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(Received for publication, November 3, 1995; and in revised form, January 30, 1996) From the
Isolation of
and
The H-1 signals of the mannose
units substituted by a 3,6-di-O-substituted unit showed a
significant upfield shift (
We have reported the presence of two types of In an earlier paper(15) , we
demonstrated the presence of a third type of a There are several
reports on the responsibility of C. guilliermondii, which is one of the
causes of human candidiasis in immunocompromised hosts, has antigenic
factors 1, 4, and 9(24, 25) . Although the structure
corresponding to antigenic factor 4 was recently identified to be the
following(26) ,
and
there is no report of the chemical structure corresponding to
antigenic factor 9 except a study by Ataoglu et
al.(27) . They showed that the factor 9 serum reacts with Saccharomyces cerevisiae X2180-1A-5 (mnn2) mutant
strain cells, which have a linear
Figure 1:
Elution patterns of oligosaccharides
obtained from fraction G by acetolysis. A, B, elution
was performed with a column (2.5
Figure 2:
The anomeric region of the
On the other hand, the
BM BM
It is obvious that BM
Figure 3:
Sequential connectivities of the mannose
units of BM
The results summarized in Table 2clearly
demonstrate that the attachment of an
Figure 4:
Assignment of H-1 and H-2 signals of
fraction G. (A) Normal
Although it is difficult to determine
the dimension of the H-1 signal of the branched mannose unit because of
the overlapping of cross-peaks 13 and 14, we can estimate it from the
dimensions of the H-1 signals of cross-peaks 2 and 5. Because the H-1
signal dimensions of cross-peaks 4 and 6 are the same as those of
cross-peaks 17 and 16 (half of the signal at 4.849 ppm), respectively,
the H-1 signal dimension of cross-peak 5 can be determined by
subtraction of the H-1 signal dimension of cross-peaks 4 and 6 from
that of the signal at 5.218 ppm as shown in Table 3. These
results indicate that the amount of the
Figure 5:
Possible structure of C.
guilliermondii IFO 10279 strain mannan. M denotes a D-mannopyranose unit. The side-chain sequence is not
specified. The molar ratio of the side chains in the mannan is
expressed as a percentage of the total side chains. The values are
calculated from the dimensions of the
Figure 6:
Inhibition of enzyme-linked immunosorbent
assay by mannooligosaccharides. To the fraction G-coated microtiter
plate, factor 9 serum pretreated with or without haptenic
mannooligosaccharides, BM
In 1988, Kogan et al.(45) reported the
presence of a 2,3-di-O-substituted mannose unit in the side
chain of C. albicans and C. guilliermondii mannans
based on the results of methylation analysis of polysaccharides. Later,
Kagaya et al.(38) suggested the presence of a
branching structure (46) in the C. guilliermondii mannan from the cross-reactivity of a monoclonal antibody against
factor 4. Recently, we found that the antigenic factor 4 corresponds to
an Ataoglu et al.(27) reported that the antigenic factor 9 corresponds to a
consecutive In 1981, Zhang and Ballou (40) reported the presence of O-linked branching mannooligosaccharides up to octaose in S. kluyveri mannoprotein. In the study, they analyzed
the structure of oligosaccharides by the methylation technique.
However, because it is impossible to determine the linkage sequence
from the methylation analysis data, they proposed the structure of
mannopentaose to mannooctaose based on that of the shorter ones. On the
other hand, the sequential assignment method of oligosaccharides
through HMBC (15, 47) or NOE (8, 26, 42) cross-peaks between the
glycosylated two mannose units was demonstrated to be suitable for
assigning and determining the linkage sequence by this and the
preceding studies. The upfield shift effect of the H-1 signal of an
From the results of this
and the preceding (12, 26) structural studies of the
mannans containing
Volume 271,
Number 16,
Issue of April 19, 1996 pp. 9259-9266
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-1,2 and 
-1,6
Linkages Corresponding to Antigenic Factor 9 in the Mannan of Candida guilliermondii(*)
-linkage-containing side chain
oligosaccharides from the mannan of Candida gilliermondii IFO
10279 strain has been conducted by acetolysis under mild conditions. A
structural study of these oligosaccharides by one- and two-dimensional
NMR and methylation analyses indicated the presence of extended
oligosaccharide side chains with two consecutive -1,2-linked
mannose units at the nonreducing terminal of 
-linked
oligosaccharides. The linkage sequence present in this mannan,
Man1
2Man13Man, has also been found in
the mannan of Saccharomyces kluyveri but not in the mannan of Candida species. Furthermore, these oligosaccharides are
branched at position 6 of the 3-O-substituted mannose units as
follows.
