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A highly branched exocellular D -glucan from Monolinia fructicola
BIOCHIMICA ET BIOPHYSICA AGTA
IO3
BBA 2 6 8 0 8
A H I G H L Y B R A N C H E D E X O C E L L U L A R D-GLUCAN FROM
M O N O L I N I A FRUCTICOLA* M I L T O N S. F E A T H E R
AND A B D U L M A L E K
Department of Agricultural Chemistry, University of Missouri, Columbia, Mo. 652or (U.S.A.) (Received O c t o b e r 26th, i97 I)
SUMMARY
An exocellular D-glucan produced by Monolinia fructicola was isolated and partially purified. The poiysacchaxide was methylated, hydrolyzed, and the resulting O-methylglucoses were identified by gas-liquid chromatography and mass spectrometry after conversion to the partially methylated, partially acetylated I-deuteroalditols. The presence of 2,6-di-0-, 2,4,6-tri-0- and 2,3,4,6-tetra-0-methyl-D-glucose in the molar ratios of 1:2.1:1.1 in the hydrolyzate indicates a highly branched polymer containing predominantly I--~ 3 linkages and lesser amounts of I - + 4 linkages. The relatively large amount of tetra-0-methyl-D-glucose indicates a highly branched polymer with each third unit containing a branch point.
Monolinia fructicola, a common plant pathogen, produces a viscous exocellular slime when maintained on synthetic media as well as when it is growing inside an infected peach. This report describes the isolation and partial purification of the major constituent of the slime, a D-g!ucan, and structural studies thereon. The organism was grown in shake culture at 25 ° C for 7 days on a synthetic medium I at which time the culture was centrifuged at IOOOO rev./min and vacuum filtered through W h a t m a n No. I filter paper. The addition of 2 vol. of ethanol to the filtrate precipitated the crude exocellular products, which were filtered off and dried in vacuo at 25 ° C. Yields of this material were about 2oo mg per 1 of culture filtrate. Visual observation indicated that a black pigment was present and qualitative tests for protein were positive. The material was hydrolyzed in o. 5 M sulfuric acid at IOO° C for 8 h. The hydrolysis solution was neutralized with BaCOn, filtered and the filtrate evaporated to a syrup which was examined b y paper chromatography using b u t a n o l - p y r i d i n e - w a t e r (6:4: 3, v/v/v) as irrigant and aniline hydrogen phthalate ~ as spray. Glucose was the major constituent along with lesser amounts of mannose, galactose and f-acose. Dissolution of the crude material in water followed by passage through a column of Dowex 5o (hydrogen form), followed by dialysis and lyophilization gave the glucan contaminated only with pigment and small amounts of protein (N = o.56 %, Kjeldahl). Hydrolysis of the material as described above followed by paper chromatography showed only glucose to be present. In addition, aliquots of the hydrolyzate also contained the same amount of glucose when analyzed with glucose oxidase " J o u r n a l P a p e r No. 622o of t h e Missouri A g r i c u l t u r a l E x p e r i m e n t Station.
