Architecture of the Yeast Cell Wall
beta (1right-arrow6)-GLUCAN INTERCONNECTS MANNOPROTEIN, beta (1right-arrow3)-GLUCAN, AND CHITIN*

(Received for publication, April 3, 1997)

Roman Kollár Dagger , Bruce B. Reinhold §, Eva Petráková , Herman J. C. Yeh par , Gilbert Ashwell **, Jana Drgonová Dagger , Johan C. Kapteyn Dagger Dagger , Frans M. Klis Dagger Dagger and Enrico Cabib Dagger §§

From the Dagger  Laboratory of Biochemistry and Metabolism, the par  Laboratory of Bioorganic Chemistry, and the ** Laboratory of Cell Biochemistry and Biology, NIDDK, Bethesda, Maryland 20892, the § Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118, the  Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, and the Dagger Dagger  Institute of Molecular Cell Biology, University of Amsterdam, 1098 SM Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

In a previous study (Kollár, R., Petráková, E., Ashwell, G., Robbins, P. W., and Cabib, E. (1995) J. Biol. Chem. 270, 1170-1178), the linkage region between chitin and beta (1right-arrow3)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with beta (1right-arrow3)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, beta (1right-arrow3)-glucan, beta (1right-arrow6)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to beta (1right-arrow6)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five alpha -linked mannosyl residues. The beta (1right-arrow6)-glucan has some beta (1right-arrow3)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a beta (1right-arrow4) or beta (1right-arrow2) linkage. Finally, the reducing end of beta (1right-arrow6)-glucan is connected to the nonreducing terminal glucose of beta (1right-arrow3)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and beta (1right-arrow6)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.


INTRODUCTION

Cell walls are essential for the survival of fungal cells. Digestion of cell walls in the absence of an osmotic protector leads to cell lysis due to the high internal turgor pressure. Thus, substances that interfere with cell wall synthesis may be considered as potential antifungal agents (1). Because of its rigidity, the cell wall determines the shape of fungal cells. For that reason, cell wall formation has been used as a model for morphogenesis (1).

The major components of fungal cell walls are polysaccharides and glycoproteins (2). In the yeast, Saccharomyces cerevisiae, the cell wall contains beta (1right-arrow3)-D-glucan, beta (1right-arrow6)-D-glucan, chitin, and mannoprotein(s) (3). The polysaccharides appear to have a structural function, whereas the mannoprotein(s) may act as "filler" and are important for the permeability of the cell wall (4, 5). How can one explain the strength and resilience of the fungal cell wall? Recent results with S. cerevisiae suggest that the answer may be found in the existence of covalent linkages between the different components of the wall that would give rise to a continuous and consequently stronger fabric. Thus, previous studies in our laboratories showed the presence of linkages between chitin and beta (1right-arrow3)-glucan (6) as well as among glycoproteins, beta (1right-arrow6)-glucan, and beta (1right-arrow3)-glucan (7).

The strategy for the investigation of interconnections between chitin and beta (1right-arrow3)-glucan consisted in the digestion of cell walls with beta (1right-arrow3)-glucanase, followed by labeling of the exposed reducing ends with borotritide and enzymatic hydrolysis of the chitin. This procedure led to the isolation of a family of tritiated oligosaccharides that contained the sought after connection, i.e. a beta (1right-arrow4)-linkage between the reducing end of a chitin chain and the nonreducing end of a beta (1right-arrow3)-glucan chain (6). The oligosaccharides were separated on a Bio-Gel P-2 sizing column. The void volume fraction of this column contained a fairly large amount of tritium-labeled material of high molecular weight (6). We have now studied the structure of this material. The results described below show that it represents a region or regions of the cell wall where all four major components, beta (1right-arrow3)-glucan, beta (1right-arrow6)-glucan, chitin, and mannoprotein, are linked together. In this complex, chitin is directly attached to a branch of beta (1right-arrow6)-glucan.


EXPERIMENTAL PROCEDURES

Materials

beta -N-Acetylhexosaminidase, beta -galactosidase, and alpha -mannosidase (all from jack bean) were from Oxford Glycosystems. beta -Glucosidase from sweet almonds and recombinant endoglycosidase-H and proteinase K were purchased from Boehringer Mannheim; glycopeptidase F was from Life Technologies, Inc.; Zymolyase 100 T was from Seikagaku; and proteinase E (Pronase) was from Sigma. Sodium [3H]borohydride (100 mCi/mmol) was obtained from ICN; [1-14C]glucose (50-60 mCi/mmol) was from American Radiolabeled Chemicals; and uridine diphospho-[U-14C]galactose (305 mCi/mmol) was from Amersham Corp. Bio-Gel P-2 (fine and extra fine) and Bio-Gel P-4 (extra fine) were from Bio-Rad; concanavalin A-Sepharose and Sephadex G-100 were from Pharmacia Biotech Inc.; Erythrina cristagalli lectin-agarose was from Vector; and Rezex RSO-Oligosaccharides HPLC1 column was from Phenomenex. Polyacrylamide gels were from Novex, and PVDF membranes were from Millipore Corp. Chitinase from S. marcescens was prepared as described (8), and endo-beta (1right-arrow6)-glucanase was prepared from B. circulans WL-12 as described (9).

Yeast Strains and Yeast Growth

The S. cerevisiae strain used was ECY36-3C (MAT a chs1-23 chs2::LEU2 trp1-1 ura3-52 leu2-2). Cells were grown in YEPD (1% yeast extract, 2% peptone, 2% glucose) at 30 °C.

Preparation of Cell Walls and Digestion with Glucanase and Chitinase

The procedure was as described previously (6), except that, after cell disintegration with glass beads, cell walls were sedimented by centrifugation, washed twice with 50 mM Tris-chloride, pH 7.5, suspended in the same buffer, and incubated in a boiling water bath for 10 min, followed by five additional washings with 50 mM Tris. The isolated cell walls from 30 g (wet weight) of yeast were suspended in 860 ml of 50 mM Tris-chloride buffer, pH 7.5, and incubated with 163 ml of phenylmethylsulfonyl fluoride-treated Zymolyase (56 mg of Zymolyase 100 T was dissolved in 154 ml of 66 mM sodium phosphate, pH 7.5, containing 0.8 M mannitol to which 250 mg of phenylmethylsulfonyl fluoride in 9.4 ml of isopropyl alcohol was added and incubated 1 h at 30 °C) for 4 h at 37 °C until the absorbance at 660 nm had decreased to about 15% of the original value. The insoluble residue was recovered by centrifugation. Washing of this material with Tris buffer, 1% SDS, and water, reduction with sodium borotritide, and digestion with chitinase were as described previously (6). Any insoluble residue after chitinase digestion was removed by centrifugation, and the supernatant fluid was used directly for Bio-Gel P-2 column chromatography (6). The material eluting in the void volume (fraction V0) was used for further analysis.

Affinity Chromatography of Fraction V0 on Sepharose-bound Concanavalin A Column

Tritiated V0 fraction (5.6 × 106 cpm) was evaporated to dryness, dissolved in 500 µl of 50 mM Tris-chloride, pH 7.4, containing 0.15 M NaCl (binding buffer), and applied to a concanavalin A (ConA)-Sepharose column (0.9 × 9 cm). Elution was with binding buffer, and 0.45-ml fractions were collected. At fraction 34, the eluting solution was changed to 0.5 M alpha -methylmannoside in 50 mM Tris-chloride containing 0.25 M NaCl to displace the bound material. A portion of each fraction (10 µl) was counted. The fractions containing radioactivity and corresponding either to the unretained (ConA-) or to the bound (ConA+) material were pooled and concentrated by evaporation.

Chromatography

High performance anionic exchange chromatography (HPAEC) and paper chromatography were performed as described previously (6).

SDS-Polyacrylamide Gel Electrophoresis

Cell wall proteins were separated by either 18% or linear gradient (2.2-20%) polyacrylamide gels according to Laemmli (10). For Western analysis, gels (2.2-20%) were blotted onto PVDF membrane, and antibody staining was carried out as described by Montijn et al. (11).

Carbohydrate Determinations

Total carbohydrate was measured with the anthrone reagent (12) or by a modified phenol sulfuric method (13). Samples were diluted to 40 µl with water, mixed with 30 µl of 5% phenol and 250 µl of concentrated sulfuric acid in 96-well microtitration plates, and incubated for 10 min at room temperature. Absorbance at 490 nm was measured in a Bio-Tek EL309 autoreader. Free GlcNAc was quantified by the method of Reissig et al. (14), and combined GlcNAc was quantified by the same method after digestion of sample with chitinase and Glusulase (6).

Transgalactosylation onto Nonreducing GlcNAc Residues

Different fractions were transgalactosylated by a modification (15) of the procedure described by Trayer and Hill (16). Sample, containing 57 µmol of total carbohydrate (as glucose) was evaporated to dryness, diluted to 3 ml with 20 mM Hepes, pH 7.3, containing 20 mM galactose, 0.5% Nonidet P-40, and 10 mM MnCl2, and incubated with 15 µl of UDP-[U-14C]galactose (0.375 µCi; specific activity, 305 mCi/mmol) and 17.1 µl (0.8 units) of autogalactosylated (17) bovine galactosyltransferase for 2 h at 37 °C in the presence of 0.02% NaN3. After the addition of unlabeled UDP-galactose (92 µl, containing 0.92 mg in 2% ethanol) incubation was continued for an additional 4 h. The volume of the incubation mixture was reduced to ~500 µl by evaporation, and the sample was applied to an appropriate gel chromatography column. The second incubation with unlabeled UDP-galactose was omitted in the case of the endo-H-released material from fraction M.