= 0.04-0.08 ppm) due
to a steric effect. The inhibition of an enzyme-linked immunosorbent
assay between the mannan of C. guilliermondii and factor 9
serum with oligosaccharides obtained from several mannans indicated
that only the oligosaccharides with the above structure were active,
suggesting that these correspond to the epitope of antigenic factor 9.
-1,2-linked
mannose units in the cell wall mannans of the genus Candida.
One is located in a phosphodiesterified oligosaccharide moiety as one
of the major epitopes for Candida albicans serotypes A and
B(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) and Candida tropicalis(12) strains. The -1,2-linked
oligosaccharides can be released selectively from these mannans by
treatment with weak acid (10 mM HCl)(1) . The
resultant acid-modified mannans of C. albicans serotype A and C. tropicalis strains still contain -1,2-linked mannose
units attaching to -1,2-linked mannotetraose side
chains(3, 4, 12) . These are the second type
of -1,2-linked mannose units corresponding to a serotype
A-specific epitope for C. albicans. The first and
second types of -1,2 linkage-containing side chains have been
identified as corresponding to antigenic factors 5 (13) and
6(14) , respectively.-1,2-linked mannose
unit attaching to an -1,3-linked one in the cell wall mannan of Saccharomyces kluyveri and speculated on the presence of the
same type of -1,2-linked unit in those of C. albicans serotype A and Candida guilliermondii based on the
presence of characteristic H-1-H-2-correlated cross-peaks in their
two-dimensional HOHAHA (
)spectra.-linked side chains(16) ,
-linked ones(9) , or complex side chains with - and
-linkages (17) of cell wall mannan for the adherence of C. albicans cells to host cells in the initial step
of Candida infection. Furthermore, mannans or
mannooligosaccharides of C. albicans cells are known to
stimulate cytokine
production(18, 19, 20, 21, 22, 23) .
Therefore, the identification of the third type of -1,2 linkage
containing mannan side chains is important for understanding the
pathogenecity of C. guilliermondii and its accurate
serodiagnosis.
-1,6-linked mannan corresponding
to the backbone in their cell wall. Therefore, we tried to detect a
novel side chain corresponding to antigenic factor 9 in the cell wall
mannan of a pathogenic yeast C. guilliermondii. For
fragmentation of the mannan, we applied a mild acetolysis, which
selectively cleaves backbone -1,6 linkages to retain -1,2 and
branched -1,6 linkages as well as -1,2 and -1,3
linkages(4, 15, 26) . Structures of the
resultant oligosaccharides were determined by one- and two-dimensional
NMR techniques. Consequently, we demonstrated the presence of the third
type of -1,2 linkage containing side chains in Candida species mannan corresponding to antigenic factor 9.
