Fungal Polysaccharides - American Chemical Society

6 Immunochemistry of Candida albicans Mannan YASUHITO OKUBO, T S U T O M U ICHIKAWA,

and S H I G E O

SUZUKI

1

Downloaded by CORNELL UNIV on May 14, 2017 | http://pubs.acs.org Publication Date: June 10, 1980 | doi: 10.1021/bk-1980-0126.ch006

The Second Department of Hygienic Chemistry, Tohoku College of Pharmacy, 4-4-1 Komatsushima, Sendai 983, Japan

Many species of yeasts are known to produce mannans containing various amounts of phosphate, which can be fractionated by anion-exchange chromatography (1, 2, 3, 4). Recently, we revealed that the mannan of Saccharomyces cerevisiae (bakers' yeast) can be fractionated on a column of diethylaminoethyl (DEAE)-Sephadex into five subfractions containing different amounts of phosphate, and that the antibody-precipitating act i v i t i e s of these mannan subfractions against their homologous antiserum were proportional to their phosphate content (5). The above findings suggest that many yeast mannans are composed of heterogeneous molecular species in which different number of phosphate groups exist, and that the mannan subfraction containing the largest amount of phosphate corresponds to the strongest antigen of the parant yeast. In the present study, the mannans from three C . albicans strains of different serotype, NIH A-207 (serotype A) (A-strain) (6,7), NIH B-792 (serotype B) (B-strain) (6, 7), and J-1012 (serotype C) (J-strain) (8) were i n vestigated to determine whether a similar relationship between phosphate content and serological activity could be observed. Also, cross-reactivity between the fractionated mannans of the C . albicans strains and anti-S. cerevisiae serum, the latter of which has been shown to be cross-reactive with the mannan of B-strain (9, 10), was investigated in order to assess density of the cross-reactive antigenic determinant groups in the fractionated mannan of B-strain. A brief communication dealing with the phosphate content and serological activity of the mannan subfractions of A - , B - , and C-strains appeared recently (11). Materials and Methods The C. albicans strains used. Three strains of C . albicans, NIH A-207, NIH B-792, and J-1012, were kindly provided by Dr. Y. Fukazawa, Meiji College of Pharmacy, Tokyo. 1

To whom a l l correspondences should be addressed. 0-8412-0555-8/80/47-126-095$05.00/0 ©

1980 A m e r i c a n C h e m i c a l Society

Sandford and Matsuda; Fungal Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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C u l t i v a t i o n of the s t r a i n . Each s t r a i n was c u l t i v a t e d i n a 500-ml f l a s k c o n t a i n i n g 0.5%-yeast extract-added Sabouraud l i q u i d medium on a r e c i p r o c a l shaker at 26 to 28°C for 60h. The c e l l s were harvested by c e n t r i f u g a t i o n , washed thoroughly with s a l i n e , and dehydrated with a l a r g e volume o f acetone. Preparation of the bulk mannans. Two batches of the bulk mannan of each s t r a i n were prepared by the method of Peat et a l . (12), modified by us as f o l l o w s : A suspension of acetone-dried whole c e l l s of each s t r a i n (300g) i n water (3 l i t e r ) was heated in an autoclave f o r 2h a t 140°C. A f t e r being c o o l e d , the mixture was c e n t r i f u g e d f o r 20min a t 7,000rpm, and the residue was s t i r r e d with water (1 l i t e r ) and c e n t r i f u g e d again for 20min a t 7,000rpm. The combined f i l t r a t e s were concentrated in vacuo to small volume (ca. 300ml), d i a l y z e d overnight against running tap water, and concentrated in vacuo to c a . 200ml. To the s o l u t i o n was added 800ml o f absolute ethanol c o n t a i n i n g 0.1% sodium a c e t a t e . A f t e r being l e f t for 30min, the p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n , then dehydrated with absolute ethanol, and d r i e d i n vacuo over P2°5 to y i e l d an amorphous powder. The above crude e x t r a c t (15g) was d i s s o l v e d i n water (150ml), and the cloudy s o l u t i o n was s t i r r e d for 2h with a 4 : 1 (v/v) mixture o f chloroform and n-butanol (200ml). Then the mixture was c e n t r i f u ged at 2,500rpm for 15min, and the supernatant c o l l e c t e d was repeatedly t r e a t e d by the same procedure u n t i l a completely c l e a r s o l u t i o n was obtained (For p r e p a r a t i o n o f the bulk mannans of Batch I I , t h i s d e p r o t e i n i z a t i o n procedure was o m i t t e d ) . The supernatant was concentrated i n vacuo to c a . 100ml and then was poured i n t o 600ml o f absolute ethanol c o n t a i n i n g 0.1% sodium a c e t a t e . The p r e c i p i t a t e was washed with ethanol, and d r i e d i n vacuo over Ρ2°5· To an aqueous s o l u t i o n o f the above crude polysaccharide f r a c t i o n (10g i n 100ml) was added 200ml o f F e h l i n g s o l u t i o n (a 1 : 1 (v/v) mixture o f aqueous s o l u t i o n o f 3.5% CuS0 -5H 0 and 17.3% Rochelle s a l t c o n t a i n i n g 5.0% NaOH), and the mixture was s t i r r e d f o r 12h a t room temperature (Batch I) or f o r 5min (Batch I I ) . The r e s u l t a n t p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n f o r 15min at 3,000rpm and then washed with 400ml of hot water (70°C) . The copper-mannan complex contained i n a c e n t r i f u g e tube was admixed with wet Amberlite IR 120 (H ) r e s i n , s t i r r i n g was then continued u n t i l the complex d i s s o l v e d and the blue c o l o r of the copper ion disappeared completely. The mixture was f i l t e r e d with a g l a s s f i l t e r , and the residue was thoroughly washed with water. The combined f i l t r a t e and washing were n e u t r a l i z e d with 10% Na2CX>3 s o l u t i o n , then concentrated _in vacuo to c a . 50ml, and the s o l u t i o n was poured i n t o absolute ethanol c o n t a i n i n g 0.1% sodium a c e t a t e . A f t e r washing with ethanol by c e n t r i f u g a t i o n , the r e s u l t a n t p r e c i p i t a t e was d r i e d over P2O5 under diminished pressure to g i v e a bulk mannan f r a c t i o n . Y i e l d s of the mannans of Batches I and II of A-, B-, and J - s t r a i n s were 6.4, 7.8, and 6.7%, and 7.5, 8.2, and 6.9%, r e s p e c t i v e l y , of the 4