Biochim. Biophys. Acta, 264 (I972) lO3-1o5
Io 4
M . S . FEATHER, A. MALEK
("glucostat", Worthington Biochemical, Freehold, N.J.) and with standard reducing sugar analyses a, ~, thus showing that the material was composed entirely of D-glucose units. The overall yield of D-glucan was IOO mg per 1 of culture filtrate. Approx. Ioo mg of the D-glucan was methylated using the methyl sulfinyl earbanion-methyl iodide reagents described by Hakamori 5 and others 6. Tile reaction solution (IO.O ml) was dialyzed overnight against running tap water and then extracted 3 times with 5o-ml portions of chloreform. After extraction of the chloroform with water, the chloroform was dried (CaC12) and evaporated to give the fully methylated D-glucan. Although the preparation had a methoxyl content of 33-5 %, considerably less than theory, it showed no hydroxyl absorption in the infrared at 33oo-36oo cm -1, and on remethylation, the methoxyl content remained unchanged. Complete hydrolysis6,7 followed by paper chromatography using butanone saturated with water as irrigant gave three components having flow rates identical to authentic samples of 2,6-di0-methyl-, 2,4,6-tri-O-methyl- and 2,3,4,6-tetra-O-methyl-D-glucose. The unequivocal identification of the mixture of sugars involved the use of gas chromatography and mass spectrometry. Both the unknown mixtures of -0-methyl sugars obtained by hydrolysis of the fully methylated D-glucan, and each authentic standard were converted to the i-deutero-alditol by reductionS, 9 with sodium borodeuteride, a procedure which specifically labels the original C-I of the 0-methyl o-glucose, and then was subjected to acetylation 8,9 to give the respective partially acetylated, partially methylated I-deuteroglucitols. These compounds were subjected to gas chromatography and, in all cases, the retention times (Table I) of the standard and unknowns were identical. Lindberg and associates ~°, 1~ have shown that such compounds can be absolutely identified from their mass spectra, obtained using an interfaced gas chromatographmass spectrometer combination. The identification depends on the appearance of peaks, having at least io °o the abundance of the acetyl peak (the most abundant in the spectrum), which correspond to either fragments produced by direct cleavage of the carbon bonds in tile alditol, or, fragments produced by loss of ketene, methanol, acetic acid or formaldehydO °,1~. When mass spectra of the alditols obtained in this study were run, the expected l°,H peaks were all present. The spectrum of the diO-methyl derivative showed peaks at role 45 corresponding to a methoxyl at C-6 and TABLE I ANALYTICAL OBTAINED
DATA ON THE
FROM
THE
PARTIALLY
METHYLATED
ACETYLATED,
PARTIALLY
METHYLATED
I-DEUTEROGLUCITOLS
D-GLUCAN
The r e t e n t i o n tin le was d e t e r m i n e d u s i n g a P e r k i n - E l n l e r Model 9o0 gas c h r o m a t o g r a p h u s i n g 3 ~, ECNSS-M oi1 Gas C h r o m Q a t I8O ° (" w i t h a gas flow of 4 ° m l / m i n . The mole r a t i o w a s determined after paper chronaatographic separation by reducing sugar determination using Somogyi's r e a g e n t s (refs. 3 an d 4). The m a s s s p e c t r a l p e a k s were o b t a i n e d u s i n g a P e r k i n - E l m e r Model 27o gas c h r o m a t o g r a p h - m a s s s p e c t r o m e t e r c o m b i n a t i o n using an i o n i z a t i o n p o t e n t i a l of 7 ° eV, a curr e n t of So HA a n d an ion source t e m p e r a t u r e of 80 ° C.
Positio*~ of methoxyl grot, ps
Retention time 3.role ratio (rain)
Significant mass spectral peaks
2,6 2,4,6 2,3,4,6
i8. 3 7.7 3.8
43, 45, 87, i o i , 118, 129 43, 45, 87, i o i , 118, 129, 161, 234 43, 45, 87, i o i , lO2, 118, 129, 145, 161, 162, 205
