Toward Understanding the Structure, Biosynthesis, and Function of a

4 Toward Understanding the Structure, Biosynthesis, and Function of a Membrane-Bound Fungal Glycopeptide Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on February 28, 2018 | https://pubs.acs.org Publication Date: June 10, 1980 | doi: 10.1021/bk-1980-0126.ch004

S t r u c t u r a l Studies J. E . G A N D E R , J O A N N A B E A C H Y , C . J. U N K E F E R , and S H E R I J. Τ Ο Ν Ν Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, MN 55108

Fungi secrete numerous glycopeptides, polysaccharides, oligo saccharides and a host of lytic enzymes which degrade the extra cellular polymers. Galactocarolose, a 5-O-β-D-galactofuranosyl -containing decasaccharide, and mannocarolose, a mannopyranosyl -containing nonasaccharide, were among the first fungal extra cellular saccharides to be examined extensively by methylation techniques (1). Both of these polymers were obtained from the filtrates of 28-day stationary cultures of P. charlesii grown on the Raulin-Thom medium (2). We have extended the early work of Haworth and colleagues (1,2,3) and that of Gorin and Spencer (4), and Hough and Perry (5,6) to show that galactocarolose and mannocarolose of P. charlesii are degradation products of a larger ethanolamine-/Ν,Ν'-dimethylaminoethanol-containing pep tidophosphogalactomannan (7-12). We have also shown that P. chrysogenum, Penicillium claviforme, Penicillium patulum and Penicillium r a i s t r i c k i i secrete galactofuranosyl-containing poly mers into their growth media (13). Peptidophosphogalactomannans have been obtained from Cladosporium werneckii (14,15) and pep tidogalactomannans have been obtained from numerous species of Aspergillus (16,17,18) and Penicillium (18). The data suggests that peptidogalactomannans, or peptidophosphogalactomannans, may be common to several genera of Ascomycetes. Peptidogalactomannans and peptidophosphogalactomannans have been extracted from powdered fungal cells and they also accumu late in the growth medium. It has been assumed by many investi gators that these polymers represent c e l l wall degradation pro ducts. However, the c e l l walls of 14-day stationary cultures of P. charlesii contain no galactofuranosyl residues (19) and the alkali-soluble, alcohol-insoluble fraction of c e l l walls of 3-day aerated cultures of P. charlesii contain only 1.6% galactofurano syl residues and no detectable mannopyranosyl residues (20). This fraction also contained glucosyl residues and ethanolamine. The results from both laboratories suggest that the c e l l walls of P. charlesii contain few galactofuranosyl residues and that the 0-8412-0555-8/80/47-126-049$07.75/0 ©

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.

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on February 28, 2018 | https://pubs.acs.org Publication Date: June 10, 1980 | doi: 10.1021/bk-1980-0126.ch004

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g a l a c t o f u r a n o s y l residues present are not r e l e a s e d by a l k a l i as a galactomannan or phosphogalactomannan as i s expected i f peptidophosphogalactomannan i s an i n t e g r a l part of the fungal c e l l w a l l . I t i s a l s o u n l i k e l y that p u t a t i v e c e l l w a l l g a l a c t o f u r a n o s y l residues were removed by the a c t i o n of exo-B-D-galactofuranosidase because n e i t h e r the growth medium nor c e l l e x t r a c t s of 3day aerated c u l t u r e s contain measurable galactofuranosidase a c t i v i t y (21,22), and galactofuranosidase i s not secreted i n t o the growth medium u n t i l s e v e r a l days l a t e r (21). Furthermore, i t has been shown that ]?. c h a r l e s i i s t a r t s s y n t h e s i z i n g peptidophosphogalactomannan, or p o s s i b l y a precursor polymer, soon a f t e r i t germinates (23) but the peptidophosphogalactomannan i s not secreted u n t i l the growth medium i s depleted of NH^ . Because of t h i s dilemma we i n i t i a t e d a search f o r i n t r a c e l l u l a r g a l a c t o f u r a n o s y l containing polymers. This paper reviews and extends our work on s t r u c t u r a l chara c t e r i z a t i o n of the e x t r a c e l l u l a r ethanolamine-/N,N -dimethylaminoethanol-containing peptidophosphogalactomannan of JP. c h a r l e s i i , and describes our s t u d i e s on i s o l a t i o n , p u r i f i c a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of a membrane-bound galactofuranosy1-containing polymer which we s h a l l r e f e r to as l i p o p e p t i d o phosphogalactomannan. +

