(Received for publication, April 3, 1997)
From the Laboratory of Biochemistry and Metabolism,
the
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
Institute of
Molecular Cell Biology, University of Amsterdam, 1098 SM
Amsterdam, The Netherlands
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 (1
3)-glucan was solubilized and
isolated in the form of oligosaccharides, after digestion of yeast cell
walls with
(1
3)-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,
(1
3)-glucan,
(1
6)-glucan, chitin, and mannoprotein are linked together.
Mannoprotein, with a protein moiety about 100 kDa in apparent size, is
attached to
(1
6)-glucan through a remnant of a
glycosylphosphatidylinositol anchor containing five
-linked mannosyl
residues. The
(1
6)-glucan has some
(1
3)-linked branches,
and it is to these branches that the reducing terminus of chitin chains
appears to be attached in a
(1
4) or
(1
2) linkage. Finally,
the reducing end of
(1
6)-glucan is connected to the nonreducing
terminal glucose of
(1
3)-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
(1
6)-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.
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 (1
3)-D-glucan,
(1
6)-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
(1
3)-glucan (6) as
well as among glycoproteins,
(1
6)-glucan, and
(1
3)-glucan (7).
The strategy for the investigation of interconnections between chitin
and (1
3)-glucan consisted in the digestion of cell walls with
(1
3)-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
(1
4)-linkage between the reducing end of a chitin chain and the
nonreducing end of a
(1
3)-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,
(1
3)-glucan,
(1
6)-glucan, chitin, and
mannoprotein, are linked together. In this complex, chitin is directly
attached to a branch of
(1
6)-glucan.
-N-Acetylhexosaminidase,
-galactosidase, and
-mannosidase (all from jack bean) were from
Oxford Glycosystems.
-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-
(1
6)-glucanase was prepared from B. circulans WL-12 as described (9).
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 ChitinaseThe 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 ColumnTritiated
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 -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.
High performance anionic exchange chromatography (HPAEC) and paper chromatography were performed as described previously (6).
SDS-Polyacrylamide Gel ElectrophoresisCell 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 DeterminationsTotal 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 ResiduesDifferent 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 DeterminationProtein was measured either spectrophotometrically or by the procedure of Lowry et al. (20).
NMR Spectroscopy1H 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 2.217 ppm; for 13C-NMR
spectra
31.07 ppm).
The ConA+ fraction was labeled with
[14C]galactose as described above and digested with
(1
6)-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
-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.
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 (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 SpectrometryThe 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.
Yeast cell walls were digested with
(1
3)-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
(1
3)-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
(1
3)-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-
(1
3)-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
-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-
(1
3)-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.
The evidence outlined above points to a linkage of the material in
fraction V0 to both (1
3)-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
-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 -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.
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.
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 (1
6)-glucan chain with some
(1
3)
branches. At the reducing terminus, the
(1
6)-glucan would be
attached to
(1
3)-glucan, a stub of which, with the terminal
glucose converted into sorbitol, remained after
(1
3)-glucanase
digestion and borohydride reduction. Chitin would be linked to one or
more
(1
3) branches of the
(1
6)-glucan through its reducing
terminal GlcNAc, in the same fashion as it is attached directly to
(1
3)-glucan (6). Finally, the nonreducing end of the
(1
6)-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.
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 (1
6)-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 (
(1
6)-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
(1
3)-linked
glucopyranosyl units. There are two more signals in the spectrum of
ConA
, at
75.46 and 73.65 ppm, which may be assigned
to
(1
6)-linked glucopyranosyl units of the main chain that
are carrying branches composed by
(1
3)-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
79.5 ppm (
-linked) or 78.4 ppm (
-linked); a branch at C-2 would have yielded signals at
82.1 ppm (
-linked) or 79.5 ppm (
-linked); finally, the signal of an
-linked branch at C-3 would have been at
99.8 ppm (26). The
only remaining possibility is a side chain composed of
(1
3)-linked glucopyranosyl units linked to a C-3 position of the
main
(1
6) chain. The signals at
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
-D-glucopyranosyl residue of a variety of branched
(1
3)-glucans (27). The
difference between the two chemical shifts (
75.46 and 73.65 ppm)
and those belonging to unbranched glucose units (
75.78 and 73.93 ppm) is probably caused by greater shielding due to the presence of the
side chain.
|
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
4.73 ppm
(J1,2 7.6 Hz) was assigned to the H-1 proton of
the side chain ((1
3)-glucopyranosyl unit), the second one, at
4.52 ppm (J1,2 7.9 Hz), belongs to the H-1
proton of the main chain, (1
6)-linked. Their coupling constants are
in good agreement with those published for
-linked D-glucopyranoses (28, 29). There is a third doublet in the anomeric region, partially overlapped by the doublet at
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
4.52 ppm)
was compared with the integral (22.2) of the doublet at
4.22 ppm,
which represents H-6a of the main chain (30). Since they
are almost identical, one may conclude that the doublet at
4.52 ppm
represents all H-1 protons from the backbone, either from branched or
unbranched units.
