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Structural determination of d -mannans of pathogenic yeasts Candida stellatoidea type I strains: TIMM 0310 and ATCC 11006 compared to IFO 1397
131
Carbohydrate Research. 214 (1991) 131-145 Elsevier Science Publishers B.V., Amsterdam
determination of D-mannans of pathogenic yeasts Candida stellatoidea Type I strains: TIMM 0310 and ATCC 11006 compared to IF0 1397*
Structural
Hidemitsu Kobayashi, Takeshi Kojimahara, Kaori Takahashi, Masashi Takikawa, Shin-ichi Takahashi, Nobuyuki Shibata, Yoshio Okawa, and Shigeo Suzuki+ Second Department
qf Hygienic
Chemistry,
Tohoku College of Pharmacy,
Miyagi 981
Sendai Aoha-ku.
IJapan I (Received
August
13th, 1990; accepted
for publication,
in revised
form December
l5th,
1990)
ABSTRACT
The structures IF0
1397, TIMM
digestion
and
their
oligosaccharides degradation
strain
products
by acid treatment
D-manno-oligosaccharides were
alkali-modified
the homologous
D-mannans
D-mannopyranose
units]
manno-oligosaccharides corresponding
lacking
‘H-n.m.r.
studied
biose
I +2)-linked
were acetolyzed residues,
were
from the parent
signals with
alkaline
whereas
above 4.900 p.p.m.
those
that
structures:
obtained
[corresponding
the longest
by alkaline
The
acid-
and
to /3-(I +2)-linked and
branches
D-Manno-
as the homologous
o-mannotriose.
(v/v) AcZO~AcOH~H2S0,,
by
The modified
analyses.
were identified and
hydrolysis,
by acetolysis.
%n.m.r.
by ‘H- and
to hexaose.
It was found
had the following
and
D-mannans
D-mannobiose
1O:lO:l
stellatoideu Type I strains,
by mild acid and,
exo-a-D-mannosidase,
from a-(
were also analyzed.
to hexaosyl
yeasts ofcandida
of pathogenic
11006, were investigated
GJM-I
degradation
released
p-(l-2)-linked
D-mannans
and ATCC
the Arthrobacter
with
D-mannans
of the cell-wall 0310,
the resultant
D-
of these D-mannans,
2-D-Manp-(l-+3)-r*-D-Manp-(
l-2)-(r-D-
l-+2)-a-D-Manp-( I +2)-D-Man and r-D-Manp-( ]+2)-rr-D-Manp-(1+3)-a-DManp-( l-2)-x-D-Manp-(l-+2)-a-D-Manp-( I +2)-D-Man. These results indicate that the D-mannans of C. stellatoideaType I strainspossess structures in common with the D-mannans of Candidu albicans serotype B Manp-(
strain
l-2)-x-D-Manp-(
(see ref. 4) containing
phosphate-bound
p-(1 -+2)-linked
oligo-D-mannosyl
residues.
INTRODUCTION
In previous papers, we reported the structures of the antigenic phospho-Dmannans of three representative Can&da albicans strains, NIH A-207 serotype A (refs. 1,2), NIH B-792 serotype B (refs. 3,4), and J- 1012 serotype A (formerly serotype C) (ref. 5) as elucidated by a sequential degradation-procedure involving treatment with hot 1OmM HCI and/or 1OOmMNaOH at 25”, acetolysis under conventional and mild conditions, enzymolysis with Arthrobacter GJM-1 cr-D-mannosidase, and ‘H- and 13C-n.m.r. analyses. These studies demonstrated a structural identity among these D-mannans in that each one possesses a long rx-(1+6)-linked-D-mannopyranose backbone having a large number of ar-(1+2)-linked D-mannotriosyl branches, each of which
* Presented ’ To whom
at the 15th International correspondence should
Carbohydrate be addressed.
Symposium,
Yokohama,
Japan,
August
12.-17, 1990.
132
II.
KOR.AYASHI
l’t
‘Ii.
