are associated with the cell wall in a reducing agent-sensitive way (10, 58). Several groups have also reported a controversial association with cell wall glucans of proteins known as abundant cytosolic proteins (reviewed by Chaffin et al. [15]).

In C. albicans, mannoproteins have been identified that mediate adhesion to host tissue and virulence (31, 70). Also, other functions related to Candida virulence, like adhesion to plastics, biofilm formation, and antigenicity, can be attributed to cell wall proteins (CWPs) (15, 27, 28, 51). For some mannoproteins, e.g., the members of the Als family and Hwp1p, which are involved in adhesion to host cells, and the structural cell wall protein Ssr1p, cell wall localization has been shown by immunological means (26, 39, 52, 70). However, the only covalently bound CWP that has been identified by using protein sequencing methods thus far is the predicted glycosylphosphatidylinositol-dependent protein (GPI protein) chitinase 2 (Cht2p) (36).

The cell wall is a dynamic structure. Wall expansion during growth requires continuous remodeling of the cell wall polysaccharide network. Further, when faced with changing conditions in the growth environment, the pleomorphic yeast C. albicans can rapidly change its morphology and adapt the composition of the newly formed wall (12, 39). Synthesis and remodeling of the cell wall polysaccharide skeleton require not only synthases, but also enzymes (hydrolases and transglycosidases) outside the plasma membrane that interconnect and remodel individual cell wall polymers to create a strong but flexible network of macromolecules (21). In humans, a similar cell wall structure is not present. The fungal cell wall and the enzymes involved in its biosynthesis thus are excellent targets for antifungal drug development.

In S. cerevisiae, two types of covalently linked CWPs have been discovered (Fig. 1). The major class, GPI-dependent CWPs (GPI-CWPs), is linked to the ß-1,3-glucan network through a relatively short, water-soluble ß-1,6-glucan moiety (40, 41, 47). The second class, a small family of proteins with internal repeats (Pir proteins [71]), is linked to ß-1,3-glucan independently of ß-1,6-glucan through a linkage that is sensitive to 30 mM NaOH (42, 58). The presence of both GPI- and Pir-CWPs in walls of C. albicans has been indicated by immunoblot analyses and database searches (19, 38, 39, 70).



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  		FIG. 1. C. albicans and S. cerevisiae contain two classes of covalently linked cell wall mannoproteins. GPI-dependent CWPs are linked with their C termini to a carbohydrate remnant of the GPI structure through a phosphodiester bond; Pir-CWPs are CWPs with internal repeats. Pir-CWPs are directly linked to ß-1,3-glucan through an alkali-sensitive linkage of unknown nature. Solid arrows indicate the orientations of macromolecules with respect to their reducing end: for example, the reducing end of polymer A and a nonreducing end of polymer B. Effects of enzymatic and chemical treatments with respect to protein release are indicated by dotted arrows.

GPI proteins are widespread among eukaryotes and are linked to the plasma membrane via a preformed lipid that is added in the endoplasmic reticulum by a transamidase enzyme complex. In fungi, a subset of GPI proteins are detached from the plasma membrane and become incorporated in the cell wall (GPI-CWPs) (25, 30). With the genome of C. albicans being fully known, the conventional approach of studying one gene at a time can now be complemented by more-global approaches to investigate the cell wall proteome directly. Recently, using a fungus-specific GPI algorithm, we identified 104 putative GPI proteins of C. albicans by in silico analysis (19). Many of the predicted GPI proteins belong to small families with enzymatic functions associated with cell wall biosynthesis and are common to yeasts and filamentous fungi (19). These enzymes are generally believed to exert their function while being linked to the plasma membrane (8). However, in line with cell wall localization of Cht2p (36), additional evidence exists that (some of) these proteins become covalently attached to cell wall glucans. For instance, immunological analysis indicated that S. cerevisiae Gas1p, which is known to be predominantly plasma membrane localized, becomes partly incorporated in the cell wall (54). This raises the question of whether such carbohydrate-active enzymes may generally become (partly) incorporated in the cell wall to modify the cell wall glycan network.

In this paper, we focus on the cell wall proteome of exponential-phase yeast cells of C. albicans. By exploiting chemical and enzymatic methods to solubilize Pir proteins and GPI proteins, combined with liquid chromatography-tandem mass spectrometry (LC/MS/MS) of tryptic digests, we identify proteins that are covalently incorporated in the cell wall. Our results validate in silico approaches used to identify such proteins and provide direct evidence for the presence of polysaccharide-modifying enzymes, adhesion proteins, defense proteins, and a minority of structural CWPs covalently bound to the cell wall glucan network.


