Existence of novel b-1,2 linkage-containing side chain in the mannanserotype A Nobuyuki Shibata1, Hidemitsu Kobayashi2, Yoshio Okawa1and Shigeo Suzuki3 1 Second Department of Hygienic Ch
Trang 1Existence of novel b-1,2 linkage-containing side chain in the mannan
serotype A
Nobuyuki Shibata1, Hidemitsu Kobayashi2, Yoshio Okawa1and Shigeo Suzuki3
1
Second Department of Hygienic Chemistry, Tohoku Pharmaceutical University, Sendai, Miyagi, Japan;2Department of Nutrition, Faculty of Home Economics, Kyushu Women’s University, Kitakyushu, Fukuoka, Japan;3Sendai Research Institute for Mycology, Sendai, Miyagi, Japan
The antigenicity of Candida lusitaniae cells was found to be
the same as that of Candida albicans serotype A cells, i.e
both cell wall mannans react with factors 1, 4, 5, and 6 sera
of Candida Check However, the structure of the mannan
of C lusitaniae was significantly different from that of
C albicansserotype A, and we found novel b-1,2 linkages
among the side-chain oligosaccharides, Manb1fi2Manb1fi
2Mana1fi2Mana1fi2Man (LM5), and
Manb1fi2Man-b1fi2Manb1fi2Mana1fi2Mana1fi2Man (LM6) The
assignment of these oligosaccharides suggests that the
mannoheptaose containing three b-1,2 linkages obtained
from the mannan of C albicans in a preceding study
con-sisted of isomers The molar ratio of the side chains of
C lusitaniae mannan was determined from the complete assignment of its H-1 and H-2 signals and these signal dimensions More than 80% of the oligomannosyl side chains contained b-1,2-linked mannose units; no a-1,3 link-ages or a-1,6-linked branching points were found in the side chains An enzyme-linked immunosorbent inhibition assay using oligosaccharides indicated that LM5 behaves as factor 6, which is the serotype A-specific epitope of
C albicans Unexpectedly, however, LM6 did not act as factor 6
Keywords: Candida lusitaniae; mannan; NMR analysis; serotype A; b-1,2-linkage
The first description of the serological heterogeneity in
Candida albicanswas reported by Hasenclever and Mitchell
[1] These researchers revealed that this species could be
divided into two serotypes, A and B, by agglutination
reaction between heat-killed whole cells of C albicans
strains and rabbit anti-C albicans whole cell sera of both
serotypes absorbed with whole cells of another serotype
Later, Tsuchiya and collaborators [2] conducted an
exten-sive serological study on a variety of yeasts based on the
ability to agglutinate rabbit antiserum of other heat-killed
fungal cells; from these data they developed a series of
absorbed factor sera ÔCandida CheckÕ, a commercially
available kit containing 10 factor sera is an effective tool for
identifying the cells of Candida species in clinical specimens
Candida lusitaniaecan be pathogenic and cause
nosoco-mial infection in immunocompromised patients,
predomin-antly in granulocytopenic patients undergoing cytoreductive
chemotherapy for acute leukemia and in bone marrow
transplantation recipients [3–6] Although this species is less
virulent than most other Candida species, it is important
because of its propensity to develop resistance to antifungal
agents, including amphotericin B [5–8] C albicans serotype
A strains have been isolated predominantly from patients
with invasive candidiasis However, C lusitaniae expresses
the same antigenic pattern as that of C albicans serotype A, i.e both cell strains have antigenic factors 1, 4, 5 and 6 [9,10] Therefore, it is difficult to distinguish these strains from the antigenic pattern alone
The cell wall mannan of C albicans serotype A is composed of an a-1,6-linked backbone moiety and an oligomannosyl side-chain moiety consisting of a-1,2-, a-1,3-, and b-1,2-linked mannose units Furthermore, a small number of a-1,6-linked branching mannose units may be present in the side chain In addition to this acid-stable structure, there is also a region composed of b-1,2-linked oligomannosyl residues linked through a phosphate group
to the side chain Because the phosphodiester linkage is acid-labile, we can selectively release the b-1,2-linked manno-oligosaccharides by treatment with 10 mMHCl We have shown that factor 6 serum reacts with acid-stable oligo-mannosyl side chains containing b-1,2 and a-1,2 linkages – the serotype A-specific structure – and that factor 5 serum reacts with the acid-labile b-1,2-linked oligomannosyl moieties [11]
We have reported the presence of three kinds of b-1,2 linkage-containing side chains in the mannans from Can-dida species [11–17] Because the b-1,2-linked mannose unit
is present among the pathogenic fungi only in the mannan
of the genus Candida and the b-1,2 linkage-containing side chains behave as strong antigens, many workers [18–25] have produced monoclonal antibodies to b-1,2-linked mannose units to protect against candidiasis, for the serodiagnosis of candidiasis, or for the identification of mechanisms of Candida infection Furthermore, studies have indicated the b-1,2-linked mannose units participate in
Correspondence to S Suzuki, Sendai Research Institute for Mycology,
1-14-34 Toshogu, Aoba-ku, Sendai, Miyagi 981–0908, Japan.