D-MANNANS
OF
Candida stellatoidea TYPE I
133
Fr. If, Ti, and Al released D-mannose corresponding to 76.8,75.2, and 56.4% of parent D-mannans on a weight basis (data not shown). The enzyme-modified Frs. If, Ti, and A 1 are designated as Frs. If-e, Ti-e, and Al-e, respectively. Acetolysis of Frs. If-ab, Ti-ab, and Al-ab. ~ This was conducted exactly as described by Kobayashi et al.“,13 in a modification of the method of Kocourek and Ballou14. Calculation of average chain-length gf the branches moieties of Frs. I’ Ti, and Al. ~ The average chain-length of the branches in Frs. If, Ti, and Al were calculated as described by Kobayashi et al.” for the partial acid-degradaded D-mannans of Pichia pastoris IF0 0948 strain. N.m.r. studies qf D-mannans and D-manno-oiigosaccharides. - ‘H-N.m.r. spectra were measured as described by Gorin and Spencer” by use of a Jeol JNM-GSX 400 spectrometer. D-Mannans and D-manno-oligosaccharides were separately dissolved in DzO in concentrations of 10 mg/0.7 mL and 5 mg/0.7 mL at 70”, respectively, and acetone was used as the internal standard (2.217 p.p.m.). The 13C-n.m.r. spectra were measured with the same spectrometer as described by Kobayashi et aZ.4The concentrations of D-mannans and D-manno-oligosaccharides in DzO were 25 and 15 mg/0.7 mL at 55”, respectively, and CD,OD was used as the internal standard (49.00 p.p.m.). Slide-agglutination reaction. - This was conducted as described by Miyakawa et al.“, using heat-killed cell suspensions of C. alhicans and C. stellatoidea strains. Other methods. - Total carbohydrate was determined by the phenol-H,SO, method of Dubois et al.‘” with D-mannose (Wako Pure Chemicals, Co. Ltd., Osaka and Tokyo, Japan) as the standard. Total protein was determined by the Folin method of Lowry et al.!“, using bovine serum albumin (Sigma Chemical Co., St. Louis, MO, U.S.A.) as the standard. Total phosphate was determined by the method of Ames and Dubin2’, using KH,PO, (Nakarai Chemicals, Ltd., Kyoto, Japan) as the standard. Specific rotations were determined by means of a JAS DIP-360 digital polarimeter. The sample was dissolved in water, and measurements made 3 h after dissolution. RESULTS
Treatment of the D-MannUnS with l&?ZMHCl. - Fractions If, Ti, and Al were first examined for the presence of acid-labile oligomannosyl residues, which were assumed to connect with phosphate groups via their reducing-terminal groups. After treatment with 1OmMHCl for 1 h at loo”, each mixture was fractionated on a column of Bio-Gel P-2. As shown in Figs. lA-lC, Frs. If, Ti, and Al released a mixture of D-mannooligosaccharides ranging from hexaose to biose, and D-mannose in amounts of 0.03, 0.72, and 1.22%, respectively, on a weight basis ofthe parent D-mannans. The ‘H-n.m.r. spectra of these D-manno-oligosaccharides were apparently identical to those of the D-manno-oligosaccharides isolated from the D-mannan of C. albicans NIH B-792 strain by Kobayashi et al4 and identified as a homologous oligosaccharide series composed solely of p-( l-+2)-linked D-mannopyranose units (data not shown).
D-MANNANS
OF Candida stellatoidea TYPE I
135
(A)
-k!T-&p.p.m.
Fig. 2. ‘H-N.m.r. spectra (anomeric regions) of intact (I), acid-modified (II), alkali-modified (III), acid and alkali-modified (IV), and enzyme-modified (V) o-mannans isolated from three C. stellatoidea strains. (A): Frs. If, If-a, If-b, If-ab, and If-e; (B): Frs. Ti, Ti-a, Ti-b, Ti-ab, and Ti-e; (C): Al, Al-a, Al-b, Al-ab, and Al-e. These were recorded using a Jeol-GSX 400 spectrophotometer in D,O solution at 70” with acetone as the standard (2.217 p.p.m.).
II.
liOHAL’ASIII
t’f
rrl.