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MATERIALS AND METHODS
 
Strains and growth conditions. The C. albicans strains used in this study were clinical isolate SC5314 and the mutants NGY23 (mnt1{Delta}::hisG-URA3-hisG/mnt1{Delta}::hisG) and CAP1-312 (pmt1{Delta}::hisG-URA3-hisG/pmt1{Delta}::hisG), both in a CAI4 background. S. cerevisiae FY834 was used for developing the HF-pyridine method. Cells were cultured overnight in synthetic complete medium containing 2% (wt/vol) glucose, 1.1% (wt/vol) Casamino Acids (Becton Dickinson and Company, Sparks, Md.), 0.8% (wt/vol) yeast nitrogen base without amino acids (Becton Dickinson and Company), 110 µg of leucine/ml, 55 µg of tyrosine/ml, 55 µg of tryptophan/ml, and 55 µg of adenine sulfate/ml at pH 5 or 5.6 and harvested at an optical density at 600 nm of 2.

Cell wall isolation. Cells were harvested by centrifugation and washed with cold H2O and with 10 mM Tris-HCl, pH 7.5. Cells were resuspended in 10 mM Tris-HCl, pH 7.5 (107 cells/µl), and disintegrated with 0.25- to 0.50-mm glass beads (Emergo BV, Landsmeer, The Netherlands) in the presence of a protease inhibitor cocktail (Sigma, St. Louis, Mo.). For breaking cells from small-scale cultures (<1 liter), we used a Bio-Savant Fast Prep 120 machine (Qbiogene, Carlsbad, Calif.), and for larger culture volumes a Bead-beater (BioSpec Products, Bartlesville, Okla.) was used. To remove noncovalently linked proteins and intracellular contaminants, isolated cell walls (hereafter designated sodium dodecyl sulfate [SDS]-treated walls) were washed extensively with 1 M NaCl and twice extracted for 5 min at 100°C with 50 mM Tris-HCl, pH 7.8, containing 2% SDS, 100 mM Na-EDTA, and 40 mM ß-mercaptoethanol. SDS-treated walls were washed three times with water and freeze dried. This procedure yields about 5 x 10–12 g (dry weight) of walls/cell.

Immunoblot analyses of CWPs. Cell wall digestions with recombinant endo-ß-1,6-glucanase (ProZyme, San Leandro, Calif.) and endo-ß-1,3-glucanase (Qbiogene) were performed as described elsewhere (43). CWPs were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, Mass.). Mannoproteins were visualized by probing the membranes with the lectin concanavalin A or polyclonal antisera directed against S. cerevisiae Cwp1p, Ssr1p/Ccw14p, or Pir2p/Hsp150p. The amounts of protein loaded on the gels per lane were released from 0.4 mg (Pir2p), 0.2 mg (Cwp1p and Ssr1p), or 0.08 mg (concanavalin A) of freeze-dried cell walls, respectively. Immunoblotting procedures and sources of the antisera were described by Kapteyn et al. (43).

Protein extraction and fractionation. GPI-CWPs were released by resuspending the cell walls in undiluted HF-pyridine (Sigma-Aldrich, Buchs, Switzerland) at 0 or 24°C and incubating them for 0, 1, 3, or 17 h. The reaction was quenched by diluting the reaction mixture with an equal amount of ice-cold H2O. HF-pyridine was removed by dialysis overnight against 20 mM bis-Tris, pH 6.0, or H2O. Pir proteins were released by incubating cell walls with 30 mM NaOH at 4°C for 17 h. The reaction was stopped by adding neutralizing amounts of acetic acid, followed by dialysis of the released protein. For enzymatic release of CWPs, cell walls were incubated with endo-ß-1,3-glucanase, followed by dialysis. For large-scale experiments, extracted proteins were fractionated by anion-exchange chromatography using a MonoQ HR 5/5 column (Amersham Biosciences, Buckinghamshire, United Kingdom). The column was equilibrated with 20 mM bis-Tris (pH 6.0), and protein extracts were loaded with a flow rate of 0.7 ml/min. After washing the column with four volumes of 20 mM bis-Tris (pH 6.0), proteins were eluted with a linear gradient of 0 to 1 M NaCl in 40 column volumes. One-milliliter eluate fractions were dialyzed against H2O, freeze dried, and subjected to electrophoresis using linear SDS-2.6-to-20% PAGE gradient gels. Proteins were visualized by staining with Coomassie brilliant blue R250. Prior to electrophoresis, NaOH-extracted protein fractions 12 to 22 were pooled to enhance staining.

Sample preparation for MS analysis. Protein from excised bands and unfractionated protein extracts was reduced with dithiothreitol and S-alkylated with iodoacetamide (64). Samples for in-solution digestions were desalted and rebuffered with 50 mM NH4HCO3 by using 0.5-kDa-cutoff centrifugal filters (Millipore). In-solution digestion with trypsin (sequencing grade; Roche Diagnostics, Indianapolis, Ind.) was carried out overnight at 37°C using a CWP/trypsin ratio of 50:1, assuming that protein accounted for approximately 2% (wt/wt) of the cell wall mass (unpublished data). In-gel digestion with trypsin and extraction of the in-gel tryptic fragments were performed as described previously (64). Extracted peptides were desalted and concentrated on a µC18 ZipTip column (Millipore).

