On the structure of the capsular polysaccharide fromCryptococcus neoformans serotype C

0019-2791,'78,'0901-Oh73 802.00/O

ON THE STRUCTURE OF THE CAPSULAR POLYSACCHARIDE FROM CR YPTOCOCCUS NEOFORMANS SEROTYPE C A. K. BHATTACHARJEE.’

K. J. KWON-CHUNG’

and C. P. J. GLAUDEMANS’*

‘National In%tilurc of Arthrttia. Infcctioua

Metabolism and Digestive Diseases, ‘National Institute of Allergy DIW~VZL National Institutes of Health. Bethesda. MD 20014. U.S.A.

and

Abstract

The capa~lar polysaccharide from (.~~,/~fo(,oc,(,lr.vW~J~W/PIWI.$ serotype C has been studied using the usual methods for the elucidation ofchemical structure. The results are conslatent with the occurrence of a polqsaccharide having an I I .3 linked backbone of D-mannopyranoside residues, every other one of which iadoubly sutxtltuted with two terminal I)-xylopqranosyl residues linked/j 1.2 and/i 1.4 respectively, and with the rcmaindcr ofthe mannobql rcslducs substituted with a terminal I>-glucuronosidopyranosyl residue linked /i 1.2. AlternativeI>. the tcrmlnal glucuronlc acid residue may he /I’ I.2 linked lo a mannosyl residue in the chain already bearing a sing11 hrnnched /j I .4 xylopyranosyl r&due. while the neighboring mannosyl residue carries a smglc branch of/i I.2 xylopyranoGdc.

is a yeast-like fungus which is pathogenic for man and animals. The cells of C. neofiwnums are encapsulated with an antigenic polysaccharide which contains determinants defining their serotypes (Evans. 1949, 19SO). Initially, there were three serotypes. A. B and C. Later. Wilson rt ul. (1968) described the fourth group, D. Recently. evidence for more fundamental heterogeneity was found among the isolates. Numerous isolates of serotypes A and D produced a perfect state called ~~lohus~di~~ll~~ rwofiwnm~.s ( Kwon-Chung. 1975) when crossed with opposite mating types. The isolates of serotypes Band C. on the other hand, produced the F. hu~~illispor~4 state upon cross. Intercrossing between A-D and B-C isolates yielded only nonviable spores. Biochemical differcnces were also noted between the A-D and B-C groups. For instance. recent studies by Bennett c’t rrl showed that L-malic acid was utilized as the sole source ofcarbon by serotypes B and C but not by A and D (Bennett (‘I al., 1978). Differences have also been discovered in the geographic distribution 01 the four serotypes. While serotype A was widespread throughout the world. D was found to be rare in the United States but common in Europe. B and C types were rare causes of infection except in Southern California (Bennett <‘I t/l.. 1977). Also the natural reservoir for the serotypes A and D seemed to be distinct from that of B and C. Serotypes A and D were most commonly found in association with pigeon droppings. while the ecological niche of B and C still remains unknown. An early report on the analysis of the chemical composition of a type A polysaccharide showed that it contained xylose. mannose, galactose and glucuronic acid, although the preparation used was shown to be heterogeneous (Rebers C! ul., 1958). Later, studies on the composition and structure of the capsular polyCr_13p/oc~~c~c~u.s rwofijr-rlwm

*To whom all correspondence

should

he sent. 673

saccharide from c‘. nrojtirman.s type B were reported (Blandamer & Danishefsky, 1966). On the basis of the composition of mono- and oligosaccharides, it was suggested that the polysaccharide consists of a mannan backbone with branches of xylose and glucuronic acid. Galactose (7.7’1,) was also found as a component sugar. A report on an untyped polysaccharide from C. r~rc~/brman.s CRD-I (Duke) showed that it consisted of o-xylose (2O.S’%;), Dmannose (61.2”,,) and D-glucuronic acid (I 8.92,). Periodate oxidation and methylation studies indicated a branched mannan structure containing mannose units linked l,2- in the main chain and 1,4- in the branch points. Xylose and glucuronic acid were present as end groups (Miyazaki, 1961). No work has been reported on the structures of the capsular polysaccharides from Cryptococcus nc,ofhrmans type C and type D. In order to find the chemical foundation for the classification of C. ncvf&mm.s in the system of Evans (Evans, 1949, 1950) it is necessary to elucidate the fine structures of the antigenic capsular polysaccharides of these organisms. It is the purpose of the present paper to report our studies on the structure of the capsular polysaccharide of C. t7~wf01.n7un.s type C. Studies on the polysaccharide of type D will be reported later.