) -<
-4
--.<
.,
OF Candida stellatoidea TYPE I
D-MANNANS
139
Acetolysis oj’D-mannans modljied h_vucid and alkali. structures
of the acid- and alkali-stable
alkali-modified
D-mannans
domains
(Frs. If-ab,
Ti-ab,
s~s’~.‘~with 10: 10: 1 Ac2@AcOHPH,S04. acetolyzates
of Frs. If-ab,
Ti-ab,
and Al-ab.
to biose, D-mannose,
and phosphate-containing
These oligosaccharides
were also investigated
identification
were neutral
through
CZI.‘~, Cohen
and Zhang
and Ballot?,
oligosaccharides
identified
D-manno-oligosaccharides,
assignment
was conducted
to acetoly-
profiles of the
As may be seen, the oligosaccharides
from these acetolyzates
D-manno-oligosaccharide
the
the acid- and
were subjected
Figs. 4A to 4C show the elution
heptaose Structural
to analyze
of the phosphomannans, and Al-ab)
isolated Al-ab).
In order
ranging
component
from
(Frs. Ti-ab
by ‘H-n.m.r.
analysis
of the anomeric-proton
and
(Fig. 5).
signals
of each
by correlation with the findings by Gorin et and Ballou24, and the structures of the D-manno-
are depicted
in formulas
1-9.
r-II-Manp-( I +2)-D-Man a-o-Manp-( 142).cc-u-May-( 1-+2)-D-Man 1 2 r-D-May-( l-2)-r-u-Mary-( I +2)-cc-D--h’kmp-( I +2)-D-Man 3 x-u-Manp-(1+2)-a-D-May-( I +2)-x-D-Manp-( 1-+2)-r-u-Manp-( I +2)-u-Man 4 a-D-Manp-( 1+3)-r-o-Manp-( I +2)-x-D-Manp-( I +2)-z-I,-Manp-( l-2)-D-Man 5 a-D-May-( 1-3)~r-u-May-( I -2).cc-D-Man/?-( 1+2)-cc-r>-Manp-( I +2)-r-D-Manp-(t -+2)-D-Man 6 a-D-Manp-( l-2)-a-u-Ma& 1+3)-cx-D-Manp-( I +2)-a-D-Manp-( I +2)-r-D-Manp-(I +2)-o-Man 7 z-D-Manp-( I-3).r-wManp-( I +2)-a-D-Manp-(l-2).a-D-Manp-( 1-2).x-u-Manp-( I+ 2)-r-u-Manp-( I -+2)-D-Man 8 cc-D-Manp-( l-2)-r-tsManp-( I -3).a-wManp-( 1+?)-r-wManp-( I +2)-rx-D-Ma&l -t2)-r-D-May-(I-+2)-D-Man 9 It is noteworthy
from
the three
oligosaccharides
different
that oligosaccharides D-mannans
higher than pentaose
of the same molecular
had the same chemical were each found
weight
structure,
to contain
obtained
although
one isomeric
the oligo-
saccharide. The D-manno-oligosaccharides resulting from the acetolysis of Fr. Ti-ab were relatively low members corresponding to structures 1, 2, 3, and 5. These four oligosaccharides lacked signals at 5.363 and/or 5.364 p.p.m., as C-l of an a-(1+3)linked
D-mannopyranose
LX-(l-2)
linkage.
saccharides
residue
However
had another
these signals
7 and 9 as isolated
D-mannopyranose
were present
from the acetolyzates
unit attached
in the higher
via an
D-manno-oligo-
of Frs. If-ab and Al-ab;
both of
these have a signal at 5.366p.p.m. (Figs. 2A and 2C). It was thereforeevident that Fr. Ti had the simplest chemical structure among the D-mannans of three C. stellutoidea strains,
in terms of average In contrast,
pentaose, saccharides
acetolysis
and hexaose
chain-length
corresponding
were investigated
and chemical
of Fr. Al-ab
to structures
by “C-n.m.r.
structure
gave homogeneous
of the branches. biose,
triose,
tetraose,
1, 2, 3, 5, and 7, and these oligo-
spectrometry.
The ‘“C-n.m.r.