Acetolysis of Mannan

[14C]Galactose-labeled ConA+ (1.2 × 106 cpm of 14C) was diluted to 200 µl with a solution containing 50 mM Tris-chloride, pH 8, and 5 mM CaCl2. After adding 15 mg of proteinase E in 4 ml of the same buffer, the mixture was incubated 24 h at 37 °C in the presence of 0.02% sodium azide. The incubated mixture was concentrated to ~500 µl by evaporation and applied to a Sephadex G-100 column (1 × 85 cm). The fraction eluting in the void volume, which was almost devoid of 14C radioactivity, was collected. A portion (6.3 mg as hexose) of this fraction was acetolyzed by a modification (18) of the method described by Kocourek and Ballou (19), followed by deacetylation with sodium methoxide (18).

Protein Determination

Protein was measured either spectrophotometrically or by the procedure of Lowry et al. (20).

NMR Spectroscopy

1H and 13C NMR spectra were recorded on a Varian XL-300 spectrometer, operating at 300 MHz (1H) and 75 MHz (13C) at a probe temperature of 35 °C. Each sample was dissolved in D2O, and a trace of acetone was used as an internal standard (for 1H-NMR spectra delta  2.217 ppm; for 13C-NMR spectra delta  31.07 ppm).

Preparation of Oligosaccharide to Be Analyzed by Mass Spectrometry

The ConA+ fraction was labeled with [14C]galactose as described above and digested with beta (1right-arrow6)-glucanase (see legend of Fig. 6). Low molecular weight material (Fig. 6b) was fractionated on an E. cristagalli lectin-agarose column (Fig. 7a), and the [14C]galactose-containing fraction (fraction B) was further chromatographed on a Bio-Gel P-2 column (Fig. 7c). Peak III (Fig. 7c, 13 nmol of carbohydrate as glucose) was evaporated to dryness, dissolved in 100 µl of 0.01 M NaOH, and reduced with 10 µl of sodium borotritide (1 mCi of NaB3H4 in 100 µl of dimethylformamide) for 2 h at room temperature, followed by an additional 2 h with 0.7 mg of unlabeled NaBH4 in 0.01 M NaOH. The excess of borohydride was eliminated by the addition of 20 µl of 1 M acetic acid followed by evaporation of the sample with methanol three times. The sample was applied to an E. cristagalli lectin-agarose column (conditions as in Fig. 2), and lectin-retarded radioactivity was applied to a Bio-Gel P-2 column (1 × 85 cm), which was eluted with 0.1 M acetic acid. The radioactive fractions were pooled and evaporated to dryness. The residue was dissolved in 100 µl of 50 mM sodium citrate, pH 3.5, and digested with 1 unit of jack bean beta -galactosidase for 16 h at 37 °C in the presence of 0.02% NaN3. The incubated mixture was applied to a Bio-Gel P-2 column and eluted as above. The tritium-containing fractions, corresponding to degalactosylated peak III, were pooled and concentrated by evaporation. This material was analyzed by mass spectrometry as outlined below. A portion of the oligosaccharide (1 nmol) was evaporated to dryness, dissolved in 25 µl of 2 M trifluoroacetic acid, and hydrolyzed for 2 h at 100 °C. After new evaporation to dryness, the residue was dissolved in 50 µl of water, and a 25-µl sample was analyzed by HPAEC.


Fig. 6. Gel chromatography of 14C-Gal-labeled ConA+ and 14C-Gal-labeled ConA- after incubation with endo-beta (1right-arrow6) glucanase. a, [14C]Gal-labeled ConA+ (1.5 × 106 cpm of tritium and 6.9 × 105 cpm of 14C) was applied to a Sephadex G-100 column (1 × 85 cm) and eluted with 25 mM Tris-chloride, pH 7.5, containing 0.02% sodium azide. Fractions of 0.45 ml were collected, and samples were counted in separate channels for tritium (bullet ) and 14C (black-triangle). b, [14C]Gal-labeled ConA+ (5.5 × 105 cpm of tritium and 2.5 × 105 cpm of 14C) was evaporated to dryness, dissolved in 0.5 ml of sodium succinate, pH 5.0, and digested with 360 µl (7.9 units) of endo-beta (1-6)-glucanase from B. circulans for 16 h at 30 °C in the presence of 0.02% NaN3. After incubation, the mixture was chromatographed on Sephadex G-100 as in a. The tritiated material eluting in the void volume is designated as fraction M. c, [14C]Gal-labeled ConA- (1.8 × 105 cpm of tritium and 3.7 × 104 cpm of 14C) was subjected to Sephadex G-100 chromatography as in a. d, [14C]Gal-labeled ConA- fraction (1.8 × 105 cpm of tritium and 3.7 × 104 cpm of 14C) was digested with endo-beta (1right-arrow6)-glucanase and separated by gel filtration as in b. Samples used for counting were 10, 15, 100, and 200 µl for a, b, c, and d, respectively. Positions of molecular mass standards are indicated in a in kDa.
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Fig. 7. Separation of tritiated and 14C-labeled oligosaccharides released from 14C-Gal-labeled ConA+ fraction by endo-beta (1right-arrow6)-glucanase. a, the low molecular weight peak, isolated from Sephadex G-100 (Fig. 6b) after glucanase digestion of [14C]Gal-labeled ConA+ fraction (2.8 × 105 cpm of tritium and 1.6 × 105 cpm of 14C), was fractionated on an E. cristagalli lectin-agarose column to separate galactose-containing (14C-labeled, B) from galactose-free (tritium-labeled, A) oligosaccharides. Conditions were as in Fig. 2. Since the 14C-labeled material was retarded rather than bound, the elution buffer was not required. Samples were counted in channels for tritium (bullet ) or 14C (black-triangle). b, fraction A was concentrated by evaporation to ~1 ml, applied to an extra fine Bio-Gel P-2 column, and eluted as in Fig. 1. In addition to radioactivity, total carbohydrate (see "Experimental Procedures") was measured in fractions (open circle ). Positions of standards are indicated with arrows. 1, [14C]glucose (internal standard); 2, hexitol; 3-8, diol to heptaol obtained by reduction of a dextran ladder (Oxford Glycosystems). c, fraction B was concentrated and chromatographed as fraction A. Standards were the same as in b, except that the internal standard was [3H]mannose. In all cases, 0.45-ml fractions were collected. Samples of 10 µl were used for counting, and samples of 40 µl were used for carbohydrate determinations.
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Fig. 2. Affinity chromatography of 14C-Gal-labeled ConA- and 14C-Gal-labeled ConA+ on E. cristagalli lectin-agarose. a, a portion of [14C]Gal-labeled ConA+ fraction (52,000 cpm of tritium and 20,500 cpm of 14C) was diluted to 500 µl with a solution containing 50 mM Hepes, pH 8.0, 100 mM KCl, 5 mM MgCl2, and 2 mM beta -mercaptoethanol (binding buffer) and applied to a column of E. cristagalli lectin-agarose (1 × 20 cm). Elution was done with the loading buffer, and 0.45-ml fractions were collected. At fraction 55 (arrow) the eluent was changed to a solution with the same composition of the binding buffer plus 0.2 M lactose and 0.5 M GlcNAc to displace the bound material. Of each fraction, a 50-µl sample was counted in separate channels for tritium (bullet ) and 14C (black-triangle). b, a portion of [14C]Gal-labeled ConA- (3.6 × 105 cpm of tritium and 7.5 × 104 cpm of 14C) was separated under the same conditions as in a, except that 200 µl of each fraction was counted, and the elution buffer was changed at fraction 60 (arrow).
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Methylation

Vacuum-desiccated samples of degalactosylated peak III (see above) were dissolved in 50-200 µl of an NaOH/Me2SO suspension prepared by vortex mixing of Me2SO and dry, finely ground NaOH pellets. After 1 h at room temperature, 10-50 µl of methyl iodide were added, and the solution was set for 1 h at room temperature with occasional vortex mixing (21). Samples were partitioned by adding 1 ml of chloroform, and the suspension was back-extracted twice with 2-3 ml of 30% acetic acid. The chloroform layer was dried, and the samples were stored at -20 °C.

Periodate Oxidation and Reduction

Periodate oxidation (22) of degalactosylated peak III was performed by using a 9 mM solution of NaIO4 (50 µl) buffered with 0.1 M sodium acetate at pH 5.5 in the dark at 4 °C for 3 days. The reaction was quenched with 1 µl of ethylene glycol, and incubation continued overnight under the same conditions. The sample was neutralized with 0.1 M NaOH and reduced by the direct addition of solid NaB2H4 (1 mg) at room temperature for 16 h. Excess reducing agent was destroyed by the addition of 5 µl of acetic acid, and the solution was dried in a vacuum centrifuge. Borate was removed by the repeated addition and drying with methanol. Periodate-oxidized and reduced oligosaccharides were then methylated as above.