MS analysis. Tryptic digests were analyzed by LC/MS/MS using an Ultimate nano-LC system (Dionex, Sunnyvale, Calif.) and a MicroMass Q-TOF mass spectrometer (Waters, Milford, Mass.). Peptide samples derived from 1 to 10 µg of CWPs were injected with a micro-autosampler (FAMOS) and concentrated on a 300-µm (inner diameter) by 5-mm precolumn. The peptides were loaded and separated on a 75-µm (inner diameter) by 150-mm PepMap column (Dionex), using a gradient program with acetonitrile and 0.1% formic acid. The outlet of the nano-LC with a flow of 300 nl/min was coupled on-line to the Q-TOF by using a coated fused-silica PicoTip (FS360-20-10CE; New Objective, Woburn, Mass.). Peptides were automatically selected from the survey spectrum for collision-induced fragmentation by using Masslynx software. The resulting MS/MS spectra were processed and analyzed with Biolynx and Masslynx Pepseq software. For protein identification, the resulting spectra and amino acid sequences were analyzed using MASCOT software and compared with in silico digests of the CandidaDB protein database (http://genolist.pasteur.fr/CandidaDB/). This database was created by the European Union-funded consortium Galar Fungail by performing independent annotation of sequence data (contig assembly 19) obtained from the Stanford Genome Technology Center (37; http://www-sequence.stanford.edu/group/candida).

Database analyses. Pattern search for proteins with a conserved cysteine motif similar to that of Pir proteins or the CFEM motif (48) was performed using the program FUZZPRO from the EMBOSS software package at the website http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The presence of an N-terminal endoplasmic reticulum import signal was analyzed using the program SignalP version 2.0 at the website http://www.cbs.dtu.dk/services/SignalP-2.0/. BLASTP searches were performed at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST).


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RESULTS
 
GPI proteins can be specifically extracted from isolated cell walls with HF-pyridine. Various covalently bound CWPs of C. albicans are instrumental in fungal adhesion and virulence, while others may play a role in cell wall expansion. Here, we aimed to unravel the covalent cell wall proteome by identifying CWPs in protein extracts from isolated cell walls. In fungi, two classes of covalently bound CWPs are known, GPI proteins and Pir proteins. For the extraction of GPI-CWPs, we have developed a new method that uses HF-pyridine. HF-pyridine specifically cleaves phosphodiester bonds, through which GPI-CWPs are linked to ß-1,6-glucan chains (Fig. 1). At room temperature it is used for protein deglycosylation (44). Release and deglycosylation of CWPs by HF-pyridine were tested using cell walls of S. cerevisiae. Western analysis using antibodies against Cwp1p and Ssr1p/Ccw14p showed an efficient release of these GPI-CWPs when cell walls were incubated with HF-pyridine for 3 h on ice (Fig. 2A). Less or no intact protein was obtained by prolonging the incubation or performing it at room temperature, probably as a result of protein degradation. Immunoblot analysis using antiserum against Pir2p indicated that Pir-CWPs were not released by HF-pyridine (data not shown). Cwp1p and Ssr1p have predicted molecular masses (mature nonglycosylated forms) of 20 and 19 kDa, respectively. The mobility of HF-pyridine-released Cwp1p and Ssr1p corresponded to proteins of approximately 60 and 140 kDa, respectively. This was only slightly faster than glycosylated Cwp1p and Ssr1p released by ß-1,6-glucanase (Fig. 2A), which also contain remnants of ß-1,6-glucan and the GPI anchor, jointly accounting for at least 1 to 2 kDa. This indicates that HF-pyridine efficiently and specifically releases GPI-CWPs but does not significantly affect their glycosylation.



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  		FIG. 2. Chemical extraction of GPI-CWPs with HF-pyridine and of Pir-CWPs with NaOH. (A) Immunoblotting of S. cerevisiae proteins with polyclonal antiserum directed against Cwp1p and Ssr1p. Left panel, proteins released by HF-pyridine. The various temperatures and times of the incubations before quenching the reactions are indicated. Right panel, proteins released from cell walls by HF-pyridine (3 h at 0°C, lane 1) or ß-1,6-glucanase (lane 2). (B) C. albicans proteins solubilized from cell walls by incubation with HF-pyridine (lane 1), 30 mM NaOH (lane 2), or Quantazyme (lane 3). Left panel, lectin staining with concanavalin A. Right panel, immunoblotting with polyclonal antiserum directed against S. cerevisiae Pir2p.

The same HF-pyridine treatment was applied to C. albicans (wild-type [WT] strain SC5314). HF-pyridine released large amounts of mannoproteins, as visualized by Western analysis using the lectin concanavalin A (Fig. 2B). Release of Pir proteins, by using the cross-reactive anti-ScPir2p antiserum (39), was not detected, indicating that release of CWPs from C. albicans with HF-pyridine is very similar to that from S. cerevisiae. As expected, Pir proteins were solubilized when cell walls were incubated with 30 mM NaOH, whereas recombinant ß-1,3-glucanase is expected to solubilize both types of known covalently linked CWPs (Fig. 1).

