©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Architecture of the Yeast Cell Wall
THE LINKAGE BETWEEN CHITIN AND beta(13)-GLUCAN (*)

(Received for publication, October 7, 1994; and in revised form, November 15, 1994)

Roman Kollár (1) Eva Petráková (2) Gilbert Ashwell (1) Phillips W. Robbins (§) Enrico Cabib (1)(¶)

From the  (1)Laboratory of Biochemistry and Metabolism and the (2)Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To isolate the putative linkage region between chitin and beta(13)-glucan, Saccharomyces cerevisiae cell walls were digested with beta(13)-endoglucanase and the reducing ends of the enzyme-resistant glucose chain stubs were labeled by reduction with borotritide. The radioactive material was further digested with exochitinase to remove the bulk of the chitin, and the liberated oligosaccharides were fractionated on a sizing column. A single peak (compound I) was found to consist of N-acetylglucosamine, glucose, and glucitol residues in the ratio 1:2:1. By digestion with beta-N-acetylglucosaminidase and by NMR spectroscopy, N-acetylglucosamine was identified as the nonreducing terminus, linked to laminaritriitol by a beta(14) bond. Five additional oligosaccharides were recovered, two being analogs of compound I, with 1 or 3 glucose units, respectively; the remaining three were shown to be the reduced analogs of laminaribiose, laminaritriose, and laminaritetraose. The presence of N-acetylglucosamine-containing oligosaccharides arises from the activity of chitinase in cleaving 2 sugar units sequentially in those chains containing an odd number of N-acetylglucosamine residues; correspondingly, oligosaccharides containing only glucose and sorbitol derive from even-numbered chitin chains, a result implying that chitinase can hydrolyze the linkage between N-acetylglucosamine and glucose. It is concluded that the terminal reducing residue of a chitin chain is attached to the nonreducing end of a beta(13)-glucan chain by a beta(14) linkage. Experiments with appropriate mutants showed that synthesis of the chitin combined with glucan is catalyzed by chitin synthetase 3. The timing and possible mechanism of formation of the chitin-glucan linkage is discussed.


INTRODUCTION

Cell walls determine the shape of fungal cells and are essential for their integrity(1) . They consist mainly of carbohydrates, some free, some linked to protein. The main components of the yeast cell wall are a beta(13)-D-glucan that also contains some beta(16)-linked branches and a mannoprotein, most of which is carbohydrate(2) . A beta(16)-D-glucan, also containing some beta(13)-linked branches(2) , is a relatively minor constituent; an even scarcer component is chitin. The latter is mostly concentrated at the septal region(2, 3) , but some of it is dispersed throughout the cell wall(4, 5) . The composition of a cell wall, however, is only a partial description of its architecture. To understand both the structure and morphogenesis of the wall, it is necessary to know how its constituents are organized in layers and cross-linked to each other to yield a fabric strong enough to resist turgor pressure. Evidence that different cell wall polysaccharides are covalently linked has been accumulating. We have argued that such a linkage must exist in the yeast cell wall between beta(13)-glucan and the mannoprotein, to explain why the latter is solubilized after treatment of the wall with beta-glucanase(6) . A fragment isolated after cell wall digestion by Van Rinsum et al.(7) , containing both mannose and glucose may represent a connecting bridge. There is also good evidence that in Schizophyllum commune(8) and in Candida albicans(9) chitin and beta-glucan are covalently bound. In the former, amino acids have been implied as participants in the bridge region(8) , whereas a direct glycosidic linkage was reported in Candida(9) ; in both cases the chemical evidence is far from compelling. In Saccharomyces cerevisiae, a covalent linkage between chitin and beta-glucan is strongly suggested by the experiments of Mol and Wessels(10) . These authors found that, after extraction of alkali-soluble glucan, treatment of the cell wall residue with chitinase rendered the remainder of the glucan alkali-soluble. We decided to look for the linkage region between chitin and glucan in S. cerevisiae by following an approach similar to that of Sietsma and Wessels (8) and of Surarit et al.(9) , i.e. by digesting cell walls with both beta(13)-glucanase and chitinase. Labeling of the enzyme-resistant core by reduction with sodium borotritide facilitated detection and purification of the mixed oligosaccharides wherein the sought after linkage was found.


EXPERIMENTAL PROCEDURES

Materials

beta-N-Acetylglucosaminidases from jack beans or from Diplococcus pneumoniae were from Oxford Glycosystems. beta-N-Acetylglucosaminidase from beef kidney and beta-glucosidase from sweet almonds were purchased from Boehringer Mannheim, Zymolyase 100 T from Seikagaku, and Glusulase from DuPont. Sodium [^3H]borohydride was obtained from American Radiolabeled Chemicals (100-500 mCi/mmol) or ICN (100 mCi/mmol) and [1-^14C]glucose (50-60 mCi/mmol) from American Radiolabeled Chemicals. Chitinase from Serratia marcescens was prepared as described(11) .

Yeast Strains and Yeast Growth

The strains used were D3C (MATalpha ura3) as wild type, ECY36-3C (MATachs1-23 chs2::LEU2 trp1-1 ura3-52 leu2-2) as Chs1- and Chs2-deficient, ECY36-3D (MATachs1-23 cal1/csd2 ura3-52 leu2-2 trp1-1) as Chs1- and Chs3-deficient, and ECY36-3D pHV9A (plasmid pHV9A carries the CAL1/CSD2 gene that restores Chs3 activity). Strains D3C and ECY36-3D were grown in YEPD (1% yeast extract, 2% peptone, 2% glucose) and strains ECY36-3C and ECY36-3D pHV9A in minimal medium (2% glucose, 0.7% Difco yeast nitrogen base without amino acids) plus nutritional requirements. In all cases growth was at 30 °C.