spectrum
of
the D-mannohexaose, 7, showed a 103.09-p.p.m. signal, which was observed in “Cn.m.r. spectra of Frs. If and Al, but was absent that of Fr. Ti.. This finding agrees well with that found
in the acetolysis
study of Fr. Ti-ab,
namely
that this D-mannohexaose
II
C-l c-2 c-3 c-4 C-5 C-6
c-2 c-3 c-4 c-5 C-6
C-l
“C Chemical
TABLE
102.92 70.97 71.32 67.81 14.25 61.93
5
96.21 12.90 72.47 69.42 74.60 63.21
I
D-Mannose
102.86 70.49 78.80 67.08 74.18 61.81
102.95 70.90 71.29 67.80 74.09 61.93
I-’
1
101.35 79.34 70.99 67.98 74.15 61.96
shifts of D-manno-oligosaccharides
101.42 79.13 70.97 68.05 74.15 61.96
93.47 79.87 70.90 68.05 73.40 61.96
1
(a anomers)
93.45 80.01 70.88 68.05 73.41 61.96
102.97 70.89 71.31 67.17 74.09 61.93
P
2
obtained
103.09 71.01 71.31 67.74 74.11 61.93
16
7
101.39 79.23 70.95 68.00 74.12 61.95
I-’ 93.44 80.54 70.95 68.04 73.40 61.97
I
102.87 70.50 79.21 67.06 74.19 61.83
I’
by acetolysis
101.41 79.37 70.95 68.00 14.26 61.93
I’
from Fr. Al-ab
101.36 79.33 70.90 68.00 74.14 61.96
1’
102.99 71.31 70.98 67.76 74.09 61.93
1’
3
101.41 79.12 70.90 68.05 74.14 61.96
I’
101.37 79.37 70.89 61.94 74.14 61.95
I’
93.44 80.02 70.90 68.05 73.41 61.96
1
101.44 79.21 70.89 68.00 74.14 61.95
1’
93.44 80.03 70.94 68.04 73.41 61.96
I
a n
D-MANNANS
OF
Candida stellatoidea
TYPE I
143
(A)
ii-l
II 2
81 t n
Fig. 7. Possible structures for the cell-wall o-mannans of C. LstelkztoideaIF0 1397 (A), TIMM 03 IO (B), and ATCC 11006 (C) strains. M (Man) and GNAc (GlcNAc) denote a D-mannopyranose and 2-acetamido-2deoxy-o-glucopyranose units, respectively. The side-chain sequence is not specified.
I-1. KOB/\YASHI
144
DIS(‘I
t7/.
SSION
AC‘K9oWl.Ft~(;Mi!h
1
The authors thank Dr istry
Pf
K
Ifisamichi.
of this college for recc>rtlillg the rl.m.r.
the First sptx‘tra.
Department
of Medicinal I’henr-
REFERENCES I N. Shibata, S. Fukasawa, H. Kobayashi, M. Tojo, T. Yonezu, A. Anbo, Y. Ohkubo, and S. Suzuki, C&x+&. Res., 187 (1989) 239-253. 2 H. Kobayashi, N. Shibata, and S. Suzuki, Program and Abstracts of JUMS Congress: 3acieri~~og~ and Mycology, Osaka, Japan, September 16-22, 1990, p. 188. 3 N. Shibata. H. Kobayashi, M. Tojo, and S. Suzuki, Arch. Biachem. Bioph~.~., 2.51 (1986) 6977708. 4 H. Kobayashi, N. Shibata, M. Nakada, S. Chaki, K. Mizugami, Y. Ohkubo, and S. Suzuki, Arch. Biockem. Biopkys., 278 (1990) 195-204. S H. Kobayashi, N. Shibata, H. Mitobe, Y. Ohkubo, and S. Suzuki, Arch. Biockem. Biopkys., 272 (1989) 364-375. 6 M. Tojo, N. Shibata, Y. Ban, and S. Suzuki, Carbokydr. Res., 199 (1990) 215-226. 7 M. Suzuki and Y. Fukazawa, Microbial. Imntlmof., 26 (1982) 387.402. 8 K. J. Kwon-Chung, B. L. Wickes, and W. 6. Merz, Infect. Immun.. 56 (1988) 1814-1819. 9 K. J. Kwon-Chung, W. S. Riggsby, R. A. Uphoff, J. 3%Hicks, W. L. Whelan, E. Reiss, B. B. Magee, and B. L. Wickes, I&v. rmrnan., 57 (1989) 527-532. IO Y. Fukazawa, T. Shinoda, and T. Tsuchiya, J. Bucteriol., 95 (1968) X4--763. 11 N. Shibata, T. Ichikawa, M. Tojo, M. Takahashi, N. fto, Y. Ohkubo, and S. Suzuki, Arch, Biorkem. 