2

+


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dry weight o f the parent whole c e l l s . Chromatographic f r a c t i o n a t i o n o f the bulk mannans. An aqueous s o l u t i o n of the bulk mannan (3g i n 40ml) was a p p l i e d to a column (4 by 25cm) of DEAE-Sephadex A-50 (acetate), and the e l u t i o n was e f f e c t e d i n a stepwise manner with water, 0.025, 0. 05, 0.1, and 0.2M NaCl. The flow r a t e was 0.5ml/min, and a 10μ1 sample of each f r a c t i o n was assayed f o r carbohydrate content with p h e n o l - s u l f u r i c acid reagent (13) . S u b f r a c t i o n s I, I I , I I I , IV, and V ( F i g s . 1 and 2) were evaporated in vacuo to dryness, and, a f t e r d i s s o l v i n g i n a minimum amount of water, the s o l u t i o n was d i a l y z e d a g a i n s t running tap water. Each mannan s u b f r a c t i o n was again concentrated to 5ml and then poured i n t o 25ml of ab s o l u t e ethanol c o n t a i n i n g 0.1% sodium acetate, and the p r e c i p i tated p o l y s a c c h a r i d e was c o l l e c t e d by c e n t r i f u g a t i o n a t 2,500rpm for 15min. A f t e r being washed with absolute ethanol, the mannan s u b f r a c t i o n s were d r i e d _in vacuo over P2°5* yi °f Subf r a c t i o n s I, I I , I I I , IV, and V from the bulk mannans o f Batch I and II are given i n Tables I and I I . T i l e

e

l

d

s

A n t i s e r a . A n t i s e r a to the C. a l b i c a n s A- and B - s t r a i n s o f Batch I were prepared by immunizing two groups o f three r a b b i t s each with the whole c e l l s o f each s t r a i n , and a n t i s e r a obtained from each group of r a b b i t s were combined. A n t i - J - s t r a i n serum, Batch I, was k i n d l y supplied by Dr. Y. Fukazawa. A l l a n t i s e r a o f Batch I had a g g l u t i n i n t i t e r s o f more than 1 : 1,280 a g a i n s t the immunizing c e l l suspensions. A n t i s e r a o f A-, B-, and J - s t r a i n s of Batch II were prepared by the same procedure as that f o r Batch 1. A g g l u t i n i n t i t e r s of a n t i s e r a o f A- and J - s t r a i n s were shown to be more than 1 : 1,280, whereas that of anti-B-serum was con s i d e r a b l y low, 1 : 320. A n t i - S . c e r e v i s i a e whole c e l l serum, Batch I, was the same specimen as that used i n the previous study (_5) . I t had a very high a g g l u t i n i n t i t e r , more than 5,120. On the other hand, that of a n t i - S . c e r e v i s i a e serum, Batch I I , was 1 : 1,280. Mannan s u b f r a c t i o n s of S. c e r e v i s i a e . The mannan s u b f r a c t i o n s o f S. c e r e v i s i a e , Batch I, were the same specimens as those employed i n the previous study (_5) , and those o f Batch II were prepared by the same procedure as the mannan s u b f r a c t i o n s o f three C. a l b i c a n s s t r a i n s o f Batch I I used f o r the present study. A n a l y t i c a l data f o r the mannan s u b f r a c t i o n s o f S. c e r e v i s i a e , Batch I I , are shown i n Table I I . Immunochemical methods. The q u a n t i t a t i v e p r e c i p i t i n r e a c t i o n was c a r r i e d out according to the previous d e s c r i p t i o n (5) as f o l l o w s : To 0.1ml o f antiserum i n a small t e s t tube (1.6 by 10.4cm) was added 0.5ml o f s a l i n e s o l u t i o n o f s e r i a l amounts of the mannan. A f t e r incubation at 37°C f o r l h , the mixture was allowed to stand a t 4°C f o r 16h and then c e n t r i f u g e d a t 2,500rpm


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0.1 M NaCI

Journal of Bacteriology Figure 1. Elution profiles of the bulk mannans of three C . albicans strains (Batch I) by DEAE-Sephadex chromatography (A-50, acetate, 4 X 25 cm) using a step wise elution system consisting of water and NaCI solutions. Ten-^L aliquots of fractions were assayed for carbohydrate content with phenol—sulfuric acid re agent (13): (A) C . albicans NIH A-207 (Α-strain); (B) C. albicans NIH B-792 (B-strain); (C) C . albicans 1-1012 (J-strain) (11). 0.1 M-

FRACTION

NUMBER

Figure 2. Elution profiles of the bulk mannans (Batch II) of three C . albicans strains by DEAE-Sephadex chromatography (A-50, acetate, 4 X 25 cm) using a stepwise elution system consisting of water and NaCI solutions. Ten-μΕ aliquots of fractions were assayed for carbohydrate content with phenoL-sulfuric acid reagent (13): (A) C. albicans NIH A-207 (Α-strain); (B) C . albicans NIH B-792 (B-strain); (C)C. albicans 1-1012 (J-strain).