Preparation of Cell Walls and Digestion with Glucanase and Chitinase

For standard preparations, strain D3C was used, except for a large-scale experiment, in which ECY36-3C was employed because of its higher chitin content(12) . In a typical preparation, 16.3 g of cells, wet weight, harvested in exponential phase of growth, were suspended in about 45 ml of 50 mM Tris chloride, pH 7.5, and added to 75 g of glass beads (0.5-mm diameter, Braun-Melsungen, Germany) in the intermediate-size vessel of a Bead-Beater (Biospec Products, Bartlesville, Ohio). After cooling the suspension to 5 °C, the Bead-Beater was operated for two 3-min periods, with a 10-min cooling period in between. The extract was aspirated from the glass beads, and the latter were washed several times with small portions of Tris buffer. Cell walls were sedimented by centrifugation at 4,000 times g for 10 min and washed five times with Tris buffer, followed by suspension in the same buffer to a final volume of 495 ml. Zymolyase 100 T (4.3 ml of a 7.5 mg/ml solution in 50 mM sodium phosphate, pH 6.3) was added, the suspension was incubated at 37 °C with shaking and the absorbance at 660 nm was monitored. After about 1 h the absorbance had decreased to about 5% of the original value. The suspension was centrifuged for 10 min at 16,000 times g. The pellet was washed twice with Tris buffer and twice with 1% SDS. In the second washing with SDS the suspension was placed for 5 min in a boiling water bath. The pellet was then washed three more times with water and suspended in water to a concentration of 10 mg of chitin/ml (about 1 ml in the preparation described). The ratio of total N-acetylglucosamine to total glucose at this step was 1:0.7. 600 µl of the suspension was treated with 600 µl of NaB^3H(4) (6 mCi) in dimethyl formamide at room temperature for 5 h. The reaction was terminated by the addition of 1 M acetic acid (300 µl) and the insoluble material was washed by repeated centrifugations followed by suspension in 50 mM potassium phosphate at pH 6.3. The washed reduced chitin was digested overnight at 30 °C with 600 µl (184 milliunits) of S. marcescens chitinase. Any insoluble residue was removed by centrifugation. The supernatant fluid was used directly for Bio-Gel P-2 chromatography (see ``Results'').

The compounds isolated from the eluate of the Bio-Gel column were again reduced with an excess of unlabeled sodium borohydride and repurified by Bio-Gel chromatography prior to further analysis.

High Performance Anionic Exchange Chromatography

Samples were analyzed on a Dionex HPAEC (^1)instrument equipped with a pulsed amperometric detector model PAD2, and pellicular anion-exchange columns (PA-1 or MA-1, 4 times 250 mm). The Dionex eluent degas module was used to sparge and pressurize the eluents with the helium set at 100 p.s.i. The flow rate was maintained at 0.8 ml/min. The applied pulse potential was 0.05 V and detector sensitivity was set at 300 nA. The system was used at ambient temperature. Samples were applied via a Dionex microinjection valve with a 50-µl loop. Areas under the curve were recorded and integrated with a Spectra-Physics integrator. Eluent contained 100-500 mM NaOH.

Paper Chromatography

The solvent used for paper chromatography was butanol/pyridine/water, 6:4:3, and the paper was Whatman No. 1 or No. 3MM.

Carbohydrate Determinations

Total carbohydrate was measured with the anthrone reagent (13) and glucose by glucose oxidase (Glucose Trinder Kit from Sigma). Free GlcNAc was quantified by the method of Reissig et al.(14) and combined GlcNAc by the same method after digestion of 60 µl of sample (0.3-0.5 µmol of combined GlcNAc) with 60 µl of Glusulase, 60 µl of chitinase, and 5 µl of 1 M sodium phosphate, pH 6.3, for 45 min at 30 °C.

Mass Spectrometry

Chemical ionization mass spectra were obtained with a Finigan 1015 D spectrometer, with the use of ammonia as the reactive gas.

NMR Spectrometry

^1H and C NMR spectra were measured at ambient temperature with a Varian FX 300 or a Varian Gemini spectrometer, operating at 300 MHz for protons and 75 MHz for C. Chemical shifts found in the spectra recorded for solutions in CDCl(3) and D(2)O are reported, respectively, with Me(4)Si and methanol ( MeOH versus Me(4)Si 49.0) as internal standards. Proton-signal assignments were done by COSY or homonuclear decoupling experiments. The nonequivalent geminal proton resonating at lower field is denoted Ha and the one at higher field Hb. Carbon signal assignments were based on heteronuclear shift-correlated two-dimensional experiments (HETCOR).

Synthesis of beta-D-GlcNAcp (16)-D-Glc; General

Optical rotations were measured at 25 °C with a Perkin Elmer automatic polarimeter, model 241 MC. All reactions were monitored by thin-layer chromatography on pre-coated slides of Silica Gel G F254 (Analtech). Detection was effected by charring with 5% sulfuric acid in ethanol or, when applicable, with UV light. Preparative chromatography was performed by gradient elution from columns of Silica Gel 60 (Merck, no. 9385). Reactions requiring anhydrous conditions were performed under dry nitrogen using common laboratory glassware and reagents and solvents were handled with gas-tight syringes. Solutions in organic solvents were dried with anhydrous sodium sulfate and concentrated under a vacuum at 40 °C. 1,2,3,4-Tetra-O-acetyl-beta-D-glucopyranose (Sigma) and bromo-2-deoxy-2-N-phtalimido-3,4,6-tri-O-acetyl-alpha,beta-D-glucopyranose (Toronto Research Chemicals Inc.) were used as supplied.

