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(Received for publication, July 11, 1994; and in revised form, November 9,
1994) From the
Isolation of side chain oligosaccharides from mannans of Candida albicans NIH B-792 (serotype B) and Candida
parapsilosis IFO 1396 strains has been conducted by acetolysis
under mild conditions. Structural study of these oligosaccharides by
and
It was observed that the H-1 proton chemical shifts of the
second and the third mannose units from the reducing terminus in each
oligosaccharide are shifted upfield by substitution with an
Yeasts of the genus Candida, especially of Candida
albicans species are known to be pathogenic in man. Candidiasis is
an opportunistic infectious disease in early childhood and in adults
with predisposing conditions such as diabetes, cancer, AIDS, and
treatment with immunosuppressive agents after organ
transplantation(1, 2) . The antigenicity of Candida cell walls resides in the mannan. Moreover, mannan is
highly soluble and can be detected in the sera of some patients with
candidiasis by various techniques, including immunologic
procedures(3, 4, 5, 6, 7, 8, 9) .
Thus, the detection of circulating mannan is important for diagnosis of
invasive candidiasis. However, the cell wall components in sera from
patients with other infections, such as Mycobacterium tuberculosis(10) , Serratia marcescens(11) , and Salmonella thompson(12) may cross-react with
anti-Candida mannan antibody if it is not specific to Candida species. On the other hand, there are many
reports(13, 14, 15, 16, 17, 18, 19, 20) about
immunomodulatory effects by the mannan or oligosaccharide of C.
albicans, including the induction of suppressive effects against
both B- and T-cell-mediated immune responses. Although the mechanism of
the immunosuppressive effects of these components is still unknown,
several reports (21, 22, 23) suggest that
side chain oligomannosyl moieties participate in adherence of the C. albicans cells to mammalian cells in the initial step of Candida infection. Therefore, the fine chemical structure of
cell wall mannans must be known to develop an accurate serodiagnostic
procedure for candidiasis and to understand these diverse host-parasite
interactions. In 1961, Hasenclever and Mitchell (24) reported two serotypes in C. albicans strains
designated serotypes A and B. Later, Tsuchiya et al.(25) proposed the relationship between antigenic
structures of many yeasts, including seven medically important species
of Candida, based on 10 cell surface antigenic factors. In
recent years, structural analysis of cell wall mannans of C.
albicans has been developed extensively(26, 27) ,
and we have demonstrated the presence of phosphodiesterified
The C. albicans serotype B and Candida parapsilosis cells used in this
study have antigenic factors 1, 4, and 5 and factors 1, 13, and 13b,
respectively(25) . Therefore, the difference in structures of
the mannans of both species correlates with the presence or the absence
of antigenic factors, 4 or 13 and 13b, although the previous study by
Funayama et al.(36) could not reveal any structural
difference. This result could be attributable to the acetolysis
conditions, since these workers prepared the side chain
oligosaccharides by a conventional acetolysis procedure that cleaves
all
Acetolysis under
conventional conditions was carried out as described by Kocourek and
Ballou (42) using a 10/10/1 (v/v) mixture of
(CH
Figure 1:
Elution patterns of oligosaccharides
obtained from C. parapsilosis (A and B) and C. albicans (C and D) mannans by acetolysis. A and C, acetolysis was performed with
(CH
Figure 2:
The
anomeric region of the
Figure 3:
Figure 4:
Partial two-dimensional HOHAHA analysis of
mannooligosaccharides PM
To identify the H-1
proton of the second mannose unit from the reducing terminal unit,
Man-B, AM
Figure 5:
Sequential connectivities of mannose units
of AM
Figure 6:
Partial two-dimensional HOHAHA spectra of
fractions P and A and other mannans of related yeasts. H-C COSY of
fractions P and A are also shown. Cross-peaks 1 and 2 indicate
The present comparative study between the structures of the
mannans of C. parapsilosis and C. albicans serotype B
strains clearly demonstrates the existence of branched side chains in
the latter mannan. Now we propose the entire chemical structures of the
mannans of C. parapsilosis IFO 1396 and C. albicans NIH B-792 strains based on the results of the present and recent
studies (29, 52) as shown in Fig. 7. There was
a report (35) on the presence of a treebranch-like structure in
the mannan of C. albicans serotype A strain. Based on our
structural studies of C. albicans mannan, we find no evidence
for such a structure. The branched structure is predominant in the
mannan of the C. albicans serotype B strain, but small amounts
occur in that of the C. albicans serotype A strain. Therefore,
the structures recognized by monoclonal antibodies specific for
antigenic factor 4 proposed by Kagaya et al.(34) were
also speculative. Funayama et al.(36) reported that
antigenic factors 13 and 13b correspond to a mannohexaose moiety with a
linear structure,
Man
Figure 7:
Possible structure of C.
