Cdohy&ate 0 Eh!‘i~

Rwe~~h, kientilk

56 (1977) 351-355 Publishing Company,

Amsterdam

P.M.R. STUDlE-S ON THE ANTITUMOUR ISOLATED FROM Cori0lu.s cersicolor YOSIIO

tNOUE

bCHIRd

POLYSACCHARIDE

CtiJd

of Polytrzv Chemistry.

Deparrment Meguro-Ku. (Received

XN?D

- Primed in Belgium

Tokyo Institute of Technolo,oy. Ookayama.

Tokyo (3apan) July 2hh,

1976: accepted for publication.

November

9th. 1976)

Application of p.m.r. spectroscopy to the aotitumour glycoprotein extracted from mycelia of Coriol~ tlersicolor showed that the pol>saccharide (o-&can) chain contains (I +3)-a, (I +4)-a, (1+6)-a, (l-+3)$, (1 -r4)-j3, and (1+6)-/I linkages. The assignments are

of the characteristic

peaks to amioo

acid residues in the protein

portion

briefly discussed.

LNTRODUC-TION

Various polysaccbarides of plant, fungal, and bacterial origin strongly inhibit the growth of transplantable tumours in animals. Thus, lentinan”2 (glucan) isolated from Lentinus edodcs (Berk.) Sing. has considerable activity against sarcoma 180. and a polysaccbaride obtained3 from the mycelium of Coriolus ~rsicolor inhjbits the growth of this tumour implanted subcutaneously in mice. Tsukagosbi and Ohashi’: showed that a glycoprotein isolated from Coriolus rersicolor (Fr.) QuPl of Basidiomycetes conrains - 15% of tightly bound protein, ‘and strongly inhibits the growth of mouse sarcoma 180 and rat ascites bepatoma AH-13 when administered orally. Miyazaki et ~1.~ reported tbat an extracellular, antitumour glucan obtained from the culture filtrate of Corioh oersicolor bad a highly branched structure including (I +3) and (l-+6) linkages. Recently, Saito et aL6 showed that an antitumour glucan (HA-3) obtained from P. ostreofur mntains (l-+3)-/3 and (1+4)-a linkages. We now report on the p.m.r.-spectral data for the antitumour glycoproteia isolated from mycelia of Coriolus uersicolor (Fr.) Quil of Basidiomycetes, as a contribution to the determination of structure-activity relationships. The signals from anomeric protons are usually readily distinguished, and have been used for the detection and quantification of a and fl anomers and various types of glycosidic linkages in mono-, oligo-, and poly-saccharides’-’ ‘. RESULTS AND DISCUSSION The

‘H-n.m.r. spectrum (D20, 900) of polysaccbaride-i, isolated from mycelia of Coriofus cersicclor (Fr.) Q&l of Basidiomyetes by extraction with hot water, is illustrated in Fig. 1. The spectral features were essentially invariant in the temperature

Y. MOUE.

352

R. CH6J6

range from 25-90”, except for the expected shift to lower field of the resonance of residual protons in the solvent, and the line broadening of al! resonances at lower temperatures. One main part of *Ais spectrum (O-3 p_p.m_) contains resonances for CH, CH2, and CH, protons of the amino acid residue in the protein moiety. The second main region (3-6 p.p_m.) contains the resonances for the CH and CH, protons of the poiysaccbaride portion, and the a-CH protons of the amino acid residues is a glycoprotein, (3-4.5 p.p_m.) ’ ‘I ’ ‘. The data in Fig. 1 confirm that polysaccharidz-i as reported by Tsukagosbi and OhashiJ. G.1.c. analysis of a hydrolysate of the polysaccharide portion of polysaccharide-I revealed mainly D-glucose4. By compnrison with the chemical shif&s”*9*” of D-gluco-oligosaccharides and -polysaccharides for solutions in D,O, the region 445.5 p.p.m_ in Fig. 1 may be assigned to anomeric protons. The intensities of si_gnals for ;r-CH protons of amino acid residues also present in this region are small, because polysaccharide-I contains’ only N 15% of protein, and a-CH prorons resonate*’ ar 3.OA.5 p.p.m. The presence of several peaks in the range 4.4-5.5 p.p_m. indicates a variety of glycosidic linkages. On the basis of litxature datasSgS’ 5, the four main peaks (p.p.m.) arc assigned to linkages as follows: 5.39, (I 44)-z and/or (1-+3)-a; 5.00, (146)-r; 4.75, (I +3)-/l; and 4.55, (1+4)-j3 and/or (I -6)-P. These assi_gnments are tentative and made on the assumption that the polyssccharide is essentially a glucan’. HDO