Identification of HF-pyridine-released CWPs. For the identification of covalently linked CWPs of C. albicans, cell walls were isolated from WT strain SC5314, grown in the yeast form. SDS-treated walls, devoid of noncovalently associated proteins, were incubated with HF-pyridine, and the extracted proteins were digested with trypsin and analyzed by LC/MS/MS. Analysis of a tryptic digest of an HF-pyridine extract generated amino acid sequences of 26 different peptides (Table 1; direct approach HF) originating from 9 different proteins (Als1p, Cht2p, Crh11p, Ecm33.3p, Phr1p, Pga24p/Ycw1p, Pga29p, Rbt5p or Pga10, and Ssr1p). All these proteins are predicted GPI proteins (Table 2), which emphasizes the specificity of the HF-pyridine extraction and validates the GPI protein prediction.


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  		TABLE 1. Identification of covalently linked C. albicans CWPs by LC/MS/MSf


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  		TABLE 2. Functional and structural classification of covalently bound CWPs of C. albicans

Identification of NaOH-released CWPs. Pir proteins can be specifically extracted from cell walls by using NaOH (42, 58). NaOH extraction yielded sequences for three tryptic peptides originating from two proteins (Table 1; direct approach A). One peptide specified IPF19968and IPF15363 two different alleles of a single gene (C. albicans is an obligate diploid). Fungal Pir proteins have several shared features, namely, an N-terminal signal peptide for secretion, a Kex2p protease cleavage site, a central domain with a variable number of characteristic tandem repeats, and a C-terminal region comprised of four cysteine residues with conserved spacing. IPF19968IPF15363 conforms to these characteristics and has homology to the Pir proteins in S. cerevisiae, indicating that it is a Pir protein. We have therefore named this protein Pir1p. IPF19968comprises seven copies of the Pir-specific internal repeats, whereas IPF15363has nine copies (Fig. 3A). Interestingly, recent studies with mutated versions of ScPir4p/Cis3p indicated that the presence of this glutamine-rich repetitive sequence is required for its covalent attachment to wall carbohydrates (13). Surprisingly, the other two peptides appeared to be derived from (both alleles of) Scw1p, which is related to the Bgl2p/Scw4p family of S. cerevisiae. These ß-1,3-glucanosyl transferases are generally known to belong to a class of CWPs that can at least partly be released by extraction with reducing agents (10). Our data suggest that part of this protein becomes covalently linked to the cell wall through a similar mechanism as that for Pir proteins. Possibly, repeat-like sequences in Scw1p (Fig. 3A) cause part of this protein to be covalently incorporated.



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  		FIG. 3. Identification of C. albicans Pir proteins by in silico analysis. (A) Alignment of Pir-specific internal repeats of S. cerevisiae Pir4p/Cis3p and C. albicans IPF19968IPF15363 and IPF7274. Repeat-like sequences of the two Scw1p alleles were added for comparison. Identical and similar residues in at least 12 repeats or repeat-like sequences are indicated by black and gray shading, respectively. (B) Alignment of the C-terminal region of S. cerevisiae Pir4p/Cis3p, IPF19968 and IPF7274. The four cysteines that are conserved in Pir proteins are numbered, and an additional cysteine in IPF7274 is indicated by an arrow. Identical and similar residues in at least two proteins are indicated by black and gray shading, respectively.

In S. cerevisiae, the Pir protein family consists of four homologous proteins. Northern and Southern analyses suggested that C. albicans contains multiple PIR-like genes as well (38), but in the NaOH extract only one Pir protein (Pir1p) was detected. Using the Pir protein characteristics as guidelines, the genome of C. albicans was searched in silico for additional Pir proteins. Besides Pir1p, only one candidate, IPF7274, was identified (Fig. 3). Homology searches with the S. cerevisiae Pir proteins showed that IPF7274 has significant homology (59 to 64%) with the C-terminal cysteine-rich domain of Pir proteins. This domain in IPF7274 deviates somewhat from the normal Pir protein structure, since it has an extra cysteine residue between Cys1 and Cys2 (Fig. 3B). Also, a Kex2p protease cleavage site is not present in IPF7274. Due to the limited number of Pir-related proteins identified to date and the lack of systematic site-directed mutagenesis studies, the exact sequence requirements for the repetitive sequence, mediating covalent attachment of Pir proteins, are presently unknown. Therefore, it is yet uncertain whether IPF7274 is covalently incorporated into the ß-1,3-glucan network.

Identification of GPI-CWPs by using a large-scale fractionation approach. LC/MS/MS analysis of tryptic peptides from unfractionated HF-pyridine and NaOH extracts proved a successful approach to solubilize and identify GPI and Pir proteins separately. To test the sensitivity of this method, we investigated whether additional, less-abundant CWPs could be identified when the amount of starting material for chemical extraction was increased 100-fold, followed by protein fractionation.