MATERIALSAND METHODS

Preparations were made from a scrotype C isolate. NIH 19 I. of C‘. I~~~O/OI.II~NII.S obtained from the cerebrospinal fluid of a patient with meningltls. The isolate has been designated as the type culture N mating type of Filohosididlu hacillisporu (Kwon-Chung. 1976). Heavy cell suspensions prepared from a Whr malt extract agar slant culture were inoculated into I liter modified sabouraud dextrose broth (Emmons (‘I rrl.. 1977). After incubation on a rotary shaker at 25 C for I week, cells were killed by addlng 10 ml ofhuffered formalin (sodium acetate Y g. H,O 900 ml and Formalin 90 ml). The culture was allowed to stand in the refrigerator for 24

674

A. K. BHATTACHARJEE,

and C. P. J. GLAUDEMANS

K. J. KWON-CHUNG

Froctloos,

20 ml

Fig. I. Chromatography of the native polysaccharide from C. neoformans type C on DEAE-cellulose (2.5 x 30 cm) using 0.01 ,V phosphate buffer. pH 7.3. Arrows indicate thechange in the salt concentration to 0.2 !M NaCl and 0.4 .1-I NaCl. respectively. hr and cells were removed by centrifugation. Procedures for the isolation of polysaccharide from the supernatant were slightly modified from the method described by Bennett (Bennett & Hasenclever. 1965). The supernatant (800 ml) was treated ulth sodium acetate (80 g) and glacial acetic acid to give pH 7 before adding 95”,, ethanol to precipitate the polysaccharide. The precipitate was removed. dissolved in 200 ml distilled water and treated with 20 g sodium acetate and 2 ml glacial acetic acid. After the sodium acetate was dissolved. the solution was deproteinated with 40 ml chloroform and 8 ml rl-butanol. The aqueous solution was then treated with sodium acetate. glacial acetic acid and 95”,, ethanol in the same proportion as above to precipitate the polysaccharide. The precipitate was redissolced in distllled water and reprecipitated with 95”,, ethanol. This procedure was repeated two more times and finally the polysaccharide was redissolved in distilled water and dialyzed against distilled water (3 I.) with toluene (3 ml) for 72 hr at 4 C. The dialyzed polqsaccharide solution was filter-sterilized and lyophilized and the white powder Has stored over calcium sulfate.

The capsular polysaccharide as obtained above was of DEAE-cellulose chromatographed on a column (Whatman DE-52) using 0.01 M phosphate buffer, pH 7.3. and a linear salt gradient of O-l M NaCI. The elution pattern showed a major peak that was eluted at a salt concentration of 0.2 M with a shoulder that followed up to a salt

concentration of 0.4 M. Therefore the polysaccharide was purified by chromatography on DEAE-cellulose (2.5 x 30 cm) by stepwjise elution with 0.01 M phosphate buffer containing 0. 0.2 and 0.4 ,V NaCl respectively. The eluted fractions were tested for the reducing sugar content by the phenol sulphuric acid method (Dubois et cl/.. 1956). A typical elution pattern is shown in Fig. I. It can be seen that about 80”,, of the material w’as eluted at a salt concentration of 0.2 ,M. This material was extensively dialyzed against distilled water and freeze dried. This preparation was homogeneous as seen by ultracentrifugation and gel filtration analysis and was used for the present study (Figs. 2 and 3). GoI filrrntior~ trnrl ult,cr~rrlt~~ftr~utio,l Gel tiltration was performed using a column (2.5 x 75 cm) of sepharose 68 (Pharmacia Fine Chemicals, Uppsala. Sweden). PBS (0.01 M phosphate buffer. pH 7.2, containing 0.15 M NaCI) was used as the eluant. Ultracentrifugation was performed on a Beckman Spinco Model E analytical ultracentrifuge at 60.000 re\l/min. The polysaccharide was dissolved in PBS at a concentration ot 0.‘” - ,I CII~oti7~lto~~u/~h~~

Paper chromatography was performed by the descending method using Whatman No. I and 3 MM paper. The solvent systems used were (A) ethyl acetate:pyridine:water (10:4:3 by vol.) and (B) ethyl acetate:acetic acid:formic acid:water (18:3:1:4 by vol.). The sugars were detected either by using

15

8

IO

8

05

4

8

12

Fractions, Fig. 2. Chromatography of the purified polysaccharide achieved with 0.01 M phosphate buffer, pH 7.3, containing

20

16

IO

24

28

ml

on Sepharose 6B (2.5 x 75 cm). Elution was 0.15 !Lf NaCI. The arrow shows the void volume.