parapsilosis IFO 1396 and C. albicans NIH B-792 strain
mannans. M denotes a D-mannopyranose unit. The side
chain sequence is not specified.
Factor 4 serum
is prepared by absorption of anti-C. albicans serotype A
strain whole-cell serum with C. parapsilosis cells(25) . Because C. albicans serotype A and C. parapsilosis cells have epitopes corresponding to antigenic
factors 1, 4, 5, and 6 and factors 1, 13, and 13b, respectively, the
factor 4 serum contains antibodies against antigenic factors 5 and 6 as
well as factor 4(32) . Therefore, some Candida species
react strongly with factor 4 serum, despite a low density of the real
antigenic factor 4 in the mannans. For example, the C. albicans serotype A strain mannan seems to contain only a small amount of
branched side chain as judged from the intensity of cross-peak 3 in the
two-dimensional HOHAHA spectrum. However, because C. albicans serotype A strain mannans contain The
upfield shift of the H-1 proton of an A strong cross-peak 3 in
two-dimensional HOHAHA spectra of the mannans of C. guilliermondii and C. stellatoidea suggests that these mannans contain
significant amounts of branched side chains. The structural analysis of
the mannans of these strains is in progress.
Volume 270,
Number 3,
Issue of January 20, 1995 pp. 1113-1122
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
STRUCTURE-ANTIGENICITY RELATIONSHIP BETWEEN THE CELL WALL
MANNANS OF CANDIDA ALBICANS AND CANDIDA PARAPSILOSIS(*)
H and C NMR and methylation analyses indicated
the presence of novel branched side chains with the following
structures in C. albicans mannan.
-linked mannose unit at position 6 of the
3-O-substituted mannose unit. An agglutination inhibition
assay between factor 4 serum and cells of Candida stellatoideaIFO 1397 lacking the 
-1,2-linked mannose unit, with
oligosaccharides obtained from these mannans, indicated that only the
branched oligosaccharides were active. This finding suggests that the
branched oligosaccharides correspond to the epitope of antigenic factor
4. The presence of the branched structure in other mannans was detected
by the characteristic H-1-H-2-correlated cross-peak of the
-1,2-linked mannose unit connected with the
3,6-di-O-substituted one by two-dimensional homonuclear
Hartmann-Hahn spectroscopy.-1,2-linked oligomannosyl residues as a group of common epitopes
throughout the two serotype strains(28, 29) .
Furthermore, a -1,2-linked mannose unit connected to an
-1,2-linked unit was found to correspond to a specific epitope for
serotype A strains(30, 31) . Although these two groups
of -1,2 linkage-containing epitopes were identified as
corresponding to antigenic factors 5 and 6(32, 33) ,
the structure of factor 4 has not been determined. From the results of
agglutination assays of monoclonal anti-factor 4 antibodies with cells
of many Candida strains, it was speculated by Kagaya et
al.(34) that the antigenic factor 4 corresponds to
treebranch-like structures proposed by Suzuki et
al.(35) . However, the results of our structural studies
for C. albicans mannans provided evidence that the mannans
have a comb-like structure with an -1,6-linked
backbone(26, 27) .-1,6 and -1,2 linkages. Our recent study (37) of Saccharomyces kluyveri mannan indicated that acetolysis under
mild conditions released oligosaccharides retaining both -1,2
linkage and part of the -1,6 linkages of the branching mannose
unit. Therefore, we applied the mild acetolysis method to analyze
structural difference(s) between oligomannosyl side chains of the two Candida mannans. Although several reports suggest the presence
of branched side chains in the mannans of C. albicans, no
isolation of any branched oligosaccharide has been
achieved(35, 38, 39, 40) . In the
present study, we demonstrate the existence of novel branched side
chains that dominate the antigenic factor 4 specificity in the mannan
of a C. albicans serotype B strain.