Materials
The C. guilliermondii IFO 10279 strain was obtained from the Institute for Fermentation,
Osaka (IFO), Japan. Linear -1,6-linked mannan prepared from the
cells of S. cerevisiae X2180-1A-5 (mnn2) strain,
which was developed by Ballou and co-workers(28, 29) ,
was the same specimen used in an earlier paper(30) . Factors 5
and 9 sera of ``Candida Check'' (lot number I751), a
commercially available kit of rabbit polyclonal antibodies against Candida cells, were purchased from Iatron (Tokyo, Japan). Jack
bean -mannosidase (EC 3.2.1.24) was obtained from Sigma.Preparation and Acetolysis of Mannan
Yeast cells
were grown at 28 °C in shaking liquid culture containing 0.5% yeast
extract, 1% peptone, and 2% glucose. Mannan was extracted from the
cells with water at 135 °C for 3 h and was separated by
precipitation with Fehling's solution(26) . The mannan
prepared from the cells of C. guilliermondii IFO 10279 strain
was designated as fraction G. Acetolysis under conventional (31) and mild (4) conditions was performed as described
in a preceding paper(26) . Fractionation of the resultant
mannooligosaccharide mixture was achieved using a column (2.5 
100 cm) of Bio-Gel P-2 (extra fine). Elution was carried out with
water, and aliquots of eluates were assayed for carbohydrate content by
the phenol-sulfuric acid method(32) . Separation of higher
oligosaccharides by HPLC was carried out with a column (10 500
mm) of YMC-Pack PA-25. Elution was done with a 52:48 (v/v) mixture of
CH
CN and water, and the eluates were monitored with a
differential refractometer. Eluates corresponding to each peak were
rechromatographed on the same column.Nuclear Magnetic Resonance Spectroscopy
All H NMR experiments were performed with a JEOL JNM-GSX 400
spectrometer at 400 MHz. 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) (33) was used as the internal
standard for H NMR.Inhibition Test of Enzyme-linked Immunosorbent
Assay
Enzyme-linked immunosorbent assay was conducted as
described in a preceding paper(13) . Enzyme-linked
immunosorbent assay-inhibition test using a factor serum was basically
conducted as described by Okawa et al.(34) . A
haptenic oligosaccharide solution (50 ml) was mixed with 5-fold-diluted
factor 9 serum (50 ml) and preincubated for 2 h at 25 °C. The
reaction mixture was then added to the wells of a fraction G-coated
microtiter plate and incubated for 2 h at 25 °C. After washing,
1000-fold-diluted goat anti-rabbit IgG antibody peroxidase-conjugate
was added to the wells and kept for 2 h at 25 °C. Finally, a
substrate solution of 0.01% o-phenylenediamine and 0.006%
HO in 150 mM citrate buffer (pH 5.0)
(100 ml) was added, and the color was measured at 492 nm after the
addition of 2 M HSO
(50 ml).Methylation Analysis
Methylation of
oligosaccharides was performed according to Ciucanu and
Kerek(35) . 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
For -mannosidase treatment, the
mannooligosaccharide mixture (200 mg) was dissolved in 50 mM sodium acetate buffer (pH 4.6) (2 ml) containing 20 units of
-mannosidase. After incubation for 48 h at 37 °C, the reaction
mixture was boiled for 5 min to inactivate the enzyme. Total
carbohydrate was determined by the phenol-sulfuric acid method of
Dubois et al.(32) with D-mannose as the
standard. Total phosphate was determined by the method of Ames and
Dubin(36) , using KHPO as the standard.
Preparation of Mannan
The cell wall mannan
prepared from the crude extract of C. guilliermondii IFO 10279
strain cells by the Fehling's solution method was designated as
fraction G. Clearly, this mannan contains no phosphodiesterified
oligosaccharides, which have been found in the mannan of C.
albicans(1, 26) , judging from the lack of an H-1
signal at about 5.55 ppm on H NMR corresponding to the
1-O--phosphorylated mannose
unit(1, 13, 37) , unreactivity to factor 5
serum(25, 38) , and a negative result for phosphate
analysis.Acetolysis of Mannan
Fig. 1A shows
the elution pattern of the acetolysate of fraction G from the Bio-Gel
P-2 column obtained under the conventional conditions. Each
oligosaccharide from biose to heptaose was rechromatographed by the
same column and designated as AM to AM
. To
obtain side chain oligosaccharides with -1,2-linked mannose units
and -1,6-linked branching ones, we also carried out the mild
acetolysis of fraction G. By this fragmentation, oligosaccharides up to
decaose were obtained (Fig. 1B). These oligosaccharides
were then digested with -mannosidase, and the enzyme-resistant
oligosaccharides were applied on a column of Bio-Gel P-2. However, it
was difficult to separate the higher oligosaccharides from each other (Fig. 1B). Therefore, these oligosaccharides were
separated by HPLC and rechromatographed by the same column. The
oligosaccharides from hexaose to decaose were designated as BM
to BM
.