Structure

Fig. 3.

Jltracentrifugation

pattern

of Cryptococcal

of the purified polysaccharide for 84 min.

alkaline silver nitrate reagent (Trevelyan rt al.. 1950) or by spraying with a solution ofp-anisidine hydrochloride in nbutanol (Hough CI ~1.. 1950). Gas-liquid chromatography (g.1.c.) was performed using a Finnigan 9500 gas chromatograph on glass columns packed with (i) 3”,, ECNSS-M on gas Chrom Q (100-120 mesh) at l7O’C for methylated alditol acetates and at 190 C for alditol acetate derivatives of free sugars, and (ii) 3”,, NPGS on Gas Chrom Q (100-120 mesh) at a temperature of l2O~C for the estimation of O-acetyl content (Bethge & Lindstrom. 1973). Gas chromatography-mass spectrometry (g.c..m.s.) was performed on a LKB Bromma-2091 gas chromatograph mass spectrometer operated at 70 eV (Jannson curul.. 1976).

Complete hydrolysis of the polysaccharide was effected bq heating with I N HCI at 100 C in sealed tubes for I6 hr. Partial hydrolysis took place by heatmg the polysaccharide with 0.5 M HCI at 100 C in sealed tubes for 2.4 and 6 hr. The cooled solutions were neutralized with powdered silver carbonate. deionized with Dowex-50 H’ resin and evaporated to dryness. The identity of the component sugars wasconfirmed by conversion into alditol acetates followed by gas-liquid chromatography using column (i) (Sawardeker ef al., 1965). Quantitative estimation of the component sugars was done by g.1.c. of the alditol acetates using inositol as the internal standard. The peak areas were obtained by triangulation.

Polysaccharides anion and methyl

Polysaccharide

were methylated using methyl sulfinyl iodide (Hakomori. 1964). In a typical

Serotype

c‘. rwofiwmurlv

675

C

type C. at 60,001 re\ min

experiment 20 mg of the dry polysaccharide has dissolved into 5 ml of DMSO by stlrring overnight and using an ultrasonic bath. Methyl sulphinyl anion m DMSO (0.5 ml) was added to the clear solution and the mixture was stlrred for 7 hr. The viscous suspension mas then treated with 0.2 ml of iodomethane (dropwise) while cooling the mixture in an ice-water bath. The reaction mixture was next sonicated for half an hour. and the methylated polhsaccharide uas recovered by dialysis against distilled water and freeze drying. The material aas further dried in a vacuum oven at 50 C overnight (yield 18 mg). Infrared spectroscopy of the methylated polysacchartde shoned the absence of any hydroxql absorption. The methylated polysaccharidc was carbox>]-reduced with lithium alummium deuterlde (Lythgoc Xr Trippett. 1950).

Periodate oxidation was performed using 0.02 &I sodium metaperiodate at room temperature in the dark. Aliquots were withdrawn at inter\als and the consumption of periodate was determined bq a spectrophotometric method (Aspinall & Ferrier. 1957). In some experiments the periodate oxldtzed poly\accharide was reduced with sodium borohqdride and then subjected to a mild hydrolysis (Goldstein PI cl/.. 1959). In one experiment I05 mg of the purified polqsaccharide was taken up in 100 ml of 0.02 n-I sodium metaperiodate solution and the mixture was allowed to stand at room temperature in the dark for 72 hr. The excess periodate was destroyed by adding ethylene glycol and the mixture was dialyzed against distilled water. Sodium borohydride (200 mg) was added to the dialyzed solution and the whole allowed

676

A.

K. ~HATT‘4~HARJEE.

K. .I. KWON-~HlJ~~

to stand at room temperature for 7 hr. The solution was neutralized with I !VHCI and then concentrated to 50 ml. 6 N HCI nas added to a final concentration of 0.5 N and the mixture M~S kept at room temperature for X hr. The solution was neutrallxd with NaOH solution and dialyzed against distilled water ;tnd freeze dried (yield 42 mg).