1,2,3,4-Tetra-O-acetyl-6-O-(3,4,6-tri-O-acetyl-2-deoxy-2-N-phtalimido-beta-D-glucopyranosyl)-beta-D-glucopyranose (3)

1,2,3,4-Tetra-O-acetylbeta-D-glucopyranose (1) (0.7 g, 2 mmol) was dissolved in dry nitromethane (30 ml) and 4-Å molecular sieve (1 g) was added. After cooling to -30 °C, the reaction mixture was stirred for 30 min and sym-collidine (0.28 ml, 2 mmol) and silver triflate (0.54 g, 2.1 mmol) were added. Finally, bromo-2-deoxy-2-N-phtalimido-3,4,6-tri-O-acetyl-alpha,beta-D-glucopyranose (2) (1 g, 2 mmol) dissolved in nitromethane (5 ml) was added dropwise. After 1 h, additional portions of sym-collidine (0.056 ml, 0.4 mmol), silver triflate (0.1 g, 0.4 mmol), and compound 2 (0.2 g, 0.4 mmol) were added. The reaction mixture was stirred for 2 h at -30 °C. At this point, compound 1 was no longer detected (TLC, toluene/acetone, 6:1). After filtration through Celite, the filtrate was washed with saturated sodium bicarbonate and water, dried with sodium sulfate, concentrated, and purified on a silica gel column (toluene/acetone, 10:1) to yield 3. [alpha](D) + 18.47° (c 7.82, chloroform); ^1H NMR (CDCl(3)) : 7.89-7.30 (m, 6 H, Ph), 5.75 (dd, 1 H, J 9.1 Hz, J 10.6 Hz, H-3`), 5.59 (d, 1 H, J 7.9 Hz, H-1), 5.44 (d, 1 H, J 8.2 Hz, H-1`), 5.16 (dd, 1 H J 9.4 Hz, H-4`), 5.13 (dd, 1 H, J 9.9 Hz, H-3), 5.01 (dd, 1 H, H-2), 4.90 (dd, 1 H, J 9.8 Hz, H-4), 4.34 (m, 2 H, H-2`, H-6`(a)), 4.17 (dd, 1 H, J 2.4 Hz, H-6), 3.88 (m, 1 H, H-5`), 3.72 (m, 1 H, H-5), 3.62 (m, 2 H, H-6(a), H-6(b)), 2.14 (s, 3 H, COCH(3)), 2.03 (s, 3 H, COCH(3)), 1.99 (s, 3 H, COCH(3)), 1.93 (s, 3 H, COCH(3)), 1.89 (s, 3 H, COCH(3)), 1.85 (s, 3 H, COCH(3)); C NMR (CDCl(3)) : 170.86, 170.27, 170.16, 169.60, 169.32, 169.21, 168.86 (COCH(3)), 97.94 (C-1`), 91.51 (C-1), 73.63 (C-5), 72.64 (C-2), 71.87 (C-5`), 70.29 (C-4), 68.82 (C-4`), 68.27 (C-3), 67.33 (C-6), 61.88 (C-6`), 54.22 (C-2`), 20.63, 20.58, 20.48, 20.37, 20.26, 20.20 (COCH(3)).

6-O-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)-alpha,beta-D-glucopyranose (4)

Disaccharide 3 (0.055 g, 0.072 mmol) was dissolved in anhydrous methanol (10 ml) and sodium methoxide in methanol (1 M, 0.1 ml) was added. The reaction mixture was stirred at room temperature for 4 h. At this time compound 3 was no longer detected (TLC, propanol/ethyl acetate/water, 4:2:1). After neutralization with Amberlite 120 (H) and filtration, the filtrate was concentrated and dried under a vacuum. Crude product was dissolved in methanol (5 ml) and anhydrous hydrazine (14 µl, 0.45 mmol) was added. The reaction mixture was kept under reflux (65 °C) for 2 h, at which point starting material was consumed (TLC, ethyl acetate/ethanol/water, 8:4:2). After cooling to room temperature, acetic anhydride (0.5 g, 0.46 ml, 4.9 mmol) was added and the mixture was stirred for 20 min. When starting material was no longer detected (TLC, propanol/ethyl acetate/water, 2:1:1) the reaction mixture was concentrated and purified on a Bio-Gel P-2 extra-fine, 2 times 90-cm column to yield 4. [alpha](D) +3.82° (c 17.00, water); ^1H NMR (D(2)O) : alpha: 5.21 (d, 1 H, J 3,7 Hz, H-1), 4.55 (d, 1 H, J 8.3 Hz, H-1`), 4.11 (dd, 1 H, J 2 Hz, J 11.4 Hz, H-6(a)), 3.94 (m, 2 H, H-6`(a), H-5), 3.81 (dd, 1 H, J 4.3 Hz, H-6(b)), 3.73 (m, H-6`(b)), 3.71 (m, H-2`, H-3), 3.68-3.46 (m, 4 H, H-3`, H-2, H-5`, H-4), 3.41 (dd, 1 H, J 6.1 Hz, J 9.6 Hz, H- 4`), 2.07 (s, 3 H, COCH3); beta: 4.63 (d, 1 H, J 7.9 Hz, H-1), 4.57 (d, 1 H, J 8.4 Hz, H-1`), 4.17 (dd, 1 H, J 1.9 Hz, J 11.5 Hz, H-6(a)), 3.75 (m, 2 H, H-2`, H-6(b)), 3.73 (m, H-6`(b)), 3.68-3.46 (m, 4 H, H-3`, H- 5, H-5`, H-3), 3.41 (dd, 1 H, J 6.1 Hz, J 9.6 Hz, H- 4`), 3.38 (dd, 1 H, J 5.6 Hz, J 9.2 Hz, H-4), 3.24 (dd, 1 H, J 9.2 Hz, H-2), 2.07 (s, 3 H, COCH3); C NMR (D(2)O) : alpha: 175.11 (COCH(3)), 101.98 (C-1`), 92.45 (C-1), 76.11 (C-5`), 73.97 (C-3`), 73.02 (C-3), 71.69 (C-2), 70.57 (C-5), 70.18 (C-4), 69.83 (C-4`), 68.71 (C-6), 55.73 (C-2`), 22.40 (COCH(3)); beta: 175.11 (COCH(3)), 102.06 (C-1`), 96.31 (C-1), 76.11 (C-5`), 76.05 (C-5), 75.06 (C-3), 74.35 (C-2), 73.97 (C-3`), 70.18 (C-4), 69.83 (C-4`), 68.95 (C-6), 55.73 (C-2`), 22.40 (COCH(3)).