100 cm) of Bio-Gel P-2 before
(
) and after (
) -mannosidase treatment. A,
acetolysis was performed with
(CHCO)O/CHCOOH/HSO (10:10:1, v/v/v) at 40°C for 12 h (conventional conditions). B, acetolysis was performed with
(CHCO)O/CHCOOH/HSO (100:100:1, v/v/v) at 40°C for 36 h (mild conditions). C, elution pattern of -mannosidase-treated acetolysate B
by HPLC with a column of YMC-Pack PA-25. AM-AM in panel A indicate mannobiose to mannoheptaose obtained
by the conventional acetolysis. BM-BM in panel C indicate mannohexaose to mannodecaose obtained by the
mild acetolysis followed by -mannosidase
treatment.
The H NMR Analysis of OligosaccharidesH NMR spectra of the oligosaccharides from AM to AM obtained by the conventional acetolysis were
the same as those obtained from the mannans of S. cerevisiae(33, 39) or S.
kluyveri(15, 40) . Namely, AM and
AM consist only of -1,2 linkages, and AM contains a nonreducing terminal -1,3-linked mannose unit.
Because AM
shows signals at 5.379 and 5.042 ppm, the
-1,3-linked mannose unit of this oligosaccharide is substituted by
an -1,2-linked mannose unit (40, 41) (Fig. 2). AM shows an
additional signal at 5.139 ppm, and the signal dimension at 5.027 ppm
was doubled compared with that of AM. This result suggests
that AM contains two -1,3-linked mannose units at the
first and the third positions from the nonreducing terminal. AM shows signals corresponding to a -1,2-linked mannose unit,
4.758 ppm, and an -1,3-linked mannose unit substituted by a
-1,2-linked one, 5.245 ppm, both of which were found in the mannan
of S. kluyveri(15) . The conventional acetolysis
conditions cleave -1,2 and -1,6 linkages; therefore, part of
these oligosaccharides seems to correspond to a degradation product of
the parent side chains.
H
NMR spectra of oligosaccharides obtained from fraction G by acetolysis
under the conventional (A) and the mild (B)
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). AM-AM and
BM-BM are designated as in the legend to Fig. 1.
H NMR
spectra of oligosaccharides higher than pentaose obtained by the mild
acetolysis commonly show signals at about 4.84 ppm corresponding to two
consecutive -1,2-linked mannose
units(3, 4, 15) . As shown in the preceding
papers(4, 15) , the H-1 proton of an -1,2-linked
mannose unit substituted by a consecutive -1,2-linked one, 5.138
ppm, appears at about 0.02 ppm upfield from that substituted by a
single -1,2-linked one, 5.160 ppm. Therefore, the signal at
5.236-5.245 ppm of the H-1 proton of an -1,3-linked mannose
unit substituted by a single -1,2-linked mannose unit (15) seems to shift to about 5.22 ppm with the addition of
consecutive -1,2-linked units. Because BM shows a
signal corresponding to an -1,2-linked mannose unit substituted by
an -1,3-linked mannose unit, 5.033 ppm, it is reasonable to assign
the signal at 5.218 ppm to the -1,3-linked mannose unit
substituted by consecutive -1,2-linked mannose units. Therefore,
we can propose that the chemical structure of BM is as
follows.
shows a new signal corresponding to an
-1,6-linked mannose unit, 4.914 ppm, in addition to the signals of
BM. Furthermore, as observed on the branched
oligosaccharides obtained from the mannans of C. albicans(26) and S. kluyveri(15) , the signal at
5.276 ppm corresponding to an -1,2-linked mannose unit, Man-B, of
BM was also shifted upfield to 5.232 ppm on BM.