Materials
Candida guilliermondii IFO
10279, C. parapsilosis IFO 1396, C. stellatoidea IFO
1397 (type I), and S. kluyveri IFO 1685 strains were obtained
from the Institute for Fermentation, Osaka (IFO), Japan. C.
albicans NIH B-792 (serotype B) and C. albicans J-1012
(serotype A) strains are stock cultures in our laboratory. Factor 1, 4,
5, 6, and 13b sera of ``Candida Check'' (lot L261), a
commercially available kit of rabbit polyclonal antibodies against Candida cells, were purchased from Iatron (Tokyo, Japan).
Except for the factor 1 serum, which is an unabsorbed rabbit whole-cell
serum against C. albicans cells, factor 4, 5, 6, and 13b sera
are the sera to C. albicans absorbed with cells of C.
parapsilosis, C. guilliermondii, C.
stellatoidea, and Candida tropicalis,
respectively(25) .Preparation of Mannans
Yeast cells were cultivated
in a 500-ml flask containing 0.5% yeast extract-supplemented Sabouraud
liquid medium on a reciprocal shaker at 28 °C for 48 h. From the
cells, which were washed and dehydrated with acetone, the crude mannan
was extracted with water at 135 °C for 3 h. After dialysis, mannan
was separated using Fehling solution to form a water-insoluble
precipitate of the Cu-mannan complex. The mannan was
recovered from the copper complex by treatment with Amberlite IR-120
(H
) resin, neutralization, dialysis, and
lyophilization. The mannans prepared from the cells of C.
parapsilosis IFO 1396 and C. albicans NIH B-792 strains
were designated as fractions P and A, respectively.
Acetolysis of Mannan
Acetolysis under mild
conditions (31) was carried out in the same manner for the
analysis of S. kluyveri mannan(37) . Namely, prior to
acetolysis, the mannan was converted into its O-acetyl
derivative. Mannan (100 mg) was dissolved in anhydrous formamide (5
ml). To the solution was added a 1/1 (v/v) mixture of
(CH
CO)
O and anhydrous pyridine (50 ml), and the
mixture was kept at 40 °C for 12 h. After evaporation under high
vacuum using an oil diffusion pump, the residual O-acetyl
mannan was dissolved in a 100/100/1 (v/v) mixture of
(CHCO)O, CHCOOH, and
HSO
(50 ml), and the resultant solution was
kept at 40 °C for 36 h. The O-acetylated
mannooligosaccharide mixture was extracted from the reaction mixture
with CHCl and de-O-acetylated with
CHONa. Fractionation of the resultant mannooligosaccharide
mixture was achieved using a column (2.5 
100 cm) of Bio-Gel P-2
(-400 mesh). Elution was carried out with water, and aliquots of
eluates were assayed for carbohydrate content by the phenol-sulfuric
acid method(41) . Eluates corresponding to each peak were
combined and rechromatographed on the same column.CO)O, CHCOOH, and
HSO, and the resultant solution was kept at 40
°C for 12 h.Nuclear Magnetic Resonance Spectroscopy
All NMR
experiments were performed with a JEOL JNM-GSX 400 spectrometer at 400
MHz for H and 100 MHz for C. The spectra were
recorded using a 1% (w/v) solution of each mannan or oligosaccharide in
0.7 ml of D
O at 45 °C. Acetone (2.217 ppm) (43) and CDOD (49.00 ppm) were used as the internal
standards for H and C NMR, respectively.
Slide Agglutination Inhibition Test
The assay was
conducted as described by Miyakawa et al.(44) as
follows. Factor 4 serum (50 µl) was preincubated for 2 h at 30
°C in the presence of serially 2-fold diluted oligosaccharide
inhibitor solution (50 µl). To this solution, heat-killed C.
stellatoidea IFO 1397 strain cells (10
/100 µl)
were added and incubated at 30 °C for 1 h.Methylation Analysis
Methylation of
oligosaccharides was performed according to Ciucanu and
Kerek(45) . Gas chromatography of O-methyl-O-acetyl-D-mannitols was performed
using a glass column (3 mm 200 cm) containing 3% OV-210 on
Supelcoport (100-200 mesh) at 185 °C using N as
the carrier gas at a flow rate of 20 ml/min.Other Methods
Total carbohydrate was determined by
the phenol-sulfuric acid method of Dubois et al.(41) with D-mannose as the standard. Total
phosphate was determined by the method of Ames and Dubin(46) ,
using KHPO as the standard.