Fax: + 81 22 2754246, Tel.: + 81 22 2754680.
(Received 7 November 2002, revised 20 March 2003,
accepted 16 April 2003)
Trang 2the adherence of Candida cells to mammalian cells as the
first step in infection [26–28] Poulain and his coworkers
[29–32] reported that the b-1,2 linkage-containing
oligosac-charides and phospholipomannans induce cytokine
pro-duction and may act as a virulent factor in candidiasis
Therefore, it is important to determine the detailed chemical
structure of the cell wall mannan, especially the b-1,2
linkage-containing side chains
In earlier structural studies by NMR, we identified
almost all of the H-1–H-2-correlated cross-peaks of the
C albicans serotype B [33], Candida stellatoidea [34],
Candida guilliermondii [17], and Candida saitoana [35]
mannans from assignment of the H-1 and H-2 signals of
many mannooligosaccharides Although the H-1 proton
signal provides the best information on the glycosidic
linkage, several H-1 signals overlap The H-2 signal, a
second structure reporter, can resolve the overlapped H-1
signals into H-1–H-2-correlated cross-peaks; therefore, we
were able to determine the dimensions of the resolved H-1
signals using the two-dimensional (2D) HOHAHA
spec-trum Because the ratio of the mannose units is
propor-tional to that of the corresponding signal dimensions, we
can estimate the molar ratio of the side chains However,
we have not completely assigned the H-1–H-2-correlated
cross-peaks of the mannan of C albicans serotype A yet,
because the mannan produces a spectrum containing a
very complicated pattern of cross-peaks However, the
2D-HOHAHA spectrum of C lusitaniae mannan was
simpler than that of the C albicans serotype A mannan,
which prompted us to assign the cross-peak s of C
lusit-aniaemannan first These accumulated data will make it
possible for us to predict the structures of other mannans
from their 2D-HOHAHA spectra
The object of this study was to determine the detailed
chemical structure of the mannan of C lusitaniae, which is
antigenically related to C albicans serotype A, using NMR
techniques with the aim of elucidating the complete
structure of the C albicans serotype A mannan
Experimental procedures
Materials
The C lusitaniae IFO 1019 strain was obtained from the
Institute for Fermentation (Osaka, Japan) The C albicans
J-1012 strain (serotype A) mannan was the same specimen
used in preceding studies [11,16] Factors 1, 4, 5, 6, 9, and
13b sera of ÔCandida CheckÕ (lot number I675), a
commer-cially available kit containing rabbit polyclonal antibodies
to Candida cells, were purchased from Iatron (Tokyo,
Japan) Except for factor 1 serum, which is unabsorbed
rabbit whole-cell serum against C albicans cells, factors 4,
5, 6, and 13b sera are anti-C albicans sera absorbed with
cells of Candida parapsilosis, C guilliermondii, C
stellato-idea, and Candida tropicalis, respectively Factor 9 serum is
anti-C guilliermondii serum absorbed with cells of
C albicans [2] Jackbean a-mannosidase (EC 3.2.1.24)
was obtained from Sigma Haptenic
oligosaccha-rides, Manb1fi2Mana1fi3Mana1fi2Mana1fi2Man and
Manb1fi2Manb1fi2Mana1fi3Mana1fi2Mana1fi2Man
were prepared from the mannan of C guilliermondii [17]
or C saitoana [35] and Manb1fi2Mana1fi2Mana1fi
2Mana1fi2Man and Manb1fi2Manb1fi2Mana1fi 2Mana1fi2Mana1fi2Man were obtained from the man-nan of C albicans J-1012 (serotype A) [16]
Preparation of mannan Yeast cells were grown at 28C with shaking in a liquid culture containing 0.5% yeast extract, 1% peptone, and 2% glucose Mannan was extracted from the cells with water at
135C for 3 h and was separated by precipitation with Fehling’s solution [33] The mannan prepared from the cells
of the C lusitaniae IFO 1019 strain was designated Fr L Acid treatment of mannan
To determine the amount of acid-labile phosphodiesterified oligosaccharides in the mannan, 500 mg of Fr L were dissolved in 50 mL of 10 mMHCl, and held at 100C for