8

7

6

5

4

3

2

I

0

Pr3m. FIN. 1. ‘H-N.m.r. A. slnglz scan;

sp.xcm of a 1096 solution B. 64 nccumuhrions.

of polysaccbtideI

in

D..O at 100 MHz

and 90”:

From these assignments, the apparent percentage of different types of linkages can he estimated, aod the results are shown in Table I. There was 2 small effect on these data by variation of conditions of sample preparation, including cultivation.

AN-lTllrnfOuR

POLYSACCHARILiE

FFtOM c,

353

Lersico~or

TABLE I CHEMICAL

CYXfPO!3TION

PWTEIN-BOUND

OF

THE

POLYMCCHARID ISOLATED

POLYSACCMARIDE

Composition

PiAfixchxide-l Polyxxcbaride-Ii

Tbe sac&aide-I

E PORTlOlr( FROV

MYCELLA

OF OF

ANTITUMOUR,

THE

Coriolus cerricofor

(96)

a&Ratio

(1+3)-z and/or (i--M-z

(I-ts)Jr

(I-+3)-8

47, 92

7 8

IO 0

31 0

49.51 1OO:o

‘H-n.m.r.

spectrum (Fig. 2) of polysncchnride-II, isolated from polyby column chromatography, showed it to contain mainly CI linkages, and/or (I +4)-r (5.39 p.p.m_), and (I +6)-1x (5.00 p.p.m.). The apparent

i.e., (1+3)-z composition of polysaccharide-II is also shown in Table I. The result is consiswnt with the generally accepted fact that a-D-glucans are more soluble than rhe /3 isomers. HDO

1

e

I

7

6

5

4

3

2

1

0 ppm.

Fig. 2. ‘H-N.m.r.

spec!rum of a 10°’,J soluLion of polq%ccharide-I!

in D-0

at 100 hlHz ami 90”.

The ‘H-n.m.r. spectra (Fig. 3) of permethylated polysaccharide-II 2nd amylose are essentially identical, except For the small signal at 3.95 p.p.m which corresponds to H-l of (! +6)+Iinked permethyiated oiigosaccharides’Ov’ ‘. Thus. the main portion of polysaccharide-II may be amylose-like nith (I -A)-cl-~ linkages, although -89/o of (I -&)-CL-D lin’kages are also present. This result indicates that the resoncmce at 5.39 p.p.m. in Fig. I may bt mainly due to H-l in (1+4)-a-linked ~-glucose residues.

Y. lIxmE,

354

R.

CHfrJB

(a)

Fig. 3. ‘H-N.m.r. SFeCk3 Ol- lo’?& =oludons _ ated mylose in CDCIJ at 100 hlHz ;+od

of

(a)

permelh~lakd polysscchwide-II

and (b) pennethyl-

70’.

spectra of iysozyme’“, the signals in the By comparison with the ‘H-•.m.r. region O-3 p.p.m_ m Fig. I may be assigned as foollows: 0.89, methyl protons of Ieucine, isoleucine, and valioe residues; 1.23, methyl protons of threonine, 1.41, mainly methyl protons of alanioe: 1.66. side-chain protons of lysine, arginine, and leucine residues; 2.06. methyl protons of methjonine residues. The relative intensities of the peaks appearing in this region are roughly compatible with the results of amino acid analysis.“. The resonances appearing at Tower Seld (6-8 p.p.m.) arise mainly from aromatic protons and from residual NH protons in the amino acid residues. The results presented indicate the structural complexity of the glycoproteio isolated from mycclia of Cor!oius wrsicofor (Fr.) QuCl of Basidiomycetes. A similarity bztv.een polysaccharide-I and the polysaccbaride T-IA-3 obtained from P. ostrtTal& is that both contain (I +4)-a and (l-+3)-P-linked ~-glucose residues.