Since CWPs are generally rich in serine and threonine residues, they are often heavily O-glycosylated (19, 68). This affects protein fractionation and SDS-PAGE analysis and may hamper protein digestion and LC/MS/MS analysis of peptides. The {alpha}-1,2-mannosyl transferase Mnt1p is one of the main enzymes contributing to addition of the second mannose residue of O-glycans (9). We have therefore chosen a homozygous mnt1 strain, grown in the yeast form, for identification of GPI-CWPs using a large-scale fractionation approach. HF-pyridine-released proteins were fractionated by anion-exchange chromatography, and the eluate was analyzed on SDS-PAGE gradient gels upon staining with Coomassie brilliant blue. This resulted in broad protein bands of about 30, 70 to 100, and 150 to 200 kDa (Fig. 4A), which were in-gel digested and analyzed by LC/MS/MS. In this way, we obtained amino acid sequences for 30 different tryptic peptides (Table 1; large-scale fractionation HF), specifying 8 GPI proteins. Seven of these proteins (18 peptides) were also detected in the unfractionated extract, and only 1 new GPI protein, Pga2p/Sod4p, was identified. Probably, the larger scale of the fractionation approach (using mnt1 cells) enabled us to identify this protein, but we cannot exclude minor strain-related differences in regulation of protein expression. Interestingly, Als1p and Phr1p were not detected in the large-scale approach. Antisera against Als1p and Gas1p (a Phr1p homologue of S. cerevisiae) produced similar patterns with (ß-1,3-glucanase and ß-1,6-glucanase released) CWP fractions of the WT and mnt1 strains (data not shown), indicating that this was not due to strain-related differences between the WT and the mnt1 strains but that these proteins were lost in the fractionation process.



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  		FIG. 4. Large-scale fractionation of C. albicans CWPs. CWP extracts were fractionated using ion-exchange (MonoQ) purification. (A) Coomassie brilliant blue (CBB) staining of SDS-PAGE shows protein solubilized from mnt1 cell walls by treatment with HF-pyridine. MonoQ fractions 14, 16, 18, and 20 (from left to right) eluted at 0.33 to 0.35, 0.38 to 0.4, 0.43 to 0.45, and 0.48 to 0.5 M NaCl, respectively. (B) CBB-stained SDS-PAGE of protein solubilized with 30 mM NaOH from pmt1 cell walls. Protein fractions 12 to 22, which eluted with 0.28 to 0.55 M NaCl, were pooled prior to electrophoresis. Excised parts of the protein smear are indicated on the left.

For Rbt5p (for repressed by Tup1p protein 5), two peptide sequences were obtained. Rbt5p is induced by treatment of WT cells with serum and also by depletion of Tup1p (6), a transcription regulator that represses hypha-specific genes in C. albicans; its identification in cell walls of yeast cells was therefore unexpected. As other Rbt5 family members, Csa1p, IPF12297 and Pga10p each contain identical tryptic peptides, it cannot be excluded that the sequenced fragments originate from these proteins. However, Rbt5p is the only one that contains both sequenced peptides (Table 1). Moreover, Csa1p and Pga10p are regulated similarly to Rbt5p (6, 59), and the C terminus of IPF12297does not follow the sequence requirements for GPI modification (19). Probably, the presence of Rbt5p in walls of cells grown in the yeast form may simply be explained by its relatively high expression levels in comparison to other CWPs, even when repressed by Tup1p (6).

Identification of NaOH-released CWPs by large-scale fractionation. For large-scale protein identification of proteins that are solubilized with NaOH, we have chosen conditions that maximize incorporation of Pir protein(s) during growth. In S. cerevisiae, Pir protein incorporation is dependent on the pH of the growth medium, with the highest levels of proteins being incorporated at acidic pH (43), and this was also found to be the case for C. albicans (unpublished data). Also, glycosylation mutants, especially the PMT1 deletion strain, were shown to have increased levels of Pir proteins in their cell walls (39).

Cell walls of a homozygous pmt1 mutant grown at pH 5 were incubated with 30 mM NaOH, and the resulting protein extract was fractionated by anion-exchange chromatography. SDS-PAGE analysis of eluate fractions produced a protein smear, with sizes ranging from 100 to 200 kDa (Fig. 4B). MS analysis of five different sections of the smear resulted in identical spectra and demonstrated the presence of Pir1p in all sections (Table 1; large-scale fractionation A). Apart from Pir1p, no other proteins were identified, and thus large-scale NaOH extraction and fractionation did not result in identification of unknown, less-abundant CWPs. Notably, mild alkali-sensitive Scw1p escaped detection, probably because it was lost in the fractionation process. The appearance on the gel of Pir1p over a wide mass range (Fig. 2B and 4B) indicates that its glycosylation is very heterogeneous, even when Pmt1p is absent. Mature unglycosylated Pir1p has calculated molecular masses of 29.3 kDa (IPF19968 and 38.7 kDa (IPF15363. Both alleles have only one potential N-glycosylation site and are rich in serine and threonine residues. The difference between the calculated and observed molecular mass cannot solely be explained by N-glycosylation; therefore, O-glycosylation of Pir1p in pmt1 is probably executed by other members of the Pmt protein family (68). The dispersed appearance on gel may also indicate that both Pir1p alleles are expressed. Unfortunately, the fragmentation data did not allow us to discriminate between the two alleles of Pir1p.