Electrospray Ionization Mass Spectrometry

The instrument used was a TSQ 700 (Finnigan-MAT, San Jose, CA) equipped with an Analytica (Analytica of Branford, CT) electrospray ion source. Methylated oligosaccharides were analyzed by direct infusion at flow rates of 0.85 µl/min of a 6:4 methanol:water electrospray buffer with 0.25 mM NaOH.


RESULTS

Preparation and General Properties of Cell Wall Complex (Fraction V0)

Yeast cell walls were digested with beta (1right-arrow3)-glucanase, labeled by reduction with borotritide, and further hydrolyzed with chitinase as described previously (6). After chromatography of the material solubilized by chitinase on a Bio-Gel P-2 column (6), the fraction emerging at the void volume (fraction V0) was collected. This early fraction, as well as later peaks, contained radioactivity (6). Initial experiments were directed to ascertain the general composition of the material and its relationships with known cell wall components. Labeling with borotritide required prior treatment with beta (1right-arrow3)-glucanase. When borotritide was used on the intact walls, only 24% of the radioactivity was incorporated into fraction V0. This result suggested that most substances in the void volume peak had been attached to beta (1right-arrow3)-glucan in the intact cell wall. Similarly, the solubilization of the material upon chitinase treatment clearly indicated that it had been linked to chitin. In our previous study of the chitin-beta (1right-arrow3)-glucan connection, it had been easy to identify remaining GlcNAc in the bridge region, which was represented by relatively small oligosaccharides. In the present case, however, chromatography of fraction V0 on Sephacryl S-400 indicated a size in the range of 200-250 kDa (results not shown), whereas sugar determinations showed the presence of glucose and mannose with very little if any N-acetylglucosamine (see below). To look for the presumably few GlcNAc groups that may have been involved in the linkage to chitin, we availed ourselves of a sensitive and specific technique that consists in labeling nonreducing GlcNAc residues with [14C]galactose by incubation with UDP-[14C]Gal in the presence of bovine galactosyltransferase (16). Treatment of fraction V0 in this fashion resulted in the incorporation of radioactivity (Fig. 1b). A control in which fraction V0 was pretreated with beta -N-acetylglucosaminidase showed no incorporation (Fig. 1a). This result confirms the covalent linkages of substances in fraction V0 to chitin. It should be kept in mind that, because Serratia chitinase cleaves two GlcNAc residues at a time, even-numbered chains may be excised completely without leaving any amino sugar, as was found for the chitin-beta (1right-arrow3)-glucan linkage (6). Therefore, assuming a random chain length, the residues labeled in the experiment of Fig. 1 may correspond to only half of the connecting chitin chains, those with an odd number of GlcNAc residues.


Fig. 1. Labeling of fraction V0 with 14C-galactose. The chitinase-solubilized fraction V0, obtained by digestion of cell walls and gel chromatography (see "Experimental Procedures") was incubated with UDP-[U-14C]galactose and bovine galactosyltransferase, with (a) or without (b) preincubation with jack bean beta -N-acetylhexosaminidase. The incubated fraction was applied to an extra fine Bio-Gel P-2 column (2 × 90 cm) and eluted with 0.1 M acetic acid. Fractions of 1.8 ml were collected, and a 10-µl portion of each sample was counted using a program that allowed determination of tritium (bullet ) and 14C (black-triangle) isotope in separate channels.
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The evidence outlined above points to a linkage of the material in fraction V0 to both beta (1right-arrow3)-glucan and chitin but does not address the question of how many different substances are present in fraction V0. Further fractionation was required to deal with this problem. Because of the presence of mannose (see below) in fraction V0, we used chromatography on ConA-Sepharose columns (see "Experimental Procedures"). Somewhat more than half of the tritium label went through the column without binding, whereas the remainder was attached and required alpha -methylmannoside for elution. The two fractions were designated ConA- and ConA+, respectively. Both fractions could be labeled with [14C]galactose in the manner outlined above. To ascertain whether the galactose (hence the GlcNAc) was attached to the same substance(s) that carried the tritium label, the derivatized fractions were applied to a column of agarose-bound E. cristagalli lectin, which binds specifically to galactosyl and Gal-GlcNAc groups (Fig. 2). Both ConA- and ConA+ were separated into three fractions, I, II, and III. The first one was not bound to the column and contained only tritium; the second one was retarded and contained tritium and a small proportion of 14C; the last fraction required lactose plus GlcNAc for elution and contained both isotopes, but with a much higher percentage of 14C. We interpret these results as follows: (a) the first fraction represents material previously joined to even-numbered chitin chains, which were completely eliminated by chitinase; (b) the second fraction contains substance(s) that were bound to a single odd-numbered chitin chain; and 3) the third fraction consists of material that had multiple linkages to chitin; therefore, it contains several galactosyl residues per molecule and binds more strongly to the lectin column.

From the results with the ConA-Sepharose and the E. cristagalli lectin-agarose columns, we conclude that the tritium-labeled chain ends, the GlcNAc to which [14C]galactose had been attached, and, in the case of ConA+, the alpha -mannosyl-containing material coexist on the same substance or series of similar substances. As will be discussed below, several lines of evidence indicated that ConA- differs from ConA+ in lacking a mannoprotein component. It may be either a precursor or a degradation product of ConA+. Therefore, most of our efforts were directed toward the elucidation of the ConA+ structure.

A Tentative Structure for ConA+

Because of the complex nature of the ConA+ and ConA- fractions, it is convenient at this point to introduce a tentative structure for them, to facilitate understanding of the experiments to be described. Such a structure is shown for ConA+ in Fig. 3.


Fig. 3. A tentative structure of ConA+. The structure within the broken line corresponds to the isolated material, whereas chains of chitin and beta (1right-arrow3)-glucan that were presumptively attached to ConA+ in the intact cell wall appear outside the line. G, glucose; M, mannose; GN, N-acetylglucosamine. Linkages between sugars are indicated with a single number, e.g. G6G stands for glucosyl(1right-arrow6)glucose. The positions at which galactose was introduced by enzymatic transfer are indicated with arrows, whereas the positions at which different hydrolytic enzymes cut the chains are shown with arrowheads. Protein is drawn as a curved line on the left. The core region of the mannan attached to the protein is not shown for simplicity. PNGaseF, glycopeptidase F. For further explanations see "Results."
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The structure inside the broken line corresponds to the isolated ConA+, whereas the connections to the remainder of the cell wall are shown outside the line. According to this hypothesis, the central portion of ConA+ would consist of a beta (1right-arrow6)-glucan chain with some beta (1right-arrow3) branches. At the reducing terminus, the beta (1right-arrow6)-glucan would be attached to beta (1right-arrow3)-glucan, a stub of which, with the terminal glucose converted into sorbitol, remained after beta (1right-arrow3)-glucanase digestion and borohydride reduction. Chitin would be linked to one or more beta (1right-arrow3) branches of the beta (1right-arrow6)-glucan through its reducing terminal GlcNAc, in the same fashion as it is attached directly to beta (1right-arrow3)-glucan (6). Finally, the nonreducing end of the beta (1right-arrow6)-glucan would be connected to a mannoprotein through a bridge region consisting of part of a GPI anchor (23). The remainder of the GPI anchor was presumably the leaving group in the transglycosylation process that gave rise to the glucan-protein linkage.

For the ConA- fraction we propose essentially the same structure, but without the mannoprotein or the GPI anchor bridge. The reasons for this attribution will be discussed below.

ConA+ Contains beta (1right-arrow6)-Glucan

Acid hydrolysis of ConA+ followed by HPAEC showed the presence of glucose and mannose (Fig. 4d) in approximately equal amounts (Glc:Man ratio of 1.3:1 in the experiment of Fig. 4, closer to 1:1 in other batches), together with a very small proportion of GlcNAc. A 13C NMR spectrum of ConA+ clearly showed the presence of signals corresponding to beta (1right-arrow6)-glucose linkages (Fig. 4c). However, the spectrum of ConA- (Fig. 4e) was chosen for detailed interpretation, because it is free from mannose signals. There are two sets of signals in this spectrum (Table I). One set of high intensity signals corresponds well with signals for pustulan (beta (1right-arrow6)-linked glucopyranosyl residues). The signals of the second set, of lower intensity, are in very good agreement with the 13C chemical shifts of laminarin; therefore, they correspond to beta (1right-arrow3)-linked glucopyranosyl units. There are two more signals in the spectrum of ConA-, at delta  75.46 and 73.65 ppm, which may be assigned to beta (1right-arrow6)-linked glucopyranosyl units of the main chain that are carrying branches composed by beta (1right-arrow3)-linked glucose residues. We arrived at this assignment by exclusion of all other possibilities, as follows: a branch linked at C-4 of the polysaccharide backbone would have resulted in signals either at delta  79.5 ppm (beta -linked) or 78.4 ppm (alpha -linked); a branch at C-2 would have yielded signals at delta  82.1 ppm (beta -linked) or 79.5 ppm (alpha -linked); finally, the signal of an alpha -linked branch at C-3 would have been at delta  99.8 ppm (26). The only remaining possibility is a side chain composed of beta (1right-arrow3)-linked glucopyranosyl units linked to a C-3 position of the main beta (1right-arrow6) chain. The signals at delta  75.46 and 73.65 ppm can be assigned to C-5 and C-2 of the branched unit, in very good agreement with the 75.4 ppm signal in spectra of the 3,6-di-O-substituted beta -D-glucopyranosyl residue of a variety of branched beta (1right-arrow3)-glucans (27). The difference between the two chemical shifts (delta  75.46 and 73.65 ppm) and those belonging to unbranched glucose units (delta  75.78 and 73.93 ppm) is probably caused by greater shielding due to the presence of the side chain.