Oxidation oi‘the acetyiated poly~dccll~r~de with chromium trioxidc v,as performed as follows (Larm L’I
All evaporations were carried out at or below 40 f. Optical rurations were measured on :i Perkin-Elmer 141 Polarimetcr at 20 C. The carboxyl-reduced polysaccharidc uas prepared b> reduction with sodium borohydride using carhodiimldc according to a known method (Taylor & Conrad. 1971). Two treatments with the reagents were required for complete reduction. The U-acetyl content ofthe polqaaccharide henry1 acetate

was determined by gas chromatography (Bethge & Lindstrom. 1973).

a\

RESULTS

Figure 2 shows the eiution pattern of the purified polysaccharide from ~‘~~~~roc~occrr.s n~~/Or~nu~s type C obtained from gel filtration on Sepharose 6B. it shows a symmetricii peak with a K,, of 0.09 indicating a very high mol. wt (cu. 8 x IO”). Analytical ultracentrifugation showed an ultrasharp peak (Fig. 3) indicating that the preparation was homogeneous. The purified polysaccharide had [r]$’ ~ 12’ (c 0.25 in water). Analysis for N and P and O-;lcetyi showed the presence ofO. I Y’,, nitrogen. 0.06”,, phosphorus and 3”,, O-acetyi groups. The infrared spectrum of the purified polysaccharide showed very weak peaks at 1740 cm ’ and 1240 cm- I regions, the characteristic absorption peaks for O-acetyl groups. The polysaccharide gave a viscous solution at a concentration of 3 mg,‘ml.

Complete hydrolysis of the poiys~ceharide followed by chromatography in solvents A and B showed the presence of three component sugars having mobiiities identical to mtinnose, xyiose and glucuronic acid. The mobilitifs of the sugars relative to D-glucose (R,) were I .09, I .30 and 0. I4 respectively in solvent A and I .07, 1.23 and 0.9 in solvent B. The identity of the component sugars was confirmed by conversion into aiditol acetates and gas chromatography using column (i). The presence of giucuronic acid was confirmed by analysis of the c~trboxyi-reduced poiysaccharide when glucose was obtained instead of glucuronic iicid. Quantitative estimation of the sugars as aiditoi

and C. P. J. ~;I,AL~~~~A~S

acetates using inositol as internal standard showed a molar ratio of xyiose:mannose as 4:3 in the polysaccharide and of xylose:mannose:giucose as 4:3: 1 in the carboxyl-reduced polysaccharide. A large-scale hydrolysis was performed using 100 mg of the poiysaccharide and the component sugars were isolated by paper chromatography using solvents A and B. Mannosc [x]$” t I2 (c I.0 in H,O), xyiosc [XI:“+ IX (c 0.5 in H,O) and eiucul-onic acid [%]?-I- 20 (c 0.4 in H,O) bvere obtlined indicating that all the sugars are present in the u-ccntrifugation. Partial hydrolysis of the polysaccharide with 0.5 N HCI at 100 C for 2 hr followed by paper chromatography showed the presence of xylose as the major component. A small amount of mannose was released but no glucuronic acid was detected. Quantitativeestinl~itiotl showed that 30”,, ofthe xyiose residues were removed by this treatment. The degraded polysaccharidr was recovered by dialysis and freeze drying. it had [x]“~ f 18 (c 0.5 in H,O). A IO mg portion of the degraded polysaccharide was methylated (sample IV) and hydrolyzed. lirst with W’,, formic acid at 100 C for I hr and then with 0.15 ill suiphuric acid at 100 for I6 hr. The product was converted into aiditoi acetates and analyzed by g.i.c. and g.c./m.s. It showed the presence of 2,X4-tri-Omethyl xyiose, 2,4.6-tri-0-rnet~lyl mannose and 4.6diO-methyl mannose as the major components. Two minor components. 2.3,4.6-tetra-O-methyl mannosc and 6-O-methyl mannose were also detected. it was found that hydrolysis of the polysaccharide with 0.5 ,V HC’I at 100 C for 6 hr yielded the maximum amount of an oligosaccharide having K,, 0.60 in solvent B. Therefore, 100 mg of the polys~lcch~tride was hydrolyzed under these co~iditions and neutral sugars were separated by passing through a short column of Amberlite IR-400 resin in the acetate form. The acidic fraction was eluted with I M formic acid. Paper chromatography showed the presence of one distinct oligosaccharide R,, 0.60 in solvent B and some sm-lll amount of monosaccharides. The oligosaccharide was isolated by prcparattvc paper chromatography in solvent B. yield 8 mg. [rli{’ .-- 20 ((, 0.S in H,O). Hydrolysis of the oligos~icch~tridc wtth 1 ,%HCi at IO0 C‘ for 16 hr gave mannose and glucuronic acid. Borohydride reduction followed by hydrolysis and paper chromatography showed the presence of mannitol and glucuronic ;Icid. No mannose was obtained. Thcrcfore. the oligosaccharide IS probably a disaccharide containing mannosc as the reducing sugar. A .5-mg samptc of the oligosaccharide was lnetl~~lated. ilydroiyzed and converted into aiditol acetates. g.l.c. using column (i) and g.c./m.s. showed the presence of X4.6tri-O-methyl mannose only. The identification of 3.4,6-tri-O-methyl mannose WBS unambiguous and shows that the giucuronic acid residue is linked to O-2 of the mannosc unit in this oligosaccharide. Although this observation does not exctude some giucuronic acid residues being linked to C-4 of ~~~~i~~~~ose units in the polys~~cc~~~~ride, it would be rather likely that all the glucuronic acid units are linked in the same manner as tbund in the acidic disacchnridc.