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for 10min. Each p r e c i p i t a t e was c a r e f u l l y washed twice with i c e c h i l l e d s a l i n e . The amounts o f p r o t e i n i n the p r e c i p i t a t e were determined by the F o l i n method o f Lowry e t a l . (14). Chemical analyses. T o t a l carbohydrate was determined by the p h e n o l - s u l f u r i c a c i d method o f Dubois e t a l . (13), and t o t a l phosphorus was q u a n t i t i a t e d by the method o f Ames and Dubin QJj), respectively.


Results F r a c t i o n a t i o n o f the bulk mannans o f three C. a l b i c a n s s t r a i n s by anion-exchange chromatography. F i g u r e s 1-A, -B, and -C shows the e l u t i o n p r o f i l e s o f DEAE-Sephadex chromatography o f the bulk mannans of Batch I using a stepwise e l u t i o n system con s i s t i n g of water, 0.025-, 0.05-, 0.1-, and 0.2M NaCI s o l u t i o n s subsequently. Under the above c o n d i t i o n , a l l bulk mannans were shown to be resolved into each f i v e s u b f r a c t i o n s designating as Subfractions I, I I , I I I , IV, and V, r e s p e c t i v e l y . The amounts of Subfractions I and I I from A- and B - s t r a i n s were r e l a t i v e l y smaller than those of the other s u b f r a c t i o n s , I I I , IV, and V, while the bulk mannan o f C - s t r a i n gave almost the same amounts of S u b f r a c t i o n I, I I , I I I , and IV. E l u t i o n p r o f i l e s of the bulk mannans of Batch II prepared by short-term treatment with F e h l i n g s o l u t i o n are e s s e n t i a l l y s i m i l a r to those of Batch I ( F i g s . 2-A, -B, and - C ) . However, the most s i g n i f i c a n t d i f f e r e n c e between the bulk mannans of Batch I and II i s that of the amounts of S u b f r a c t i o n V of B - s t r a i n , suggesting that the cause i s a t t r i b u t able to the d i f f e r e n t c o n d i t i o n s of treatment with F e h l i n g s o l u t i o n , as pointed out by Thieme and B a l l o u on the phosphomannan of Kloeckera b r e v i s (16). Chemical composition o f the mannan s u b f r a c t i o n s of three C. a l b i c a n s s t r a i n s . Table I shows the a n a l y t i c a l data f o r each f i v e mannan s u b f r a c t i o n s , Batch I, obtained from the corresponding bulk mannans of three C. a l b i c a n s s t r a i n s . I t i s c l e a r that three s e r i e s o f mannan s u b f r a c t i o n s contain i n c r e a s i n g amounts of phosphate. With the exception of B - s t r a i n mannan s u b f r a c t i o n s e r i e s , p r o t e i n contents o f the s u b f r a c t i o n s are a l s o propor t i o n a l to the NaCI concentration used i n the e l u t i o n . Among the three mannan s u b f r a c t i o n s e r i e s , those of the J - s t r a i n show the l a r g e s t Man/P r a t i o i n comparison with those of each c o r r e sponding s u b f r a c t i o n s of the two other s e r i e s . The mannan subf r a c t i o n s o f B - s t r a i n s e r i e s e x h i b i t e d the smallest Man/P r a t i o . Furthermore, the order of the sum of the y i e l d s of Subfractions I and I I can be represented as J ^ A > B, i n d i c a t i n g that the parent bulk mannan of J - s t r a i n c o n s i s t s o f r e l a t i v e l y l a r g e r amounts of molecular species c o n t a i n i n g fewer phosphate groups. On the other hand, the order o f the sum o f Subfractions IV and V i s shown to be Β > A ^ J , demonstrating that the mannan o f the


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Table I . Chemical composition o f mannan s u b f r a c t i o n s (Batch I) i s o l a t e d from C. a l b i c a n s NIH A-207 ( A - s t r a i n ) , C. a l b i c a n s NIH B-792 ( B - s t r a i n ) , and C. a l b i c a n s J-1012 (J-strain). ( l l ) Mannan subfractions

Carbohydrate (%)

. b Protein (%)

c Phosphate (%)

Man/P molar ratio

Yield (%)

A-1 A-II A-III A-IV A-V

96 94 96 93 92

0.8 0.8 1.1 1.4 2.3

0.00 0.18 0.44 0.84 1.20

>1000 224 94 48 34

14.5 23.5 19.0 19.5 3.5

B-I B-II B-III B-IV B-V

97 95 95 96 93

1.3 1.1 1.3 1.4 1.4

0.00 0.21 0.60 1.15 1.83

>1000 198 69 36 22

12.5 17.0 19.5 21.0 12.0

J-I J-II J-III J-IV J-V

95 94 92 92 91

1.0 1.3 2.2 2.6 4.1

0.00 0.10 0.29 0.57 0.63

>1000 392 139 70 63

16.6 24.8 17.6 16.7 3.2

a

Determined by the p h e n o l - s u l f u r i c a c i d method (13).

b

Determined by the Lowry-Folin method (14).

c

Quantitated by the Ames-Dubin method (15) as

d

Weight b a s i s o f the corresponding bulk mannan.