T

Experimental Pénicillium c h a r l e s i i G. Smith ATCC 1887 was maintained (7) and c u l t u r e d (24) at 20°C w i t h constant a e r a t i o n as described previously. I s o l a t i o n and F r a c t i o n a t i o n of Membranes. M y c e l i a from 48 hr c u l t u r e s were separated from the growth medium by f i l t r a t i o n and the mycelia were washed with s e v e r a l volumes of d i s t i l l e d , deionized ^ 0 at 4°C and the water removed from the mycelia by p r e s s i n g between paper towels. The mycelia were p u l v e r i z e d at 4°C i n 50 mM T r i s - H C l , pH 7.5 b u f f e r with A 1 0 ; r a t i o of mycelia:alumina:buffer, 1:0.3:2 (w/w/v). Membranes were i s o l a t e d from the homogenate e s s e n t i a l l y as described p r e v i o u s l y (25). The supernatant s o l u t i o n s from the i n i t i a l c e n t r i f u g a t i o n and the membrane wash were pooled, d i a l y z e d against d i s t i l l e d deionized water f o r at l e a s t 24 hr at 4°C, and used as a source of "cytoplasmic" peptidophosphogalactomannan. The membranes were separated by i s o p y c n i c sucrose gradient u l t r a c e n t r i f u g a t i o n (25). The i n d i v i d u a l membrane f r a c t i o n s were p e l l e t e d by c e n t r i f u g a t i o n at 120,000xg i n a Ti-75 r o t o r of a Beckman L2-65B u l t r a c e n t r i f u g e and the p e l l e t resuspended i n 50 mM T r i s - H C l , pH 7.5 b u f f e r . 2

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Chromatography Gel permeation. Bio-Gel A-5m (57 χ 2.5 cm column) was e q u i l i b r a t e d i n the appropriate b u f f e r and packed i n a column. Three ml sample of membranes was a p p l i e d to the top of the column and the column i r r i g a t e d with 40 mM Tris-HCl-40 mM NaCl-0.24% deoxycholate (DOC) at a flow r a t e of 10 drops rnin"^-. F r a c t i o n s con t a i n i n g 100 drops (approximately 1.7 ml) were c o l l e c t e d . Ion exchange. The borate complexes of v a r i o u s glycopeptide preparations were f r a c t i o n a t e d on Whatman DE-23 (10). The polypeptides obtained by t r e a t i n g the glycopeptide(s) with anhydrous HF were f r a c t i o n a t e d on Whatman DE-23 i n the acetate form. The sample was a p p l i e d i n 0.02 M p y r i d i n i u m ace t a t e , pH 5 and the column was washed with 70 ml of 0.02 M p y r i dinium acetate (pH 5.0) followed by a gradient from 0.03 to 1.0 M a c e t i c a c i d . Each f r a c t i o n contained 3.5 ml. A f f i n i t y chromatography. Concanavalin A was l i n k e d to cyano gen bromide a c t i v a t e d Sepharose 4B by standard procedures (26). The remaining a c t i v e groups on the cyanogen bromide a c t i v a t e d Sepharose 4B were blocked by treatment with 1 M ethanolamine, pH 7.0 f o r 2 hr at 4°C. Approximately 90% of the concanavalin A was l i n k e d c o v a l e n t l y to Sepharose 4B. Immobilized concanavalin A was a c t i v a t e d by i n c u b a t i o n overnight at 4°C i n 40 mM T r i s - H C l 40 mM NaCl-1 mM M g C l - l mM M n C l - l mM C a C l . Before use the con canavalin A-Sepharose 4B p r e p a r a t i o n was e q u i l i b r a t e d with 40 mM Tris-HCl-40 mM NaCl-0.24% DOC. F r a c t i o n s c o n t a i n i n g DOC-soluble glycopeptide obtained from the column of Bio-Gel A-5m were a p p l i e d to a column of concana v a l i n A-Sepharose 4B and the column was washed with 40 mM T r i s HCl-40 mM NaCl-0.24% DOC u n t i l the UV-absorbing m a t e r i a l was removed. The DOC-soluble glycopeptide was e l u t e d with 40 mM T r i s HCl-40 mM NaCl-0.24% D0C-1% methyl-a-D-mannopyranoside. 2

2

2

Thin l a y e r chromatography. Chromatography on Brinkman s i l i c a g e l p l a t e s was conducted with the s o l v e n t systems given i n the appropriate t a b l e s . A f t e r development of the chromatogram, the p o s i t i o n of ^ C determined by s c r a p i n g the s i l i c a g e l i n 2 cm s e c t i o n s i n t o s c i n t i l l a t i o n v i a l s . S c i n t i l l a t i o n f l u i d was added and the -^C was determined i n a l i q u i d s c i n t i l l a t i o n spec trometer. w

a

s

Hydrogen f l u o r o l y s i s . Carbohydrate was removed from the glycopeptide with anhydrous HF (27) without cleavage of the p o l y peptide. The products were d i s s o l v e d i n p y r i d i n e - H 0 , the a n i s o l e g l y c o s i d e s were p r e c i p i t a t e d with water, and the polypeptides were r e s o l v e d by i o n exchange chromatography. 2


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Carbon-13 NMR spectroscopy. N a t u r a l abundance carbon-13 NMR s p e c t r a were taken on a V a r i a n XL-100 spectrometer operated i n the F o u r i e r transform mode at a frequency of 25.2 MHz. Samples of phosphogalactomannan were u s u a l l y i n excess of 100 mg m l " of oO. The f i e l d was locked on the deuterium s i g n a l and the spec t r a were recorded with proton n o i s e decoupled. Chemical s h i f t s are reported i n ppm from an e x t e r n a l reference s o l u t i o n of 5% sodium ( t r i m e t h y l s i l y l ) - l - p r o p a n e s u l f o n a t e (TSP) i n a c o a x i a l tube. The spectrum shown represents data from 64,000 t r a n s i e n t s .