1 h [13] After cooling, the reaction mixture was neutralized with 100 mM NaOH and was separated by column chromatography (2.5· 100 cm) using Bio-Gel P-2 (extra fine) Elution was conducted with water, and aliquots of the eluates were assayed for carbohydrate content using the phenol–sulfuric acid method [36] Because this treatment cleaves the phosphodiester linkage, the acid-stable moiety of the mannan is eluted in the void volume and the manno-oligosaccharides released from the mannan are retained by the column The acid-stable moiety of the mannan was designated Fr L-a
Acetolysis of mannan Acetolysis under mild conditions [37] was performed as previously described [38] Briefly, acetylated mannan was dissolved in 100 : 100 : 1 (v/v/v) acetic anhydride–acetic acid–sulfuric acid and held at 40C for 36 h After deacetylation using sodium methoxide, fractionation of the resultant mannooligosaccharide mixture was achieved
by HPLC This treatment selectively cleaves the backbone a-1,6 linkages and yields an oligosaccharide mixture that originates from the oligomannosyl side-chain moieties HPLC of oligosaccharides
HPLC was carried out using a column (10· 500 mm) of YMC-PackPA-25 Elution was carried out with 52 : 48 (v/v)
CH3CN–water, and the eluates monitored using a differen-tial refractometer [17] Eluates corresponding to each peak were rechromatographed on the same column
Nuclear magnetic resonance spectroscopy All1H 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 D2O at 45C with acetone (2.217 p.p.m.) [39] as the internal standard
Enzyme-linked immunosorbent inhibition assay The enzyme-linked immunosorbent assay was conducted
as described previously [11] Assays using factor sera were
Trang 3conducted basically as described by Okawa et al [40] A
haptenic oligosaccharide solution (50 lL) was mixed with a
100-fold dilution of factor 6 serum (50 lL) and
preincu-bated at 25C for 2 h The reaction mixture was then added
to the wells of a Fr L-a-coated microtiter plate and
incubated at 25C for 2 h After washing, a 1000-fold
dilution of goat anti-(rabbit IgG) antibody–peroxidase
conjugate (100 lL) was added to the wells and held at
25C for 2 h Finally, a substrate solution of 0.01%
o-phenylenediamine and 0.03% H2O2 in 150 mM citrate
buffer (pH 5.0) (100 lL) was added, followed by the
addition of 2M H2SO4 (50 lL), and the color measured
at 492 nm
Other methods
For a-mannosidase treatment, the mannooligosaccharide
mixture (200 mg) was dissolved in 50 mMsodium acetate
buffer (pH 4.6; 2 mL) containing 20 U of a-mannosidase
After incubation at 37C for 48 h, the reaction mixture was
boiled for 5 min to deactivate the enzyme Total
carbohy-drate content was determined by the phenol–sulfuric acid
method of Dubois et al [36] with D-mannose as the
standard
Results
Prediction of the structure of Fr L from its 2D-HOHAHA
spectrum
Fr L showed strong reactivity with factor 1, 4, 5 and 6 sera
on ELISA (Fig 1), indicating that this mannan possesses
the same antigenic determinants as those of C albicans
serotype A [11] Therefore, we compared the structures
of the two mannans using their 2D-HOHAHA spectra Figure 2 shows the H-1 region ( 4.7–5.6 p.p.m.) of the one-dimensional (1D) NMR spectrum and the 2D-H-1– H-2-correlated cross-peaks of Fr L and the mannan of
C albicansJ-1012 The H-1 region of Fr L gave relatively simple signals in the a-anomeric region ( 4.9–5.6 p.p.m.) and signals at the b-anomeric region ( 4.7–4.9 p.p.m.) that were two times larger than the 1D-NMR signals of the mannan of C albicans J-1012 Several structural differences between the mannans are apparent The differences between cross-peaks 5, 6, 7 and 8, which correspond to a-1,2-linked mannose units in the mannans, suggests differences in the lengths of the a-1,2-linked side-chain moieties The spec-trum of Fr L does not show cross-peaks 3, 4, 9 or 17, which correspond to a-1,3-linked mannose units or 3-O-substi-tuted ones Furthermore, Fr L does not show cross-peaks