This result suggests that the -1,6-linked mannose unit is attached
to the 3-O-substituted one, Man-C, of BM. 
shows new signals at 5.223, 4.846, and 4.838 ppm in
addition to those of AM. This indicates that the structure
of BM was that of AM with two -1,2-linked
mannose units at the nonreducing terminal as
follows.
and BM contain
one and two -1,6-linked mannose units, respectively, judging from
the dimension of the signals at about 4.91 ppm. In the spectrum of
BM, about two-thirds of the signal at 5.368 ppm
corresponding to Man-D seems to be shifted upfield to 5.311 ppm by the
addition of an -1,6-linked mannose unit to Man-E of
BM. Furthermore, about one-third of the signal at 5.269 ppm
corresponding to Man-B is also shifted upfield to 5.231 ppm as the
result of the attachment of an -1,6-linked mannose unit to Man-C.
Namely, BM seems to be a mixture of two isomers with a
difference in the branching point. Finally, the two -1,6-linked
mannose units of BM seem to attach on Man-C and Man-E
judging from the presence of two upfield-shifted signals at 5.291 and
5.226 ppm.Determination of Branching Point
From the
structural study of S. kluyveri(15) and C.
albicans(26) mannans, we can speculate that the -1,6
branching point of the side chain of C. guilliermondii mannan
is the 3-O-substituted mannose units. Although the H-1 signal
of a mannose unit does not shift by phosphorylation or glycosylation at
the O-6 position, the attachment affects the chemical shift of some
ring proton signals allocated around the substituted
position(15, 26, 42, 43, 44) .
Therefore, to detect the branching point of oligosaccharides, we
recorded one- and two-dimensional HOHAHA spectra, and we found that the
shifted ring protons correlated only with the 3-O-substituted
mannose units of BM, BM, and BM compared with those of BM and BM (data
not shown). These findings demonstrate that the branching point of
these oligosaccharides is the 3-O-substituted mannose unit.
This result was confirmed by methylation analysis of the
oligosaccharides as shown in Table 1. Namely, BM,
BM, and BM gave
2,4-di-O-methyl-1,3,5,6-tetra-O-acetyl mannitol,
which corresponds to the 3,6-di-O-substituted mannose unit. On
the other hand, 2,4,6-tri-O-methyl-1,3,5-tri-O-acetyl
mannitol, which corresponds to the 3-O-substituted mannose
unit, was obtained from BM, BM, and BM but was not obtained from BM and BM.
Sequential NMR Assignment
A sequential assignment
study of the H-1 and H-2 signals of these oligosaccharides was
performed to confirm the structure by the method described by Hernandez et al.(42) with slight
modification(8, 26) . The right side of the diagonal of each panel in Fig. 3shows the
relayed COSY, whereas the left side shows the rotating frame
NOE spectroscopy. In this figure, cross-peaks labeled with primed
letters indicate through-space interresidue H-1-H-2` or H-1-H-3`
connectivities between two adjacent mannose units. On the other hand,
cross-peaks labeled with unprimed letters indicate
intraresidue H-1-H-2- or H-1-H-3-correlated cross-peaks caused by J-coupling. By this procedure, the H-1 and H-2 signals of
BM were sequentially assigned from the H-1 of the Man-A,
A-A`-B-B`-C-C`-D-D`-E-E`-F (Fig. 3). Similarly, the H-1 and H-2
signals of BM, BM, BM, and
BM were assigned sequentially (Fig. 3). Because an
-1,3 linkage gives a weak H-1-H-2` NOE cross-peak in addition to a
strong H-1-H-3` NOE cross-peak, we can use the H-1-H-2` NOE cross-peak
c` instead of the H-1-H-3` NOE cross-peak C` for the assignment or to
confirm the connectivity. Especially for BM, two
H-1-H-2-correlated cross-peaks c and e of 3-O-substituted
mannose units, Man-C and Man-E, were better separated than the
corresponding two H-1-H-3-correlated ones, C and E. Therefore, it was
effective to use the cross-peaks c and e and H-1-H-2` NOE cross-peaks
c` and e`.
, BM, BM, and
BM. The right side of the diagonal shows
the relayed COSY, and the left side of the diagonal shows the rotating frame NOE spectroscopy. Primed letters indicate interresidue H-1-H-2` or H-1-H-3` NOE cross-peaks, and unprimed letters indicate the H-1-H-2- and the
H-1-H-3-correlated cross-peaks, caused by J-coupling; e.g.