1

Enzymic and chemical assays, a) Galactofuranosy1-containing chains. Exo-3-D-galactofuranosidase (21) was used to r e l e a s e g a l a c t o f u r a n o s y l residues from galactan chains and the q u a n t i t y of galactose r e l e a s e d was q u a n t i f i e d (28). C o n t r o l s without s u b s t r a t e or g a l a c t o f u r a n o s i d a s e or with i n a c t i v a t e d enzyme were c a r r i e d out and appropriate c o r r e c t i o n s were made. b) Carbohydrate. T o t a l carbohydrate p h e n o l - s u l f u r i c a c i d method (29).

was

determined by

the

c) T o t a l phosphate. Samples were ashed according to the procedure of Ames and Dubin (30) and phosphate was determined by the method of F i s k e et a l - (31) . d) P r o t e i n . P r o t e i n was determined by the procedure of Lowry et_ _al. (32) with bovine serum albumin as a reference. e) Free amino groups. The f r e e amino groups were reacted with 5-dimethylaminonaphthalene-l-sulfonyl c h l o r i d e (dansyl c h l o r i d e ) (33) and the low molecular weight substances were removed. The d e r i v a t i z e d glycopeptides were hydrolyzed with 6 Ν HC1 f o r 22 hr at 110°C i n sealed, evacuated tubes, and the dansylamino acids were chromatographed on polyamide l a y e r sheets (11). f) Low molecular weight polypeptides. The low molecular weight polypeptides were q u a n t i f i e d by r e a c t i n g with f l u o r e s camine and measuring the f l u o r e s c e n c e emission at 460 nm f o l l o w ing i r r a d i a t i o n at 390 nm on a Farrand spectrofluorometer. The r e l a t i v e f l u o r e s c e n c e emission was compared to l e u c i n e as a reference. g) Carbon-14 l a b e l e d substances. Carbon-14 was q u a n t i f i e d with a Beckman LS-235 l i q u i d s c i n t i l l a t i o n spectrometer (25). Results E x t r a c e l l u l a r peptidophosphogalactomannan. The growth medium of aerated c u l t u r e s of _P. c h a r l e s i i becomes depleted of NH^ a f t e r about 2.5 days but glucose i s not depleted u n t i l about the eighth day ( F i g . 1). The growth medium used (34) i s unbalanced +


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+

with an excess of carbohydrate with respect to NH^ . At the onset of NH^+ s t a r v a t i o n the r a t e of glucose uptake decreases as does the r a t e of i n c r e a s e i n dry weight, and the organism s t a r t s s e c r e t i n g a n o n - d i a l y z a b l e hexosy1-containing polymer. This polymer accumulates i n the growth medium u n t i l the medium i s depleted of glucose. The polymer from 8- to 10-day aerated c u l tures was i s o l a t e d by p r e c i p i t a t i o n from 50 mM borate at pH 9.5 as the c e t y l t r i m e t h y l ammonium-borate complex (35) and p u r i f i e d by ion-exchange chromatography (7)· Nearly a l l of the carbohy drate was e l u t e d from DEAE-cellulose (borate) with 0.01 Ν HC10.06 Ν L i C l ; the same c o n d i t i o n s which e l u t e d the g l u c o s y l - , g a l a c t o f u r a n o s y l - , mannopyranosy1-containing polymers obtained from 28-day s t a t i o n a r y c u l t u r e s of _P. c h a r l e s i i (7). P r e l i m i n a r y chemical analyses showed that the aerated c u l t u r e s contained a saccharide composed of g a l a c t o f u r a n o s y l and mannosyl r e s i d u e s . The saccharides obtained from the aerated c u l t u r e f i l t r a t e s was f u r t h e r p u r i f i e d by gel-permeation chromatography. The p a r t i a l l y p u r i f i e d polymer was subjected to i s o e l e c t r i c f o c u s i n g and over 90% of the polymer showed an i s o e l e c t r i c p o i n t near pH 2 (10). However, the f r a c t i o n s obtained from gel-permeation chromatography showed c o n s i d e r a b l e heterogeneity with respect to the molar r a t i o s of hexose:phosphate and percentage of g a l a c t o s e i n the saccharides ( F i g . 2) (21). The polymers w i t h the l a r g e s t mass ( f r a c t i o n 13, F i g . 2) contained 70% g a l a c t o s e and a h e x o s e p h o s phate r a t i o of greater than 30 which i s i n c o n t r a s t to that ( f r a c t i o n 20) c o n t a i n i n g l e s s than 30% g a l a c t o s e and a hexose: phosphate r a t i o of 12. These and other r e s u l t s showed a propor t i o n a l i n c r e a s e i n mass with increase i n percentage of g a l a c t o s e . Furthermore, we showed that the polymer from 6-day c u l t u r e s had a weight average molecular weight of about 68,000 and 69.9% g a l actose as compared to the polymer from 12-day c u l t u r e s with a weight average molecular weight of 22,600 and 12.1% g a l a c t o s e ( F i g . 3). The percentage of g a l a c t o s e i n e x t r a c e l l u l a r sacchar ides decreased from about 70% i n 6-day c u l t u r e s to 30% i n 12-day c u l t u r e s . T h i s decrease i n mass and percentage g a l a c t o s e c o i n cided with the appearance of exo-3-D-galactofuranosidase i n the growth medium (21). Galactofuranosidase degrades the g a l a c t o mannan r a p i d l y u n t i l galactose accounts f o r only about 15% of the t o t a l saccharide. Galactomannans from other species of f u n g i a l s o c o n t a i n approximately 15% g a l a c t o s e . We have obtained g a l actofuranosidase a c t i v i t y from c u l t u r e f i l t r a t e s of s e v e r a l Pénicillium species and one species of A s p e r g i l l u s (36). Treatment of the phosphogalactomannan with e i t h e r 0.1 N HC1 f o r 90 min at 100°C or with exo-B-D-galactofuranosidase for 2 days removed e s s e n t i a l l y a l l of the g a l a c t o s y l r e s i d u e s . The a c i d treatment a l s o removed some of the phosphorus, but treatment with g a l a c t o f u r a n o s i d a s e removed only the g a l a c t o s y l r e s i d u e s . These r e s u l t s suggest that the g a l a c t o s y l residues occur i n the f u r a n o s y l form and that there are no g a l a c t o f u r a n o s y l residues w i t h i n the mannan backbone. Gel permeation chromatography