i.o 2.1 1.1
Biochim. Biophys. ,4cta, 264 (1972) lO3-1o 5
A D-GLUCAN FROM Monoliniafructicola
lO 5
a peak at 118 corresponding to the C-I + C-2 fragment (C-I being acetylated and deuterated, C-2 being methoxylated). These and the additional peaks (Table I) serve to uniquely identify1°, n the compound as 1,3,4,5-tetra-0-acetyl-I-deutero-2,6-diO-methyl-D-glucitol, and, hence the original sugar as 2,6-di-0-methyl-D-glucose. The tri-0-methyl derivative gave peaks at role 45 (C-6), 118 (C-I + C-2), 161 (C-4 + C-5 C-6) and 234 (C-I + C-2 + C-3 + C-4) as well as others (Table I) which identify the original sugar as 2,4,6-tri-0-methyl-D-glucose. In the case of the tetra-0-methyl derivatives, peaks at 45 (C-6), 118 (C-I + C-2), 161 (C-4 + C-5 + C-6) and 162 /C-I + C-2 + C-3) and others (Table I) identifyl°, n the original sugar as 2,3,4,6-tetra0-methyl-D-glucose. In all cases the standards when so treated, gave identical mass spectra. The molar ratios (Table I) of the sugars indicate a highly branched polysaccharide (as evidenced by the presence of significant quantities of both the tetra-0and di-0-methylglucoses) containing approx. 67 % I--~ 3 linkages and 33 % I--> 4 linkages. The polysaccharide may be viewed as a I---> 3 linked chain of anhydroglucose residues which contain branches at each third residue and which are attached by i--~ 4 linkages. Structurally, this material is quite different from many other fungal glucans which contain only I--> 3 and I--> 4 linkag es12, since the latter have been shown to be essentially linear in structure. ACKNOWLEDGMENT
We wish to acknowledge the advice and assistance of Drs. B. G. Tweedy and J. Ross of the Department of Plant Pathology for help in culturing the organism and for making the mass spectrographic facilities available. REFERENCES I , 3 4 5 6 7 S 9 lO IE I2
P. R. B a r k h o l d e r and E. W. Sinnott, Am. j . Bot., 32 (i945) 124. S. M. Partridge, Nature, 164 (1949) 443. M. J. Somogyi, J. Biol. Chest., 195 (1952) 19. N. Nelson, J. Biol. Chem., 153 (1944) 375. S. H a k a m o r i , J. Biochem. Tokyo, 55 (1964) 2o5. P. A. Sanford and H. E. Conrad, Biochemistry, 5 (1966) 15o8. P. J. Garegg and B. Lindberg, Acta Chem. Scand., 14 (196o) 871. J. s. Sawardeker, J. H. Sloneker and A. Jeanes, Anal. Chem., 37 (1965) 16o2. H. Bjornbal, B. Lindberg and S. Svensson, Aeta Chem. Scand., 21 (1967) 18Ol. H. Bjorndal, B. Lindberg, A. Pilotti and S. Svensson, Carbohydrate Res., 15 (197 o) 339. H. Bjorndal, C. G. Hellerquist, B. Lindberg and S. Svensson, Angew. Chem., 82 (197 o) 643. P. A. J. Gorin and J. F. T. Spencer, Adv. Carbohydrate Chem., 23 (I968) 367 .
Biochim. Biophys. Acta, 264 (1972) lO3-1o 5
IO3
BBA 2 6 8 0 8
A H I G H L Y B R A N C H E D E X O C E L L U L A R D-GLUCAN FROM
M O N O L I N I A FRUCTICOLA* M I L T O N S. F E A T H E R
AND A B D U L M A L E K
Department of Agricultural Chemistry, University of Missouri, Columbia, Mo. 652or (U.S.A.) (Received O c t o b e r 26th, i97 I)
SUMMARY
An exocellular D-glucan produced by Monolinia fructicola was isolated and partially purified. The poiysacchaxide was methylated, hydrolyzed, and the resulting O-methylglucoses were identified by gas-liquid chromatography and mass spectrometry after conversion to the partially methylated, partially acetylated I-deuteroalditols. The presence of 2,6-di-0-, 2,4,6-tri-0- and 2,3,4,6-tetra-0-methyl-D-glucose in the molar ratios of 1:2.1:1.1 in the hydrolyzate indicates a highly branched polymer containing predominantly I--~ 3 linkages and lesser amounts of I - + 4 linkages. The relatively large amount of tetra-0-methyl-D-glucose indicates a highly branched polymer with each third unit containing a branch point.