All these data indicate the presence in ConA of a
(1
6)-linked glucopyranose polysaccharide with
(1
3)
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
(1
6)-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
(1
6)-glucanase treatment indicates that chitin is linked to
(1
6)-glucan.
Fractionation of the low molecular weight
material released by (1
6)-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
(1
6)-glucan chain (see Fig. 3)
and consist exclusively of glucose, whereas the tritiated compounds are
derived from the reducing terminus previously attached to
(1
3)-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 (1
6)-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
-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
-N-acetylglucosaminidase,
followed by
-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.
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.
[View Larger Version of this Image (25K GIF file)]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 -N-acetylhexosaminidase. All hexoses are
-linked glucose residues, as shown by the action of
-glucosidase
and by HPAEC (see above). The acetylhexosamine is attached to the next
glucose by a
(1
2) or a
(1
4) 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
(1
3)-linked. In the branched structure,
there is a
(1
6)-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
(1
3) to the main chain of
(1
6)-glucan. Cutting at different locations by the
(1
6)-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 (1
3)-linked glucose in the linear isomer being
directly attached to sorbitol. However, this would imply that
(1
3) linkages would be occasionally present in the main
(1
6) chain. There is no evidence for this; in fact, all four
internal oligosaccharides from
(1
6)-glucan that we analyzed after
reduction were completely hydrolyzed to glucose and sorbitol by
-glucosidase (see example below in Fig. 9b), an indication that there were no
(1
3) linkages between glucose and sorbitol (6). Furthermore,
13C NMR spectra indicate the presence of branches attached
to the main
(1
6) chain by a
(1
3) linkage, as discussed
above. Another piece of evidence for attachment of chitin to a
(1
3) branch comes from a study of oligosaccharide I (Fig.
7c), a tetrasaccharide. After reduction with borohydride and
incubation with
-galactosidase and
-N-acetylglucosaminidase,
-glucosidase was unable to
hydrolyze the residual reduced disaccharide (data not shown), a result
suggestive of a
(1
3) linkage between glucose and sorbitol (6). We
also considered the possibility that the GlcNAc was directly attached to a glucose residue of the main
(1
6) chain, but we found that all structures that would have resulted in that case were incompatible with the mass spectrometry results.
Scheme 3.
The Reducing End of the
In a previous study on the
chitin-(1
3)-glucan linkage, we showed that treatment of cell wall
with
(1
3)-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
(1
6)-glucanase
suggested that we had here a similar situation, but with the reducing
end of
(1
6)-glucan, rather than of chitin, attached directly to
(1
3)-glucan. To investigate this possibility, we made use of the
previous finding that sweet almond
-glucosidase can hydrolyze a
glucose residue attached to sorbitol in a
(1
6) but not in a
(1
3) linkage (6). When tritiated oligosaccharides from the
(1
6)-glucanase digest of ConA+ were treated with
-glucosidase, the final labeled product was laminaribiitol, as
determined by paper chromatography. On the other hand, when unlabeled
oligosaccharides from the
(1
6)-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
(1
6)-glucan chains are directly attached to
(1
3)-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
material eluting in the void volume of the Sephadex G-100 column after
(1
6)-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 (1
6)-linked mannosyl chain with
(1
2) and
(1
3) branches 1-4 mannoses long (35). Acetolysis
of mannan results in breakage of the main chain
(1
6) 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-
-1
2-Manp
for M2, Manp-
-1
2-Manp-
-1
2-Manp
for M3, and Manp-
-1
3-Manp-
-1
2-Manp-
-1
2-Manp
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.
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 MannoproteinSince 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.