Acid Treatment of Mannans
Acid treatment of C. albicans mannans with 10 mM HCl at 100 °C for
1 h selectively cleaves phosphodiester linkage to release
-1,2-linked mannooligosaccharides(28) . The C.
albicans serotype B mannans contain -1,2-linked mannose units
in only acid-eliminable oligosaccharide moieties, and therefore the
resultant acid-modified mannans consist of -linked mannose units.
Since the -1,2-linked oligosaccharides correspond to one of the
major epitopes(29) , antigenic factor 5(32) , for C. albicans mannans, those of C. albicans NIH B-792
strain, fraction A, and C. parapsilosis IFO 1396 strain,
fraction P, were subjected to treatment with 10 mM HCl to
compare the ratios of these oligosaccharides(28, 47) .
Although acid hydrolysis of fraction A gave mannose and
mannooligosaccharides from biose to heptaose as reported (28) ,
release of neither mannose nor oligosaccharides from fraction P
occurred under the same conditions (data not shown). The absence of
acid-labile oligosaccharides in fraction P is consistent with the lack
of an H-1 H NMR signal at about 5.55 ppm corresponding to
the 1-O--phosphorylated mannose
unit(28, 32) , unreactivity to factor 5
serum(32) , and a negative result for phosphate analysis.Acetolysis of Mannans
The polysaccharides
recovered from the partial hydrolysates of fractions P and A were
subjected to acetolysis under conventional and mild conditions. Fig. 1shows elution patterns of the acetolysates of the
acid-modified mannans from a Bio-Gel P-2 column. It is apparent that
the amounts of higher oligosaccharides (heptaose and either hexaose or
pentaose) in the acetolysates obtained under the mild conditions were
increased as compared with those obtained under the conventional
conditions. The oligosaccharides from tetraose to heptaose obtained
from the C. parapsilosis and C. albicans mannans by
mild acetolysis were designated as PM, PM
,
PM
, and PM
and AM, AM,
AM, and AM, respectively.
CO)O, CHCOOH,
HSO (10:10:1, v/v) at 40 °C for 12 h
(conventional conditions). B and D, acetolysis was
performed with (CHCO)O, CHCOOH,
HSO (100:100:1, v/v) at 40 °C for 36 h
(mild conditions). M, M, M, M,
M, M, and M indicate mannose,
mannobiose, mannotriose, mannotetraose, mannopentaose, mannohexaose,
and mannoheptaose, respectively. Vo, void
volume.
H NMR Analysis of
OligosaccharidesH NMR spectra of the
oligosaccharides obtained from the two acid-modified mannans by
conventional acetolysis were essentially the same as those reported by
Funayama et al.(36, 48) . The
oligosaccharides consisted of -1,2- and -1,3-linked mannose
units (data not shown). On the other hand, as shown in Fig. 2,
the H NMR spectra of the oligosaccharides higher than
tetraose, except for PM, obtained by mild acetolysis gave
other signals at about 4.91 ppm corresponding to -1,6-linked
mannose units(49) . Furthermore, signals at 5.22 and 5.24 ppm
were present only in AM and AM. These chemical
shifts are only slightly different from that of an -1,2-linked
mannose unit connected with a 3,6-di-O-substituted mannose
unit in the branched oligosaccharides obtained from the mannan of S. kluyveri, 5.23 ppm(37) . The spectra of
AM, AM, and AM indicated that these
oligosaccharides were mixtures of isomers with or without an
-1,6-linked mannose unit, judging from the ratio of dimensions of
signals corresponding to the reducing terminal and -1,6-linked
mannose units. Thus, we tried to separate the isomers by high pressure
liquid chromatography on a normal or a reverse phase column after
modification of the oligosaccharides to 2-aminopyridyl derivatives by
the method of Hase et al.(50, 51) .