Monolinia fructicola, a common plant pathogen, produces a viscous exocellular slime when maintained on synthetic media as well as when it is growing inside an infected peach. This report describes the isolation and partial purification of the major constituent of the slime, a D-g!ucan, and structural studies thereon. The organism was grown in shake culture at 25 ° C for 7 days on a synthetic medium I at which time the culture was centrifuged at IOOOO rev./min and vacuum filtered through W h a t m a n No. I filter paper. The addition of 2 vol. of ethanol to the filtrate precipitated the crude exocellular products, which were filtered off and dried in vacuo at 25 ° C. Yields of this material were about 2oo mg per 1 of culture filtrate. Visual observation indicated that a black pigment was present and qualitative tests for protein were positive. The material was hydrolyzed in o. 5 M sulfuric acid at IOO° C for 8 h. The hydrolysis solution was neutralized with BaCOn, filtered and the filtrate evaporated to a syrup which was examined b y paper chromatography using b u t a n o l - p y r i d i n e - w a t e r (6:4: 3, v/v/v) as irrigant and aniline hydrogen phthalate ~ as spray. Glucose was the major constituent along with lesser amounts of mannose, galactose and f-acose. Dissolution of the crude material in water followed by passage through a column of Dowex 5o (hydrogen form), followed by dialysis and lyophilization gave the glucan contaminated only with pigment and small amounts of protein (N = o.56 %, Kjeldahl). Hydrolysis of the material as described above followed by paper chromatography showed only glucose to be present. In addition, aliquots of the hydrolyzate also contained the same amount of glucose when analyzed with glucose oxidase " J o u r n a l P a p e r No. 622o of t h e Missouri A g r i c u l t u r a l E x p e r i m e n t Station.
Biochim. Biophys. Acta, 264 (I972) lO3-1o5
Io 4
M . S . FEATHER, A. MALEK
("glucostat", Worthington Biochemical, Freehold, N.J.) and with standard reducing sugar analyses a, ~, thus showing that the material was composed entirely of D-glucose units. The overall yield of D-glucan was IOO mg per 1 of culture filtrate. Approx. Ioo mg of the D-glucan was methylated using the methyl sulfinyl earbanion-methyl iodide reagents described by Hakamori 5 and others 6. Tile reaction solution (IO.O ml) was dialyzed overnight against running tap water and then extracted 3 times with 5o-ml portions of chloreform. After extraction of the chloroform with water, the chloroform was dried (CaC12) and evaporated to give the fully methylated D-glucan. Although the preparation had a methoxyl content of 33-5 %, considerably less than theory, it showed no hydroxyl absorption in the infrared at 33oo-36oo cm -1, and on remethylation, the methoxyl content remained unchanged. Complete hydrolysis6,7 followed by paper chromatography using butanone saturated with water as irrigant gave three components having flow rates identical to authentic samples of 2,6-di0-methyl-, 2,4,6-tri-O-methyl- and 2,3,4,6-tetra-O-methyl-D-glucose. The unequivocal identification of the mixture of sugars involved the use of gas chromatography and mass spectrometry. Both the unknown mixtures of -0-methyl sugars obtained by hydrolysis of the fully methylated D-glucan, and each authentic standard were converted to the i-deutero-alditol by reductionS, 9 with sodium borodeuteride, a procedure which specifically labels the original C-I of the 0-methyl o-glucose, and then was subjected to acetylation 8,9 to give the respective partially acetylated, partially methylated I-deuteroglucitols. These compounds were subjected to gas chromatography and, in all cases, the retention times (Table I) of the standard and unknowns were identical. Lindberg and associates ~°, 1~ have shown that such compounds can be absolutely identified from their mass spectra, obtained using an interfaced gas chromatographmass spectrometer combination. The identification depends on the appearance of peaks, having at least io °o the abundance of the acetyl peak (the most abundant in the spectrum), which correspond to either fragments produced by direct cleavage of the carbon bonds in tile alditol, or, fragments produced by loss of ketene, methanol, acetic acid or formaldehydO °,1~. When mass spectra of the alditols obtained in this study were run, the expected l°,H peaks were all present. The spectrum of the diO-methyl derivative showed peaks at role 45 corresponding to a methoxyl at C-6 and TABLE I ANALYTICAL OBTAINED
DATA ON THE
FROM
THE
PARTIALLY
METHYLATED
ACETYLATED,
PARTIALLY
METHYLATED
I-DEUTEROGLUCITOLS
D-GLUCAN
The r e t e n t i o n tin le was d e t e r m i n e d u s i n g a P e r k i n - E l n l e r Model 9o0 gas c h r o m a t o g r a p h u s i n g 3 ~, ECNSS-M oi1 Gas C h r o m Q a t I8O ° (" w i t h a gas flow of 4 ° m l / m i n . The mole r a t i o w a s determined after paper chronaatographic separation by reducing sugar determination using Somogyi's r e a g e n t s (refs. 3 an d 4). The m a s s s p e c t r a l p e a k s were o b t a i n e d u s i n g a P e r k i n - E l m e r Model 27o gas c h r o m a t o g r a p h - m a s s s p e c t r o m e t e r c o m b i n a t i o n using an i o n i z a t i o n p o t e n t i a l of 7 ° eV, a curr e n t of So HA a n d an ion source t e m p e r a t u r e of 80 ° C.