Structure

Methylation

of Cryptococcal

analysk

The methylated polysaccharide (I) has [c(]g - 28’ fc 0.25 in CHCI,). The infrared spectrum showed the absence of any hydroxyl absorption. The methylated polysaccharide was hydrolyzed and the mixture of component sugars converted into alditol acetates. g.1.c. and g.c./m.s. showed the presence of 2.3,4-tri-Omethyl xylose (having major m.s. fragments m/e 161, 129, 117, 101, 87, 71), 4,6-di-O-methyl mannose (m/e 261,201. 187, 161. 159, 129, 127, 101,99,87,85)and6O-methyl mannose (m/e 259 231, 217, 187, 170, 159, 147, 139, 129, 115, 103. 99, 97. 87, 85) as the only components. A 5-mg portion of the methylated polysaccharide was carboxyl reduced with LiAID, and the resulting product was isolated by extraction with chloroform and evaporated to dryness. The product (II) was hydrolyzed and the components analyzed as their alditol acetate by g.1.c. and g.c.:m.s. This showed the presence of 2,3,4-tri-O-methyl glucose in addition to the above three components. The latter component therefore arose by carboxyl reduction of the methylated polysaccharide. The identification of 2,3.4-tri-O-methyl glucose was unambiguous and some of the fragments were 2 m.u. larger than usual. The following major fragments were obtained (m/e 247, 235, 191,. 187. 175, 161. 131, 129, 117, 101, 99). Methylation of the carboxyl-reduced polysaccharide (Ill) followed by hydrolysis and analysis as the alditol acetates showed the presence of 2,3,4-tri-Omethyl xylose. 2,3.4,6-tetra-O-methyl glucose, 4,6-diO-methyl mannose and 6-O-methyl mannose in the approximate molar proportions of 6:1:2:4. The 2.3,4.6-tetra-O-methyl glucose obviously came from the reduction and remethylation of the glucuronic acid unit present in the polysaccharide.

677

Polysaccharide Scrotype C

Goldstein et al., 1959) gave a periodate-resistant polysaccharide which was isolated by dialysis and freeze drying. This material was insoluble in water but soluble in DMSO. It had [XI*; + 103 (c 0.4 in DMSO:H,O, 3:l). Hydrolysis of the degraded polysaccharide followed by paper chromatography in solvents A and B showed the presence of mannose only. A IO-mg sample of the Smith-degraded polysaccharide was methylated in dimethyl sulfoxide and was then hydrolyzed and analyzed as alditol acetates by g.1.c. and g.c./m.s. It showed the presence of only methylated sugar, i.e. 2,4.6-tri-O-methyl one mannose. indicating that the periodate resistant backbone was a 1.3-linked mannan. Chromium

trioridc~ osida tion

Chromium trioxide oxidation of the polysaccharide for I hr resulted in the loss of 307,, of the xylose residues as indicated by quantitative estimation of the component sugars before and after oxidation. Treatment with the oxidizing reagent for 3 hr resulted in the loss of 80’:,, of the xylose residues indicating that the xylose residues had a P-linkage in the polysaccharide.