RESULTS

To isolate the linkage region between chitin and beta-glucan, yeast cell walls were digested with a beta(13)-endoglucanase (zymolyase), with the expectation of forming short glucose oligosaccharide stubs attached to the chitin. After removal of all the solubilized material, the insoluble fraction was reduced with sodium borotritide, to label the reducing ends of the stubs. This treatment also reduces and labels GlcNAc residues at the reducing end of chitin chains not bound to glucan. The labeled material was then digested with S. marcescens exo-chitinase, an enzyme that sequentially cleaves diacetylchitobiose residues from chitin, starting from the nonreducing end(11) . The material solubilized by this treatment was applied to a Bio-Gel P-2 column (Fig. 1). Two of the major radioactive peaks correspond to diacetylchitobiitol (Fig. 1, Peak 4) and triacetylchitotriitol (Fig. 1, Peak 2), which originate from free chitin chains containing an even and an odd number of GlcNAc residues, respectively(15) . In addition to the above, a large peak in the void volume and some additional minor peaks were detected (Fig. 1). The latter appeared to be candidates for the linkage region and were named Peaks A, B, and C. It may be mentioned that essentially the same technique was previously used for determination of chitin chain length(15) . It is in those experiments that some of the minor peaks were first detected.


Figure 1: Gel chromatography of cell wall fraction solubilized by glucanase and chitinase digestion. Digestion of cell walls was carried out as described under ``Experimental Procedures.'' The chitinase-solubilized fraction from 6 mg of glucanase-resistant insoluble residue was applied to an extra-fine Bio-Gel P-2 column (2 times 90 cm) and eluted with 0.1 M acetic acid. Fractions of 1.8 ml were collected and a 20-µl portion of each sample was counted. 1 refers to the void volume position and 2-8 are the position of standards, as follows: 2, triacetylchitotriitol (or laminarihexaitol); 3, laminaripentaitol; 4, diacetylchitobiitol (or laminaritetraitol); 5, laminaritriitol; 6, GlcNAc-ol (or laminaribiitol); 7, glucitol; 8, Glc ([^14C]glucose was added as internal standard).



For an understanding of the chromatographic procedures involving Bio-Gel P-2 columns, it is important to realize that each GlcNAc residue, whether free or combined, counts as 2 hexose residues in determining relative elution positions(16) . Thus, diacetychitobiose elutes in the same volume as a glucose tetrasaccharide. This is not true of paper chromatography, where the mobility of each monosaccharide depends on the solvent used and, within the same series, is inversely proportional to molecular weight.

Early in this study, it was realized that Peak A contained both N-acetylglucosamine and glucose, whereas Peaks B and C contained only glucose. For that reason, the initial effort was devoted to elucidate the structure of the substance contained in Peak A (compound I).

Structure of Compound I

Upon acid hydrolysis, I gave rise to glucosamine, glucose, and glucitol, in the ratio 1.1:2:0.9 (Fig. 2). The molecular weight of the acetylated compound, as measured by mass spectrometry (Fig. 3a), was 1316, compared to a value of 1315 for an acetylated tetrasaccharide consisting of a residue each of GlcNAc and glucitol and 2 glucose residues (includes the weight of ammonium ion). This composition is also reflected in the elution volume of Peak A which corresponds to a reduced glucose pentasaccharide standard.


Figure 2: Analysis of I, II, and III (Peaks A, B, and C, respectively) by acid hydrolysis followed by HPAEC. Five nmol each of I, II, or III was evaporated to dryness under nitrogen and hydrolyzed with 25 µl of 2 M trifluoroacetic acid for 2 h at 100 °C. After new evaporation to dryness, the residue was dissolved in 250 µl of water; a 50-µl sample was analyzed by HPAEC in a PA-1 column, as described under ``Experimental Procedures.'' The standards and their retention time were glucitol (3.01 min), glucosamine (3.96 min), and Glc (4.64 min). The early-emerging peak in C is glucosaminitol, derived from some N-acetylglucosaminitol contaminating III. The N-acetylglucosaminitol was detected before hydrolysis of III (data not shown).




Figure 3: Determination of the molecular weight of I, II, and III by mass spectrometry. 100 nmol each of I, II, or III were evaporated to dryness under nitrogen. The residue was dissolved in 200 µl of pyridine and 200 µl of acetic anhydride were added. After overnight incubation at room temperature, a few drops of toluene and methanol were added and the solution was evaporated to dryness. The dissolution-evaporation was repeated several times, first with toluene, then with methanol. Finally, the samples were dissolved in 20 µl of dichloromethane and analyzed by mass spectrometry. Because ammonia was used as the reactive gas, the ammonium ion weight must be added to the calculated molecular weight. a, I; b, II; c, III.



To explore in which manner and position the GlcNAc was attached, I was treated with N-acetyl-beta-glucosaminidase and beta-glucosidase (Fig. 4). Whereas beta-glucosidase treatment had no effect on the elution of I (Fig. 4c), incubation with beta-N-acetylglucosaminidase moved it to the position of laminaritriitol (Fig. 4b). This result showed the GlcNAc in I to be at the nonreducing end and to be attached to a trisaccharide by a beta-linkage. This structure was consistent with the results of partial hydrolysis by either acid or enzymes (Fig. 5). After partial acid hydrolysis three additional peaks were detected, which migrated as Glc(beta1-3)Glc(beta1-3)Glc-ol (or Glc(beta1-6)Glc-ol), Glcbeta(1-3)Glc-ol, and Glc-ol (Fig. 5a). All these compounds would be expected if GlcNAc were attached to a reduced laminaritriose. It should be kept in mind that only those products that still retain the labeled sorbitol can be detected by the radiometric assay. Partial hydrolysis with beta-N-acetylglucosaminidase gave rise to a peak in the position of the original material and another one moving as Glc(beta1-3)Glc(beta1-3)Glc-ol or Glc(beta1-6)Glc-ol (Fig. 5b). This result already indicates that the glucose trisaccharide cannot be gentiotriitol since the latter (standard 6 in Fig. 5b) moves much more slowly than the product of the reaction. When both beta-N-acetylhexosaminidase and beta-glucosidase were allowed to act on I, the product comigrated with Glc(beta1-3)Glc-ol and was clearly different from the 1-2, 1-4, and 1-6 isomers (Fig. 5b). In separate experiments it was found that laminaribiitol is resistant to beta-glucosidase. Taken together, the above results are consistent with a structure in which GlcNAc is beta-linked to reduced laminaritriose. To confirm the linkage between the 2 glucose residues as 1-3, the reduced trisaccharide obtained by N-acetylglucosaminidase digestion of I was oxidized with periodate and hydrolyzed with acid. Any configuration of the glucose to glucose linkage other than 1-3 would have resulted in the destruction of both Glc residues. However, glucose was recovered after oxidation, indicating that the terminal and penultimate glucose residues are bound in a 1-3 linkage (Fig. 6).