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Archives of Microbiology

Figure 1. Time course of Ό-glucose and NHf uptake from the growth medium and increase in dry weight of mycelia of P. charlesii cultures. Pénicillium charlesii was cultured in a modified Raulin-Thom medium (24). The contents of the flasks were removed at intervals,filteredand thefiltratewas assayed for (O—O) total carbohydrate, (Α—Δ) NHf, ( · — φ ) dry weight, and (A—A) pPGM (23). The initial concentrations of Ό-glucose andiNH^ were 278 and 36.3mM, respectively.

Figure 2. Evidence for heterogeneity in peptidophosphogalactomannan (21). A 50-mg sample was fractionated on BioGel P-60 column (1.5 X 82 cm). The elution profile is shown in the bottom panel. The percentage of galactose as a function of total carbohydrate is given in the middle panel. The (A) mannose .phosphate ratio and (A) hexose:phosphate ratio are shown in the top panel.

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showed that the phosphomannan had a mass of about 20,000 d a l tons. Proton nuclear magnetic resonance spectrum of a 5% s o l u t i o n of phosphomannan was taken i n ^ ^ 0 at 29°C and the spectrum of the protons attached to the anomeric carbon atoms show resonance s i g n a l s a t 5.04, 5.10 and 5.22 p.p.m. ( F i g . 4 ) . The s i g n a l a t 5.04 p.p.m. l i k e l y represents an a(l->6) linkage and those a t 5.10 and 5.22 represent a(l->2) linkages s i m i l a r to those observed f o r yeast mannan o l i g o s a c c h a r i d e (37). I n t e g r a t i o n of the area under these peaks suggests a r a t i o of mannosyl (l->6) : mannosy 1(1*2) of 1:3. Treatment of the galactomannan with a l k a l i r e s u l t e d i n a time-dependent i n c r e a s e i n absorbance at 241 nm, t y p i c a l of that observed when g l y c o s y l residues undergo ^ - e l i m i n a t i o n from s e r y l or t h r e o n y l groups of a p r o t e i n (38). I t was shown that the galactomannan was attached to a polypeptide containing about 30 amino a c y l residues with a mass of about 3,000 daltons (11). The polymer has the chemical composition of a peptidophosphogalacto mannan. A reasonably homogenous p r e p a r a t i o n c o n t a i n i n g 15% galactose i s composed of 110 mannosyl r e s i d u e s , 18 g a l a c t o s y l r e s i d u e s , 10 atoms of phosphorus and 30 amino a c y l residues per molecule of peptidophosphogalactomannan. Treatment of the pep tidophosphogalactomannan with 0.4 Ν NaOH changes the molar r a t i o s of galactose:mannose and P:mannose from 1:5.4 to 1:4.6 and 1:10.1 to 1:8.6, r e s p e c t i v e l y . However, there was no change i n the r a t i o of Ρ : g a l a c t o s e . T h i s suggests that the phosphogalactoman nan r e l e a s e d from the polypeptide contained 90 mannosyl residues; a l o s s of about 20 mannosyl residues from that c a l c u l a t e d f o r peptidophosphogalactomannan. Phosphogalactomannan. Yeast glycopeptide has mannosy1-con t a i n i n g o l i g o s a c c h a r i d e s attached by O - g l y c o s i d i c linkage to s e r y l and t h r e o n y l residues o f the polypeptide (39). However, the phosphomannan i s attached to an a s p a r a g i n y l group of the polypeptide through an N - g l y c o s i d i c l i n k a g e . Therefore, the o l i g o s a c c h a r i d e s are r e a d i l y r e l e a s e d by 0.4 Ν NaOH a t 25°C while the N - g l y c o s i d i c l i n k a g e i s s t a b l e under these c o n d i t i o n s . Pep tidophosphogalactomannan from P_. c h a r l e s i i was t r e a t e d with NaB%4 i n a l k a l i followed by n e u t r a l i z i n g the r e a c t i o n mixture and d i a l y s i s to separate the low molecular weight substances from the n o n - d i a l y z a b l e ones. The n o n - d i a l y z a b l e f r a c t i o n was t r e a t e d with 2 Ν HÔ^ at 100°C f o r 3 hr, the r e a c t i o n mixture was n e u t r a l i z e d , deionized by passage over Amberlite MB-3 r e s i n , and analyzed by paper chromatography f o r the presence of % sugar a l c o h o l s . A l l of the % was found i n the r e g i o n of the chromatogram corresponding to mannitol (11). We conclude that the phosphogalactomannan r e g i o n i s attached to the polypeptide by an O - g l y c o s i d i c linkage which when t r e a t e d with a l k a l i r e s u l t e d i n the r e l e a s e of a p o l y s a c c h a r i d e with a reducing terminal mannose r e s i d u e .