4, 8, 15, 18, 19 or 30, suggesting that the mannan does not have b-1,2-substituted a-1,3-linked mannose units or a-1,6-linked branching mannose units [17,33–35,38,41] In con-trast, both mannans contain cross-peaks 25, 27, and 28, which correspond to the b-1,2-linked mannose units [17,35,38]
Acid treatment of Fr L
To determine the structures and amounts of the phospho-diesterified oligosaccharides, we treated Fr L with 10 mM HCl at 100C for 60 min As shown in Fig 3, the released oligosaccharide was predominantly triose The 1H NMR spectrum of the triose was identical to that obtained from the mannan of C albicans [14,42–44], indicating that it is the b-1,2-linked mannotriose The polysaccharide moiety eluted
in the void volume fraction was designated Fr L-a Acetolysis of Fr L-a
Figure 4A shows the HPLC elution pattern of the aceto-lysate of Fr L-a from a YMC PA-25 column Oligosac-charides up to hexaose were obtained from this fractionation These oligosaccharides were then digested with a-mannosidase, and the reaction products separated by HPLC (Fig 4B) The resistance of tetraose, pentaose, and hexaose to a-mannosidase degradation indicates that these oligosaccharides contain b-linkages The oligosaccharides from tetraose to hexaose obtained by a-mannosidase treatment were designated LM4 to LM6
1
H NMR analysis of oligosaccharides Figure 5 shows the H-1 region of the1H NMR spectra of M3 and LM4 to LM6 As we expected, the signals corresponding to the b-1,2-linked mannose units from 4.776 to 4.918 p.p.m were present in LM4, LM5 and LM6 Although the signals corresponding to the b-1,2-linked mannose units of LM4 and LM5 (4.776–4.853 p.p.m) were the same as those of the one and two b-1,2 linkage-containing oligosaccharides, respectively, obtained from Candida mannans, the 2 : 1 ratio of signal intensities at 4.918 and 4.845 p.p.m of LM6 was different from the corresponding three b-1,2 linkage-containing mannohepta-ose and mannopentamannohepta-ose obtained from the mannans of
Fig 1 Enzyme-linked immunosorbent assay of Fr L The assay was
carried out using the factor sera of Candida Check s, factor 1 serum;
d, factor 4 serum; n, factor 5 serum; m, factor 6 serum; h, factor 9
serum; j, factor 13b serum These reactivities were exactly the same as
those of the mannan of C albicans serotype A [11].
Trang 4Fig 2 Two-dimensional HOHAHA spectra of
Fr L and the mannan of C albicans J-1012 (serotype A) The boxed or circled regions in the spectra indicate the H-1–H-2-correlated cross-peaks, except for cross-peak 30, which indicates an H-1–H-4 correlated one The circled regions correspond to the b-mannose units in the phosphodiesterified acid-labile b-1,2-linked mannooligosaccharide Assign-ments of these cross-peaks are shown in Table 2.
Fig 3 Determination of the phosphodiesterified acid-labile
oligosac-charide Fr L was treated with 10 m M HCl at 100 C for 1 h, and the
degradation products were separated using a column (2.5 · 100 cm) of
Bio-Gel P-2 (A) In addition to a peakin the void volume (Fr L-a),
one oligosaccharide peak(fractions 109–118) was recovered The 1 H
NMR spectrum (B) of the released oligosaccharide was identical to
that of the b-1,2-linked mannotriose [43].
Fig 4 Elution patterns of oligosaccharides obtained from Fr L-a by acetolysis HPLC was performed with a YMC PA-25 column (10 · 500 mm) (A) before and (B) after a-mannosidase treatment Elution was carried out with 52 : 48 (v/v) CH 3 CN–water Acetolysis was performed with (CH 3 CO) 2 O–CH 3 COOH–H 2 SO 4 (100 : 100 :
1 v/v/v) at 40 C for 36 h M1, M2, M3 and M4 indicate mannose, a-linked mannobiose, mannotriose, and mannotetraose, respectively LM4, LM5, and LM6 indicate the b-linkage-containing mannotetra-ose, mannopentamannotetra-ose, and mannohexamannotetra-ose, respectively.