3iopky.y.. 243 (1985) 338+348. 12 H. Kobayashi. N. Shibata, and S. Suzuki, Arch. B&hem. Biopkys., 245 (1986) 494503. I3 H. Kobayashi, N. Shibata, T. Yonezu, and S. Suzuki, Arch. Biochent. BiophJv.. 256 (1987) 381-396. 14 J. Kocourek and C. E. Ballou, J. Bucferiol.. 100 (1969) 1175-I 181. 15 H. Kobayashi, N. Shibata, T. Yonezu, and S. Suzuki, Ckem. Pkurm. Bull., 36 (1988) 3168-3172. 16 P. A. J. Gorin and J. F. T. Spencer, Adu. Appl. Microbiof., 13 (1970) 2.$&9. 17 Y. Miyakawa. K. Kagaya, and Y. Fukazawa, J. C’lin.Microbial., 23 (1986) 881-886. 18 M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. R&em. and F. Smith, Anal. Ckem., 28 (1956) 350-3.56, 19 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Bioi. Chem., 193 (1951) 265.-275. 20 B. N. Ames and D. T. Dubin, J. Biol. Chem., 235 (1960) 769-775. 21 J. C. Speck, jr., A& Carbohydr. Chem., 13 (1958) 63-103. 22 P. A. J. Gorin, J. F. T. Spencer, and R. J. Magus, Canad. 3. Chem.. 47 (1969) 3569-3576. 23 R. E. Cohen and C. E. Ballou, Biachembrry. 19 (I 980) 43454358. 24 W. Zhang and C. E. Ballou, J. B&l. C%em.. 256 (1981) 10073.-10079. 25 P. A. J. Gorin, Caned. J. Ckem., 51 (1973) 2375-2383. 26 T. Ogawa and H. Yamamoto, Carbokydr. Res., 104 (1982) 271-283. 27 A. Allerhand and E. Berman, 1. Am. Ckem. Sot., 106 (1984) 2400-2412. 28 M. Funayama. A. Nishikawa, T. Shinoda, M. Suzuki, and Y. Fukazawa, ~icrab~ol. ~~trn~~o~.,28 (1984) 1359-1371. 29 E. R&s, L. de Repentigny, R. J. Kuykendall, A. W. Carter, R. Galindo, P. Auger, S. L. Bragg, and L. Kaufman, J. Cl&t. Microbial., 24 (1986) 796-802. 30 M. Tojo, N. Shibata, M. Kobdyashi, T. Mikami, M. Suzuki, and S. Suzuki, 0%. C&m.. 34 {1988) 539.-543.
Carbohydrate Research. 214 (1991) 131-145 Elsevier Science Publishers B.V., Amsterdam
determination of D-mannans of pathogenic yeasts Candida stellatoidea Type I strains: TIMM 0310 and ATCC 11006 compared to IF0 1397*
Structural
Hidemitsu Kobayashi, Takeshi Kojimahara, Kaori Takahashi, Masashi Takikawa, Shin-ichi Takahashi, Nobuyuki Shibata, Yoshio Okawa, and Shigeo Suzuki+ Second Department
qf Hygienic
Chemistry,
Tohoku College of Pharmacy,
Miyagi 981
Sendai Aoha-ku.
IJapan I (Received
August
13th, 1990; accepted
for publication,
in revised
form December
l5th,
1990)
ABSTRACT
The structures IF0
1397, TIMM
digestion
and
their
oligosaccharides degradation
strain
products
by acid treatment
D-manno-oligosaccharides were
alkali-modified
the homologous
D-mannans
D-mannopyranose
units]
manno-oligosaccharides corresponding
lacking
‘H-n.m.r.
studied
biose
I +2)-linked
were acetolyzed residues,
were
from the parent
signals with
alkaline
whereas
above 4.900 p.p.m.
those
that
structures:
obtained
[corresponding
the longest
by alkaline
The
acid-
and
to /3-(I +2)-linked and
branches
D-Manno-
as the homologous
o-mannotriose.
(v/v) AcZO~AcOH~H2S0,,
by
The modified
analyses.
were identified and
hydrolysis,
by acetolysis.
%n.m.r.
by ‘H- and
to hexaose.