Fig. 4. 13C NMR spectra of different fractions and corresponding HPAEC chromatograms of their acid hydrolysates. 13C NMR spectra of fraction V0, ConA+, ConA-, and fraction M are shown in a, c, e, and g, respectively; the corresponding HPAEC profiles of their acid hydrolysates are presented in b, d, f, and h. For hydrolysis, a sample of each fraction (10-20 nmol of carbohydrate, as glucose) was evaporated to dryness, dissolved in 50 µl of trifluoroacetic acid, and heated for 2 h at 100 °C. After a new evaporation to dryness, the residue was redissolved in water (5 µl/nmol) and analyzed by HPAEC.
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Table I. 13C NMR chemical shifts for ConA-


C-1 C-2 C-3 C-4 C-5 C-6

Strong signals
  ConA- 103.83 73.93 76.49 70.44 75.78 69.74
  Pustulana 103.83 73.93 76.50 70.44 75.78 69.72
Weak signals
  ConA- 103.58 74.35 85.35 68.99 76.86 61.64
  Laminarinb 103.39 74.10 85.18 68.99 76.49 61.58

a Our measurements.
b Our measurements, which coincide with published results (24, 25).

The results of the 1H NMR spectrum of ConA- (Fig. 5) were in agreement with those of the 13C spectrum. In the anomeric region of this spectrum there are two resolved doublets. The first one, at delta  4.73 ppm (J1,2 7.6 Hz) was assigned to the H-1 proton of the side chain ((1right-arrow3)-glucopyranosyl unit), the second one, at delta  4.52 ppm (J1,2 7.9 Hz), belongs to the H-1 proton of the main chain, (1right-arrow6)-linked. Their coupling constants are in good agreement with those published for beta -linked D-glucopyranoses (28, 29). There is a third doublet in the anomeric region, partially overlapped by the doublet at delta  4.52 ppm. We assume that this doublet belongs to the branched glucopyranosyl residues (3,6-linked). The sum of the integrals (22.6 at delta  4.52 ppm) was compared with the integral (22.2) of the doublet at delta 4.22 ppm, which represents H-6a of the main chain (30). Since they are almost identical, one may conclude that the doublet at delta  4.52 ppm represents all H-1 protons from the backbone, either from branched or unbranched units.


Fig. 5. Proton NMR spectrum of ConA- fraction.
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All these data indicate the presence in ConA- of a beta (1right-arrow6)-linked glucopyranose polysaccharide with beta (1right-arrow3) branches. All of the signals in the NMR spectra of ConA- could be found in those of ConA+ and of fraction V0; therefore, these fractions contain the same or a similar polysaccharide.

In confirmation of these results, digestion of [14C]Gal-labeled ConA+ or ConA- with bacterial beta (1right-arrow6)-glucanase followed by chromatography on Sephadex G-100 showed extensive degradation of the material, with all of the 14C and part of the tritium in a low molecular weight wide peak, whereas the remainder of the 3H was still in the void volume fraction (Fig. 6b). When the three fractions isolated by chromatography of ConA+ on the E. cristagalli lectin-agarose column (Fig. 2a) were individually incubated with the glucanase, all of them showed a similar pattern of degradation, with part of the tritium eluted in the void volume fraction (results not shown). Since the 14C represents galactose attached to GlcNAc, the generation of 14C-labeled low molecular substances by beta (1right-arrow6)-glucanase treatment indicates that chitin is linked to beta (1right-arrow6)-glucan.

Chitin Is Directly Attached to beta (1right-arrow6)-Glucan, Probably through a beta (1right-arrow3) Branch

Fractionation of the low molecular weight material released by beta (1right-arrow6)-glucanase on a E. cristagalli lectin-agarose column completely separated the 14C-containing material (B), which was retarded, from the tritium-labeled substances (A), which were not absorbed by the column (Fig. 7a). Each one of the E. cristagalli lectin-agarose fractions was rechromatographed on a Bio-Rad P-2 column (Fig. 7, b and c). The tritium-containing fraction gave rise to several labeled peaks, at positions corresponding to oligosaccharides in the 2-6 hexose residue range (Fig. 7b). Carbohydrate determinations revealed the presence of unlabeled oligosaccharides in a similar range of sizes but eluting at slightly different positions (Fig. 7b). The unlabeled oligosaccharides presumably originate in inner portions of the beta (1right-arrow6)-glucan chain (see Fig. 3) and consist exclusively of glucose, whereas the tritiated compounds are derived from the reducing terminus previously attached to beta (1right-arrow3)-glucan and contain sorbitol. This difference in composition explains the slight variation in elution volume (6).

The 14C-labeled oligosaccharides fractionated in the P-2 column into three main peaks, I, II, and III (Fig. 7c). These oligosaccharides represent linkage points of beta (1right-arrow6)-glucan to chitin, because they contain GlcNAc. Since the linkage points are very few relative to the total length of the glucan chain, this fraction contains very little carbohydrate, which was undetectable under our conditions (Fig. 7c). Peak III was subjected to further analysis. After labeling the reducing end by reduction with borotritide, the [14C]galactose at the nonreducing end was cleaved off with beta -galactosidase (see "Experimental Procedures"). The liberated tritiated oligosaccharide emerged from the P-2 column at a position consistent with the composition GlcNAcGlc3Glcol. The same composition was indicated by separation of the sugars on HPAEC after acid hydrolysis. Incubation of the oligosaccharide with beta -N-acetylglucosaminidase, followed by beta -glucosidase, resulted in the liberation of tritiated sorbitol (results not shown).

Further studies of degalactosylated peak III were carried out by mass spectrometry before and after derivatization, a technique that provides structural information with very small amounts of material. Electrospray mass spectrometry (ES-MS) of methylated degalactosylated peak III produced two major peaks at m/z 584.7 and 1146.6 (Fig. 8a) corresponding to doubly and singly charged ions (charge arising by the adduction of sodium), with a mass that is consistent with a methylated oligosaccharide alditol containing one N-acetyl hexosamine and four hexose residues. Since oligosaccharide permethylation eliminates acyl modifications, the underivatized oligosaccharide was also examined by ES-MS in the negative ionization mode, and this was also consistent with a composition of a oligosaccharide alditol containing one N-acetyl hexosamine and four hexose residues (results not shown). The CID spectrum of the derivatized oligosaccharide was examined to obtain information about the sequencing and branching structure. The collision spectrum of the doubly charged molecular ion at m/z 584 (Fig. 8c) produced a prominent B1/Y3 ion pair (31) at m/z 282 and 887 (Scheme 1a, Fig. 8c). The loss of an additional hexose from the m/z 887 Y4 ion is observed (m/z 683, Y3), as is another hexose loss (m/z 479, Y2), to form a partial set of sequence ions containing the reducing end. The final Y1 ion is not observed, since the reducing end glucose had been reduced, and the pyranose ring structure that would stabilize the adduction of a sodium cation on this fragment was destroyed. The set of Y ions at m/z 887, 683, and 479 indicates a linear topology; however, there is another sequence of Y ions that indicates a branched structure. These are the tri- and disaccharide Y/Z fragments at m/z 669/651 and m/z 465/447, showing multiple glycosidic losses (Scheme 1b, Fig. 8c). Although the fragmentation pattern does indicate both a linear and branched structure, it provides a poor quantification of the relative amounts of these structures.


Fig. 8. Mass spectrometry of degalactosylated peak III. a, ES-MS spectrum of permethylated sample. The m/z 584.7 peak is doubly charged, adducting two sodium ions, and the m/z 1146.6 peak is singly charged, adducting a single sodium. The mass is consistent with a permethylated oligosaccharide alditol having one N-acetyl hexosamine and 4 hexose residues. b, ES-MS spectrum of the periodate-oxidized, NaB2H4-reduced and permethylated degalactosylated peak III. A triplet of ion peaks is observed in the single and double charge states. c, ES-MS-CID-MS spectrum of the doubly natriated, permethylated oligosaccharide alditol at m/z 584.7. The spectrum is plotted as a relative entropy surface (34), which suppresses noise spikes and allows the graphical presentation of high and low amplitude peaks in a single scale, similar to a log scale. d, ES-MS-CID-MS of the singly natriated peak at m/z 939 from the periodate-oxidized, NaB2H4-reduced and permethylated oligosaccharide. The spectrum is again an entropy surface.
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Scheme 1.