Figure 4: Treatment of I with beta-N-acetylglucosaminidase or beta-glucosidase. Aliquots (130 pmol, 130,000 cpm) of I were evaporated to dryness and redissolved: a, in 50 µl of 100 mM citrate/phosphate buffer, pH 5.0; b, in the same buffer plus 5 µl (135 milliunits) of beta-N-acetylglucosaminidase from jack beans; and c in 60 µl of 0.1 M acetate buffer, pH 4.5, containing 0.1 mg of sweet almonds beta-glucosidase. All mixtures were incubated 16 h at 37 °C, then diluted with 300 µl of water and subjected to Bio-Gel P-2 chromatography as described in the legend of Fig. 1, except that the column size was 1 times 90 cm and the fraction size 450 µl. Standards used were: 1, triacetylchitotriitol; 2, diacetylchitobiitol; 3, laminaritriitol; 4, laminaribiitol; 5, glucitol; 6, glucose ([^14C]glucose was the internal standard). The positions of Peaks A, B, and C are also indicated.




Figure 5: Products of partial acid or enzymatic digestion of I. a, a sample of I (25 pmol, 25,000 cpm) was evaporated to dryness under nitrogen and hydrolyzed with 50 µl of 0.05 M trifluoroacetic acid for 2 h at 100 °C. The hydrolysate was subjected to paper chromatography. Segments (1-cm) of the paper were counted. Standards: 1, glucose ([^14C]glucose internal standard); 2, glucitol; 3, laminaribiitol; 4, laminaritriitol or gentiobiitol; 5, laminaritetraitol; 6, gentiotriitol; 7, laminaripentaitol. b, aliquots of I (10 pmol, 10,000 cpm each) were evaporated to dryness and dissolved into 30 µl of 100 mM citrate/phosphate buffer, pH 6.0. Both aliquots were incubated 16 h at 37 °C, one with 7.5 milliunits of beta-N-acetylglucosaminidase from D. pneumoniae and the other with the same enzyme plus 0.02 mg of beta-glucosidase from sweet almonds. Both samples were subjected to paper chromatography as in a. bullet, incubated with N-acetylglucosaminidase; circle, incubated with both enzymes. Standards: 1, glucose; 2, laminaribiitol; 3, sophoritol; 4 cellobiitol; 5, laminaritriitol or gentiobiitol; 6, gentiotriitol. To facilitate understanding of the explanations in the text, the tentative structure of I and of the hydrolysis products are shown, where an open square stands for GlcNAc, an open circle for Glc, and a filled circle for glucitol.




Figure 6: Periodate oxidation of the trisaccharide resulting from N-acetylglucosaminidase digestion of I. A portion of I (60 nmol) was digested with jack bean beta-N-acetylglucosaminidase and subjected to Bio-Gel P-2 chromatography essentially as described in the legend of Fig. 4. The recovered trisaccharide, in a total volume of 50 µl, was oxidized with 700 nmol of sodium metaperiodate for 70 h at 4 °C in the dark. Ethylene glycol (1%, 23 µl) was added. After 2 h at room temperature, 40 µl of 0.1 M NaOH and 50 µl of sodium borohydride in 0.01 M NaOH were added, and incubation was continued for 3 h. The sample was evaporated to dryness under nitrogen, dissolved in 100 µl of 2 M trifluoroacetic acid, and heated at 100 °C for 2 h. After evaporation to dryness, the residue was dissolved in 350 µl of water and a 50-µl portion was subjected to HPAEC on a PA-1 column with 0.2 M NaOH as solvent. Laminaribiitol and laminaritriitol (50 nmol of each) were subjected to the same treatment and chromatographed. a, oxidized laminaribiitol; b, oxidized laminaritriitol; c, trisaccharide from I, not treated with periodate but hydrolyzed with trifluoroacetic acid; d, oxidized trisaccharide from I. The large peak at 2.34-2.36 min in a, b, and d is ethylene glycol. Glucose (retention time 5.15-5.25 min in b and d and 4.93 min in c) was present in the samples resulting from oxidation of laminaritriitol (b) or of the trisaccharide from I (d), but not in the one from laminaribiitol (a). The non-oxidized control of the trisaccharide from I (c) shows both glucose and sorbitol (retention time 3.49 min). The small peaks emerging after ethylene glycol in a and b have not been identified and may be due to impurities in the commercial samples of laminaribiose and laminaritriose used.



On the basis of the tentative structure of I it was possible to interpret the proton NMR spectrum of this compound in a way that reinforces the conclusion that GlcNAc is linked to the remainder of the molecule by a beta-linkage. In that spectrum, we expected to find signals for three protons in the region of the anomeric protons (4.00-6.00 ppm). Actually, only two doublets were found, with chemical shifts of 4.58 and 4.48 ppm (data not shown). From ^1H NMR studies of reduced laminaritriose, it was concluded that the anomeric proton of the internal residues (H`-1 from ring C`, see Fig. 7, top) resonates at approx4.65 ppm. From this it may be inferred that the 4.58 ppm doublet corresponds to the anomeric protons H`-1 and H"-1 of I. Because the doublet is partially overlapped by the HOD signal, the signal cannot be quantified by integration to confirm that is originated in two protons. The corresponding coupling constant J = J is 7.9 Hz, as expected for a beta-linkage.


