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.