P 0

H 3

2

d

*

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f i r s t s t r a i n contains r e l a t i v e l y l a r g e r amounts of the h i g h l y phosphorylated mannans. Table II summarizes the r e s u l t s of chemical a n a l y s i s of the mannan s u b f r a c t i o n s e r i e s of Batch I I . Although c o n s i d e r a b l e d i f f e r e n c e s are observed between the y i e l d s of the mannan s u b f r a c t i o n s of the two batches, e s p e c i a l l y i n Subf r a c t i o n s V of the B - s t r a i n s e r i e s , the existence of an ess e n t i a l l y c o n s i s t e n t r e l a t i o n s h i p between t h e i r phosphate contents and the concentrations of NaCI s o u l t i o n s used for e l u t i o n can be d i s c e r n e d . Q u a n t i t a t i v e p r e c i p i t i n r e a c t i o n s between the mannan subf r a c t i o n s and a n t i s e r a of three C a l b i c a n s s t r a i n s . The r e s u l t s of the q u a n t i t a t i v e p r e c i p i t i n r e a c t i o n between the mannan subf r a c t i o n s of Batch I and t h e i r corresponding a n t i s e r a are depicted i n F i g s . 3-A, -B, and -C. In the homologous antigen-antibody systems, the amounts of antibody p r e c i p i t a t e d with each mannan s u b f r a c t i o n of A- and B - s t r a i n s are found to be p r o p o r t i o n a l to the phosphate content of the mannan. These r e s u l t s are obviously s i m i l a r to those obtained with the mannan s u b f r a c t i o n s and the homologous antiserum o f S. c e r e v i s i a e (J5) . On the other hand, the q u a n t i t a t i v e p r e c i p i t i n curves o f the J - s t r a i n mannan s u b f r a c t i o n s and anti-J-serum s i g n i f i c a n t l y d i f f e r from those of A- and Bs t r a i n s , _i.e., S u b f r a c t i o n J - I I I shows n e a r l y i d e n t i c a l p r e c i p i t i n a c t i v i t y with that of Subractions J-IV and -V, although sub f r a c t i o n s J - I , - I I , and - I I I afforded p r o p o r t i o n a l amounts of antibody nitrogen to t h e i r phosphate contents. Furthermore, the d i f f e r ence i n the maximal amounts of p r e c i p i t a t e d nitrogen by J-I and -V appears to be s i g n i f i c a n t l y smaller than those of the c o r r e sponding two mannan s u b f r a c t i o n s of A- and B - s t r a i n s , r e f l e c t i n g a smaller d i f f e r e n c e i n the d e n s i t i e s of the a n t i g e n i c determinants i n the mannan of J - s t r a i n . The l a r g e r d i f f e r e n c e s i n a n t i b o d y - p r e c i p i t a t i n g a c t i v i t i e s of S u b f r a c t i o n I and V i n both the A- and B - s t r a i n s e r i e s than that observed i n the J - s t r a i n s e r i e s can a l s o be regarded to c o r r e l a t e with t h e i r large d i f f e r ences i n phosphate content, suggesting the presence of a l a r g e r d i f f e r e n c e i n the d e n s i t y of the a n t i g e n i c determinant groups. In the c r o s s - p r e c i p i t i n r e a c t i o n against a n t i s e r a of the other serotype s t r a i n s , a l l mannan s u b f r a c t i o n s o f A- and J s t r a i n s were found to be capable of d i s p l a y i n g strong r e a c t i v i t i e s to the a n t i s e r a of d i f f e r e n t serotype s t r a i n s , a f f o r d i n g the amounts o f antibody n i t r o g e n p r o p o r t i o n a l to t h e i r phosphate cont e n t s . T h i s f i n d i n g i n d i c a t e s that the d e n s i t i e s of the c r o s s r e a c t i v e a n t i g e n i c determinant groups i n each s e r i e s of mannan s u b f r a c t i o n s are a l s o p r o p o r t i o n a l to t h e i r phosphate contents. The mannans of B - s t r a i n are shown to be c r o s s - r e a c t i v e to r e l a t i v e l y weaker extents with a n t i s e r a of A- and J - s t r a i n s ; JL.e., only Subfractions B-IV and -V are c r o s s - r e a c t i v e with a n t i s e r a of the A- and J - s t r a i n s * S u b f r a c t i o n B-III can be regarded to be a h i g h l y s p e c i f i c antigen f r a c t i o n of B - s t r a i n because t h i s subf r a c t i o n i s devoid of any c r o s s - r e a c t i v e c o n s t i t u e n t and shows a



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Table I I .

Chemical isolated albicans strain),

Mannan subfractions

Carbohydrate(%)

POLYSACCHARIDES

composition of mannan s u b f r a c t i o n s (Batch II) from C. a l b i c a n s NIH A-207 ( A - s t r a i n ) , Ç . NIH B-792 ( B - s t r a i n ) , Ç. a l b i c a n s J-1012 ( J and S. c e r e v i s i a e . . b Protem(%)

Q

Phosphate(%)

Man/P molar ratio

Yield(%)

A-1 A-II A-III A-IV A-V

94 92 92 91 90

1.9 2.5 2.6 2.5 3.0

0.03 0.35 0.72 1.23 1.76

>1000 114 56 32 22

3.9 14.3 13.7 30.3 12.7

B-I B-II B-III B-IV B-V

95 92 93 92 90

2.2 2.5 2.6 2.6 2.3

0.06 0.38 0.76 1.38 2.36

688 105 53 29 17

2.1 5.1 11.4 31.9 24.9

J-I J-I I J-III J-IV J-V

94 91 91 90 89

2.1 3.0 3.5 4.1 4.3

0.03 0.20 0.50 0.95 1.70

>1000 202 81 42 23

15.1 17.5 13.7 22.1 9.7

S. c e r e v i s i a e A 96 Β 94 92 c D 91 90 Ε

1.5 1.9 2.3 3.1 3.9

0.04 0.22 0.47 0.78 1.45

>1000 186 85 51 27

4.8 8.7 12.0 23.4 25.5

a

Determined by the phenol - s u l f u r i c a c i d method (13) .

b

Determined by the Lowry-F o l i n method (14) ·

c

Quantitated by the Ames-Dubin method (15) as -P 0 H .

d

Weight b a s i s o f the corresponding bulk mannan.