The glycosidic linkages in the oligosaccharide were also investigated by CID. The glycosidic bond on the reducing side of N-acetyl hexosamine residues is rapidly cleaved under the collision conditions of the triple quadrupole, and this results in the prompt loss of a nonreducing terminal GlcNAc. This, unfortunately, largely prevented the formation of ring-opening fragments that can be used for linkage assignment with tandem mass spectrometry of permethylated oligosaccharides (32, 33). The only ring-opening fragments observed were a minor m/z 301 (0,4A) and 329 (3,5A) pair (Fig. 8c), and these suggest a terminal 6-linked hexose.

Periodate oxidation, reduction, and permethylation of the oligosaccharide can be used to obtain linkage information at high sensitivity (22). The ES-MS spectrum of the periodate-treated and methylated oligosaccharide (Fig. 8b) shows a triplet of ions, adducting one or two sodium cations. The increments of 4 atomic mass units exhibited by the oxidation products (e.g. the singly adducted triplet at m/z 931, 935, and 939 (Fig. 8b, inset) correspond to the oxidation of additional diols by the periodate anion (4 atomic mass units, since NaB2H4 was used as the reducing agent). The m/z 939 peak is consistent with the presence of both a branched and a linear structure, and in this case periodate oxidation cannot directly assign the molar fraction of the two structures by a specific mass shift. The oxidation product total mass, together with the knowledge that the reducing end is a hexose alditol, does indicate specific combinations of oxidized and unoxidized residues; however, their sequence in the oligosaccharide requires tandem mass spectrometry (Scheme 2: a, linear structure, m/z 939; b, branched structure, m/z 939). The m/z 939 ion was dissociated by collisional activation, and the mass spectrum of the product ions was taken (Fig. 8d). The ion fragments at m/z 818, 676, and 468 are common to both structures and indicate an oxidized nonreducing terminal N-acetyl hexosamine, 2- or 4-linked to a hexose (Scheme 2). The m/z 761 fragment corresponds to the loss of an oxidized hexose and could only arise from the branched structure. The hexose linked to the sorbitol was not oxidized, and this is consistent with it being linked at the 3-position (or branched). The tandem mass spectrometry spectra of the m/z 935 and 931 peaks show that the 4-atomic mass unit decrement arises from a failure to oxidize either the terminal N-acetyl hexosamine or the adjacent hexose (m/z 935) or both (m/z 931). Failure to oxidize the terminal N-acetyl hexosamine suggests that incomplete oxidation chemistry and not linkage arrangement is responsible for these peaks.


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Scheme 2.


Both the mass spectrometry results and the chemical and enzymatic determinations indicate the presence in degalactosylated peak III of a pentasaccharide, containing one GlcNAc and four hexose residues, one of which had been reduced to the corresponding alditol. The fragmentation pattern shows the GlcNAc at the nonreducing terminus, as confirmed by its cleavage by beta -N-acetylhexosaminidase. All hexoses are beta -linked glucose residues, as shown by the action of beta -glucosidase and by HPAEC (see above). The acetylhexosamine is attached to the next glucose by a beta (1right-arrow2) or a beta (1right-arrow4) linkage. CID data, both of the permethylated material and of the periodate-oxidized, reduced, and permethylated material indicate the presence of two isomers, one linear and the other branched (Schemes 1 and 2). In both structures, one of the hexoses appears to be beta (1right-arrow3)-linked. In the branched structure, there is a beta (1right-arrow6)-linked glucose. All of these results are compatible with a linkage of the GlcNAc at the reducing end of a chitin chain to a glucose linked beta (1right-arrow3) to the main chain of beta (1right-arrow6)-glucan. Cutting at different locations by the beta (1right-arrow6)-glucanase used in the preparation of peak III would give rise either to the branched or to the linear isomer (Scheme 3; arrows show two possible cutting patterns). Notice that the mass spectrometry results would also be compatible with the (1right-arrow3)-linked glucose in the linear isomer being directly attached to sorbitol. However, this would imply that beta (1right-arrow3) linkages would be occasionally present in the main beta (1right-arrow6) chain. There is no evidence for this; in fact, all four internal oligosaccharides from beta (1right-arrow6)-glucan that we analyzed after reduction were completely hydrolyzed to glucose and sorbitol by beta -glucosidase (see example below in Fig. 9b), an indication that there were no beta (1right-arrow3) linkages between glucose and sorbitol (6). Furthermore, 13C NMR spectra indicate the presence of branches attached to the main beta (1right-arrow6) chain by a beta (1right-arrow3) linkage, as discussed above. Another piece of evidence for attachment of chitin to a beta (1right-arrow3) branch comes from a study of oligosaccharide I (Fig. 7c), a tetrasaccharide. After reduction with borohydride and incubation with beta -galactosidase and beta -N-acetylglucosaminidase, beta -glucosidase was unable to hydrolyze the residual reduced disaccharide (data not shown), a result suggestive of a beta (1right-arrow3) linkage between glucose and sorbitol (6). We also considered the possibility that the GlcNAc was directly attached to a glucose residue of the main beta (1right-arrow6) chain, but we found that all structures that would have resulted in that case were incompatible with the mass spectrometry results.


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Scheme 3.



Fig. 9. beta -Glucosidase digestion of oligosaccharides released from ConA+ by beta (1right-arrow6)-glucanase. a, a sample of tritiated pentasaccharide (2 × 104 cpm) isolated by Bio-Gel P-2 (Fig. 7b) was evaporated to dryness, dissolved in 280 µl of 50 mM sodium acetate, pH 5.0, and digested with 0.3 mg of sweet almond beta -glucosidase for 16 h at 37 °C in the presence of 0.02% NaN3. After incubation, the sample was applied to a Bio-Gel P-2 column, and the digestion product, eluting at the position of a reduced hexose disaccharide, was recovered. Pooled fractions were evaporated to dryness, dissolved in 30 µl of water, and subjected to paper chromatography. Segments (1 cm) of the paper were counted. Standards were glucose ([14C]glucose, internal standard) (1), glucitol (2), laminaribiitol (3), sophoritol (4), cellobiitol (5), and gentiobiitol (6). b, a sample of nonreduced tetrasaccharide, isolated from the same chromatography (Fig. 7b), was labeled by reduction with sodium borotritide. Part of the recovered reduced tetrasaccharide (1 × 105 cpm) was evaporated to dryness, dissolved in 200 µl of 50 mM sodium acetate, pH 5.0, and digested with 1.2 mg of sweet almond beta -glucosidase as in a. A portion of incubation mixture (20,000 cpm) was subjected to paper chromatography as in a. Standards were the same for both panels.
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The Reducing End of the beta (1right-arrow6) Chain Is Attached to beta (1right-arrow3)-Glucan

In a previous study on the chitin-beta (1right-arrow3)-glucan linkage, we showed that treatment of cell wall with beta (1right-arrow3)-glucanase followed by reduction of the exposed ends with borotritide left stubs consisting of sorbitol and one or a few glucoses attached to chitin (6). These stubs could then be released by chitinase, with or without an attached GlcNAc residue, depending on the even or odd numbering of the chitin chain. The liberation of tritiated oligosaccharides from ConA+ by beta (1right-arrow6)-glucanase suggested that we had here a similar situation, but with the reducing end of beta (1right-arrow6)-glucan, rather than of chitin, attached directly to beta (1right-arrow3)-glucan. To investigate this possibility, we made use of the previous finding that sweet almond beta -glucosidase can hydrolyze a glucose residue attached to sorbitol in a beta (1right-arrow6) but not in a beta (1right-arrow3) linkage (6). When tritiated oligosaccharides from the beta (1right-arrow6)-glucanase digest of ConA+ were treated with beta -glucosidase, the final labeled product was laminaribiitol, as determined by paper chromatography. On the other hand, when unlabeled oligosaccharides from the beta (1right-arrow6)-glucanase digest, originated from internal chains of the polysaccharide, were reduced with borotritide and then subjected to the glucosidase treatment, the tritiated product was sorbitol. Examples of both experiments are shown in Fig. 9. We conclude that beta (1right-arrow6)-glucan chains are directly attached to beta (1right-arrow3)-glucan. The type of linkage between the last glucose of one of the polysaccharides and the first glucose of the other has not been established.

The High Molecular Weight Material Remaining after beta (1right-arrow6)Glucanase Digestion Contains Mannan

The material eluting in the void volume of the Sephadex G-100 column after beta (1right-arrow6)-glucanase digestion (fraction M, Fig. 6b) contains all of the mannose and very little of the glucose associated with ConA+ (Fig. 4h). This fraction also contains part of the original tritium label (Fig. 6b). The nature of the radioactive component will be discussed below.