Figure 7: NMR spectra of I, laminaritriitol, and laminaribiitol. Approximately 1 µmol of I was evaporated to dryness several times with D(2)O, and finally dissolved in 600 µl of D(2)O. The spectrum was measured continuously for 5 days, as described under ``Experimental Procedures.'' Interpretation of the spectra was based on identification of the signals in two-dimensional COSY and HETCOR spectra of standard laminaribiitol, laminaritriitol, and GlcNAc(beta1-6)Glc. Attribution of peaks to different carbons are shown for the spectrum of Compound I. For each bracketed group of peaks, the carbon listed on top refers to the first peak from left under the bracket. The other carbons follow from top to bottom and from left to right, respectively. The position at which the C4" peak was expected is shown, together with that where it was actually found (arrow). For explanations, see text.



Given this attribution of the 4.58 ppm signal, the doublet at 4.48 ppm must represent the anomeric proton (H‴-1) of the GlcNAc unit. This chemical shift is in good agreement with those published (17) for branched penta- and hexasaccharides bearing GlcNAc at the nonreducing end (4.45-4.58 ppm). The coupling constant J of the 4.48 ppm doublet is 8.3 Hz, a value typical of beta-linked units.

The position of glucose that is beta-substituted with the anomeric carbon of the terminal GlcNAc remained to be determined. We attempted the synthesis of analogous compounds for comparison, but were successful only with GlcNAc(beta1-6)Glc. This, however, allowed us to eliminate the 1-6 linkage as a possibility, because the synthetic compound was decomposed by beef kidney beta-N-acetylglucosaminidase, whereas I was resistant (data not shown). The possibility that GlcNAc was attached to Glc by a 1-2, 1-3, or 1-4 linkage still remained. Since the amount of material available was insufficient for methylation analysis, we sought the answer to this question by the use of NMR spectroscopy.

Model oligosaccharides laminaribiitol and laminaritriitol and the above-mentioned synthetic compound GlcNAc(beta1-6)Glc were studied by two-dimensional NMR spectroscopy (COSY, HETCOR). In this fashion, the groups of signals belonging to the different carbons of D-sorbitol (C), D-glucopyranosyl units (C` and C"), and 2-acetamido-2-deoxy-D-glucopyranosyl (C‴) could be identified (Fig. 7). Next, it was necessary to analyze the chemical shifts of the D-glucopyranosyl unit marked C in the structure of I (Fig. 7), which participates in the glycosidic linkage. The largest deviation of chemical shifts of the C" unit of I compared to the corresponding unit of reduced laminaritriose is expected at the carbon involved in the glycosidic bond. C"-2 and C"-6 can be eliminated as participants in the bond, because their chemical shifts, 73.55 and 73.35 ppm for C"-2 and 60.65 and 60.86 ppm for C"-6, are the same for I and for reduced laminaritriose. If the glycosidic linkage were at position C"-3, one of the signals in the region 75.41-75.97 ppm would move to lower field in the spectrum of I, because this is the area in which carbons C`-5, C"-5, and C"-3 are located. This shift did not occur (Fig. 7). Also, in all three cases considered so far, in the spectrum of I, signals for 5 carbons should appear in the region 68.35-70.83 ppm, representing C-5 and all four C-4 carbons (C-4, C`-4, C"-4, and C‴-4). However, only four signals were found in this area, and they were assigned to carbons C-4, C-5, C‴-4, and C`-4. This means that carbon C"-4 signal was moved to lower field, where it can be found at 78.68 ppm (Fig. 7), due to the large positive alpha-effect of the glycosidic linkage at that position(18) . We conclude that the glycosidic linkage between GlcNAc and Glc in I is beta(14) and that the complete structure of the substance is GlcNAc(beta1-4)Glc(beta1-3)Glc(beta1-3)Glc-ol.

Structure of Compounds in Peaks B and C

Complete acid hydrolysis of II (Peak B) gave rise to glucose and sorbitol in a 2.0:1.0 ratio (Fig. 2). The molecular weight, as measured by mass spectrometry (Fig. 3b) was in agreement with this result (calculated, 1028; found, 1028). Digestion of II with beta-glucosidase followed by paper chromatography showed that the labeled material had moved to the position of laminaribiitol (results not shown). The NMR spectrum of II is identical to that of reduced laminaritriose (Table 1). We conclude that II is laminaritriitol.



Acid hydrolysis of III (Peak C) liberated glucose and sorbitol in a 1.1:1.0 ratio (Fig. 2). The molecular weight of the compound (Fig. 3c) was as expected from the analysis (calculated, 739.4; found, 740). The material behaved on paper chromatography or HPAEC (results not shown) like laminaribiitol and could be distinguished from the 1-2, 1-4, and 1-6 isomers. Although the anomeric configuration of the linkage was not directly determined, on the basis of these data and by analogy with I and II we attribute to III the structure of laminaribiitol.

Other Mixed Oligosaccharides Containing N-Acetylglucosamine and Glucose

The isolation of oligosaccharides containing only glucose after treatment of cell walls with beta(13)-glucanase and chitinase was unexpected and was first attributed to some contaminating endoglucanase activity in the chitinase preparation. Such an activity, however, was not detected. An alternative explanation (Fig. 8) is that chitinase is able to cut between a GlcNAc and a Glc, at least so long as the linkage between the two sugars is beta(14). In this fashion, I and II would be derived from chitin chains with an odd or even number of GlcNAc, respectively, both attached to a reduced laminaritriose. Similarly, III would result from hydrolysis of an even-numbered chain linked to laminaribiitol. Where is, then, the corresponding oligosaccharide from an odd-numbered chain (Fig. 8, IV)? According to its composition it should behave in the P-2 column either as a reduced tetrasaccharide of glucose or as reduced diacetylchitobiose. Thus, it may be hidden under the large peak of diacetylchitobiitol (Fig. 1). Accordingly, material from that peak was subjected to paper chromatography and found to contain, in addition to the reduced disaccharide, some slowly moving labeled material (results not shown). The latter was eluted and re-chromatographed on P-2, where it eluted at the expected position (Fig. 9a). Treatment of the material with beta-N-acetylglucosaminidase caused displacement of a large portion of the label to the position where either laminaribiitol or N-acetylglucosaminitol would elute (Fig. 9b), whereas incubation with beta-glucosidase had the same effect on a minor portion of the radioactive material (Fig. 9c). Finally, with a mixture of both enzymes, all of the radioactivity moved to the new position (Fig. 9d). The results are consistent with the hypothesis that the material eluted from paper contained a mixture of GlcNAc-beta-Glc-beta-Glc-ol (IV) and Glc-beta-Glc-beta-Glc-beta-Glc-ol (V in Fig. 8), originated as depicted in the figure. The isolation of V suggested that the corresponding compound VI (Fig. 8) should also have been formed. According to its composition, this compound would be eluted in the P-2 column together with reduced triacetylchitotriose. Again, material from the reduced trisaccharide peak was analyzed by paper chromatography and found to contain a small amount of slower moving labeled material (Fig. 10a). This substance was resistant to beta-glucosidase, but was digested by beta-N-acetylglucosaminidase, with concomitant displacement to the laminaritetraitol position in the P-2 column (Fig. 10, b and c). Therefore, the substance has the expected properties of VI (GlcNAc-beta-Glc-beta-Glc-beta-Glc-beta-Glc-ol) and completes the series of oligosaccharides.