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r2(cm2)

Figure 3. Sedimentation equilibrium ultracentrifugation of peptidophospho galactomannan (21). Solutions containing 0.05% peptidophosphogalactomannan in 0.1 M NaCl were centrifugea for 24 hr at 20.6°C in an AnD rotor. Galactose ac counted for (O) 69.6% and (Φ) 12.1% of the total carbohydrate in the two samples. Centrifugation was conducted at 30,000 rpm in a Spinco Model Ε analytical ultracentrifuge.

Figure 4. Proton magnetic resonance spectroscopy of the anomeric proton region of peptidophosphomannan (10). Fifty mg of peptidophosphomannan was dissolved in H 0 and held at room tem perature for 1 hr. The solvent was removed under reduced pressure and the residue redissolved in 1 mL H 0. The NMR spec trum was taken on a Varian XL-100 MHz spectrometer at 29°C with the instrument locked on deuterium and referenced to an internal standard of sodium 2,2-dimethyl-2sûapentane-5-sulfonate. 2

r (cm ) 2

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2

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5.4

5.2 ppm Journal of Biological Chemistry



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57

Chemical a n a l y s i s of phosphogalactomannan showed that each molecule contained about 9 galactan chains and 9 atoms of phosphorus. Phosphogalactomannan from peptidophosphogalactomannan containing 15% galactose has a r a t i o of Prmannose of 1:8.6 and 18% galactose. Each galactan chain contains about 2-galactos y l residues. In a separate experiment the saccharides were 3-eliminated i n the presence of NaOH and phosphogalactomannan separated from the low molecular weight substances by g e l permeation chromatography. The low molecular weight substances which were r e f r a c tionated on Bio-Gel P-2, were r e s o l v e d i n t o f r a c t i o n s c o n t a i n i n g mannose, mannobiose, and mannotriose with s m a l l q u a n t i t i e s of l a r g e r saccharides ( F i g . 5) (10). The linkages of mannobiose and mannotriose have not been determined, but p r e l i m i n a r y experiments suggest that the mannotriose f r a c t i o n contains p r i m a r i l y Dmannopyranosyl-D-mannopyranosyl-(1*2)-D-mannose (36). Methylation a n a l y s i s . Peptidophosphogalactomannan and i t s degradation products were q u a n t i t a t i v e l y methylated and the methylated polymers were converted to t h e i r permethylated a l d i t o l acetates i n an attempt to determine the types of linkages i n the polymer. The permethylated a l d i t o l acetates were separated and q u a n t i f i e d by g a s - l i q u i d chromatography and they were i d e n t i f i e d by t h e i r fragmentation patterns i n a mass spectrometer. A q u a n t i t a t i v e recovery of galactose was obtained, but 10 to 15 mannosyl residues were l o s t during the chemical manipulations. This probably occurred because of the a l k a l i l a b i l i t y of some of the mannose-containing o l i g o s a c c h a r i d e s . Thus, only 2 to 3 nonreducing terminal mannosyl residues were observed i n c o n t r a s t to the 12 to 14 expected based on the q u a n t i t y of o l i g o s a c c h a r i d e s released with a l k a l i . Therefore, the data on the phosphogalactomannan, and phosphomannan are probably the most r e l i a b l e . The data (Table I) show 8-9 moles of nonreducing t e r m i n a l g a l a c t o s y l residues and v a r i a b l e q u a n t i t i e s of i n t e r n a l g a l a c t o s y l residues per mole of phosphogalactomannan. The mannan appears to contain about 3 - f o l d more (1*2) than ( 1 * 6 ) - l i n k e d mannosyl r e s i d u e s . In a d d i t i o n , the occurrence of approximately 9 moles of 4,6-dimethyl mannose, a number equivalent to the number of nonreducing t e r m i n a l g a l a c t o s y l r e s i d u e s , and the l o s s of 4,6-dimethyl mannose when the phosphogalactomannan i s t r e a t e d with d i l u t e mineral a c i d or peptidophosphogalactomannan i s t r e a t e d with g a l a c t o f u r a n osidase, suggests that the galactan chains are attached by (1*3)l i n k a g e to a mannosyl residue which a l s o has another mannosyl residue attached to i t by a (1*2)-linkage. 13

13 C-NMR spectroscopy. We have now examined the C spectrum of phosphogalactomannan. The polymer was f i r s t t i a l l y degraded with galactofuranosidase to increase the ence of the resonance s i g n a l s from the mannosyl residues


NMR parprominand to


Sugar

2 12 38 22 62 14 8

1 1 11 26 75 4 4

+ 2 8 60 17 45 11 3

B

PGM

3

2 8-9 114 18 51 9 4

A

4

26 61 _

10 _ _

A

pPM

3

pPGM, peptidophosphogalactomannan; PGM, phosphogalactomannan; pPM, peptidophospho-

2,3,4,6 2,3,5,6 2,3,6 2,3,4 3,4,6 4,6 3,4

z

pPGM* Galactofuranosidase Treatment

2 The permethylated a l d i t o l acetates were separated on Chromosorb W with 3% polypheny1 ether (6 r i n g s ) as the l i q u i d phase. 3 The permethylated a l d i t o l acetates were separated on ECNSS-M and OV-225.