Mannoprotein in ConA+ Is Attached to
The simultaneous binding of the
mannoprotein and of (1
6)-glucan to ConA-Sepharose indicated that
these two components are covalently linked, because ConA has no
affinity for
-linked glucose. The nature of the linkage remained to
be determined. Several cell wall proteins are attached to
(1
6)-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
(1
6)-linked glucoses left
over after
(1
6)-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
-glucosidase (Fig. 14b). They were, however, hydrolyzed
by
-mannosidase to compounds with the size of oligosaccharides
containing 3 (A) and 2 (B) hexoses, respectively (Fig. 14c). Both products were now degraded by
-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
-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
(1
6)-endoglucanase.
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 (1
6)-glucan (11). The amount of immunoreactive material
was much diminished after the HF treatment and completely eliminated by
incubation with
(1
6)-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
(1
6)-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
(1
6)-glucan (results not shown).
After degradation of ConA+ with
(1
6)-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+.
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
(1
3)-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
(1
6)-glucanase (Fig. 6,
c and d). (b) The 13C NMR
spectrum of ConA
shows the presence of
(1
6)-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
(1
6)-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
(1
6)-glucan chains.
The fraction
solubilized from the cell wall by successive treatments with
(1
3)-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
(1
3)-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
(1
6)-glucanase in a similar fashion. Furthermore, some of the
fractions obtained by chromatography of ConA+ contain
mannoprotein,
(1
6)-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
(1
6)-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.
The main components of the
ConA+ fraction are (1
6)-glucan and mannoprotein.
(1
6)-glucan was identified by the NMR spectrum and by its
susceptibility to hydrolysis by
(1
6)-glucanase. The proton NMR
spectrum provided evidence that
(1
3) branches are attached to the
main
(1
6) chain. The size of the glucan molecules is somewhat in
doubt. In ConA
only
(1
6)-glucan makes a significant
contribution to the molecular weight, because the mannoprotein is
missing and chitin and
(1
3)-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,
(1
6)-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
(1
6)-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 (1
3)-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).
As found in other cases
(7), the connection between mannoprotein and (1
6)-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
-agglutinin (49). In the present study,
ConA+ was digested with
(1
6)-glucanase, reduced with
borotritide, and hydrolyzed with HF. The resulting oligosaccharides
were isolated and shown to consist of five
-linked mannose residues,
attached to the nonreducing end of
(1
6)-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-(1
6)-glucan complex to
(1
3)-glucan and chitin were postulated on account of the need of both
(1
3)-glucanase and chitinase for solubilization of the complex and were confirmed by the presence of remnants of both polysaccharides in the complex. Thus,
(1
3)-linked
oligosaccharides were connected to the reducing end of
(1
6)-glucan, and GlcNAc that survived chitin degradation by
chitinase was found to be attached to the same glucan, probably onto
(1
3) side branches. The GlcNAc at the reducing end of the chitin
chain is connected to glucose either by a
(1
2) or a
(1
4)
linkage. We favor the latter possibility by analogy to the bond between
chitin and
(1
3)-glucan (6).
If our hypothesis that chitin is attached to a (1
3)-linked
glucose residue is correct, one may ask whether the previously found
linkage of chitin to glucan might not have been to a
(1
3) side
chain of
(1
6)-glucan rather than to the long
(1
3)-linked polysaccharide. This notion seems unlikely for the following reasons: first, the endo-
(1
3)-glucanase activity in Zymolyase requires a
minimum of 5 glucose residues to cut the chain (50), whereas
(1
3)
branches of
(1
6)-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
(1
6)-glucan (51), shows a normal complement of oligosaccharides indicative of a chitin-
(1
3)-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+.
From the foregoing analysis, it appears that
(1
6)-glucan is the central molecule or "glue" that keeps
together the other components of the cell wall, including
(1
3)-glucan, mannoprotein, and part of the chitin (Fig. 3). Thus,
it is not surprising that defects in
(1
6)-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
(1
6)-glucan synthesis, if such can be found, would behave as
antifungal agents.
The chitin participating in linkages to both (1
3)- and
(1
6)-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
(1
6)-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
(1
6)-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
(1
6)-glucan, whereas another portion
of the ring chitin is directly linked to
(1
3)-glucan (6). It is
possible that the
(1
6)-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 (1
3)-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, (1
6)-glucan,
(1
3)-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).
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 (1
3)-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
(1
6)-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.
We thank H. Bussey for yeast strains, R. C. Montijn for the (1
6)-glucan antibody, A. Llobell for a sample of
(1
6)-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.