Preliminary experiments on digestion of ConA+ with Pronase2 followed by Sephadex G-100 chromatography suggested that the mannose could be a component of a large polysaccharide, such as yeast cell wall mannan. This mannan consists of a core region that includes the N-linkage to protein and of an extended alpha (1right-arrow6)-linked mannosyl chain with alpha (1right-arrow2) and alpha (1right-arrow3) branches 1-4 mannoses long (35). Acetolysis of mannan results in breakage of the main chain alpha (1right-arrow6) bonds and release of the short side chains (35). High molecular weight carbohydrate-containing material was isolated from a Pronase digest of ConA+ by Sephadex G-100 chromatography and subjected to acetolysis followed by deacetylation (see "Experimental Procedures"). The products were separated by chromatography on a Bio-Gel P-2 column into four sugar peaks, eluting at the position of free hexose and di-, tri-, and tetrasaccharide (M2, M3, and M4, respectively; see Fig. 10). The structure of the oligosaccharides was studied by 1H NMR and 13C NMR. Comparison of spectra with those reported in the literature (36-44) resulted in the assignment of the structure Manp-alpha -1right-arrow2-Manpalpha for M2, Manp-alpha -1right-arrow2-Manp-alpha -1right-arrow2-Manpalpha for M3, and Manp-alpha -1right-arrow3-Manp-alpha -1right-arrow2-Manp-alpha -1right-arrow2-Manpalpha for M4 (data not shown). Signals for the same linkages were found in the 1H NMR spectrum (not shown) and 13C NMR spectrum (Fig. 4g) of fraction M. 


Fig. 10. Acetolysis of fraction M. Fraction M was subjected to acetolysis (see "Experimental Procedures") and subsequently applied to a Bio-Gel P-2 column (1 × 85 cm) and eluted with 0.1 M acetic acid. Fractions of 0.45 ml were collected, and a 40-µl portion of each fraction was used for assay of total carbohydrates by the phenol-sulfuric acid method (see "Experimental Procedures"). Standards were glucose ([14C]glucose, internal standard) (1), laminaribiitol (2), laminaritriitol (3), and laminaritetraitol (4).
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Based on all of these results, it is clear that fraction M contains a high molecular weight mannose polysaccharide of the type normally found in yeast cell walls.

ConA+ Contains Mannoprotein

Since yeast mannan is invariably found attached to protein, the presence of mannan in fraction M suggested that protein should also be a component of this fraction. Mannan is N-linked to protein, and it can be cleaved off with either glycopeptidase F or endoglycosidase H. When fraction M was relabeled by borotritide reduction and treated with glycopeptidase F, chromatography of the digest on Sephadex G-100 separated unlabeled carbohydrate that emerged in the void volume from most of the tritiated material that was eluted in later fractions and presumably contained the protein (Fig. 11). Similar results were obtained with endoglycosidase H (data not shown). The latter finding suggested a procedure to label specifically the protein, by transgalactosidation onto the GlcNAc that remains attached to protein after endo-H hydrolysis. Accordingly, fraction M was treated with endo-H (45) and fractionated on Sephadex G-100. The liberated material containing the intrinsic tritium label (similar to the tritiated peak of Fig. 11) was concentrated and incubated with UDP[14C]Gal in the presence of galactosyltransferase (see "Experimental Procedures"). Upon Sephadex G-100 chromatography, the incorporated 14C radioactivity eluted in the same position as the tritiated material. Electrophoresis of the 14C-labeled material on polyacrylamide in the presence of SDS, followed by autoradiography, showed a broad band at about 100 kDa and some material that trailed behind (Fig. 12a). Fraction M or ConA+ remained at the origin.


Fig. 11. Treatment of 3H-relabeled fraction M with glycopeptidase F. A portion of fraction M (40 µg of protein) was evaporated to dryness, dissolved in 40 µl of 0.01 M NaOH, and reduced with 100 µCi of NaB3H4. The excess NaB3H4 was eliminated by concanavalin A and Sephadex G-100 chromatography. After new evaporation to dryness, the sample (2.5 × 105 cpm) was incubated with 8 µl (4000 units) of glycopeptidase F under the conditions specified by the supplier (Life Technologies, Inc.). The mixture was incubated for 16 h at 37 °C in the presence of 0.02% NaN3 and subsequently applied to a Sephadex G-100 column (1 × 85 cm) and eluted with 0.1 M acetic acid. Fractions of 0.45 ml were collected, and a 40-µl portion of every second fraction was either counted (bullet ) or used for the assay of total carbohydrates (open circle ) (see "Experimental Procedures"). Positions of molecular mass standards (in kDa) are indicated.
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Fig. 12. Electrophoresis and Western blots of different fractions. a, SDS-polyacrylamide gel electrophoresis of 14C-Gal-labeled ConA+, of fraction M and of material released from fraction M by endo-H and relabeled with 14C-Gal. Samples (20,000-30,000 cpm) were evaporated to dryness, dissolved in sample buffer containing 0.5% beta -mercaptoethanol, incubated for 5 min in a boiling water bath, and separated on an 18% gel, together with 14C-labeled molecular mass standards, in Tris-glycine running buffer. The gel was fixed for 45 min in 15% trichloroacetic acid, washed 3 times for 30 min in Me2SO, and soaked for 45 min in 100 ml of Me2SO containing 50 g of 2,5-diphenyloxazole. After overnight washing in water and drying, the gel was subjected to fluorography with 1 week of exposure. Lane 1, 14C-labeled ConA+ fraction; lane 2, fraction M; lane 3, 14C-labeled endo-H-released material from fraction M. Positions of molecular mass standards (in kDa) are indicated. b and c, Western blot and autoradiography of the [14C]Gal-labeled ConA+ fraction after incubation with beta (1right-arrow6)-glucanase or hydrofluoric acid. b, portions (4 × 104 cpm) of [14C]Gal-labeled ConA+ were either incubated with endo-beta (1right-arrow6) glucanase in 45 µl of 111 mM sodium acetate with 20 milliunits of enzyme for 17 h at 37 °C (lane 2) or with 50 µl of 50% hydrofluoric acid for 24 h on ice (lane 3). Lane 1, unincubated control. After incubation, samples were separated by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF membrane, and blotted with antibody against beta (1right-arrow6)-glucan (11). c, lane 2, a portion (3 × 104 cpm) of [14C]Gal-labeled ConA+ fraction was treated with 50% HF under the same conditions as in the legend of Fig. 13 and separated on SDS-polyacrylamide gel. Lane 1, unincubated control. The gel was transferred to a PVDF membrane. The membrane was dried and exposed for autoradiography for 96 h.
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Mannoprotein in ConA+ Is Attached to beta (1right-arrow6)-Glucan through a Portion of a GPI Anchor

The simultaneous binding of the mannoprotein and of beta (1right-arrow6)-glucan to ConA-Sepharose indicated that these two components are covalently linked, because ConA has no affinity for beta -linked glucose. The nature of the linkage remained to be determined. Several cell wall proteins are attached to beta (1right-arrow6)-glucan through what seems to be a remnant of a former GPI anchor (7), part of which was lost in a putative transglycosylation. To verify whether that was the case here, fraction M was relabeled by reduction with borotritide and treated in the cold with hydrofluoric acid, under conditions that lead to hydrolysis of the linkage between phosphate and mannose in a GPI anchor (46, 47). When the reaction mixture was applied to a Sephadex G-100 column, a large portion of the tritium label was eluted at a position corresponding to low molecular weight material (Fig. 13b). This material contained the tritium introduced in the relabeling of fraction M, because when the original fraction M was treated with hydrofluoric acid, almost all of the tritium remained in the void volume fraction (Fig. 13a). If the anchor hypothesis were correct, the low molecular weight material released by HF would consist of a mannose oligosaccharide attached to a stub of beta (1right-arrow6)-linked glucoses left over after beta (1right-arrow6)-glucanase hydrolysis of ConA+ (Fig. 3). This proved to be the case. The tritiated material yielded one peak, corresponding to an oligosaccharide of 8 residues upon HPLC on a Rezex RSO-Oligosaccharides column (Fig. 14a). However, when this material was rechromatographed on a Bio-Gel P-4 column, it split into two peaks eluting at positions corresponding to oligosaccharides of 7 and 8 residues (results not shown). These substances were resistant to beta -glucosidase (Fig. 14b). They were, however, hydrolyzed by alpha -mannosidase to compounds with the size of oligosaccharides containing 3 (A) and 2 (B) hexoses, respectively (Fig. 14c). Both products were now degraded by beta -glucosidase, giving rise to a free hexitol (Fig. 14d). The hexitol was identified as sorbitol by paper chromatography (results not shown). These results are consistent with the HF-released material being made of a chain of five alpha -linked mannosyl units (a number found in yeast GPI anchors (47)), attached to a reduced glucose disaccharide or trisaccharide, respectively. The latter was left over after hydrolysis with the beta (1right-arrow6)-endoglucanase.