A indicates the H-1-H-2-correlated cross-peak of the reducing
terminal mannose unit, Man-A, and A` indicates the
interresidue NOE cross-peak between the H-2 of Man-A and the H-1 of an
adjacent mannose unit, Man-B. By this procedure, the H-1 and H-2
signals were sequentially assigned from the H-1 of Man-A,
A-A`-B-B`-C-C`-(or c-c`-)D-D`-E-E`-F for
BM.
-1,6-linked mannose unit to
Man-C and Man-E causes an upfield shift of the H-1 signals of Man-B and
Man-D, respectively, due to a steric effect(15, 26) .
Two-dimensional Homonuclear Hartmann-Hahn Spectroscopy of
Mannan
The molar ratio of the mannan side chains was calculated
from the dimensions of the H-1 and H-2 signals of fraction G (Fig. 4A) based on the assignment result of the cross-peaks
on the two-dimensional HOHAHA spectrum (Fig. 4B) followed
by the method described previously(15) . As shown in Table 3, the cross-peaks 1 and 3 in the two-dimensional HOHAHA
spectrum of fraction G were assigned to correspond to
2-O-substituted -1,3- and -1,2-linked mannose units,
respectively. The cross-peaks 2 and 5 were found to correspond to the
upfield-shifted ones of cross-peaks 1 and 3, respectively, by the
effect of the presence of an -1,6-linked branching mannose unit as
shown in Table 2. Cross-peaks 4 and 6 correspond to
-1,3-linked mannose units substituted with a -1,2 linkage by
mannose and -1,2-linked mannobiose unit(s), respectively. On the
other hand, cross-peaks 7 and 8 correspond to -1,2-linked mannose
units substituted by -1,2-linked one(s)(15) . The absence
of these cross-peaks indicates that this mannan has no side chains
corresponding to the C. albicans serotype A-specific epitope,
antigenic factor 6. Cross-peak 12 indicates the presence of the
3-O-substituted -1,2-linked mannose unit. Although the
H-1 signal at 5.037 ppm overlaps those of cross-peaks 11 and 12, the
H-2 signal at 4.213 ppm corresponds only to the cross-peak 12.
Therefore, we can determine the molar ratio of the
3-O-substituted -1,2-linked mannose units or that of the
-1,3-linked ones in mannan as shown in Table 3. To
distinguish the NMR spectra of the two kinds of -1,6-linked
mannose units, branch and backbone forming ones, the signals of the
ring protons of both mannose units were compared. Fig. 4C indicates the one-dimensional HOHAHA spectrum of BM recorded by the irradiation of the signal at 4.914 ppm
corresponding to the branching -1,6-linked mannose unit. On the
other hand, Fig. 4D shows a normal H NMR
spectrum of a linear -1,6-linked backbone mannan prepared from S. cerevisiae X2180-1A-5 (mnn2) mutant strain cells.
As shown in Fig. 4C, the H-4 signal of the former
mannose unit appeared at a characteristic region, 3.66-3.68 ppm,
and gave cross-peaks 18 and 19 corresponding to the H-1-H-4- and
H-2-H-4-correlated ones, respectively. This finding suggests that the
cross-peaks 13, 18, and 19 correspond to the branching -1,6-linked
mannose unit, and cross-peak 14 corresponds to the backbone
-1,6-linked mannose unit.
H NMR spectrum of fraction G, (B)
two-dimensional HOHAHA spectrum of fraction G, (C) one-dimensional
HOHAHA spectrum of BM recorded by the irradiation of the
signal at 4.914 ppm corresponding to the branching -1,6-linked
mannose unit, (D) normal H NMR spectrum of linear
-1,6-linked mannan obtained from the cells of the S.
cerevisiae X2180-1A-5 (mnn2)
strain.
-1,6-linked branching
mannose units is slightly smaller than that of the total
-1,3-linked ones but is sufficient to attach to all of the
3-O-substituted ones in the -1,2 linkage-containing side
chains. Namely, BM, BM, and BM correspond to the degradation products of BM or
BM on mild acetolysis. From these results, we can propose
the chemical structure of the cell wall mannan of C. guilliermondii IFO 10279 strain as shown in Fig. 5.