3


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considerably strong r e a c t i v i t y to i t s homologous antiserum. Thus, the r e s u l t s o f the q u a n t i t a t i v e p r e c i p i t i n assay against a n t i - B - s t r a i n serum i n d i c a t e that the a n t i b o d y - p r e c i p i t a t i n g a c t i v i t i e s o f two mannan s u b f r a c t i o n s e r i e s of A- and J - s t r a i n s are c l o s e l y s i m i l a r to those o f B - s t r a i n , while the l a s t l a t t e r showed d i s t i n c t l y lower c r o s s - r e a c t i v i t i e s against a n t i s e r a o f the two former s t r a i n s , A and B. The mannan s u b f r a c t i o n s and a n t i s e r a of Batch II gave the c o n s i s t e n t r e s u l t s with those ob tained i n the r e a c t i o n o f the mannans and a n t i s e r a o f Batch I (Figs. 4-A, -B, and - C ) . C r o s s - p r e c i p i t i n r e a c t i o n between the mannan s u b f r a c t i o n s o f S. c e r e v i s i a e and three C. a l b i c a n s s t r a i n s against t h e i r a n t i sera, v i c e versa. As depicted i n F i g . 3-A, none o f the mannan s u b f r a c t i o n s of S. c e r e v i s i a e (Batch I) were shown to c r o s s react with a n t i - A - s t r a i n serum. A l l heterologous mannan subf r a c t i o n s , i n c l u d i n g those o f S. c e r e v i s i a e , were found to be c r o s s - r e a c t i v e with a n t i - B - s t r a i n serum i n the extents propor t i o n a l to t h e i r phosphate contents ( F i g . 3-B), i n d i c a t i n g that the d e n s i t i e s o f the c r o s s - r e a c t i v e a n t i g e n i c determinant groups i n these mannan s u b f r a c t i o n s are a l s o p r o p o r t i o n a l to t h e i r phos phate contents, and f u r t h e r i n d i c a t i n g that the d e n s i t i e s o f the c r o s s - r e a c t i v e a n t i g e n i c determinant groups i n these mannan subf r a c t i o n s are a l s o p r o p o r t i o n a l to t h e i r phosphate contents. The order o f the extents o f c r o s s - r e a c t i v i t i e s o f three hetero logous mannan s u b f r a c t i o n s e r i e s can be represented as A 4=j > S. c e r e v i s i a e , p r o v i d i n g a f i n d i n g that the c r o s s - r e a c t i v i t i e s between the mannans o f S. c e r e v i s i a e and a n t i - B - s t r a i n serum are s i g n i f i c a n t l y lower than those observed between antigens and a n t i s e r a o f C. a l b i c a n s . Against a n t i - S . c e r e v i s i a e serum, the mannan s u b f r a c t i o n s o f B- and J - s t r a i n s were found to be c r o s s r e a c t i v e i n low extents, and those o f Α-strain were completely unreactive to t h i s serum as shown i n F i g . 3-D. The above f i n d i n g s a l s o i n d i c a t e that the extent o f c r o s s - r e a c t i v i t y between the mannans o f C. a l b i c a n s and a n t i - S . c e r e v i s i a e serum i s a l s o lower than those o f antigen and a n t i s e r a o f C. a l b i c a n s . A d d i t i o n a l l y , the d i f f e r e n c e between the amounts o f p r e c i p i t a t e d antibody with Subfractions I and V of B- and J - s t r a i n s was not s i g n i f i c a n t , thus i n d i c a t i n g that the d e n s i t i e s o f the c r o s s r e a c t i v e a n t i g e n i c determinant groups i n these mannan subfrac t i o n s , o f which Hasenclever and h i s co-workers (9 10) assumed to be the al->3 l i n k e d D-mannopyranosyl residues, do not c o r r e l a t e with t h e i r phosphate contents. The r e s u l t s o f c r o s s p r e c i p i t i n r e a c t i o n s between the mannans o f S. c e r e v i s i a e and a n t i s e r a o f three C. a l b i c a n s s t r a i n s o f Batch I I gave essen t i a l l y s i m i l a r r e s u l t s to those o f the corresponding antigens and a n t i s e r a o f Batch I, excepting the f a c t that the a n t i - S . c e r e v i s i a e serum o f Batch I I d i d not show any c r o s s - r e a c t i v i t y with a l l mannan s u b f r a c t i o n s o f three C. a l b i c a n s s t r a i n s o f Batch I I ( F i g s . 4-A, -B, -C and -D). r



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Figure 3. Quantitative precipitin reactions between the mannan subfractions (Batch I) and antisera of three C . albicans strains and S. cerevisiae: (A) anti-C. albicans NIH A-207 (Α-strain) serum; (B) anti-C. albicans NIH B-792 (Bstrain) serum; (C) anti-C. albicans J-1012 (J-strain) serum; (D) anti-S. cerevisiae serum. Mannan subfractions are of: (O) Α-strain; (Φ) B-strain; (A) J-strain; (A) S. cerevisiae.

Figure 4. Quantitative precipitin reactions between the mannan subfractions (Batch II) and antisera of three C . albicans strains and S. cerevisiae: (A) anti-C. albicans NIH A-207 (Α-strain) serum; (B) anti-C. albicans NIH B-792 (B-strain) serum; (C) anti-C. albicans J-1012 (J-strain) serum; (D) anti-S. cerevisiae serum. Mannan subfractions are of: (O) Α-strain; (Φ) B-strain; (A) J-strain; (A) S. cere visiae. Dilutions of anti-S. cerevisiae serum are homologous system, X 6; heter ologous system, X 1.5.