Unfortunately, however, the isomers could not be separated from each
other by these procedures. Therefore, we attempted structural analysis
of these oligosaccharides without further separation. Assignment of the H NMR signals for the isomeric oligosaccharides that did
not contain the -1,6 linkage, which had been obtained by the
conventional acetolysis in the present and the preceding (29) studies, was readily achieved. Therefore, it seemed
possible to assign the signals of other -1,6-linkage-containing
oligosaccharides in the mixture.
H NMR spectra of oligosaccharides
obtained from C. parapsilosis (A) and C. albicans (B) mannans by acetolysis under the mild conditions.
Spectra were recorded using a JEOL JNM-GSX 400 spectrometer in
DO solution at 45 °C using acetone as the standard
(2.217 ppm). M-M are designated as in the
legend to Fig. 1.
To
confirm the existence of the branched structure, DEPT (C NMR Analysis of Oligosaccharides
)135 C NMR spectra of the oligosaccharides were recorded (Fig. 3). Negative signals in these spectra imply that the
carbon atom must have two protons and therefore correspond to the C-6
of a mannose unit(52) . The downfield shifted negative signals
at 66.14 and 66.39 ppm in the spectra of AM
,
AM, and AM indicate that the mannose unit is
substituted at the 6-O-position. Reduction of AM with NaBH caused a downfield shift of the two signals
from 61.96 ppm to 64.09 ppm and from 66.39 ppm to 69.79 ppm, whereas
the signal at 66.14 ppm did not shift (Fig. 3E). This
result indicates that the mannose unit with a C-6 signal at 66.14 ppm
is not located on the reducing terminus and that AM and
AM each contain one branching -1,6-linked mannose unit
with a C-1 signal at 100.30 ppm. It was further demonstrated from the
presence of cross-peaks in the HMBC spectrum between an H-1 proton at
4.907 ppm and the downfield shifted negative signals from 61.96 ppm in
DEPT 135, based on a three-bond coupling across an O-glycosidic linkage(53) , that the shifted signals
should correspond to the C-6 carbons of the 6-O-substituted
mannose unit (Fig. 3F). On the other hand, PM and PM gave only one downfield shifted negative
signal at 66.39 ppm (data not shown). These results indicate that the
-1,6-linked mannose unit in PM and PM must
be connected to a reducing terminus. Therefore, it is apparent that the
C-1 signals at 100.50 ppm of PM and PM correspond to -1,6-linked mannose units that originated from
the backbone of this mannan.
C NMR spectra of
oligosaccharides obtained from fraction A. DEPT 135
C NMR
spectra of AM
(A), AM (B),
AM (C), AM (D), and
AM-H (E), and HMBC of AM-H (F) are shown. Spectra were recorded using a JEOL JNM-GSX 400
spectrometer in DO solution at 45 °C using
CDOD as the standard (49.00 ppm). Negative signals on DEPT
135 spectra correspond to C-6 carbon. The dashed lines among
those spectra indicate downfield shift of C-6 signals by substitution
with another mannose unit or by reduction on the reducing terminal
mannose unit.
Two-dimensional Homonuclear Hartmann-Hahn Spectroscopy of
Oligosaccharides
To determine the location of a mannose unit
substituted by an -1,6-linked mannose unit, two-dimensional HOHAHA (54) analysis of PM, PM,
AM, and AM was carried out. It has been shown
in the previous papers that the H-1 proton chemical shift of a mannose
unit does not change by phosphorylation or glycosylation at the
6-O-position but affects the chemical shift of some proton
signals allocated around the substituted
position(37, 52, 55, 56) . In the
present study, it was revealed that cross-peaks of PM that
correlated with H-1 protons at 5.348 and 5.264 ppm, which have been
assigned to those of Man-A and Man-B, respectively (Table 1),
were significantly shifted as compared with those of PM (Fig. 4). Although two-dimensional HOHAHA spectra of
PM-H and PM-H, the reduction products of
PM and PM with NaBH, respectively,
show an upfield shift of the H-1 signals corresponding to Man-B, the
difference between the cross-peaks has disappeared. This finding also
indicates that the -1,6-linked mannose unit is attached to the
reducing terminal unit, Man-A, and that the difference in the
cross-peaks correlated with the H-1 protons of Man-B of PM and PM results from a steric effect. Despite some
difference between cross-peaks correlated with the H-1 proton at about
5.03 ppm (corresponding to the 3-O-substituted mannose unit,
Man-D, of PM and AM), it is not significant
enough to determine the substituting position.