"^"Samples under heading A and B were derived from pPGM which contained 53% and 40% g a l a c t o s e , respect i v e l y . PGM was c a l c u l a t e d t o c o n t a i n 90 mannosyl r e s i d u e s . The number of hexosyl residues per mole i s c a l c u l a t e d on the b a s i s of 90 mannosyl residues and 20 residues i n the phosphogalactomannan and o l i g o s a c c h a r i d e regions r e s p e c t i v e l y .

Âbbreviations: mannan .

Mannose Galactose Galactose Mannose Mannose Mannose Mannose

Parent

P o s i t i o n of O-Methyl Group

PERMETHYLATED ALDITOL ACETATES IN PEPTIDOPHOSPHOGALACTOMANNAN AND ITS DERIVATIVES

Table I


4.

GANDER E T

AL.

Glycopeptide

Structural

Studies

59

decrease the s i g n a l s from the i n t e r n a l g a l a c t o s y l r e s i d u e s . The spectrum ( F i g . 6) contains four s i g n a l s (A through D) i n the region which i s c h a r a c t e r i s t i c of anomeric carbon atoms (90 to 110 ppm), numerous s i g n a l s (E through M) i n the region of 65 to 85 ppm, and the doublet at 42.7 ppm which i s c o i n c i d e n t with the C ( 2 ) methylene group of L-a-glycerophosphorylethanolamine. The s p e c t r a of reference compounds c o n s i s t i n g of the a- and 3-anomers of methyl g a l a c t o f u r a n o s i d e and D-mannopyranosyl-a(l->2)-D-mannopyranosyl-a(l-*2)-D-mannose were compared to that of phosphogalactomannan ( F i g . 6) and to that of phosphomannan (not shown). The resonance s i g n a l (A) at 109.5 ppm i s that from the anomeric carbon of the i n t e r n a l 5-0-3-D-galactofuranosyl residues and that (A ) at 110.4 ppm was probably from the anomeric carbon of the 9 nonreducing t e r m i n a l g a l a c t o f u r a n o s y l r e s i d u e s . The l a r g e s i g n a l (E) at 84.0 ppm i s derived from C(2) and C(4) of the 5-0-3-Dg a l a c t o f u r a n o s y l r e s i d u e s . The resonance s i g n a l of the C(5) atom of methyl-3-D-galactofuranoside i s at 73.2 ppm and the s i g n a l at 78.2 ppm s h i f t e d downfield by 5 ppm i s strong evidence f o r the occurrence of a g l y c o s i d i c attachment to C(5) of the g a l a c t o s y l residue. The resonance s i g n a l s at 79.1 (G) and 63.7 ppm (M) are those from the C(3) and C(6) carbon atoms, r e s p e c t i v e l y . Treatment of the polymer with d i l u t e m i n e r a l a c i d causes the l o s s of s i g n a l s at p o s i t i o n s A , A, E, G and H, and the s i g n a l (M) at 63.7 ppm i s decreased to the same peak height as s i g n a l K. The s i g n a l at 63.7 ppm represents the C(6) atom of the f r e e hydroxymethyl groups of both mannopyranosyl and g a l a c t o f u r a n o s y l r e s i dues .


f

T

f

The resonance s i g n a l s (B-D) at 104.9, 103.3 and 100.9 ppm represent those from the anomeric carbon of three species of pyranosides. The s i g n a l s at 104.9 and 103.3 c o i n c i d e with the nonreducing t e r m i n a l mannopyranosyl and i n t e r n a l a(l->2)-D-mannopyranosyl r e s i d u e s , r e s p e c t i v e l y . The s i g n a l at 100.9 ppm has not been i d e n t i f i e d , but i t i s known to be e l i m i n a t e d upon r e moval of the phosphorus at pH 3.5. However, the chemical s h i f t of the anomeric carbon atom of an a-D-mannopyranosyl-l-phosphoryl residue should be around 97.1 ppm (40) which seems to e l i m i n a t e an a-D-mannopyranosy1-1-phosphoryl group as being attached to the mannan backbone as occurs i n the glycopeptide from C. w e r n e c k i i (15). The resonance s i g n a l s (F, I-M) a l l c o i n c i d e w e l l with resonance s i g n a l s from i n t e r n a l mannopyranosyl r e s i d u e s . The s i g n a l from the C(3) atom of the mannopyranosyl residues to which the g a l a c t a n chains are attached should appear at about 5 ppm downf i e l d from unsubstituted C(3) atom of i n t e r n a l mannopyranosyl residues; that i s at about 78 ppm. The s i g n a l from t h i s C(3) atom may be buried i n that of the C(5) s i g n a l from 5-0-3-D-galactof u r a n o s y l residues ( s i g n a l H). The s i g n a l (L) at 65.7 ppm and the u n l e t t e r e d one which appear as shoulders on other major resonance species may represent some of the species to which the