Trang 5C albicans serotype A [16] and Citeromyces matoritensis
[45], respectively As the a-1,6-linked mannose unit also
shows a signal at about 4.91 p.p.m [17,33,34], we cannot
assign the signal from 1D-NMR Therefore, we analyzed
these b-1,2 linkage-containing oligosaccharides using
2D-NMR
Sequential NMR assignment
Sequential assignment of the H-1 and H-2 signals of LM4,
LM5, and LM6 was performed using the NOE cross-peaks
of the NOESY spectrum by the method described by
Her-nandez et al [46] with slight modifications [17,43] (Fig 6)
The NOE cross-peaks labeled with primed letters indicate
through-space inter-residue H-1–H-2¢ connectivities be-tween two adjacent mannose units The boxed cross-peaks labeled with unprimed letters indicate intraresidue H-1– H-2-correlated cross-peaks Inter-residue cross-peaks due to dipolar coupling are identified by their absence from a COSY spectrum (not shown), which shows only the intraresidue cross-peaks due to J coupling Using this procedure, the H-1 and H-2 signals of LM4 were sequen-tially assigned as shown in the leftmost panel of Fig 6 The spectrum in this panel shows inter-residue H-1–H-2¢ connectivities between a reducing terminal mannose unit (Man-A) with an H-1 signal at 5.348 p.p.m and a mannose unit with an H-1 signal at 5.276 p.p.m (A2–A2¢–B2), and between the latter (Man-B) and a mannose unit with an
Fig 5 The anomeric region of the1H NMR
spectra of oligosaccharides obtained from
Fr L-a by acetolysis Spectra were recorded
using a JEOL JNM-GSX 400 spectrometer in
D 2 O at 45 C using acetone as the internal
standard (2.217 p.p.m.) LM4, LM5 and LM6
are designated as indicated in the legend of
Fig 4.
Fig 6 Partial NOESY spectra of LM4, LM5,
and LM6 Sequential assignment of the H-1
and H-2 signals was performed using NOESY
and COSY spectra Primed letters indicate
inter-residue H-1–H-2¢-NOE cross-peaks and
unprimed letters indicate intraresidue H-1–
H-2-correlated cross-peaks, caused by J
coup-ling, e.g A2 indicates the H-1–H-2-correlated
cross-peakof the reducing terminal mannose
unit, Man-A, and A2¢ indicates the
inter-resi-due NOE cross-peakbetween 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, A2–A2¢–B2–B2¢–C2–C2¢–D2
for LM4.
Trang 6H-1 signal at 5.160 p.p.m (B2–B2¢–C2) Moreover,
inter-action between Man-C and the b-linked mannose unit with
an H-1 signal at 4.776 p.p.m (C2–C2¢–D2) is seen,
indica-ting that LM4 has the following structure:
Manb1!2Mana1 !2Mana1!2Man (LM4)
The H-1 and H-2 signals of LM5 and LM6 were also
sequentially assigned from the H-1 of Man-A, A2–A2¢–B2–
B2¢–C2–C2¢–D2–D2¢–E2 and A2–A2¢–B2–B2¢–C2-C2¢–
D2–D2¢–E2–E2¢–F2, respectively Although the H-1 of
Man-E and Man-F of LM6 gave almost the same chemical
shifts, we could assign the H-2 signal (not shown) of the
nonreducing terminal b-1,2-linked mannose unit as the
signal with a chemical shift at about 4.14 p.p.m from
the assignments of the phosphodiesterified b-1,2-linked
mannooligosaccharides [42,43] Therefore, we could
unam-biguously assign the H-1–H-2-correlated cross-peaks E and
F These results indicate that LM5 and LM6 have the
following structures:
Manb1!2Manb1!2Mana1!2Mana1!2Man(LM5)
Manb1!2Manb1!2Manb1!2Mana1!2Mana1!2Man
(LM6)
These assignment results indicate that cross-peaks 22, 23, and 24 on the 2D-HOHAHA spectrum in Fig 2 corres-pond to Man-E, -F, and -D of LM6, respectively
We also assigned other ring protons using the 2D-HOHAHA spectra; the results are shown in Table 1 Although a small number of side chains with the same structure of LM4 has been found in the mannan of
C glabrata[47], LM5 and LM6 are novel oligosaccharides
Determination of the molar ratio of mannan side chains
The molar ratio of the mannan side chains was calculated using the dimensions of the specific H-1 and H-2 signals corresponding to each side chain based on assignment results of the cross-peaks on the 2D-HOHAHA spectra (Fig 2) as previously described [17,34,35] The H-1 signal of the mannan from C lusitaniae at 5.152 p.p.m overlaps two signals at 5.160 p.p.m and 5.138 p.p.m corresponding to cross-peaks 20 and 21, respectively, as shown in Fig 2, and the amount of the two signals was 15.5% of the total H-1 signal dimension (Table 2) These signals were assigned as H-1 of the mannose unit substituted by b-1,2 linkages in LM4, LM5, and LM6 The H-1 signal at 4.778 p.p.m and H-2 signal at 4.401 p.p.m (not shown) simply correspond
to cross-peaks 28 and 22, respectively, and both were 1.9% of the total dimension Cross-peaks 28 and 22 were identified as the one and three b-1,2 linkage-containing oligomannosyl side chains, LM4 and LM6, respectively Therefore, the amount of the two b-1,2 linkage-containing
Table 1 Chemical shifts of the oligosaccharides obtained by acetolysis ND, not determined.