Positio*~ of methoxyl grot, ps
Retention time 3.role ratio (rain)
Significant mass spectral peaks
2,6 2,4,6 2,3,4,6
i8. 3 7.7 3.8
43, 45, 87, i o i , 118, 129 43, 45, 87, i o i , 118, 129, 161, 234 43, 45, 87, i o i , lO2, 118, 129, 145, 161, 162, 205
i.o 2.1 1.1
Biochim. Biophys. ,4cta, 264 (1972) lO3-1o 5
A D-GLUCAN FROM Monoliniafructicola
lO 5
a peak at 118 corresponding to the C-I + C-2 fragment (C-I being acetylated and deuterated, C-2 being methoxylated). These and the additional peaks (Table I) serve to uniquely identify1°, n the compound as 1,3,4,5-tetra-0-acetyl-I-deutero-2,6-diO-methyl-D-glucitol, and, hence the original sugar as 2,6-di-0-methyl-D-glucose. The tri-0-methyl derivative gave peaks at role 45 (C-6), 118 (C-I + C-2), 161 (C-4 + C-5 C-6) and 234 (C-I + C-2 + C-3 + C-4) as well as others (Table I) which identify the original sugar as 2,4,6-tri-0-methyl-D-glucose. In the case of the tetra-0-methyl derivatives, peaks at 45 (C-6), 118 (C-I + C-2), 161 (C-4 + C-5 + C-6) and 162 /C-I + C-2 + C-3) and others (Table I) identifyl°, n the original sugar as 2,3,4,6-tetra0-methyl-D-glucose. In all cases the standards when so treated, gave identical mass spectra. The molar ratios (Table I) of the sugars indicate a highly branched polysaccharide (as evidenced by the presence of significant quantities of both the tetra-0and di-0-methylglucoses) containing approx. 67 % I--~ 3 linkages and 33 % I--> 4 linkages. The polysaccharide may be viewed as a I---> 3 linked chain of anhydroglucose residues which contain branches at each third residue and which are attached by i--~ 4 linkages. Structurally, this material is quite different from many other fungal glucans which contain only I--> 3 and I--> 4 linkag es12, since the latter have been shown to be essentially linear in structure. ACKNOWLEDGMENT
We wish to acknowledge the advice and assistance of Drs. B. G. Tweedy and J. Ross of the Department of Plant Pathology for help in culturing the organism and for making the mass spectrographic facilities available. REFERENCES I , 3 4 5 6 7 S 9 lO IE I2
P. R. B a r k h o l d e r and E. W. Sinnott, Am. j . Bot., 32 (i945) 124. S. M. Partridge, Nature, 164 (1949) 443. M. J. Somogyi, J. Biol. Chest., 195 (1952) 19. N. Nelson, J. Biol. Chem., 153 (1944) 375. S. H a k a m o r i , J. Biochem. Tokyo, 55 (1964) 2o5. P. A. Sanford and H. E. Conrad, Biochemistry, 5 (1966) 15o8. P. J. Garegg and B. Lindberg, Acta Chem. Scand., 14 (196o) 871. J. s. Sawardeker, J. H. Sloneker and A. Jeanes, Anal. Chem., 37 (1965) 16o2. H. Bjornbal, B. Lindberg and S. Svensson, Aeta Chem. Scand., 21 (1967) 18Ol. H. Bjorndal, B. Lindberg, A. Pilotti and S. Svensson, Carbohydrate Res., 15 (197 o) 339. H. Bjorndal, C. G. Hellerquist, B. Lindberg and S. Svensson, Angew. Chem., 82 (197 o) 643. P. A. J. Gorin and J. F. T. Spencer, Adv. Carbohydrate Chem., 23 (I968) 367 .
Biochim. Biophys. Acta, 264 (1972) lO3-1o 5