Identification of enzymatically released CWPs. To investigate the efficiency and specificity of the chemical extraction methods described above, the ß-1,3-glucan cell wall network was enzymatically digested. This was expected to result in the solubilization of both types of CWPs (Fig. 1 and 2). Also, by solubilizing proteins from wall extracts with glucan-degrading enzyme mixtures, a tight association with cell wall glucans has been suggested for several abundant cytosolic proteins (2, 60). To test this, SDS-treated walls (WT) were incubated with a recombinant ß-1,3-glucanase (Quantazyme), and the released proteins were directly trypsinized and analyzed by LC/MS/MS. This yielded 33 peptide sequences specifying 13 proteins, including all CWPs that had been found in unfractionated HF-pyridine and NaOH extracts. Interestingly, we identified two additional GPI proteins: Als4p, a member of the Als adhesin family, and Pga4p, a new Gas/Phr family member (Table 1; direct approach Q). Both proteins were only detected in Quantazyme extracts, and their identifications resulted from sequencing of a single peptide, suggesting that they are relatively rare in our samples. Consistent with this, Als4p expression has been shown to correlate with growth phase; it begins to increase at mid-log phase and reaches maximal levels at stationary phase (33).

Incubation with Quantazyme did not result in the release of detectable amounts of nonsecretory proteins. Also, a mock treatment in Quantazyme buffer only (50 mM Tris-HCl [pH 7.4], 40 mM ß-mercaptoethanol) did not release any detectable protein, indicating that in this buffer, spontaneous desorption of proteins from SDS-treated cell walls did not occur.

Fragmentation spectra reveal posttranslational modifications. Detailed analysis of the fragmentation spectra revealed interesting additional results (Table 1). For example, some peptides are not preceded by K or R, behind which trypsin normally cleaves. For Cht2p, Pga29p, Pir1p, and Ssr1p, the N termini of peptides of 1,935.0, 1,107.6, 1,702.6, and 1,270.7 Da, respectively, coincided with their predicted signal peptidase cleavage sites, thus establishing the N termini of the mature proteins at amino acid positions 22, 16, 19, and 23, respectively. The presence of the N-terminal Pir1p peptide also indicated that, in contrast to S. cerevisiae Pir proteins, CaPir1p was not further processed (efficiently) by a Kex2p peptidase, which is in agreement with established substrate site preferences of Kex2p (7). In other cases, trypsin cleavage after residues other than K/R may point to either impurities of the used enzyme batch or aspecificity of the trypsin, possibly caused by autolysis. Additionally, for several tryptic peptides we found derivatives with one or multiple 162-Da mass additions, which represent glycosylated forms of the peptides. Corresponding MS/MS spectra showed large peaks of 162 Da and the unglycosylated unfragmented peptide, indicating that protein-glycan linkages are more susceptible to argon-induced fragmentation than peptide bonds. In all cases, except for the tryptic fragment representing Rbt5p residues 37 to 46, the number of added sugar moieties did not exceed the number of potential O-glycosylation sites. Possibly, this limited glycan addition is caused by the lack of Mnt1p function.


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DISCUSSION
 
By MS analysis of chemically and enzymatically extracted CWPs, we have made an inventory of the covalently linked CWPs from exponential-phase yeast cells of C. albicans. To remove contaminants, cell wall preparations were extensively washed with hot SDS in the presence of reducing agents. This excluded from our analyses noncovalently bound proteins and proteins that are exclusively linked through disulfide bridges with other proteins (58). In total, we have identified 14 proteins, of which only Cht2p has been previously identified from cell wall extracts by using protein sequencing methods (36). Ten of the identified CWPs were present in HF-pyridine extracts, indicating that they are GPI modified. All these proteins, plus two proteins that were only identified in Quantazyme extracts, have structural features that are typical of GPI proteins, which validates the in silico approach that has been used to predict the C. albicans GPI proteome (19). Additionally, two proteins, one a predicted Pir protein, were identified in mild alkali extracts. The other protein, Scw1p, is homologous to the members of the Bgl2 family of S. cerevisiae, which have been reported to be released from cell walls, at least partly, by extraction with reducing agents (10). The fact that Scw1p is present in mild alkali extracts suggests that part of this protein becomes covalently incorporated in a Pir-like manner.