, PM, AM,
and AM, obtained by mild acetolysis and the reduction
products PM-H, PM-H, AM-H, and
AM-H. The cross-peaks for all ring protons, H-2 to H-6, are
indicated by a vertical line correlated with the H-1 signal of
each mannose unit. The dashed line between the cross-peaks of
parent and reduced oligosaccharides indicates a shift of the H-1 signal
caused by the reduction by
NaBH.
and AM were reduced with
NaBH. The H-1 and H-2 signals of Man-B were easily assigned
based on their upfield shift, 
=
0.06 and 0.1
ppm, respectively(37) . Namely, the signals at 5.24 and 5.27
ppm of AM
and AM were assigned to the H-1
proton of Man-B. These results indicate that the upfield shift of the
H-1 signal of Man-C from 5.28 to 5.22 ppm, = 0.06
ppm, by the attachment of an
-1,6-linked mannose unit to Man-D is
larger than that of Man-B from 5.27 to 5.24 ppm, =
0.03 ppm. The shift value of the H-1 signal of Man-C is similar to that
observed on the branched oligosaccharide from S. kluyveri mannan,
= 0.05 ppm (37) . Therefore, we
propose that the chemical structures of the branched oligosaccharides
in the isomer mixtures, AM
and AM, are as
follows (Structures 1 and 2, respectively).
Sequential NMR Assignment
To confirm the
structure of the branched oligosaccharides in AM and
AM, a sequential assignment study of the H-1 and H-2
signals of their reduction products, AM-H and
AM-H, was performed by the method described by Hernandez et al.(55) with slight modification(47) . The right side of the diagonal of each panel in Fig. 5shows COSY, whereas the left side shows rotating frame NOE
spectroscopy. In this figure, cross-peaks labeled with primed letters indicate through-space interresidue H-1-H-2`
connectivities between two adjacent mannose units except D`,
which indicates interresidue H-1-H-3` connectivities. On the other
hand, cross-peaks labeled with unprimed uppercase and lowercase letters indicate intraresidue H-1-H-2- and
H-2-H-3-correlated cross-peaks, respectively, caused by J-coupling. By this procedure, H-1 and H-2 signals were
sequentially assigned from the H-1 of the Man-B, B-B`-C-C`-D-d-D`-E for
AM-H (Fig. 5A) and B-B`-C-C`-D-d-D`-E-E`-F
for AM-H (Fig. 5B). The results summarized
in Table 1clearly demonstrate that attachment of an
-1,6-linked mannose unit to Man-D causes an upfield shift not only
of the H-1 proton of Man-C but also that of Man-B by a steric effect,
such as that observed on an -1,3-linked mannose unit in S.
cerevisiae mannan(57) .
-H (A) and AM-H (B). The right side of the diagonal shows COSY, and the left
side of the diagonal shows rotating frame NOE spectroscopy. Primed
letters indicate interresidue H-1-H-2` or H-1-H-3` NOE
cross-peaks, and unprimed uppercase and lowercase letters indicate the H-1-H-2 and the H-2-H-3-correlated cross-peaks,
respectively, caused by J-coupling; e.g.B indicates the H-1-H-2-correlated cross-peak of the second mannose
unit, Man-B, and B` indicates the interresidue NOE cross-peak
between H-2 of Man-B and H-1 of an adjacent mannose unit, Man-C. By
this procedure, H-1 and H-2 signals were sequentially assigned from the
H-1 of the Man-B, B-B`-C-C`-D-d-D`-E for AM-H and
B-B`-C-C`-D-d-D`-E-E`-F for
AM-H.
Methylation Analysis
The presence of branching
points in these oligosaccharides was further confirmed by methylation
analysis. The results of PM-H, PM-H,
AM-H, and AM-H indicate that almost all of the
-1,6-linked mannose units in the latter two oligosaccharides were
connected to a 3-O-substituted mannose unit, i.e. only 1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl
mannitol was detected as a di-O-methyl mannitol derivative by
gas-liquid chromatography (Table 2).