F U N G A L POLYSACCHARIDES

60


0.375

ε 4. ! 0.250

χ ο Figure 5. Release of mono- and oligo saccharides from the glycopeptide in 0.125 0.4N NaOH (10). Approximately 20 mg of peptidophosphogalactomannan were treated in the dark in 0.4N NaOH in an atmosphere of N for 72 hr. The saccha rides were fractionated, after neutralization, on a Bio-Gel P-2 column (3 X 92 cm). Ref erence substances of glucose, maltose, raffinose, and stachyose showed maximum con centrations in Fractions 103, 81, 85, and 79, respectively. Carbohydrate was quantified by the phenol-sulfuric acid assay (29) 2

80 FRACTION

100


CHEMICAL C ATOM

SHIFT

C(l) - 1 , 5 - B - D - G A L

F

C(l) -L3-«-D-MANp C(l) -1,2-a-D-MANp C(l) -1,6-a-D-MANp C(2)

CCD

C(2)

-

1,5-B-D-GAL

-1,2-"-D-MAN

F

D

D-MANp & l,5-e-D-GAL

F

60

40 Sc IN PPM

Figure 6. Proton decoupled natural abundance C-NMR spectrum of galacto furanosidase treated phosphogalactomannan. Pictured is the spectrum, taken on a Varian XL-100 spectrometer, from 0 to 3000 Hz downfield from an external reference of 5% TSP. The spectrum of a sample (100 mg mL' ) was taken at 37°C in a 5-mm diame ter NMR tube. The spectrum shown represents data accumulated from 64,000 transients. 13

1


4.

GANDER

ET

AL.

Glycopeptide

Structural

Studies

61


phosphoryl groups are attached. Resonance s i g n a l s of some C atoms from α(1*6)mannopyranosyl residues may be n e a r l y the same as those from some C atoms of α(1*2)mannopyranosyl u n i t s . For instance the J s i g n a l probably represents two unresolved reson ance s i g n a l s as do s i g n a l s at F and K. A c e t o l y s i s of the Glycopeptide. Conditions of a c e t o l y s i s developed i n B a l l o u ' s l a b o r a t o r y (41,42) provide s e l e c t i v e c l e a v age of (l*6)mannopyranosidic l i n k a g e i n mannans c o n t a i n i n g both (1*6) and (1*2) g l y c o s i d i c l i n k a g e s . The g a l a c t o f u r a n o s y l r e s i dues w i l l be cleaved a l s o because of t h e i r a c i d l a b i l i t y . Samples of peptidophosphogalactomannan, phosphogalactomannan and phospho mannan were a c e t o l y s i z e d , the a c e t y l a t e d sugars were extracted i n t o methanol, and deacetylated with m e t a l l i c sodium i n methanol and the products of the r e a c t i o n were f r a c t i o n a t e d on Bio-Gel P-2. A t y p i c a l e l u t i o n p a t t e r n shows f i v e saccharide c o n t a i n i n g peaks. The moles of o l i g o s a c c h a r i d e per mole of polymer was c a l c u l a t e d f o r each p r e p a r a t i o n . The data show that a p r e p a r a t i o n c o n t a i n i n g 53% galactose r e l e a s e d 12 moles of mannotetraose, 6 moles of mannotriose, 14 moles of mannobiose and 17 moles of mannose during a c e t o l y s i s (Table I I ) . A c e t o l y s i s of the phosphogalactomannan gave s i m i l a r r e s u l t s except that t h i s polymer contained 2, 9 and 6 moles l e s s of mannotriose, mannobiose and mannose, r e s p e c t i v e l y , than peptidophosphogalactomannan. T h i s suggests that a c e t o l y s i s a l s o cleaves the O - g l y c o s i d i c linkages between the mannopyranosyl residues and the s e r y l and t h r e o n y l r e s i d u e s . The 17 p o i n t s of cleavage between the o l i g o s a c c h a r i d e s and the polypeptide estima ted by t h i s i n d i r e c t procedure i s i n reasonably agreement with the number of residues estimated by ^ - e l i m i n a t i o n . I t was shown i n separate experiments with ^2p l a b e l e d peptidophosphogalactomannan that the ^2p extracted i n t o the aqueous phase f o l l o w i n g aceto lysis. There was n e g l i g i b l e q u a n t i t y of carbohydrate i n t h i s phase. w

a

s

Polypeptide. The r e l e a s e of phosphogalactomannan, mannose, mannobiose and mannotriose from the polymer by a l k a l i suggests that these saccharides are attached to a polypeptide (11). The amino a c i d composition of a peptidophosphogalactomannan prepara t i o n was determined (Table I I I ) . T h i s t a b l e shows that approxi mately one-half of the amino a c y l residues of the polypeptide are e i t h e r s e r y l or t h r e o n y l residues and that the polypeptide has no aromatic or s u l f u r - c o n t a i n i n g amino a c i d s . Treatment of the polymer with a l k a l i followed by r e d u c t i o n of the α,$-dehydroaminoa c y l residues formed r e s u l t e d i n a l o s s of a l l but 2 of the s e r y l residues and e s s e n t i a l l y a l l of the t h r e o n y l residues (Table I V ) . Furthermore, the number of a l a n y l residues increased from 4 to 8 and 4 residues of α-aminobutyric a c i d were obtained. These were d e r i v e d from the r e d u c t i o n products of the a,$-dehydroserine (aa m i n o a c r y l i c acid) and a,3-dehydrothreonine (a-aminocrotonic acid), r e s p e c t i v e l y , f o l l o w i n g 3 - e l i m i n a t i o n of saccharides from s e r y l