6. OKUBO ET AL.

Immunochemistry

of Candida

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105


Discussion I t i s well-known that C. a l b i c a n s i s one o f the most important pathogenic fungi causing a s e r i o u s i n f e c t i o u s disease, C a n d i d i a s i s , i n humans. In order to provide a s e r o l o g i c a l c l a s s i f i c a t i o n system f o r yeasts i n c l u d i n g genus Candida, antigen s t r u c t u r e analyses o f C. a l b i c a n s and i t s c l o s e l y r e l a t e d species have been conducted by s e v e r a l workers, Martin et a l . (17), Tsuchiya e t a l . (18) , P o s p i S i l (19) , and Biguet e t a l . {20), r e s p e c t i v e l y , although assignment o f symbols i n the antigen formulae proposed by these workers to the chemical s t r u c t u r e s o f the i s o l a t e d antigen has not been achieved as y e t . In 1961, Hasenclever and M i t c h e l l (6, 1) reported that species C. a l b i c a n s can be c l a s s i f i e d i n t o two groups e x h i b i t i n g d i s t i n c t l y d i f f e r e n t serotypes designated A and B, r e s p e c t i v e l y . Hasenclever and h i s co-workers a l s o provided the f o l l o w i n g f i n d i n g s that the a n t i g e n i c i t y o f yeasts i n c l u d i n g genera Candida, Saccharomyces, and Hansenula, i s a t t r i b u t a b l e to t h e i r c e l l w a l l mannans (9, 10). On the s t r u c t u r a l feature of the a n t i g e n i c determinant groups o f the mannans o f B - s t r a i n , they assumed that ocl-*3 l i n k e d D-mannopyranosyl residues occupying the non-reducing terminal groups i n t h i s mannan dominate the serotype s p e c i f i c i t y , because the mannan o f S. c e r e v i s i a e , i n which the existence o f the above type o f linkages had been confirmed by Peat e t a l . (2^1) , was shown to be c r o s s - r e a c t i v e with the mannan of B - s t r a i n against a n t i - S . c e r e v i s i a e serum (10). T h e r e a f t e r , the presence o f an a d d i t i o n a l serotype, C, was proposed by Tsuchiya and h i s co-workers i n the C. a l b i c a n s s t r a i n s i s o l a t e d from p a t i e n t s o f C a n d i d i a s i s i n Japan (Q). A n a l y s i s o f the chemical s t r u c t u r e o f the mannan o f a C. a l b i c a n s s t r a i n , f i r s t achieved by Bishop and h i s co-workers, provided evidence that t h i s mannan possesses a h i g h l y branched s t r u c t u r e c o n s i s t i n g o f D-mannopyranosyl residues connecting with od-*2 and od -*6 linkages (22) . In 1965, an important m o d i f i c a t i o n o f the chemical s t r u c t u r e of bakers yeast mannan was made by B a l l o u and h i s co-workers. They pointed out that the longest branching moieties o f t h i s mannan correspond to a t e t r a o s e , O-orD-mannopyranosyl (1 -»3) O-aD-mannopyranosyl (l-*2) O-oc-D-mannopyranosyl (l-*2) mannose (23) . In 1967, the Canadian workers and Hasenclever published the modified f i n d i n g s o f s t r u c t u r a l a n a l y s i s o f the mannan o f s e v e r a l Candida s t r a i n s i n c l u d i n g those o f C. a l b i c a n s serotype A, C. a l b i c a n s serotype B, C. t r o p i c a l i s , C. s t e l l a t o i d e a , and C. p a r a p s i l o s i s , a f f o r d i n g evidence that a l l mannans contain small amounts o f al->3 l i n k e d D-mannopyranosyl residues i n a d d i t i o n to the large amounts o f ocl->2 and ocl ->6 linkages (24) . Basing on the above f i n d i n g s provided by the workers above mentioned, we s t a r t e d a s e r i e s o f immunochemical s t u d i e s o f yeast mannans. In 1968, we reported a n a l y s i s o f the a n t i g e n i c d e t e r minant groups o f the mannan o f S. c e r e v i s i a e , i n which the mannan 1



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was fragmented by two methods, a c e t o l y s i s and p a r t i a l a c i d h y d r o l y s i s , separately, and the r e s u l t a n t o l i g o s a c c h a r i d e s were assayed for t h e i r p r e c i p i t i n - i n h i b i t o r y a c t i v i t i e s to an antigen antibody system c o n s i s t i n g of the parent mannan and a n t i - S . c e r e v i s i a e whole c e l l serum. The r e s u l t of t h i s assay c l e a r l y i n d i c a t e d that a mannotetraose, Man a l -» 3Manal 2Mancd -»2Man, i s o l a t e d from the a c e t o l y s a t e of the parent mannan showed the strongest p r e c i p i t i n - i n h i b i t o r y a c t i v i t y , p r o v i d i n g evidence that the tetraose moieties i n the mannan correspond to the a n t i g e n i c determinant groups (25). Correctness of t h i s f i n d i n g was confirmed by B a l l o u {26.) . As subsequent work i n t h i s s e r i e s , we have analyzed the a n t i g e n i c determinant groups of the mannan of a C. a l b i c a n s s t r a i n i s o l a t e d i n Japan by means of s i m i l a r procedure to that of the a n a l y s i s of the a n t i g e n i c determinants of the mannan of S. c e r e v i s i a e , and a mannohexaose c o n s i s t i n g of one ocl-*3 and four a l - * 2 linkages obtained from the a c e t o l y s a t e of the parent mannan was shown to r e v e a l the strongest p r e c i p i t i n - i n h i b i t i o n a g a i n s t the homologous mannan-anti-C. a l b i c a n s whole c e l l serum system (27, 28). Further a n a l y t i c a l studies of the a n t i g e n i c d e t e r minant groups were conducted on the mannans of A- and B - s t r a i n s , r e s p e c t i v e l y , to obtain the f o l l o w i n g f i n d i n g s ; 1) a mannoheptaose connecting s o l e l y with l->2 linkages i s o l a t e d from the a c e t o l y s a t e of the parent mannan corresponds to the a n t i g e n i c determinant groups of the mannan of C. a l b i c a n s NIH A-207 (As t r a i n ) (29) and 2) a hexaose f r a c t i o n c o n t a i n i n g od->2 and ocl->3 linkages i n a r a t i o of 10 : 1 revealed the strongest i n h i b i t o r y a c t i v i t y against the homologous mannan-antiserum system of the Bs t r a i n (_30) . However, the mannans of S. c e r e v i s i a e and C. a l b i c a n s so f a r used i n our previous study were the n e u t r a l f r a c t i o n s excluded from phosphorus and p r o t e i n , c o n s i s t i n g of minor parts of the parent bulk mannans, and r e l a t i v e l y l i t t l e information was a v a i l able regarding the immunochemical p r o p e r t i e s of the a c i d i c mannan f r a c t i o n s , the major p a r t of the bulk mannans, d e s p i t e various important f i n d i n g s on the s t r u c t u r a l feature of the l o c a t i o n s i t e of phosphate groups i n s e v e r a l yeast mannans accumulated through the e f f o r t s of B a l l o u and h i s co-workers (_31, 32/ .33) · Recently, we published the r e s u l t s of an immunochemical study of the bulk mannan of S. c e r e v i s i a e with s p e c i a l reference to the s e r o l o g i c a l a c t i v i t y o f the a c i d i c f r a c t i o n (_5) . Namely, the bulk mannan of S. c e r e v i s i a e was f r a c t i o n a t e d on a column of DEAE-Sephadex i n t o f i v e s u b f r a c t i o n s c o n t a i n i n g d i f f e r e n t amounts of phosphate groups according to the method described by Thieme and B a l l o u on the f r a c t i o n a t i o n of the phosphomannan of Kloeckera b r e v i s (16) ( F i g . 5), and these mannan s u b f r a c t i o n s were assayed for t h e i r a n t i b o d y - p r e c i p i t a t i n g a c t i v i t i e s against the homologous anti-whole c e l l serum ( F i g . 6). From the r e s u l t of the q u a n t i t a t i v e p r e c i p i t i n r e a c t i o n , i t was evident that the a n t i b o d y - p r e c i p i t a t i n g a c t i v i t y of these mannan s u b f r a c t i o n s was