H NMR signals in Fig. 4.
Haptenic Activity of Side Chain
Oligosaccharides
C. guilliermondii cells have antigenic
factors 1, 4, and 9(24, 25) . Therefore, we examined
the inhibitory effect of BM to BM on the
reactivity of factor 9 serum to fraction G on enzyme-linked
immunosorbent assay. As shown in Fig. 6, these oligosaccharides
showed the same strong inhibitory effect despite the presence or the
absence of the -1,6-linked branching mannose unit. On the other
hand, Man12Man12Man12Man (antigenic
factor 5),
Man12Man12Man12Man1 2Man,
and
Man12Man12Man12Man12Man1
2Man (antigenic factor 6) showed no inhibitory effect. These results
indicate that the third type of -1,2 linkage containing
oligosaccharide moieties,
Man12Man12Man1 3Man1, in
fraction G behaves as the antigenic factor 9.
(), BM (),
BM (
), BM (
), M4
(Man12Man1 2Man12Man) (),
M5
(Man12Man12Man12Man1 2Man)
(), and M6
(Man12Man12Man12Man1
2Man12Man) (
), for 2 h, at 25 °C, was added.
After the mixture was allowed to stand for 2 h, 1000-fold-diluted goat
anti-rabbit IgG antibody was added, and binding was detected as
described under ``Experimental
Procedures.''
-1,6-branched side chains of the mannan with a comb-like
structure(26) .-1,6-linked mannose unit from the reactivity of factor
9 serum to the cells of the S. cerevisiae X2180-1A-5 (mnn2) mutant strain, which have linear -1,6-linked
mannan. It is true that fraction G exposes about 50% of the
-1,6-linked backbone mannose units (Fig. 5). Because factor
9 serum was prepared simply by absorption of the anti-C.
guilliermondii whole cell serum with C. albicans serotype
A cells(24) , it is reasonable to expect that it contains
antibodies against several epitopes, including the backbone mannose
units. In this study, however, we could demonstrate the existence of
novel side chains containing a third type of -1,2-linked mannose
unit, BM and BM, as the specific structure for C. guilliermondii mannan corresponding to antigenic factor 9. -1,2-linked mannose unit substituted by a
3,6-di-O-substituted one, the effect of which was first found
on the branched side chains of the mannan of S. kluyveri ( = 0.047 ppm) (15) and later on that of C. albicans ( = 0.055 ppm)(26) , was
observed on BM, BM, and BM ( of Man-B = 0.044-0.049 ppm). In this
study, we also found the same effect of the H-1 signal of an
-1,3-linked mannose unit, Man-D, substituted by a
3,6-di-O-substituted one ( of Man-D = 0.062
ppm for BM and 0.082 ppm for BM). The large
upfield shift effect found in Man-D of BM seems to be due
to the attachment of two -1,6-linked mannose units to the
neighboring 3-O-substituted ones of Man-D at the reducing and
the nonreducing sides. The upfield shift of the H-1 signals seems to be
the result of a steric effect; therefore, it is of interest to identify
the conformation of these oligosaccharides.-1,6-branched side chains, we can speculate
that the -1,6-mannosyltransferase responsible for the biosynthesis
of branched side chains requires oligosaccharides containing an
-1,3 linkage as an acceptor. This hypothesis is supported by the
results of Pang et al.(48) . They reported that the
mannan of an S. kluyveri (mnn1) mutant strain that
lacks -1,3-mannosyltransferase activity also lacks the branching
-1,6-linked mannose unit. To understand the timing of the transfer
of an -1,6-linked mannose unit to the side chain, however, we need
to determine the substrate specificity of the
-1,6-mannosyltransferase. Recently, we detected a
-1,2-mannosyltransferase responsible for the synthesis of the
second type of -1,2 linkage (antigenic factor 6)(49) . Now
we are interested in an -1,6-mannosyltransferase and a
-1,2-mannosyltransferase responsible for the synthesis of the
branch and the third type of -1,2 linkage (antigenic factor 9),
respectively.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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