It was found
had the following
and
D-mannans
D-mannobiose
1O:lO:l
stellatoideu Type I strains,
by mild acid and,
exo-a-D-mannosidase,
from a-(
were also analyzed.
to hexaosyl
yeasts ofcandida
of pathogenic
11006, were investigated
GJM-I
degradation
released
p-(l-2)-linked
D-mannans
and ATCC
the Arthrobacter
with
D-mannans
of the cell-wall 0310,
the resultant
D-
of these D-mannans,
2-D-Manp-(l-+3)-r*-D-Manp-(
l-2)-(r-D-
l-+2)-a-D-Manp-( I +2)-D-Man and r-D-Manp-( ]+2)-rr-D-Manp-(1+3)-a-DManp-( l-2)-x-D-Manp-(l-+2)-a-D-Manp-( I +2)-D-Man. These results indicate that the D-mannans of C. stellatoideaType I strainspossess structures in common with the D-mannans of Candidu albicans serotype B Manp-(
strain
l-2)-x-D-Manp-(
(see ref. 4) containing
phosphate-bound
p-(1 -+2)-linked
oligo-D-mannosyl
residues.
INTRODUCTION
In previous papers, we reported the structures of the antigenic phospho-Dmannans of three representative Can&da albicans strains, NIH A-207 serotype A (refs. 1,2), NIH B-792 serotype B (refs. 3,4), and J- 1012 serotype A (formerly serotype C) (ref. 5) as elucidated by a sequential degradation-procedure involving treatment with hot 1OmM HCI and/or 1OOmMNaOH at 25”, acetolysis under conventional and mild conditions, enzymolysis with Arthrobacter GJM-1 cr-D-mannosidase, and ‘H- and 13C-n.m.r. analyses. These studies demonstrated a structural identity among these D-mannans in that each one possesses a long rx-(1+6)-linked-D-mannopyranose backbone having a large number of ar-(1+2)-linked D-mannotriosyl branches, each of which
* Presented ’ To whom
at the 15th International correspondence should
Carbohydrate be addressed.
Symposium,
Yokohama,
Japan,
August
12.-17, 1990.
132
II.
KOR.AYASHI
l’t
‘Ii.
D-MANNANS
OF
Candida stellatoidea TYPE I
133
Fr. If, Ti, and Al released D-mannose corresponding to 76.8,75.2, and 56.4% of parent D-mannans on a weight basis (data not shown). The enzyme-modified Frs. If, Ti, and A 1 are designated as Frs. If-e, Ti-e, and Al-e, respectively. Acetolysis of Frs. If-ab, Ti-ab, and Al-ab. ~ This was conducted exactly as described by Kobayashi et al.“,13 in a modification of the method of Kocourek and Ballou14. Calculation of average chain-length gf the branches moieties of Frs. I’ Ti, and Al. ~ The average chain-length of the branches in Frs. If, Ti, and Al were calculated as described by Kobayashi et al.” for the partial acid-degradaded D-mannans of Pichia pastoris IF0 0948 strain. N.m.r. studies qf D-mannans and D-manno-oiigosaccharides. - ‘H-N.m.r. spectra were measured as described by Gorin and Spencer” by use of a Jeol JNM-GSX 400 spectrometer. D-Mannans and D-manno-oligosaccharides were separately dissolved in DzO in concentrations of 10 mg/0.7 mL and 5 mg/0.7 mL at 70”, respectively, and acetone was used as the internal standard (2.217 p.p.m.). The 13C-n.m.r. spectra were measured with the same spectrometer as described by Kobayashi et aZ.4The concentrations of D-mannans and D-manno-oligosaccharides in DzO were 25 and 15 mg/0.7 mL at 55”, respectively, and CD,OD was used as the internal standard (49.00 p.p.m.). Slide-agglutination reaction. - This was conducted as described by Miyakawa et al.“, using heat-killed cell suspensions of C. alhicans and C. stellatoidea strains. Other methods. - Total carbohydrate was determined by the phenol-H,SO, method of Dubois et al.‘” with D-mannose (Wako Pure Chemicals, Co. Ltd., Osaka and Tokyo, Japan) as the standard. Total protein was determined by the Folin method of Lowry et al.!“, using bovine serum albumin (Sigma Chemical Co., St. Louis, MO, U.S.A.) as the standard. Total phosphate was determined by the method of Ames and Dubin2’, using KH,PO, (Nakarai Chemicals, Ltd., Kyoto, Japan) as the standard. Specific rotations were determined by means of a JAS DIP-360 digital polarimeter. The sample was dissolved in water, and measurements made 3 h after dissolution. RESULTS
Treatment of the D-MannUnS with l&?ZMHCl. - Fractions If, Ti, and Al were first examined for the presence of acid-labile oligomannosyl residues, which were assumed to connect with phosphate groups via their reducing-terminal groups. After treatment with 1OmMHCl for 1 h at loo”, each mixture was fractionated on a column of Bio-Gel P-2. As shown in Figs. lA-lC, Frs. If, Ti, and Al released a mixture of D-mannooligosaccharides ranging from hexaose to biose, and D-mannose in amounts of 0.03, 0.72, and 1.22%, respectively, on a weight basis ofthe parent D-mannans. The ‘H-n.m.r. spectra of these D-manno-oligosaccharides were apparently identical to those of the D-manno-oligosaccharides isolated from the D-mannan of C. albicans NIH B-792 strain by Kobayashi et al4 and identified as a homologous oligosaccharide series composed solely of p-( l-+2)-linked D-mannopyranose units (data not shown).