Shown below are two octasaccharide repeating unit structures for the capsular polysaccharide of C. ncwfbrmcms serotype C. These structures are based on the results obtained from the present study as outlined below. The data do not allow us to distinguish between (1) and (2) at this time.

Periodate oxidation of the polysaccharide showed that approximately 1.4 mole of periodate was consumed per mole of sugar (based on an average mol. wt of 165) in 72 hr. The results are shown in Fig. 4. Periodate oxidation followed by borohydride reduction and mild hydrolysis (Smith degradation,

I

1;:

I>-XqlP

,i,‘r

+

II-GlucAP ’ ~,I 12

3).1.,I-ManP-(

I + ikr-I,-M,inP(

I,-\ylP

hr

I -

‘5

T :i

Time.

(1)

I,-QIP

Id

I

Fig. 4. Periodate oxidation of polysaccharide type C. The consumption of periodate is based on an average mol. wt of I65 for a sugar molecular.

/{

I

IXilUCAP

I,-U>lP

q _

IJ

I I,-*iP

i)-Y-wManP(

I -

,I

;f

3)-x-l+

(2)

I>-\hIP

Complete hydrolysis of the polysaccharide gave xylose, mannose and glucuronic acid as the component sugars. Quantitative estimation of the component sugars in the carboxyl-reduced polysaccharide showed xylose. mannose and glucose in the ratio of 4:3:1. Since no glucose was present in the unreduced polysaccharide. this component must have been formed by reduction of the glucuronic acid unit. This was confirmed by LiAID, reduction of the methylated polysaccharide, hydrolysis and isolation of deuterated glucose (see below). Hydrolysis of the polysaccharide followed by isolation of the component sugars and measurement of their optical rotation indicated that all the sugars were present in the o-configuration.

678

A. K. BHATTACHARJEE,

K. J. KWON-CHUNG

The optical rotation of the polysaccharide was - 12‘ in water and - 4” in DMSO:H,O (3:l). Partjally-hyd~iyzed polysaccharide (which had lost 30”,, of the xylose residues) had a rotation of + 18” in water indicating that the xylose residues were p-linked. This was further confirmed by chromium trioxide oxidation when 80% of the xylose residues were destroyed in 3 hr. Smith-degraded polysaccharide, which contained themannan backbone, had an optical rotation of + 102’ showing that the mannose units were x-linked. Finally, an aldobiouronic acid was isolated from the partially-hydrolyzed polysaccharide and was found to be composed of D-mannose and L)glucuronic acid. This material had @]g of - 20”, indicating that the glucuronic acid was P-linked to the mannose unit. Methylation analysis of the polysaccharide (Table I) showed the presence of 2,3,4-tri-U-methyl xylose, 4,6-di-O-methyl mannose and GO-methyl mannose. Methylation of the carboxyl-reduced polysaccharide followed by hydrolysis and analysis as alditol acetates showed the presence of 2,3,4-tri-O-methyl xylose, 2,3,4.6-tetra-O-methyl glucose, 4,6,-di-O-methyl mannose and 6-O-methyl mannose in the approximate molar proportion of 6: 1:2:4. This is close to the ratio of these sugars in the original polysaccharide and quite consistent with the structure in scheme (1). The ideal ratio of these sugars consistent with the structure would be 8:2:2:4, and it shows that the proportion of the first two components are less than expected. This is understandable, however, since these two components are more volatile and therefore subject to loss during evaporations in the work-up. It is also possible that a slightly incompletecarboxyl-reduction would result in a low value for the tetra-O-methyl glucose obtained. Isolation of methylated xyloses and glucoses only as 2.3,4-tri-O-methyl xylose and 2,3,4,6-tetra-O-methyl glucose from the methylated carboxyl-reduced polysaccharide (II) indicates that both the xvlose and glucuronic acid residues are present as single end groups in the polysaccharide. The failure to obtain any-tri-O-methyl mannose from the methylated Table

1. Methylation

Methylated

analysis of the capsular of C. rteoforrncms type C” -

-

suga?