Fig. 13. Release of an oligosaccharide from fraction M by hydrofluoric acid. a, a portion of fraction M (3 × 104 cpm) was evaporated to dryness, dissolved in 200 µl of precooled 50% hydrofluoric acid, and incubated at 0 °C for 72 h. The digest was pipetted onto frozen 10 M NaOH (400 µl) in a microcentrifuge tube placed in ice. The resulting suspension was spun down, and the NaF precipitate was washed twice with 100 µl of water. Supernatants were combined and applied to a Sephadex G-100 column (1 × 85 cm), which was eluted with 25 mM Tris-chloride, pH 7.5, containing 0.02% NaN3. Fractions of 0.45 ml were collected, and a 400-µl portion of every second fraction was counted. Positions of molecular mass standards (in kDa) are indicated. b, a portion (3 × 104 cpm) of 3H-relabeled (see legend of Fig. 14) fraction M was treated with hydrofluoric acid and chromatographed as in a.
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Fig. 14. Treatment of HF-released oligosaccharides from 3H-relabeled fraction M with beta -glucosidase and alpha -mannosidase. a, a portion (10,000 cpm of 3H) of HF-released oligosaccharide (Fig. 13b) was chromatographed on a Rezex RSO-Oligosaccharides column (10 × 200 mm, guard column 10 × 20 mm) at 75 °C with a Gilson model 303 HPLC pump at 0.2 ml/min. Fractions of 100 µl were collected, and a 25-µl portion of every second sample was counted. b, a similar portion of oligosaccharide was incubated with 0.1 mg of beta -glucosidase in 50 µl of 50 mM sodium acetate, pH 5.0, for 16 h at 37 °C in the presence of 0.02% NaN3, followed by chromatography on the Rezex column. c, another portion of oligosaccharide was incubated with alpha -mannosidase (250 milliunits) in 50 µl of a reaction mixture containing 100 mM sodium acetate, pH 5.0, 2 mM Zn2+, and 0.02% NaN3 for 16 h at 37 °C and applied to the Rezex column. d, peaks A and B from c were individually pooled and incubated with beta -glucosidase as in b, followed by chromatography on a Bio-Gel P-2 column (see legend of Fig. 12). Standards were [14C]glucose (internal standard) (1); hexitol (2); diol to octaol obtained by reduction of a dextran ladder (Oxford Glycosystems) (3-9).
[View Larger Version of this Image (20K GIF file)]

The hydrofluoric acid treatment was also applied to the intact ConA+. After HF hydrolysis, the material was subjected to polyacrylamide electrophoresis and Western blotting with an antibody against beta (1right-arrow6)-glucan (11). The amount of immunoreactive material was much diminished after the HF treatment and completely eliminated by incubation with beta (1right-arrow6)-glucanase (Fig. 12b). Similarly, ConA+ that had been labeled with [14C]galactose was incubated with HF, subjected to electrophoresis on polyacrylamide gel, and transferred to a PVDF membrane. The membrane was exposed for autoradiography under conditions in which only 14C but not tritium would be detected. The sample treated with HF yielded a negative result, whereas an intact sample showed a band at about the same position as found before for intact ConA+ (Fig. 12c). In both cases, the results indicate that the beta (1right-arrow6)-glucan had been separated from the protein and therefore was lost in the transfer to the PVDF membrane. The split between the two moieties of the complex could also be shown by ConA-Sepharose chromatography, but it was found that hydrofluoric acid, even at the low temperature used in the experiment, led to rather extensive degradation of beta (1right-arrow6)-glucan (results not shown).

Hexitol Residues Are Linked to Protein in ConA+

After degradation of ConA+ with beta (1right-arrow6)-glucanase, tritiated material was still found in the Sephadex G-100 void volume peak (fraction M; see Fig. 6b). This result was unexpected, because most of the glucose had been liberated by the glucanase (Fig. 4h); therefore, all of the labeled sorbitol attached to the glucose should be in the low molecular weight fraction. The puzzle was solved when fraction M was hydrolyzed with trifluoroacetic acid and the labeled material was tentatively identified by paper chromatography as mannitol, rather than sorbitol (results not shown). The alditol appears to be directly or indirectly attached to protein, because it remains with the protein after treatment of fraction M with glycopeptidase F (Fig. 11), endo-H, or HF (Fig. 13). Treatment of fraction M with proteinase K, followed by Sephadex G-100 chromatography, yielded a peak containing about 45% of the radioactivity in a position corresponding to low molecular weight material, whereas the remainder was still in the void volume fraction (results not shown). The structure of the alditol-labeled material and the manner in which it is attached to protein remain undetermined. From the practical point of view, it served as a convenient label during the isolation of the protein moiety of ConA+.

ConA- Is Similar to ConA+ but Lacks Mannoprotein

Although most of the emphasis of this study was on the ConA+ fraction, many experiments were also done with ConA-. The structure of ConA- appears to be similar to that of ConA+, except for the absence of mannoprotein in ConA-, on the strength of the following evidence. (a) ConA- is linked to chitin and to beta (1right-arrow3)-glucan, based on the incorporation of [14C]galactose and binding to the E. cristagalli lectin-agarose column (Fig. 2b) as well as on the production of both 14C- and tritium-labeled oligosaccharides after incubation with beta (1right-arrow6)-glucanase (Fig. 6, c and d). (b) The 13C NMR spectrum of ConA- shows the presence of beta (1right-arrow6)-glucan, but the signals for mannan are missing (Fig. 4e). Accordingly, ConA- contains no mannose (Fig. 4f). The absence of this sugar is also consistent with the inability of ConA- to bind to ConA-Sepharose. (c) No "mannitol tritium" was found after beta (1right-arrow6)-glucanase digestion of ConA- (Fig. 6d).

From the Sephadex G-100 elution pattern of intact ConA- it is clear that this material has a lower average molecular weight than ConA+ and is quite heterogeneous in size. This heterogeneity presumably reflects variability in length of the beta (1right-arrow6)-glucan chains.


DISCUSSION

A Family of Compounds in the Isolated Complex

The fraction solubilized from the cell wall by successive treatments with beta (1right-arrow3)-glucanase and chitinase and isolated in the void volume of a P-2 column turned out to be surprisingly complex. This material, expected to represent a linkage region between chitin and beta (1right-arrow3)-glucan, was separated on a ConA-Sepharose column into the ConA+ and ConA- fractions. After transferring [14C]galactose to terminal GlcNAc residues in ConA+ and ConA-, each one of them could be further fractionated on an E. cristagalli lectin-agarose column into portions containing different amounts of galactose, hence of GlcNAc. Added to this complexity is a certain variability in size evidenced during chromatography of ConA+ on Sephacryl S-400 or of ConA- on Sephadex G-100. Despite all these signs of heterogeneity, there are many indications that the substances comprising either the ConA+ or the ConA- fraction share the same basic structure. Thus, the NMR spectra are relatively uncomplicated and of straightforward interpretation, the sugar composition is simple, and all fractions are degraded by beta (1right-arrow6)-glucanase in a similar fashion. Furthermore, some of the fractions obtained by chromatography of ConA+ contain mannoprotein, beta (1right-arrow6)-glucan, tritiated sorbitol residues, and GlcNAc (indicated by the presence of [14C]galactose): these components could be separated only after treatment of the complex with either enzymes or hydrofluoric acid. The heterogeneity in size can be accounted for by variability in length of the beta (1right-arrow6)-glucan chains. Still, the complexity of the material, together with its scarcity, reflected often in samples at the nanomolar level, severely limited the range of usable methodologies and prevented the complete determination of some of the sugar to sugar linkages.

Components of the Complex

The main components of the ConA+ fraction are beta (1right-arrow6)-glucan and mannoprotein. beta (1right-arrow6)-glucan was identified by the NMR spectrum and by its susceptibility to hydrolysis by beta (1right-arrow6)-glucanase. The proton NMR spectrum provided evidence that beta (1right-arrow3) branches are attached to the main beta (1right-arrow6) chain. The size of the glucan molecules is somewhat in doubt. In ConA- only beta (1right-arrow6)-glucan makes a significant contribution to the molecular weight, because the mannoprotein is missing and chitin and beta (1right-arrow3)-glucan are represented by only one or a few sugar residues. Chromatography of ConA- on Sephadex G-100 (Fig. 6c) shows a quite heterogenous distribution, with sizes between approximately 10 and 100 kDa, which would correspond to chains of between 60 and 600 glucose residues, with an average of perhaps 300-350 residues. By comparison, beta (1right-arrow6)-glucan isolated from yeast by alkali and acetic acid extraction seems to consist on average of 140 glucose units (48). For ConA+, it is more difficult to estimate the glucan size because of the presence of mannoprotein. Hydrofluoric acid cuts the linkage between glucan and protein but also partially hydrolyzes the glucan (see "Results"). An attempt was made to degrade the mannoprotein by sequential incubation of ConA+ with glycopeptidase F and proteinase K, followed by chromatography on Sephadex G-100. The treatment caused a decrease in size, with part of the material now entering the included volume (results not shown). However, we lacked criteria to determine whether the action of the enzymes used had been complete; therefore, the size of beta (1right-arrow6)-glucan in ConA+ remains in doubt.

The presence of protein in ConA+ was inferred indirectly from the finding that mannan was a component. The polysaccharide is a typical yeast cell wall mannan, as judged from its NMR spectrum, the oligosaccharides generated by acetolysis and their NMR spectra, and the size shown in gel filtration after excision from protein with glycopeptidase F or endo-H. Use of the latter enzyme allowed us to label the protein by transfer of [14C]galactose onto the residual GlcNAc and thus detect it after the subsequent gel electrophoresis. The apparent molecular weight of the deglycosylated protein, in the vicinity of 100,000, is remarkably similar to that we previously found (94,000) for the main component of cell wall proteins after beta (1right-arrow3)-glucanase and endo-H treatment (4). In addition to the N-linked mannan, it is possible that the protein has O-linked mannose oligosaccharides, but the small amounts of material available did not allow investigation of this point. The radioactivity incorporated into the protein upon reduction of the complex with borotritide was found to be in an alditol residue tentatively identified as mannitol, an indication that mannose with its reducing group exposed was attached, either directly or indirectly, to the untreated protein. This is a very surprising result, because in glycoproteins both N-linked and O-linked sugars are attached through their reducing group, making the latter unavailable to borohydride. The exceptions are GPI anchors (see below), but the alditol could not be part of an anchor because it was not released by hydrofluoric acid (Fig. 13). Preliminary results indicate that a substantial portion of the tritium incorporated by reduction of intact cell walls is in the alditol, confirming that the reducing carbon of the original sugar, presumably mannose, was unlinked (results not shown).