D-MANNANS
OF Candida stellatoidea TYPE I
135
(A)
-k!T-&p.p.m.
Fig. 2. ‘H-N.m.r. spectra (anomeric regions) of intact (I), acid-modified (II), alkali-modified (III), acid and alkali-modified (IV), and enzyme-modified (V) o-mannans isolated from three C. stellatoidea strains. (A): Frs. If, If-a, If-b, If-ab, and If-e; (B): Frs. Ti, Ti-a, Ti-b, Ti-ab, and Ti-e; (C): Al, Al-a, Al-b, Al-ab, and Al-e. These were recorded using a Jeol-GSX 400 spectrophotometer in D,O solution at 70” with acetone as the standard (2.217 p.p.m.).
II.
liOHAL’ASIII
t’f
rrl.
) -<
-4
--.<
.,
OF Candida stellatoidea TYPE I
D-MANNANS
139
Acetolysis oj’D-mannans modljied h_vucid and alkali. structures
of the acid- and alkali-stable
alkali-modified
D-mannans
domains
(Frs. If-ab,
Ti-ab,
s~s’~.‘~with 10: 10: 1 Ac2@AcOHPH,S04. acetolyzates
of Frs. If-ab,
Ti-ab,
and Al-ab.
to biose, D-mannose,
and phosphate-containing
These oligosaccharides
were also investigated
identification
were neutral
through
CZI.‘~, Cohen
and Zhang
and Ballot?,
oligosaccharides
identified
D-manno-oligosaccharides,
assignment
was conducted
to acetoly-
profiles of the
As may be seen, the oligosaccharides
from these acetolyzates
D-manno-oligosaccharide
the
the acid- and
were subjected
Figs. 4A to 4C show the elution
heptaose Structural
to analyze
of the phosphomannans, and Al-ab)
isolated Al-ab).
In order
ranging
component
from
(Frs. Ti-ab
by ‘H-n.m.r.
analysis
of the anomeric-proton
and
(Fig. 5).
signals
of each
by correlation with the findings by Gorin et and Ballou24, and the structures of the D-manno-
are depicted
in formulas
1-9.
r-II-Manp-( I +2)-D-Man a-o-Manp-( 142).cc-u-May-( 1-+2)-D-Man 1 2 r-D-May-( l-2)-r-u-Mary-( I +2)-cc-D--h’kmp-( I +2)-D-Man 3 x-u-Manp-(1+2)-a-D-May-( I +2)-x-D-Manp-( 1-+2)-r-u-Manp-( I +2)-u-Man 4 a-D-Manp-( 1+3)-r-o-Manp-( I +2)-x-D-Manp-( I +2)-z-I,-Manp-( l-2)-D-Man 5 a-D-May-( 1-3)~r-u-May-( I -2).cc-D-Man/?-( 1+2)-cc-r>-Manp-( I +2)-r-D-Manp-(t -+2)-D-Man 6 a-D-Manp-( l-2)-a-u-Ma& 1+3)-cx-D-Manp-( I +2)-a-D-Manp-( I +2)-r-D-Manp-(I +2)-o-Man 7 z-D-Manp-( I-3).r-wManp-( I +2)-a-D-Manp-(l-2).a-D-Manp-( 1-2).x-u-Manp-( I+ 2)-r-u-Manp-( I -+2)-D-Man 8 cc-D-Manp-( l-2)-r-tsManp-( I -3).a-wManp-( 1+?)-r-wManp-( I +2)-rx-D-Ma&l -t2)-r-D-May-(I-+2)-D-Man 9 It is noteworthy
from
the three
oligosaccharides
different
that oligosaccharides D-mannans
higher than pentaose
of the same molecular
had the same chemical were each found
weight
structure,
to contain
obtained
although
one isomeric
the oligo-
saccharide. The D-manno-oligosaccharides resulting from the acetolysis of Fr. Ti-ab were relatively low members corresponding to structures 1, 2, 3, and 5. These four oligosaccharides lacked signals at 5.363 and/or 5.364 p.p.m., as C-l of an a-(1+3)linked
D-mannopyranose
LX-(l-2)
linkage.
