(Received for publication, November 3, 1995; and in revised form, January 30, 1996)
From the
Isolation of -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,
Man
1
2Man
1
3Man
, 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.
and
The H-1 signals of the mannose
units substituted by a 3,6-di-O-substituted unit showed a
significant upfield shift ( = 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.
We have reported the presence of two types of -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.
In an earlier paper(15) , we
demonstrated the presence of a third type of a -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.
There are several
reports on the responsibility of -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.
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 -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.
Figure 1:
Elution patterns of oligosaccharides
obtained from fraction G by acetolysis. A, B, elution
was performed with a column (2.5 100 cm) of Bio-Gel P-2 before
(
) and after (
)
-mannosidase treatment. A,
acetolysis was performed with
(CH
CO)
O/CH
COOH/H
SO
(10:10:1, v/v/v) at 40°C for 12 h (conventional conditions). B, acetolysis was performed with
(CH
CO)
O/CH
COOH/H
SO
(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.
Figure 2:
The anomeric region of the 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 D
O 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.
On the other hand, the 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.
BM 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
.
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.
It is obvious that BM 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.
Figure 3:
Sequential connectivities of the mannose
units of BM, 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
.
The results summarized in Table 2clearly
demonstrate that the attachment of an -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) .
Figure 4:
Assignment of H-1 and H-2 signals of
fraction G. (A) Normal 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.
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 -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.
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 H NMR signals in Fig. 4.
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 (
), BM
(
),
BM
(
), BM
(
),
M4
(Man
1
2Man
1
2Man
1
2Man) (
),
M5
(Man
1
2Man
1
2Man
1
2Man
1
2Man)
(
), and
M6
(Man
1
2Man
1
2Man
1
2Man
1
2Man
1
2Man) (
), 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.''
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 -1,6-branched side chains of the mannan with a comb-like
structure(26) .
Ataoglu et al.(27) reported that the antigenic factor 9 corresponds to a
consecutive -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.
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
-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.
From the results of this
and the preceding (12, 26) structural studies of the
mannans containing -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.