Cross-links between the Components

As found in other cases (7), the connection between mannoprotein and beta (1right-arrow6)-glucan is through a portion of GPI anchor. Previously, the presence of anchor material was inferred somewhat indirectly, from the susceptibility of the protein-glucan linkage to hydrofluoric acid and phosphodiesterase (7) and from the attachment to cell wall of hybrid proteins containing the anchor-bearing domain of alpha -agglutinin (49). In the present study, ConA+ was digested with beta (1right-arrow6)-glucanase, reduced with borotritide, and hydrolyzed with HF. The resulting oligosaccharides were isolated and shown to consist of five alpha -linked mannose residues, attached to the nonreducing end of beta (1right-arrow6)-linked reduced disaccharide and trisaccharide, respectively. This result identifies the residual portion of the original GPI anchor as ethanolamine-phosphate-Man5. The glucosaminyl residue was eliminated together with the phosphatidylinositol group in the putative transglycosylation reaction that originated the protein-glucan connection.

Connections of the mannoprotein-beta (1right-arrow6)-glucan complex to beta (1right-arrow3)-glucan and chitin were postulated on account of the need of both beta (1right-arrow3)-glucanase and chitinase for solubilization of the complex and were confirmed by the presence of remnants of both polysaccharides in the complex. Thus, beta (1right-arrow3)-linked oligosaccharides were connected to the reducing end of beta (1right-arrow6)-glucan, and GlcNAc that survived chitin degradation by chitinase was found to be attached to the same glucan, probably onto beta (1right-arrow3) side branches. The GlcNAc at the reducing end of the chitin chain is connected to glucose either by a beta (1right-arrow2) or a beta (1right-arrow4) linkage. We favor the latter possibility by analogy to the bond between chitin and beta (1right-arrow3)-glucan (6).

If our hypothesis that chitin is attached to a beta (1right-arrow3)-linked glucose residue is correct, one may ask whether the previously found linkage of chitin to glucan might not have been to a beta (1right-arrow3) side chain of beta (1right-arrow6)-glucan rather than to the long beta (1right-arrow3)-linked polysaccharide. This notion seems unlikely for the following reasons: first, the endo-beta (1right-arrow3)-glucanase activity in Zymolyase requires a minimum of 5 glucose residues to cut the chain (50), whereas beta (1right-arrow3) branches of beta (1right-arrow6)-glucan are quite short, probably not more than one or two residues in length (48); second, we found that mutant kre5, despite its low content of beta (1right-arrow6)-glucan (51), shows a normal complement of oligosaccharides indicative of a chitin-beta (1right-arrow3)-glucan linkage (results not shown).

The ConA- material is similar to ConA+, except for the absence of mannoprotein. Thus, it may be either a precursor or a degradation product of ConA+.

The role of beta (1right-arrow6)-Glucan in the Organization of the Yeast Cell Wall

From the foregoing analysis, it appears that beta (1right-arrow6)-glucan is the central molecule or "glue" that keeps together the other components of the cell wall, including beta (1right-arrow3)-glucan, mannoprotein, and part of the chitin (Fig. 3). Thus, it is not surprising that defects in beta (1right-arrow6)-glucan formation, as found in several mutants (52, 53), can interfere with cell wall assembly and have severe effects on cell growth (52, 53). For this reason, it is possible that compounds that specifically inhibit beta (1right-arrow6)-glucan synthesis, if such can be found, would behave as antifungal agents.

The chitin participating in linkages to both beta (1right-arrow3)- and beta (1right-arrow6)-glucan is synthesized by chitin synthase 3 (6). This chitin is found both in the ring formed at the base of an emerging bud and, in dispersed form, throughout the cell wall (54). It is of interest to compare the results of the present investigation with those of an earlier study on the distribution of chitin in the cell wall (55). In that study, we found that most of the chitin present in lateral walls and part of the bud scar chitin could be solubilized by incubation of alkali-treated cell ghosts with a beta (1right-arrow6)-glucanase preparation that was contaminated with a small amount of chitinase. The same amount of chitinase without the glucanase had no effect. Those results are in good agreement with the present findings about chitin synthase 3 involvement in chitin synthesis and about the role of beta (1right-arrow6)-glucan in chitin cross-linking. From both sets of results it may be concluded that the chitin interspersed in the cell wall and possibly part of that in the ring are attached to beta (1right-arrow6)-glucan, whereas another portion of the ring chitin is directly linked to beta (1right-arrow3)-glucan (6). It is possible that the beta (1right-arrow6)-glucan molecules attached to a single chitin chain come from a different region than those bearing several chains.

It is difficult to estimate what proportion of the cell wall is involved in the linkages studied in this work. Fraction V0 contained about 4% of the total cell wall carbohydrate. To this, one should add the chitin and beta (1right-arrow3)-glucan chains that had been eliminated by treatment with the corresponding hydrolytic enzymes.

From this and previous studies (6, 7), the concept emerges of a "flexible building block" of the yeast cell wall (Fig. 15). The complete structure would comprise mannoprotein, beta (1right-arrow6)-glucan, beta (1right-arrow3)-glucan, and chitin. Part of the blocks (ConA-) would miss mannoprotein, especially in inner layers of the wall (4), or chitin, mostly in the outer layer (7).


Fig. 15. Proposed flexible building block of the yeast cell wall. Some of the components may be missing at different locations; e.g. absence of mannoprotein would result in a ConA--like structure, absence of mannoprotein and beta (1right-arrow6)-glucan would leave a chitin-beta (1right-arrow3)-glucan polymer, and so on.
[View Larger Version of this Image (23K GIF file)]

Biosynthesis of Cross-linkages

How are the components of the yeast wall building blocks joined together in the cell? From the available evidence it appears that chitin (54) and beta (1right-arrow3)-glucan (56) are synthesized at the plasma membrane with simultaneous secretion into the periplasmic region. On the other hand, mannoprotein is synthesized at the ER and modified during its transport through the secretory pathway (57). Finally, at least part of beta (1right-arrow6)-glucan synthesis may occur in the ER or Golgi (53). It is clear that all of the components will only meet in the periplasmic region, which is then the location where the linkage reactions must take place. Because it is unlikely that high energy compounds are present in that location and on account of the nature of the connecting bonds, transglycosidation is the most likely reaction leading to formation of the cross-linkages. Identification of the enzymatic activities involved in these reactions awaits further work.

Although the mechanism of biosynthesis of the cross-linkages remains obscure, we have some information about its timing in the cell cycle. The chitin interspersed in the cell wall that is synthesized by chitin synthase 3 is laid down after septum formation, in the final phase of bud growth (58). As discussed before (6), that must be the phase of the cell cycle in which cross-links involving chitin are formed. Walls of mutants lacking chitin synthase 3 have been found to be less resistant to osmotic shock than those of wild type (59). This invites speculation that the cross-links are not created during most of the period of active growth lest they interfere with the plasticity of the developing wall, but are instead added at the end to increase rigidity of the final product.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§§   To whom correspondence should be addressed: Bldg. 10, Rm. 9N-115, National Institutes of Health, 10 Center Dr., Bethesda, MD 20892.
1   The abbreviations used are: HPLC, high performance liquid chromatography; HPAEC, high performance anion exchange chromatography; ConA, concanavalin A; ConA+, cell wall fraction that binds to ConA-Sepharose; ConA-, cell wall fraction that does not bind to ConA-Sepharose; fraction M, high molecular weight material remaining after beta (1right-arrow6)-glucanase digestion of ConA+; endo-H, endo-beta -N-acetylglucosaminidase H; GPI, glycosylphosphatidylinositol; ES-MS, electrospray mass spectrometry; CID, collision-induced decomposition; ER, endoplasmic reticulum; PVDF, polyvinylidene difluoride; Manp, mannopyranosyl.
2   In the course of this study it was found that the commercial preparation of Pronase used contained endo-beta (1right-arrow6)-glucanase activity. Therefore, Pronase could not be used in most experiments. Here, however, the contaminating activity was beneficial, because it allowed us to isolate mannan free of beta (1right-arrow6)-glucan by collecting the void volume fraction in gel columns.

ACKNOWLEDGEMENTS

We thank H. Bussey for yeast strains, R. C. Montijn for the beta (1right-arrow6)-glucan antibody, A. Llobell for a sample of beta (1right-arrow6)-glucanase, and A. Murphy for amino acid analysis. We are also grateful to T. Drgon, R. Ford, and V. Pozsgay for a critical reading of the manuscript and to W. Berlin for useful discussions. Special thanks are due to T. Drgon for help with the figures.


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