6.

OKUBO E T A L .

Immunochemistry

of C a n d i d a

107

Mannan


0.1M NaCI

0

100

200 FRACTION NUMBER

300

Journal of Bacteriology Figure 5. Elution profile of 3 g of bulk mannan on a DEAE-Sephadex A-50 column (acetate, 4 X 25 cm) by stepwise elution with water and NaCI solutions. Samples (10 μg) of fractions were assayed for carbohydrate content with phenolsulfuric acid reagent (13): O.D., optical density (5).

0

5 10 15 20 MANNAN ADDED (jjg)

25


Figure 6. Quantitative precipitin curves of five mannan subfractions against ho mologous anti-whole-cell serum of parent bakers yeast: (O) Fraction Α; (Φ) Frac tion B; (A) Fraction C; (A) Fraction D; (Q) Fraction E; O.D., optical density (5).



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p r o p o r t i o n a l to t h e i r phosphate contents (Table III) . Therefore, i t i s reasonable to mention that the mannan s u b f r a c t i o n cont a i n i n g the l a r g e s t amount o f phosphate groups corresponds to the strongest antigen of the parent y e a s t . The s t r u c t u r a l a n a l y s i s o f f i v e mannan s u b f r a c t i o n s by the c o n t r o l l e d a c e t o l y s i s technique afforded a f i n d i n g that the d e n s i t y of the longest branching moiety corresponding to the a n t i g e n i c determinant group i n these s u b f r a c t i o n s was p r o p o r t i o n a l to t h e i r phosphate content ( F i g . 7 ) . C o n t r i b u t i o n of phosphate groups to the s e r o l o g i c a l a c t i v i t y as an a n t i g e n i c determinant group i s deniable, because inorganic phosphate was shown to be completely i n a c t i v e to i n h i b i t the homologous antigen-antibody system. These f i n d i n g s i n d i c a t e that two types of microheterogeneities, r e l a t i n g to phosphate content and to d e n s i t y of the branching moieties, c o e x i s t i n a bulk mannan, and the f u n c t i o n of phosphate groups can therefore be assumed to a c t as a marker evoking s t r u c t u r a l complexity by elongation of the branching moieties, or terminating the extending branching moieties, or both. In the present study, we revealed the existence of a microheterogeneity i n three C. a l b i c a n s s t r a i n s o f d i f f e r e n t serotypes, A, B, and C, s i m i l a r to that observed i n the bulk mannan of S. c e r e v i s i a e (5). From the r e s u l t s obtained i n the previous (_5) and present s t u d i e s , the f o l l o w i n g f i n d i n g s can be obtained: 1) Microheterogeneity r e s i d i n g i n the bulk mannans of S. c e r e v i s i a e and C. a l b i c a n s s t r a i n s can be regarded to c o r r e l a t e to t h e i r phosphate contents which dominate the d e n s i t i e s of the branching moieties i n c l u d i n g the a n t i g e n i c determinant groups. 2) The f a c t that the r e a c t i v i t i e s of the mannan s u b f r a c t i o n s of three C. a l b i c a n s s t r a i n s against a n t i - A - s t r a i n serum were strong and p r o p o r t i o n a l to t h e i r phosphate contents i n d i c a t e s that the d e n s i t i e s of the c r o s s - r e a c t i v e a n t i g e n i c determinant groups, corresponding to a mannoheptaose moiety c o n s i s t i n g s o l e l y of ocl-»2 l i n k e d D-mannopyranosyl residues (29), are c o r r e l a t i v e with the phosphate content of the mannan s u b f r a c t i o n s of three C. a l b i c a n s s t r a i n s . 3) On the other hand, the f i n d i n g that c r o s s - r e a c t i v i t i e s between antigens and a n t i s e r a of three C. a l b i c a n s s t r a i n s and S. c e r e v i s i a e were weak and not n e c e s s a r i l y p r o p o r t i o n a l to the phosphate contents of the antigens i n d i c a t e s that the d e n s i t i e s of the c r o s s - r e a c t i v e a n t i g e n i c determinant groups, c o r r e sponding to the branching moieties possessing non-reducing terminal 3 l i n k e d D-mannopyranosyl residues (25, 26., 30) i n the mannan s u b f r a c t i o n s of C. a l b i c a n s are not c l o s e l y c o r r e l a t e to t h e i r phosphate contents. 4) I t w i l l a l s o be worthy to note that the mannan s u b f r a c t i o n of each s t r a i n c o n t a i n i n g the l a r g e s t amount of phosphate corresponds to the strongest antigen of the parent yeast, allowing an assumption that such a molecular species d i s p l a y i n g antibody response i n the strongest extent should l o c a t e the utmost surface of the c e l l w a l l of the parent yeast. Further immunochemical i n v e s t i g a t i o n of yeast mannans r e l a t i n g to t h e i r a n i o n i c microheterogeneity can be expected to


6.