Materials. - The polysaccharides were isolated and purified as described in the literature’. Pclysacchnride-I was isolated from mycelia ofCoriolu.s cwsicolor (Fr.) Q.tel of Basidiomycetes by extraction with hot water; the protein and polysaccharide portions are chemically linked’, and the latter (-85%) ccrnbins mainly D-glucose residues

gave

together

with

minor

amounts

of mannose

and

xylcse

residuesa.

Elution of polyjacchan’de-T from a co!umn of DEAE-ceTluTose with hot water polysaccbaride-TT as the most soluble fraction. The polysaccharide-LI was

methylated

by Hakomori’s

procedure

I’.

-OUR

POLYsACCHARt DE FROM c.

ZWSiC0h

355

Method. - ‘H-N.m.r. spectra of polysaccharides were obtained with a JEOL JNM PS-100 spectrometer operated at 100 MHz. Samples were subjected to deuterium exchange by repeamzd dissolution in D,O and freeze-drying, and then examined as IO?/, solutions in D,O. Sodium S,&dimethyl4silapentane-I-sulpbonate (DSS) and tetrametbylsilane (Mc,Si) were used as internal standards for solutions in D,O and CDCI,, respectively. Signal intensities obtained by integration are accurate to + 3%. The effective signal-to-noise ratio was improved by means of the multiscan average technique with a JEOL EC-6 computer. REFERENCES 1 2 3 4 5 6 7 8 9 10 II I? I3

I4 15 I6

Y. MXEDA AND G. CHIHXM. h’urure (London). 229 (1971) 634. Y. MAEDA. J. HA~TURO, AND G. CHTH>R*. inr. J. Cm~crr. 8 (1971) Al-%. S. ~~ARUSE, IL Fum, AND H. In>. Proc. Japan Concw Assoc.. 30/h rlnn. Meeting. 1971. p. 171. S. Tsu~cos~+r AND F. OHASHI. Gann, 65 (197-1) 557-558. T. MIYAZAM, T. YADOME, hf. SUG~URA. H. ITO, K. Fum, S. NARUSE, AXJ hf. K~.IHEA, Chcm. Pharm. Bull., 22 (197-t) 1739-l 742. H. SAITO, Y. YOSHJOKA,AND F. Furtuoic~. Pnper read at the 14Lh NMR DiscussIonal hleeung. Fukuoba, Japan, Oclober 1975. R. U. LEMIEUX AND J. D. STEVEIWB, Gun. /. Chum.. 4-I (1966) 2-!9-262. T. USUI, hf. YOKOYAMA, N. YAMAOL+, K. hIr\rsuo4. Ii. TUJIWJ~M, H. SIIGIYMW. ANT) 5. SETO, Corboh~dr. Res.. 33 (1971) 105-l 16. J. N. C. WHn-E. CarbohJ&. Rex., I6 (1971) 2’0-22-1. D. E. hlI?.iMfilN, CUrbo/lJ&. Rcs., 23 (1972) 139-I-13. N. FR\~K, H. F~ZIEBOLM. G. KEILICH. J. P. h1ERI-E. AND E. SIEFERT, Org. ~\Juqn. Resonance. 4 (1972) 725-732. J N. WC. hf. J A. DE WE, AND 3. F. G. VLLECENTHART. T~rruhtidron. 33 (1972) 3037-3047. G. C. K. ROBERTX AND 0. JARDETZKI’, A&. Protein Chem., 24 (1970) 447-545. C. C. XICDONARD, W. D. PHILLIE., AND J. D. GLICKSON, J. Am. Chem. Sot.. 93 (1971) 233-246. W. M. PAXIKAAND H. CRAGG, Coil. J. Chem., -II (1963) 293-299. S. HAIWMORI, 1. Bmchem.. 53 (l9M) 20%20%