Sugar residue Chemical shift
LM4 Manb1fi2Mana1fi2Mana1fi2Mana
H-1 4.776 5.160 5.276 5.348 H-2 4.035 4.274 4.118 3.932 H-3 3.644 3.858 3.956 3.945 H-4 3.579 3.695 3.673 3.693 H-5 3.379 ND ND ND H-6 3.919 3.858 3.871 3.860 H-6¢ 3.738 3.759 3.738 3.499 LM5 Manb1fi2Manb1fi2Mana1fi2Mana1fi2Mana
H-1 4.841 4.853 5.140 5.262 5.340 H-2 4.148 4.261 4.251 4.113 3.933 H-3 3.610 3.655 3.887 3.954 3.944 H-4 3.567 3.598 3.605 3.667 3.692 H-5 3.348 3.389 ND ND ND H-6 3.914 3.916 3.854 3.866 3.854 H-6¢ 3.728 3.756 3.731 3.733 3.780 LM6 Manb1fi2Manb1fi2Manb1fi2Mana1fi2Mana1fi2Mana
H-1 4.918 4.914 4.845 5.150 5.267 5.345 H-2 4.147 4.399 4.245 4.265 4.119 3.940 H-3 3.613 3.631 3.690 3.898 3.959 3.950 H-4 3.575 3.589 3.503 3.592 3.675 3.702 H-5 3.379 3.362 3.395 ND ND ND H-6 3.929 3.916 3.936 3.854 3.866 3.865 H-6¢ 3.737 3.750 3.715 3.760 3.738 3.769
Trang 7oligomannosyl side chain, LM5, can be estimated using the
following formula
15:5ðlM4 + lM5 + lM6Þ 2 1:9 ðlM4 + lM6Þ
¼ 11:7 ðlM5Þ
Thus, the one, two, and three b-1,2 linkage-containing side
chains, which correspond to LM4, LM5, and LM6,
respect-ively, are in the ratio of 1.00 : 6.15 : 1.00 Similarly, we could
estimate the molar ratio of the a-linked side chains
From these results, we propose the chemical structure and the molar ratio of the side chains of the cell wall mannan of C lusitaniae as shown in Fig 7 Surprisingly, more than 80% of the oligomannosyl side chains were substituted by b-1,2-linked mannose units We could also identify that the circled cross-peaks a and b in Fig 2 correspond to the middle and the nonreducing terminal b-1,2-linked mannose units, respectively, of phosphodieste-rified b-1,2-linked mannotriose
Table 2 Assignments and dimensions of the NMR signals of Fr L Chemical shifts are for mannose residues shown in bold Signal dimensions were determined by integration of the peakarea in the 1 H NMR spectra or by calculation.