The 14 identified CWPs can be categorized into different functional groups (Table 2). Remarkably, five proteins, Cht2p, Crh11p, Pga4p, Phr1p, and Scw1p, form a group of proteins predicted to belong to carbohydrate-active enzyme families that are also found in other yeasts and filamentous fungi (18). They are likely to be involved in cell wall biosynthesis or remodeling (21) but could conceivably also be a factor in biofilm formation. Scw1p belongs to carbohydrate-active enzyme family 17, which mainly consists of ß-1,3-glucanases and ß-1,3-1,4-glucanosyl transferases. It was earlier reported as mannoprotein MP65 (28), which was found to cause a cell-mediated host immune response (50). Crh11p belongs to carbohydrate-active enzyme family 16; in S. cerevisiae it is cell cycle regulated and has been localized to the cell surface using green fluorescent protein as a fusion marker (63). Cht2p is a chitinase and has previously been identified in cell walls of C. albicans by Iranzo et al. (36). Phr1p has been shown to have ß-1,3-glucanosyl transferase activity, indicating that this protein may also play an active role in cell wall expansion (56). PHR1 and the homologous PHR2 genes are reversibly regulated by external pH. PHR1 is induced at neutral and alkaline pHs, which promote filamentation in C. albicans. We hypothesize that these enzymes may be functional while being covalently bound to the cell wall. This would allow the organism to retain them at the cell surface and to more efficiently expand the cell wall during growth. In contrast, soluble enzymes might be easily lost by diffusion into the surrounding medium. For the GPI proteins, the connection via a GPI anchor remnant to the nonreducing end of a flexible and water-soluble ß-1,6-glucan chain may provide diffusional freedom, restricted by the length of the ß-1,6-glucan tether. This would allow these carbohydrate-active enzymes to operate within a substantial radius of action.

A second category of CWPs are adhesion proteins. We have identified Als1p and Als4p, which belong to a large protein family of Als (for agglutinin-like sequence) proteins. Evidence for the involvement of such adhesins in pathogenesis has been obtained by using deletion mutants. Strains that did not produce the glycoproteins Als1p or Als5p were less adhesive (31) and, recently, a novel GPI protein involved in binding to human epithelial cells, Eap1p, has been identified (51). Moreover, deletion of the gene encoding HWP1, another adhesin, caused attenuated virulence in mouse models of systemic infection (67). Interestingly, Pga24p shows similarity with flocculins of S. cerevisiae. Increased expression of Als1p and Pga24p during biofilm formation suggests an important role for these adhesins in this medically important process (27).

Third, we have identified the superoxide dismutase Pga2p/Sod4p. Superoxide dismutases have a role in protection against reactive oxygen species. C. albicans contains superoxide dismutases, of which Sod4p, Sod5p, and Sod6p are predicted GPI-anchored proteins (19) and have significant similarity with the copper- and zinc-containing S. cerevisiae Sod1p, which is partially localized in the cytosol and in the intermembrane space of mitochondria (69). However, consistent with cell wall localization of Sod4p/Pga2p, the similarity with extracellular Cu/Zn Sod proteins from other eukaryotes is much stronger, especially with the predicted GPI protein NCU030131 of the filamentous fungus Neurospora crassa. The fungicidal activity of macrophages and neutrophils is based on the production of as part of the oxidative burst (73). Possibly, extracellular Sod proteins protect C. albicans from reactive oxygen species produced by the host. Interestingly, transcript levels of SOD5 are elevated in hyphae (53, 59) and in C. albicans cells incubated in the presence of human blood (24).

The functions of the other identified proteins (Ecm33.3p, Pga29p, Pir1p, Rbt5, and Ssr1p) are not clearly known. The homologue of Ecm33.3p in S. cerevisiae (Ecm33p) was picked up in a deletant screen aimed at identifying genes involved in cell wall biosynthesis, and its loss results in a strong cell wall defect (20). Pir proteins are cell cycle regulated in S. cerevisiae. Their transcript levels are elevated during G1 phase when the cells grow isotropically (66). They are also strongly upregulated during cell wall stress (5, 27, 49). Conceivably, CaPir1p may behave similarly. Ssr1p and Pga29p are small proteins and may have a structural role. Deletion of SSR1 results in increased sensitivity to the cell wall-perturbing compounds Calcofluor and Congo Red and to ß-1,3-glucanase (26). Interestingly, Ssr1p, as well as all Rbt5p family members, possesses the CFEM motif, an eight-cysteine motif of unknown function commonly found in cell surface proteins of pathogenic fungi (48). Furthermore, Rbt5p (and family members) also contain short tandem repeats which are typically present in fungal adhesins, for instance Hwp1p and Als proteins of C. albicans and Epa1p of Candida glabrata, raising the question of whether Rbt5p may also have adhesin-like properties.