2.3.4.6-Me,-Man 2,3.4.6-Me,-Glc 2.3.4-Me,-Xyl 2.4.6-Me,-Man 2.3.4-Me,-Glc 4,6-Me,-Man

6-Me-Man

T’ I.00 I.00 0.66 2.09 2.44 3.30 4.50

I

polysaccharide

Sample” II III

IV &

+

t (1.0)” + (6)

+ +

+ (2) t (4)

+

and C. P. J. GLAUDEMANS

polysaccharide shows that all the mannose units are substituted. The presence of both 4,6-di-O-methyl mannose and 6-O-methyl mannose indicates that some of the mannose units are substituted at positions 2 and 4 and a lesser number are substituted at position 2 only. The ratio of mono:disubstituted mannose units was approximately 1:2. The resistance of the mannose backbone of the polysaccharide to periodate oxidation indicated that the mannose units were 1,3-linked. This was further confirmed by methylation of the Smithdegraded polysaccharide followed by analysis of the methylated sugars as alditol acetates when only 2,4,6tri-O-methyl mannose was obtained. The latter finding also shows that no mannose residues occur in side chains. The position of linkage of the glucuronic acid residues to the mannose units was obtained by methylation of the isolated aldobiouronic acid followed by analysis of the component sugars as alditol acetates. it is conceivable that the doublysubstituted mannosyl residues in the backbone carry a glucuronic acid residue (at O-2) and a xylose residue (at O-4). The structure of the capsular polysaccharide of C. rz~(~f~r~ans type C; as shown in schemes (1) or (2) has some similarity with the structure of the polysaccharide from Tremella mesenferica NRRL Y6158 (Fraser ~?f al., 1973) in having a 1,3-a-linked mannan backbone with xylose and glucuronic acid in the side chains. However, there were two important differences in that the mannan backbone was more heavily substituted in type C polysaccharide and also all the side chains were present as single units, as opposed to multiple-unit side chains in the polysaccharide of Tremella. The structure of C. ~~o~#rrn~ns type C also differs from the structure reported for an untyped polysaccharide C. neojbrnrans CRD-1 (Duke) which contains mannose linked 1,2- in the main chain and 1,4- in the side chain. As mentioned in the Introduction, some structural work has been reported (Blandamer & Danishefsky, 1966) on the capsular polysaccharide from C. neqfbrmans type B which suggest that it consists of a mannan backbone with branches of xylose and glucuronic acid, but the detailed structure has not been worked out. A~~knoil,(~rl~epilt~?~.~-.Weare indebted to Dr. J. E. Bennett for many discussions, to Mr. W. B. Hill for his technical assistance and to Mr. W. E. Comstock for obtaining the mass spectral data.

+ +

f

+

+

+

+

“All the sugars were analyzed as methylated alditol acetates using column (i) at ‘I70-C. *2.3.4,6-Me,-Man = 2,3,4,6-tetra-O-methyl mannose etc. ‘ Retention times are relative to 1.5-di-O-acetyl 2.3.4,6tetra-0-methyl-o-glucitol. d Methylated polysaccharide (I), methylated carboxylreduced polysaccharide (II). methylated poiysacchdride carboxyl-reduced (III) and methylated partially hydrolyzed polysaccharide (IV). “The numbers in parenthesis show the relative moles of the respective sugars.

REFERENCES Aspinall G. 0. & Ferrier R. J. (lY57) A spectrophotometric method for the determination of periodate consumed during the oxidation of carbohydrates. Che~r,r I&. 1216. Bennett J. E. & Hasenclever H. F. (1965) ~~~~~~~~~~~2~~ ~&ornrans polysaccharide: Studies of serologic properties and role in infection. J. fnmtun. 94, 916-920. _’..-” Bennett J. E.. Kwon-Chune K. J. & Theodore T. S. 097Qi , Biochemical differentiation between serotypes of CrJpt<~i.<~ccvs nrr~fontrcms. .~u~o~~~uL~~~~~ 16. Bennett J. E.. Kwon-Chung K. J. & Howard D. H. (1477) ._. ., Epidemiologic differences among serotypes of Cr.vpf”(‘o~(Y.Fnrofbimcm.v. Ant. J. Epid. 105, 5X2-586. Bethge P. 0. & Lindstrom K. (1973) Determination of O/“,