saccharides
residue
However
had another
these signals
7 and 9 as isolated
D-mannopyranose
were present
from the acetolyzates
unit attached
in the higher
via an
D-manno-oligo-
of Frs. If-ab and Al-ab;
both of
these have a signal at 5.366p.p.m. (Figs. 2A and 2C). It was thereforeevident that Fr. Ti had the simplest chemical structure among the D-mannans of three C. stellutoidea strains,
in terms of average In contrast,
pentaose, saccharides
acetolysis
and hexaose
chain-length
corresponding
were investigated
and chemical
of Fr. Al-ab
to structures
by “C-n.m.r.
structure
gave homogeneous
of the branches. biose,
triose,
tetraose,
1, 2, 3, 5, and 7, and these oligo-
spectrometry.
The ‘“C-n.m.r.
spectrum
of
the D-mannohexaose, 7, showed a 103.09-p.p.m. signal, which was observed in “Cn.m.r. spectra of Frs. If and Al, but was absent that of Fr. Ti.. This finding agrees well with that found
in the acetolysis
study of Fr. Ti-ab,
namely
that this D-mannohexaose
II
C-l c-2 c-3 c-4 C-5 C-6
c-2 c-3 c-4 c-5 C-6
C-l
“C Chemical
TABLE
102.92 70.97 71.32 67.81 14.25 61.93
5
96.21 12.90 72.47 69.42 74.60 63.21
I
D-Mannose
102.86 70.49 78.80 67.08 74.18 61.81
102.95 70.90 71.29 67.80 74.09 61.93
I-’
1
101.35 79.34 70.99 67.98 74.15 61.96
shifts of D-manno-oligosaccharides
101.42 79.13 70.97 68.05 74.15 61.96
93.47 79.87 70.90 68.05 73.40 61.96
1
(a anomers)
93.45 80.01 70.88 68.05 73.41 61.96
102.97 70.89 71.31 67.17 74.09 61.93
P
2
obtained
103.09 71.01 71.31 67.74 74.11 61.93
16
7
101.39 79.23 70.95 68.00 74.12 61.95
I-’ 93.44 80.54 70.95 68.04 73.40 61.97
I
102.87 70.50 79.21 67.06 74.19 61.83
I’
by acetolysis
101.41 79.37 70.95 68.00 14.26 61.93
I’
from Fr. Al-ab
101.36 79.33 70.90 68.00 74.14 61.96
1’
102.99 71.31 70.98 67.76 74.09 61.93
1’
3
101.41 79.12 70.90 68.05 74.14 61.96
I’
101.37 79.37 70.89 61.94 74.14 61.95
I’
93.44 80.02 70.90 68.05 73.41 61.96
1
101.44 79.21 70.89 68.00 74.14 61.95
1’
93.44 80.03 70.94 68.04 73.41 61.96
I
a n
D-MANNANS
OF
Candida stellatoidea
TYPE I
143
(A)
ii-l
II 2
81 t n
Fig. 7. Possible structures for the cell-wall o-mannans of C. LstelkztoideaIF0 1397 (A), TIMM 03 IO (B), and ATCC 11006 (C) strains. M (Man) and GNAc (GlcNAc) denote a D-mannopyranose and 2-acetamido-2deoxy-o-glucopyranose units, respectively. The side-chain sequence is not specified.
I-1. KOB/\YASHI
144
DIS(‘I
t7/.
SSION
AC‘K9oWl.Ft~(;Mi!h
1
The authors thank Dr istry
Pf
K
Ifisamichi.
of this college for recc>rtlillg the rl.m.r.
the First sptx‘tra.
Department
of Medicinal I’henr-
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