OKUBO E T A L .

Immunochemistry

of C a n d i d a Mannan

109

Table I I I . Chemical composition o f mannan s u b f r a c t i o n s % Composition

Optical

Subfrac tion

Carbo hydrate


A B c D Ε

99 96 98 94 93

Protein

b

Phosphate

3

0.0 0.1 0.1 0.1 0.3

0.00 0.23 0.57 0.99 2.09

0

Man/P

specific^

molar . e ratio

+ 80° +78° +77° +76° +72°

>1,000 178 73 41 19

rotation,

a

Determined by the p h e n o l - s u l f u r i c a c i d method (13).

b

Determined by the F o l i n method o f Lowry e t a l . (14).

c

Quantitated by the methpd o f Ames and Dubin (15) as P0 H,

d

c l . 0 , 11.0, water.

e

The r a t i o o f mannose residues to phosphate groups.

3



Figure 7. Acetolysis fingerprints of Subfractions Α (Α), Β (B), C (C), D (D), and Ε (Ε) (5). Each acetolysate, obtained from 100 mg of the mannan subfractions, was applied onto a column (2 X 100 cm) of Bio-Gel P-2 and eluted with water (7 mL/hr). Samples (20 μΕ) of eluates corresponding to Fractions I, II, III, and IV were assayed for the amounts of carbohydrate, and 50 μΕ of Fraction Vo was used for the determination of carbohydrate and phosphorus in the void volume regions: (Φ) carbohydrate at 490 nm; (X) phosphorus at 820 nm; O.D., optical density.


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provide much i n t e r e s t i n g information on the a r c h i t e c t u r e o f yeast c e l l walls and the f u n c t i o n o f phosphate groups e x i s t i n g i n many polysacchar ides·


Abstract The mannans i s o l a t e d from Candida a l b i c a n s s t r a i n s , NIH A207 (serotype A ) , NIH B-792 (serotype Β ) , and J-1012 (serotype C ) , were f r a c t i o n a t e d on a column o f DEAE-Sephadex i n t o f i v e s u b f r a c t i o n s c o n t a i n i n g d i f f e r e n t amounts o f phosphate. A n t i b o d y - p r e c i p i t a t i n g a c t i v i t i e s o f the mannan s u b f r a c t i o n s of s t r a i n s NIH A-207 and NIH B-792 were p r o p o r t i o n a l to t h e i r phosphate content, while those o f s t r a i n J-1012 d i d not show regularly proportional p r e c i p i t i n a c t i v i t y . A s i m i l a r tendency was a l s o observed i n the c r o s s - r e a c t i o n between the mannan s u b f r a c t i o n s o f s t r a i n s NIH A-207 and J-1012 and t h e i r heterologous a n t i s e r a . The mannans o f s t r a i n NIH B-792 showed lower c r o s s - r e a c t i v i t i e s against a n t i s e r a of s t r a i n s NIH A-207 and J-1012, i.e., only two s u b f r a c t i o n s c o n t a i n i n g l a r g e r amounts o f phosphate were able t o c r o s s - r e a c t with these antisera. The mannan s u b f r a c t i o n s o f Saccharomyces c e r e v i s i a e , pre pared by the s i m i l a r chromatographic f r a c t i o n a t i o n o f the c o r r e sponding bulk mannan, showed no or very weak c r o s s - r e a c t i v i t y with a n t i s e r a o f s t r a i n s NIH A-207 and NIH B-792, while the mannan s u b f r a c t i o n s o f s t r a i n s NIH B-792 and J-1012 were weakly c r o s s - r e a c t i v e with a n t i - S . c e r e v i s i a e serum, a f f o r d i n g almost i d e n t i c a l amounts o f p r e c i p i t a t e d antibody nitrogen r e g a r d l e s s o f the phosphate content o f these mannan s u b f r a c t i o n s . From the above f i n d i n g s , it is reasonable to conclude that mannan molecular species c o n t a i n i n g the l a r g e s t amount o f phos phate groups corresponds t o the strongest antigen o f the parent yeast, and that such a molecular species i s assumed to occupy the utmost surface layer o f the cell w a l l o f the parent y e a s t . Acknowledgment We thank Dr. Y. Fuazawa, Department o f Microbiology, M e i j i C o l l e g e o f Pharmacy, f o r generously supplying the Ç . a l b i c a n s s t r a i n s and a n t i - J - s t r a i n serum. We are a l s o g r a t e f u l to Mr. Nobuyuki Shibata of our Department f o r h i s t e c h n i c a l a s s i s t a n c e s . T h i s work was supported i n p a r t by a G r a n t - i n - A i d from the M i n i s t r y o f Education, Science, and C u l t u r e , Japan.

Literature Cited (1) (2) (3)

Sakaguchi, O.; Suzuki, S.; Suzuki, M . ; Sunayama, H . ; Jpn. J. Microbiol., 1967, 11, 119. Lloyd, Κ. O.; Biochemistry, 1970, 9, 3446. Thieme, T. R.; Ballou, C. Ε . ; Biochemistry, 1971, 10, 4121.


6.

OKUBO ET AL.

(4) (5) (6) (7) (8) (9)


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RECEIVED August 6, 1979.


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