Figure 8: Scheme for the generation of different oligosaccharides by chitinase digestion. Vertical lines indicate bonds hydrolyzed by chitinase. The final product, indicated with a roman numeral, consists of the units found on the right side of the last vertical line. Symbols for the sugars are the same as in the legend of Fig. 5.




Figure 9: Treatment of material separated from diacetylchitobiitol with beta-N-acetylglucosaminidase and/or beta-glucosidase. The original material was from the slow moving band in the paper chromatogram of the diäcetylchitobiitol peak (see text), which was eluted with water and concentrated. All panels show chromatographic profiles from Bio-Gel P-2 columns (1 times 90 cm), eluted as in Fig. 1. Aliquots of the original material were subjected to the following treatments. a, no treatment. b, incubation with beta-N-acetylglucosaminidase as in Fig. 4b. c, incubation with beta-glucosidase, as in Fig. 4c. d, incubated with both beta-N-acetylglucosaminidase and beta-glucosidase. Standards: 1, triacetylchitotriitol; 2, diacetychitobiitol; 3, laminaritriitol; 4, laminaribiitol; 5, glucitol; 6, glucose.




Figure 10: Effect of beta-N-acetylglucosaminidase and beta-glucosidase on a pentasaccharide eluting together with triacetylchitotriitol in Bio-Gel P-2 columns. a, separation of the pentasaccharide from triacetylchitotriitol on paper chromatography. The slow moving radioactive material was eluted with water from paper and concentrated. b, treatment of the pentasaccharide with beta-N-acetylglucosaminidase followed by Bio-Gel P-2 chromatography. Procedure was as described in the legend to Fig. 4b. c, incubation of the pentasaccharide with beta-glucosidase followed by Bio-Gel P-2 chromatography. Procedure as in Fig. 4c. Standards: 1, triacetychitobiitol or laminarihexaitol; 2, laminaripentaitol; 3, diacetychitobiitol or laminaritetraitol; 4, laminaritriitol; 5, III or laminaribiitol; 6, glucose. The nature of the small peak between Standards 4 and 5 in c is unknown.



The Glucose Oligosaccharides Do Not Pre-exist in the Intact Cell Wall

The results described above do not eliminate the possibility that the glucose oligosaccharides attached to chitin pre-existed as such in the intact cell wall before glucanase digestion, rather than being part of a larger chain. If this were the case, they should be labeled if the borotritide reduction were performed before, rather than after treatment with glucanase. However, when the experiment was carried out in that fashion, only triacetylchitotriitol and diacetylchitobiitol, resulting from free chitin chains, were labeled (results not shown). It may be concluded that the oligosaccharides were originally part of an extended glucan chain and can only be exposed to reduction by degradation of the chain with glucanase.

Synthesis of the Glucan-linked Chitin Requires Chitin Synthetase 3

Since three different chitin synthetases (Chs1, Chs2, and Chs3) participate in different aspects of chitin synthesis in yeast(12, 19) , it was of interest to establish which one was involved in the synthesis of the chitin attached to glucan. Mutants in each of the three synthetases are available. Strain ECY36-3C, with mutations in Chs1 and Chs2, still shows a full array of oligosaccharides (Fig. 11b), i.e. it is not impaired in the formation of chitin-glucan chains. However, strain ECY36-3D, that lacks Chs1 and Chs3, showed only the two reduced chitooligosaccharide peaks, derived from free chitin (Fig. 11c). Transformation of this strain with a plasmid carrying the CAL1/CSD2 gene, required for Chs3 activity, restored a full complement of oligosaccharides (Fig. 11d). Clearly, Chs3 is the synthetase involved in the formation of the glucan-linked chitin.


Figure 11: Presence of oligosaccharides in cell wall digests from different mutant strains. In all cases, cell walls were treated with endoglucanase, reduced, incubated with chitinase, and chromatographed on P-2 columns as described under ``Experimental Procedures'' and in the legend of Fig. 1. a, wild type strain D3C. b, strain ECY36-3C (chs1 chs2::LEU2). c, strain ECY36-3D (chs1 cal1/csd2). This strain is deficient in Chs3. d, strain ECY36-3D pHV9a. The plasmid contains the CAL1/CSD2 gene and restores Chs3 activity. Standards: 1, void volume; 2, triacetylchitotriitol; 3, diacetylchitobiitol; 4, diacetylchitobiose; 5, laminaritriitol; 6, laminaribiitol; 7, GlcNAc; 8, Glc.



Tritium-labeled Void Volume Peak

When the material solubilized by glucanase and chitinase digestion was fractionated on P-2 columns, a fairly large amount of radioactivity emerged at the void volume (Fig. 1). Rechromatography of this material on Sephacryl S-200 and Sephacryl S-300 columns indicated that it is heterogeneous and of high molecular weight, in the 200,000-300,000 range. NMR spectra were similar to those of pustulan, a beta(1-6)-linked glucan, although other components appeared to be present (data not shown). Acid hydrolysis released glucose and some mannose.

