(Received for publication, October 7, 1994; and in revised form, November 15, 1994)
From the
To isolate the putative linkage region between chitin and
(1
3)-glucan, Saccharomyces cerevisiae cell walls
were digested with
(1
3)-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
-N-acetylglucosaminidase and by NMR spectroscopy, N-acetylglucosamine was identified as the nonreducing
terminus, linked to laminaritriitol by a
(1
4) 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
(1
3)-glucan chain by a
(1
4)
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.
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 (1
3)-D-glucan that also contains
some
(1
6)-linked branches and a mannoprotein, most of which
is carbohydrate(2) . A
(1
6)-D-glucan, also
containing some
(1
3)-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
(1
3)-glucan and the mannoprotein, to explain why the latter
is solubilized after treatment of the wall with
-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
-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
-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
(1
3)-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.
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.
To isolate the linkage region between chitin and
-glucan, yeast cell walls were digested with a
(1
3)-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 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
([
C]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).
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--glucosaminidase and
-glucosidase (Fig. 4). Whereas
-glucosidase treatment
had no effect on the elution of I (Fig. 4c),
incubation with
-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
-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(
1-3)Glc(
1-3)Glc-ol (or
Glc(
1-6)Glc-ol), Glc
(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
-N-acetylglucosaminidase gave rise to a peak in the
position of the original material and another one moving as
Glc(
1-3)Glc(
1-3)Glc-ol or
Glc(
1-6)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
-N-acetylhexosaminidase and
-glucosidase were allowed to act on I, the product
comigrated with Glc(
1-3)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
-glucosidase. Taken together, the
above results are consistent with a structure in which GlcNAc is
-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
-N-acetylglucosaminidase or
-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
-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
-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
90 cm and the
fraction size 450 µl. Standards used were: 1,
triacetylchitotriitol; 2, diacetylchitobiitol; 3,
laminaritriitol; 4, laminaribiitol; 5, glucitol; 6, glucose ([
C]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
([
C]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
-N-acetylglucosaminidase from D. pneumoniae and the other with the same enzyme plus 0.02 mg of
-glucosidase from sweet almonds. Both samples were subjected to
paper chromatography as in a.
, incubated with N-acetylglucosaminidase;
, 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
-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 -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
H 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
4.65 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
-linkage.
Figure 7:
NMR spectra of I, laminaritriitol, and
laminaribiitol. Approximately 1 µmol of I was evaporated to dryness
several times with DO, and finally dissolved in 600 µl
of D
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(
1-6)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
-linked units.
The position of glucose
that is -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(
1-6)Glc. This, however, allowed us to eliminate the
1-6 linkage as a possibility, because the synthetic compound was
decomposed by beef kidney
-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(1-6)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
-effect of the glycosidic linkage at that
position(18) . We conclude that the glycosidic linkage between
GlcNAc and Glc in I is
(1
4) and that the complete
structure of the substance is
GlcNAc(
1-4)Glc(
1-3)Glc(
1-3)Glc-ol.
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.
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 -N-acetylglucosaminidase and/or
-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
90 cm), eluted as in Fig. 1. Aliquots of the original material were subjected to the
following treatments. a, no treatment. b, incubation
with
-N-acetylglucosaminidase as in Fig. 4b.
c, incubation with
-glucosidase, as in Fig. 4c.
d, incubated with both
-N-acetylglucosaminidase and
-glucosidase. Standards: 1, triacetylchitotriitol; 2, diacetychitobiitol; 3, laminaritriitol; 4, laminaribiitol; 5, glucitol; 6,
glucose.
Figure 10:
Effect of
-N-acetylglucosaminidase and
-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
-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
-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.
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.
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.
The oligosaccharides analyzed in this study were not
solubilized until cell walls were digested with both -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
(1
4) 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
(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
(1
3) linkage
hydrolyzed by the zymolyase preparation. The main activity in zymolyase
appears to be a
(1
3)-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(
1
4)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-(1
3)-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. ()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--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.