1
Cross peakChemical shift (p.p.m.) Residue Signal dimension (%)
1 5.554 4.210 Manb1fi2Mana1fiphosphate 0
2 5.542 4.189 Manb1fi2Manb1fi2Mana1fiphosphate 1.7
4 5.302 4.100 a1fi3Mana1fi2Mana1fi3Mana1fi2
Mana1 Mana1
0
8 5.233 4.096 a1fi3Mana1fi2Mana1fi2Mana1fi2
›6 Mana1
0
10 5.121 4.017 a1fi6Mana1fi6Mana1fi6Mana1fi6
›2 Mana1
11 5.104 4.029 a1fi6Mana1fi6Mana1fi6Mana1fi6
›2 ›2 ›2 Mana1
12 5.093 3.997 a1fi6Mana1fi6Mana1fi6Mana1fi6
›2 a1fi2Mana1
18.9
13 5.074 4.008 a1fi6Mana1fi6Mana1fi6Mana1fi6
›2 ›2 ›2 a1fi2Mana1
19 5.218 4.250 Manb1fi2Manb1fi2Mana1fi3 0
21 5.138 4.256 Manb1fi2Manb1fi2Mana1fi2 13.6
22 4.915 4.401 Manb1fi2Manb1fi2Manb1fi2Mana1fi2 1.9
23 4.918 4.147 Manb1fi2Manb1fi2Manb1fi2Mana1fi2 1.9
24 4.844 4.247 Manb1fi2Manb1fi2Manb1fi2Mana1fi2 1.9
25 4.857 4.263 Manb1fi2Manb1fi2Mana1fi2 11.7
26 4.849 4.269 Manb1fi2Manb1fi2Mana1fi3 0
27 4.844 4.152 Manb1fi2Manb1fi2Mana1fi2(3) 11.7
a 4.906 4.290 Manb1fi2Manb1fi2Mana1fiphosphate 1.7
b 4.850 4.173 Manb1fi2Manb1fi2Mana1fiphosphate 1.7
9
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Trang 8Haptenic activity of the b-1,2 linkage-containing
oligosaccharides
The reactivity of factor 6 serum to several b-1,2
linkage-containing oligosaccharides was tested using an
enzyme-linked immunosorbent inhibition assay between Fr L-a
and factor 6 serum As we expected, the b-1,2-substituted
a-1,3-linked mannose-containing oligosaccharides,
Man-b1fi2Mana1fi3Mana1fi2Mana1fi2Man and Manb1fi
2Manb1fi2Mana1fi3Mana1fi2Mana1fi2Man, obtained
from the mannan of C guilliermondii [17] or C saitoana
[35] did not behave as factor 6 (Fig 8) In contrast,
LM4 showed the same inhibition activity as that of
the C albicans-derived pentaose, Manb1fi2Mana1fi
2Mana1fi2Mana1fi2Man The inhibition activity of
LM5 was twice that of LM4, but was the same as that
of the C albicans-derived hexaose, Manb1fi2Manb1fi
2Mana1fi2Mana1fi2Mana1fi2Man Unexpectedly, LM6
showed no inhibition activity at all, suggesting that the
three-b-1,2 linkage-containing oligomannosyl side chain
does not behave as factor 6
Discussion
In previous papers, we reported two different lengths of
three b-1,2 linkage-containing oligosaccharides,
Man-b1fi2Manb1fi2Manb1fi2Mana1fi2Mana1fi2Mana1fi
2Man (AM7), and Manb1fi2Manb1fi2Manb1fi
2Mana1fi2Man (CM5), from the mannans of C albicans
serotype A [15,16] and Cit matritensis [45], respectively In
these reports, the ratio of the signals at 4.91 and 4.84 p.p.m
were about 1 : 2 However, the sequential assignment study
of LM6, which also contains three b-1,2-linked mannose
units, indicated that both Man-E and Man-F gave a signal
at 4.918 p.p.m., Man-D showed a signal at 4.845 p.p.m.,
and the ratio of the dimensions of the two signals was 2 : 1
These results suggest that both AM7 and CM5 were
mixtures of a two-b-1,2 linkage-containing and a three-b-1,2
linkage-containing oligosaccharide and that the assignment
results for the b-mannose units were incorrect
Because we could assign cross-peaks 22, 23, and 24 of
Fr L, we could also calculate the molar ratios of the three-b-1,2 linkage-containing oligosaccharide side chains of the mannan of C albicans serotype A However, this mannan also includes both b-1,2-substituted a-1,3-linked mannose-containing oligomannosyl side chains and a-1,6-branched oligomannosyl side chains, which is apparent from cross-peaks 8, 15, 18, 19, and 30 Therefore, we need to
Fig 7 Possible structure of C lusitaniae IFO 1019 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 1 H NMR signals shown in Fig 2.