Structure

of Cryptococcal

acetyl groups in wood. Svrt~s/i Pup/~-Tit/n. 76, 645-649. Blandamer A. & Danishefsky I. (1966) Investigations on the structure of the capsular polysaccharide from Cry/~rococcus nrofb~n~url.c type B. Biodtirw. hioplij,s. Actu 117, 305-313. Dubois M.. Giles K. A.. Hamilton J. K.. Rebers P. A. & Smith F. (1956) Calorimetric method for determination of sugars and related substances. Anrr/vr. Chm,. 28, 35&356. Emmons C. W., Binford C. H., Utz J. P. & Kwon-Chung K. J. (1977) Met/i& ,v~.co/ogj,, 3rd Edn. p. 206. Lea & Febyer. Philadelphia. PA. Evans E. E. (1949) An Immunologic comparison of twelve strains of C‘~J~I~ON~L.(~I(.\ rwc!for~fzans (Torula histolytica). Proc,. Sot. <‘.Y,,. B/o/. ,Mrd. 71, 644-646. Evans E. E. (1950) The antigenic composition of Cr!,/~roc,oc,c I,.S r~c,c~fo~r~~un.s. I. A serologic classification by means of the capsular and agglutination reactions. J. Itntnun. 64, 423-430. Fraser C. G., Jennings H. J. & Moyna P. (1973) Structural analysis of an acidic polysaccharide from Trwnrllu n~~.w~rcl.i<~c/N RRL Y-6 158. Cart. .I. Biochrm. 51, 2 19-224. Goldstein I. J.. Hay B. W.. Lewis B. A. & Smith F. (1959) A new approach to the determination of the fine structure of polysaccharides. Ahstr. Pup. Am. dwn~. SW. 135, 3D. Hakomori S. (1964) A rapid permethylation ofglycolipid and polysaccharide catalqsed by methylsulfinyl carbanion in dimethyl sulfoxide. J. Bioch~m., T&JYJ 55, 205-208. Hough L.. Jones J. K. N. & Wadman W. H. (1950) Quantitative analysis of mixtures of sugars by the method of partition chromatography. V. Improved methods for the sugars and their methylated derivatives on the paper chromatogram. J. chenl Sot. 1702-1706. Jansson P.. Kenne L., Liedgren H.. Lindberg B. & Lonngren J. (1976) A practical guide to the methylation analysis of carbohydrates. Chen~. Conln71o7. C;~ii,. Stockh. No. 8.

Polysaccharide

Serotype

C

679

Kwon-Chung K. J. (1975) A new genus, Fi/ohusic/id/a. the perfect state of Cry/~tococcus rleq$~rn~uns. M,~,c~o/o~icr67, 1197-1200. Kwon-Chung K. J. (1976) A new species of Fi/ohrrsit/ir//o. the sexual state of Cr~,ptococcus neoforn~rrit.c- B and C serotypes. M~cologin 68, 942-946. Larm 0.. Lindberg B. & Svensson S. ( 1973) Further studies of the capsular polysaccharide of Pneumococcus type II. Ccrrhoh.~/. Res. 31, 12&l 26. Lythgoe B. & Trippett S. (1950) The constitution of the disaccharide of glycyrrhinic acid. J. &?I?. Sot. 1983-I 99 I. Miyazaki T. (1961) Studies on fungal polysaccharides. II. On the componental sugars and partlal hydrolysis of the capsular polysaccharide from C‘r~~rococ~c~rs )ICO~O~~~?NII.\. Chrnl. p/irrmr~ Bull 9, 826-833. Rebers P. A.. Barker S. A., Heidelberger M.. Dische Z. & Evans E. E. (1958) Precipitation of the specific polysaccharide of C~~~~,rococcus IIPO~O~~NNII.S A by type II and XIV antipneumococcal sera. J Am. them. Sot,. 80, 1135-1137. Sawardeker J. S., Sloneker J. H. & Jeanes A. (1965) Quantitative determination of monosaccharldes as their alditol acetates by gas liquid chromatography. Anrr/).. Ckm. 37, 1602-I 604. Taylor R. L. & Conrad H. E. (1972) Stolchiometric depolymerisatlon of polyuronides and glycosaminoglycuronans to monosaccharides following reduction of their carbodiimide-activated carboxyl groups. Bioc~hrmi.~rr_l~ I I, 1383-1388. Trevelyan W. E.. Procter D. P. & Harrison J. G. (1950) Detection of sugars on paper chromatograms. n’crru~,, Nr\r Bid. 166, 444445. Wilson D. E.. Bennett J. E. & Bailey J. W. (1968) Serologic grouping of Cr~~prococ~cus ncoformum~. Ptw. SW. t’.x-p. Bid. ML’c/. 127, 82&823.