The void volume material was barely detectable in the Chs3 mutant (Fig. 11c) and was restored by the CAL1/CSD2 plasmid (Fig. 11d), which indicates that it was originally bound to chitin synthesized through the agency of Chs3. Surprisingly, the void volume labeled material was also somewhat reduced in the chs1-chs2 mutant (Fig. 11b). A detailed study of the void volume fraction is now in progress.


DISCUSSION

The oligosaccharides analyzed in this study were not solubilized until cell walls were digested with both beta-glucanase and chitinase, which points to their origin in the linkage region of glucan and chitin. This is confirmed by the presence of both N-acetylglucosamine and glucose in some of the compounds. The short glucose chains were originally part of the glucan, because they are protected from reduction when the polysaccharide is intact.

The determination of oligosaccharide structure was hampered by the availability of only very small amounts of these compounds. For instance, a typical Bio-Gel P-2 column yielded about 5 nmol of compound I and two such columns were needed to fractionate material from a 16-g batch of yeast such as in the example described under ``Experimental Procedures.'' This precluded the use of alternate conventional methods, such as methylation analysis, in the study of these oligosaccharides. On the other hand, advantage was taken of the fact that amounts of material in the subnanomolar range could be detected by chromatographic analysis, due to the high specific activity of the borotritide used in the reduction.

The structure of I, the oligosaccharide studied in most detail, was elucidated by a combination of chemical and enzymatic methods as well as NMR spectroscopy. It corresponds to an original oligosaccharide (before reduction) containing one N-acetylglucosaminyl group linked in beta(14) to laminaritriose. To our knowledge, this is the first time that such a linkage has been described in nature. I and the other five compounds studied can be arranged in two homologous series, one containing 2, 3, or 4 beta(1-3)-linked glucose units and the other with the same units plus an N-acetylglucosaminyl group at the nonreducing end. The different length of the glucose moieties is due to some variability in the position of the beta(13) linkage hydrolyzed by the zymolyase preparation. The main activity in zymolyase appears to be a beta(13)-endoglucanase that gives rise to laminaripentaose as the major product(20) , therefore one does not expect the remaining stubs attached to chitin to be much more than 5 glucose units long. As for the presence or absence of GlcNAc, it is readily explained by the existence of chitin chains with an odd or even number of GlcNAc residues and by the apparent ability of Serratia exochitinase to hydrolyze a GlcNAc(beta14)Glc residue.

An interesting question arises as to the percentage of the cell wall chitin that is involved in linkages with glucan. The sum of reduced diacetylchitobiose and triacetylchitobiose is equivalent to the free chitin chains, whereas the sum of oligosaccharides should be equivalent to the glucan-linked chains. A rough calculation based on these assumptions and also including the void volume peak suggests that between 40 and 50% of the chitin chains are engaged in linkage with glucan. The chitin to glucan ratio in the cell wall is about 1:10 (our data for strain D3C). How such a small amount of chitin can affect the solubilization in hot alkali of about 70% of the glucan (10) is not immediately clear. The explanation may reside in the different chain length of chitin and glucan. The reported values are 100 for the former (15) and 1500 for the latter(21, 22) . Thus, a relatively small number of chitin molecules may suffice to affect the properties of a 15-fold higher amount of glucan.

As for the number of chitin-beta(13)-glucan linkages compared to the total number of linkages in the cell wall, the result of our calculations is about one chitin-glucan bond per 8,000 hexose units.

Another intriguing question relates to how the linkages between chitin and glucan are synthesized. The results with chitin synthetase mutants clearly point to Chs3 as the enzyme responsible for the formation of the glucan-linked chitin. This is not surprising, because Chs3 is involved in the synthesis of 80-90% of the cell wall chitin including that present in a ring at the base of an emerging bud and that dispersed throughout the wall(12, 23) . This chitin is incorporated into the wall late in the cell cycle, after cytokinesis and during bud maturation(12) . Therefore, the glucan of the bud cell wall formed until that moment could not be bound to chitin and must represent the alkali-soluble glucan alluded to in the Introduction. This notion is supported by the finding that soluble glucan is the precursor of insoluble glucan (24) and that bud walls disappear after prolonged alkali extraction. (^2)Thus, chitin is attached to pre-existing glucan. The chitin-glucan bond may be formed in the periplasmic space by transglycosylation from a newly formed chitin chain, as previously suggested(1) . In that case, a portion of the chitin chain would be released in the reaction. An alternative mechanism is possible if chitin chains grow from the reducing end, as it was found for the O-antigen of Gram-negative bacteria(25) . In that case, the GlcNAc residue at the reducing end would remain activated during synthesis and the whole nascent chain could be transferred directly to glucan.

Whatever the mechanism used for synthesis of the chitin-beta-glucan linkage, this reaction may be a target for inhibitors functioning as antifungal agents. It is true that mutants lacking the linkage, such as cal1/csd2, cal2, and cal3, all defective in Chs3, are viable. However, their wall may be weaker and more prone to damage by other agents. Furthermore, defects in the linkage may be more serious for fungi that have a higher chitin content than S. cerevisiae, an organism that has only a minute amount of this polysaccharide.


FOOTNOTES

*
This work was supported in part under Cooperative Research and Development Agreement DK0016 between the National Institutes of Health and American Cyanamid Co. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Center for Cancer Research, Massachusetts Institute of Technology, E17-233, Cambridge, MA 02139.

To whom correspondence should be addressed: National Institutes of Health, Bldg. 10, Rm. 9N-115, Bethesda, MD 20892.

(^1)
The abbreviations used are: HPAEC, high performance anion exchange chromatography; silver triflate, silver trifluoromethanesulfonate; Chs1, Chs2, and Chs3, chitin synthetase 1, 2, and 3, respectively; HETCOR, heterocorrelated spectroscopy.

(^2)
E. Cabib, unpublished results.


ACKNOWLEDGEMENTS

We thank H. Yeh and W. White for help with NMR spectrometry, N. Whittaker for mass spectra, and W. Berlin for many useful suggestions. We are also grateful to W.-J. Choi, R. Ford, and J. Hanover for a critical reading of the manuscript.


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