Fig 8 Enzyme-linked immunosorbent inhibition assay of b-1,2 linkage-containing oligosaccharides An inhibition assay of the reaction between Fr L-a and factor 6 serum was performed Haptenic oligo-saccharides; h, LM4; , LM5; j, LM6; s, Manb1fi2Mana1fi 2Mana1fi2Mana1fi2Man; d, Manb1fi2Manb1fi2Mana1fi2Man a1fi2Mana1fi2Man; n, Manb1fi2Mana1fi3Mana1fi2Mana1fi 2Man; m, Manb1fi2Manb1fi2Mana1fi3Mana1fi2Mana1fi 2Man.
Trang 9separate the side-chain oligosaccharides for precise structure
determination and for the calculation of the molar ratio of
the side chains We will clarify the structure of the mannan
of C albicans serotype A based on these results in a later
study
Enzyme-linked immunosorbent inhibition assay of the
reaction system between Fr L-a and factor 6 serum using
several pure b-1,2 linkage-containing oligosaccharides was
performed to identify the antigenic activity Although
reaction was not inhibited by the b-1,2 and a-1,3
link-age-containing oligosaccharides, Manb1fi2Mana1fi
3Mana1fi2Mana1fi2Man and Manb1fi2Manb1fi
2Mana1fi3Mana1fi2Mana1fi2Man, strong inhibition
was observed with LM4 and LM5 In a preceding paper
[48], we tested the haptenic activity of the b-1,2
linkage-containing oligosaccharides using a cell agglutination
inhibition assay, but had not used the b-1,2-substituted
a-1,3-linked mannose-containing oligosaccharides
There-fore, in this quantitative inhibition assay, we confirmed that
the antibody to factor 6 recognizes the a-1,2 linkage of the
mannose unit substituted by the b-1,2-linked mannose unit
Furthermore, the lackof reactivity of the three-b-1,2
linkage-containing oligosaccharide, LM6, indicates that
factor 6 serum does not react with the intermediary linkages
fi2Manb1fi2Mana1fi2, but contains at least two kinds of
antibodies which recognize LM4 and LM5 from the
nonreducing terminal The weakagglutination inhibition
activity of the b-1,2 linkage-containing heptaose obtained
from C albicans serotype A mannan in a preceding paper
[48] also suggests that the heptaose was comprised of a
mixture of two and three b-1,2 linkage-containing
oligosac-charides The above findings indicate that the recognition
size limit of the antibody to these oligosaccharides is four
mannose units, Manb1fi2Manb1fi2Mana1fi2Mana1fi,
and that more than two b-1,2 linkage-containing
oligosac-charides behave as factor 5 [11] instead of as factor 6
Nevertheless, these side chains correspond to a serotype
A-specific structure because b-1,2-substituted a-1,2-linked
mannose-containing oligomannosyl side chains are not
present in the serotype-B mannan
A series of studies on the epitope structure of factor 5
has been carried out by Han and Cutler and his coworkers
where they isolated monoclonal antibodies B6.1 (IgM) [22]
and C3.1 (IgG) [25], both of which react with
phospho-diesterified b-1,2-linked oligomannosyl moieties (factor 5)
These monoclonal antibodies enhance the resistance of
mice to disseminated candidiasis and protect against
Candida vaginal infection [22–25] They determined the
minimal epitope of these monoclonal antibodies to be
b-1,2-linked mannotriose by agglutination inhibition
assays [23,25] Furthermore, they showed, using synthetic
oligomers, that these epitopes adopt a compact helical
conformation [49]
In a preceding study [50], we detected and characterized
b-1,2-mannosyltransferase II in the cell homogenate of
C albicans, but could not detect b-1,2-mannosyltransferase
I, which is responsible for the transfer of the first
b-1,2-linked mannose unit to an a-1,2-b-1,2-linked mannose unit and is
the key enzyme for the synthesis of factor 6 Because the
a-1,2-linked oligomannosyl moieties of b-1,2
linkage-containing side chains of the mannans of C albicans,
C lusitaniae, and Cit matritensis are strictly fixed at
tetraose, triose and biose, respectively, there is a possibility that the b-1,2-mannosyltransferase I responsible for the synthesis of these side chains recognizes the length of the side chain from the a-1,6-linked backbone mannose units
If we use the backbone a-1,6-linked mannose-containing side-chain oligosaccharide as the substrate, we may be able
to detect b-1,2-mannnosyltransferase I
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