Comparison of Two-dimensional Homonuclear Hartmann-Hahn
Spectroscopy of Several Mannans
Fig. 6shows the
two-dimensional HOHAHA spectra of fractions P and A and four other
mannans obtained from related yeasts, C. stellatoidea IFO
1397, C. albicans J-1012 (serotype A), S. kluyveri IFO 1685, and C. guilliermondii IFO 10279 strains.
Cross-peak 1 in the two-dimensional HOHAHA of fractions P and A, those
connected with a dotted line to a cross-peak in H-C COSY with
a C-1 signal at 100.49 ppm, corresponds to the consecutive
-1,6-linked mannose units of the mannan backbone. On the other
hand, cross-peak 2, connected to a C-1 signal at 100.29 ppm through a
cross-peak in H-C COSY, was not present in the spectrum of fraction P
but existed in that of fraction A. Although cross-peak 1 is observed in
the two-dimensional HOHAHA spectra of all six tested mannans,
cross-peak 2 was absent in that of C. parapsilosis.
Furthermore, cross-peak 3, corresponding to an -1,2-linked mannose
unit substituted by a 3,6-di-O-substituted unit with an
-1,2 linkage, was observed distinctly in the two-dimensional
HOHAHA spectra of mannans of C. albicans serotype B, C.
stellatoidea, C. guilliermondii, and S.
kluyveri, and faintly in that of C. albicans serotype A,
but it was absent from the spectrum of fraction P.
-1,6-linked backbone and -1,6-linked branching
mannose units, respectively. Cross-peak 3 indicates an
-1,2-linked mannose unit substituted by a
3,6-di-O-substituted mannose unit.
Haptenic Activity of Branched Oligosaccharides
The
presence of cross-peaks 2 and 3 in the two-dimensional HOHAHA spectra
of mannans correlates well with the reactivities of the cells of these
strains with factor 4 serum of Candida Check. Therefore, we examined
the inhibitory effect of oligosaccharides obtained by acetolysis of the
two acid-modified mannans on the agglutination reaction between factor
4 serum and cells of the C. stellatoidea IFO 1397 strain
expressing only factors 1 and 4. As shown in Table 3, only
AM and AM showed inhibitory effects at an
amount of 0.1 µmol or greater. These results indicate that the
branched oligosaccharide side chains present in fraction A behave as
the antigenic factor 4.
12Man
13Man
12Man
12Man
1
2Man. Therefore, it is apparent that the antigenic factor 4
corresponding to the branched mannoheptaose moiety would be degraded to
structures corresponding to antigenic factor 13b by conventional
acetolysis(29, 30, 36, 58) . The
above results suggest that the C. parapsilosis IFO 1396 strain
cannot synthesize branched side chains and express antigenic factors 13
and 13b but not factor 4. However, in the cells of the C. albicans serotype B strain, these side chains are branched by the addition
of
-1,6-linked mannose units to make the epitope corresponding to
antigenic factor 4 instead of 13b. Therefore, we can say that the
relationship between the structures of antigenic factors 13b and 4 is
the same as that seen in blood groups H and A or B.
-1,6-Linked mannose units in
brackets indicate partial absence of this
unit.
-1,2-linked mannose units
connected to an -1,2-linked mannose to give antigenic factor 6,
strong cross-reactions with factor 4 serum can be observed. As
additional evidence, Kobayashi et al.(59) and Okawa et al.(60) observed a significant decrease in the
reactivity of C. albicans serotype A strain cells to factor 4
serum in addition to the disappearance of reactivities to factor 5 and
6 sera when the cells were cultivated at low pH or at high temperature.
This result suggests that the presence of two types of
-1,2-linkage-containing side chains, corresponding to antigenic
factors 5 and 6, are responsible for the strong reactivity of the C. albicans serotype A strain cells to factor 4 serum.-1,2-linked mannose unit by
a steric effect, found in branched mannooligosaccharides obtained from
the mannan of S. kluyveri(37) , was also observed in
the branched oligosaccharides of C. albicans mannan. In the
case of the latter oligosaccharides, the -1,6-linked branching
mannose unit affects the H-1 proton chemical shifts of both the third
mannose unit and the second one from the reducing terminal. Therefore,
the three-dimensional structure of these branched oligosaccharides in
aqueous solution would be of interest.
)H-H- correlated spectroscopy;
NOE, nuclear Overhauser effect.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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