In silico analysis of the C. albicans genome by de Groot et al. (19) has identified 104 putative GPI proteins. This number far exceeds the 12 GPI-CWPs that were detected by LC/MS/MS. Nevertheless, we have reason to believe that the 14 identified CWPs represent almost all proteins that are covalently bound to cell wall glycans of exponential-phase yeast cells of C. albicans. First, only a subset of genes encoding GPI proteins is expressed under defined growth conditions. In C. albicans, several hypha-specific CWPs are known, such as Hwp1p, Hyr1p, and Als3p (3, 32, 67). Conversely, for the genes encoding Pga24p, Pir1p, and Cht2p, transcript analysis has shown that they are expressed in yeast extract-peptone-dextrose at 30°C but are repressed under hypha-inducing growth conditions (59, 65). Many other GPI proteins are also differentially regulated (16, 27, 59, 65). A striking example is seen in S. cerevisiae cells growing under hypoxic conditions, which use a very different set of CWPs compared to those growing under fully aerobic conditions (1). Second, only a subset of the expressed GPI proteins probably gets incorporated in the cell wall. In S. cerevisiae approximately 30 of the about 70 GPI proteins are predicted to remain predominantly attached to the plasma membrane rather than becoming covalently linked to the cell wall (46). Third, we roughly estimate that we can already identify CWPs when there are about 500 molecules present per wall (~0.05% of the covalently bound CWP molecules). This is based on the notion that, under our conditions, approximately 100 fmol of a peptide is sufficient for obtaining good fragmentation spectra with LC/MS/MS and by assuming an average CWP size of 50 kDa. This indicates that our method is very sensitive and allows identification of relatively less-abundant CWPs. Fourth, use of a 100-fold-increased amount of cell walls, followed by chemical fractionations and protein separations, did not increase the number of identified CWPs. Fifth, the number of proteins we have identified is in agreement with the limited number of CWPs that can be visualized by SDS-PAGE in exponential-phase yeast cells of C. albicans (Fig. 2) (39). Sixth, in an extensive search for CWPs using a negatively charged biotinylation reagent to specifically label cell surface proteins, Tanner's group detected only 13 different CWPs on SDS gels in exponential-phase cells of S. cerevisiae (10, 57, 58). On the other hand, we cannot rule out that we may have missed proteins because their tryptic peptides fall out of the range detectable by LC/MS/MS, especially when they are glycosylated.

As mentioned above, only a subset of GPI proteins is targeted to the cell wall. In S. cerevisiae, this is controlled, at least partly, by the region immediately upstream of the GPI attachment site (25, 29, 30). The presence of (adjacent) basic residues in this region was suggested to be confined to proteins that are predominantly localized in the plasma membrane (11, 74). This rule also seems valid in C. albicans (70). Of the 12 GPI-CWPs that we have identified, predicted GPI attachment sites are not preceded by adjacent basic residues in 9 of the proteins (Fig. 5). A dibasic structure is present in Als4p, Pga4p, and Ecm33.3p, which may indicate that part of these proteins stays attached to the plasma membrane. However, the majority of GPI proteins have multiple putative sites for GPI attachment (19). In Als4p, Pga4p, and Ecm33.3p, residues upstream of the two lysines may potentially serve as sites for GPI attachment.



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  		FIG. 5. CWPs released by HF-pyridine have predicted GPI signal peptides. The C-terminal 40 amino acids of all identified HF-pyridine-released CWPs are shown. Putative GPI attachment sites are underlined. Predicted GPI signal peptides conform to the algorithm [NSGDAC]-[GASVIETKDLF]-[GASV]-X(4,19)-[FILMVAGPSTCYWN](10)>, in which [NSGDAC] describes the GPI attachment site and > denotes the C terminus of the protein (19). Dibasic sequences in Als4p, Ecm33.3p, and Pga4p are marked in boldface.

Several authors have reported cell wall localization of proteins that are generally known to be located in the cytosol, for example, heat shock proteins (Hsp70p and Hsp90p), numerous glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, phosphoglycerate kinase, and enolase), and thiol-specific antioxidant protein Tsa1p (2, 15, 60, 72). In our experiments, we did not identify cytosolic proteins in any cell wall preparation. This was consistent with our SDS-PAGE analysis of protein extracts of SDS-treated walls, in which we observed broad protein bands that were characteristic of glycosylated proteins but no sharp bands that were typical of unglycosylated proteins. This indicates that cytosolic proteins have been efficiently removed during the cell wall isolation procedure and are not covalently linked to the cell wall glucan network. Our results, however, do not exclude the possibility that cytosolic proteins are present at the cell surface as noncovalently bound proteins.

Finally, most or all Ascomycotina—both filamentous fungi and fungi able to grow in the yeast form—seem to possess an external protein layer covalently linked to the underlying skeletal layer (22). Many species of the Basidiomycotina and Zygomycotina also seem to possess external glycoproteins (4), as has recently been demonstrated in the human pathogen Cryptococcus neoformans (34). It is tempting to predict that, like in C. albicans, these externally located CWPs may not just have a structural function but have additional roles related to cell wall expansion and maturation, binding to substrates, and host-fungus interactions. This is particularly relevant for other human pathogens, which interact in various ways with the extracellular matrix of human cells (14).


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ACKNOWLEDGMENTS
 
We thank Qing Yuan Yin and Jaap Willem Back of the Biomacromolecular Mass Spectrometry group for valuable discussions and Neil Gow (University of Aberdeen) and Joachim Ernst (Universität Düsseldorf) for kindly providing glycosylation mutants. CandidaDB was created using sequence data for C. albicans obtained from the Stanford Genome Technology Center website at http://www-sequence.stanford.edu/group/candida.

Sequencing of C. albicans was accomplished with the support of the NIDR and the Burroughs Wellcome Fund. This work was supported by the European Commission (QLRT-1999-30795; Galar Fungail consortium).


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FOOTNOTES
 
* Corresponding author. Mailing address: Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands. Phone: 31-20-525 7834. Fax: 31-20-525 7056. E-mail: pgroot{at}science.uva.nl. Back


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Eukaryotic Cell, August 2004, p. 955-965, Vol. 3, No. 4
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.4.955-965.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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