62

FUNGAL

POLYSACCHARIDES

Table I I O l i g o s a c c h a r i d e s Derived from Peptidophosphogalactomannan and i t s D e r i v a t i v e s

Glycan A c e t y l a t e d

Moles/mole Glycan M

pPGM* PGM PM

5

M

1.9 1.9 1.6

4 11.7 10.1 8.8

M

M

5.6 3.7 4.8

13.8 4.7 7.1

2

M 17.6 11.8 14

Âbbreviations: pPGM, peptidophosphogalactomannan; PGM, phosphogalactomannan; PM, phosphomannan; M^, mannopentaose; M^, mannot e t r a o s e ; M3, mannotriose; M , mannobiose; M, mannose. 2

A c e t o l y s i s was c a r r i e d out on pPGM and PGM which contained 53% and 62% g a l a c t o s e , r e s p e c t i v e l y . A value of 110 mannosyl r e s i dues/mole i s used f o r pPGM and 90 mannosyl residues/mole i s used f o r both PGM and PM.



4.

GANDER E T

AL.

Glycopeptide

Structural

63

Studies

Table I I I Amino A c i d Composition of _P. c h a r l e s i i Glycopeptide

Amino A c i d Residues' Amino Acid

Ser Thr Ala Gly Val Pro Asx Glx His He Leu Lys Arg Trp Phe Met Tyr

a) b) c) d)

b 4 hrs

8.47 5.80 2.62 1.96 0.79 1.21 0.76 0.83 0.24 0.21 0.29 0.25 Trace d

Trace Trace

d

Based on a Average of Average of Determined

11 hrs

7.54 6.32 3.22 2.40 1.21 1.54 0.88 1.07 0.77 0.45 0.38 0.26 Trace

Trace Trace

-

22 hrs°

7.19 5.62 3.87 2.52 1.66 2.21 1.01 1.43

0.53 0.53 0.36 Trace

Trace Trace

-

44 hrs

5.93 5.63 4.05 2.76 1.48 1.39 1.08 1.04 0.68 0.45 0.36 0.25 Trace

Integral Maximal or E x t r a p o l a t e d Number Values 8.60 6.32 4.06 2.76 1.66 2.21 1.08 1.43 0.77 0.53 0.53 0.36 Trace

Trace Trace

Trace Trace

-

-

molecular weight of 26,500. two determinations. three determinations. by the spectrophotometric method.


9 6 4 3 2 2 1 1 1 0-1 0-1 0-1 Trace Trace Trace Trace Trace

64

FUNGAL

POLYSACCHARIDES

Table IV Changes i n Amino A c i d

Composition

Following A l k a l i n e


Borohydride Treatment of the Glycopeptide

Amino Acid

Untreated polymer

Serine Threonine Alanine ot-Aminobutyric A c i d

a) b)

Residues /mole A l k a l i n e Borohyd r i d e Treated polymer

7. 28 5. 66 3 82

1.92 0.30 7.82

0

4.02

Increase or Decrease

(-> (-) (+) (+)

5.36 5.36 4.00 4.02

Quantity present a f t e r 22 hours h y d r o l y s i s i n constant b o i l i n g HC1 a t 110° i n a s e a l e d evacuated tube. Based on a molecular weight of 26,500.

31 0

1

1

20

.

1

40

1

.

60

•

1

80

.

ν/ 1 100 120

Figure 7. Separation of polypeptides, derived from peptidophosphogalactoman nan by treatment with anhydrous HF, by anion exchange chromatography. Τeptidophosphogalactomannan was treated with anhydrous HF, the polypeptide that remained was passed through a Bio-Gel P-6 column and the fraction containing the poly peptide was lyophylized and was dissolved in pyridinium acetate (ph 5.0). The sample was applied to a Whatman DE cellulose (DEAE cellulose) that had been pre-equilibrated in 0.02M pyridinium acetate buffer. The DEAE cellulose was washed with 0.02M pyri dinium acetate buffer until the first peak came off, then a gradient of 0.02M pyridinium acetate (pH 5)-lM acetic acta was started. About 20-30% of the material that was applied to the column was not eluted from the column.



4.

GANDER

ET

AL.

Glycopeptide

Structural

Studies

65

and t h r e o n y l r e s i d u e s . The amino a c y l composition of the p o l y peptide obtained from P . c h a r l e s i i peptidophosphogalactomannan i s unique i n c o n t a i n i n g such a l a r g e p r o p o r t i o n of s e r y l and t h r e o n y l r e s i d u e s , and i n c o n t a i n i n g e s s e n t i a l l y no aromatic or s u l f u r c o n t a i n i n g amino a c i d s . However, s e v e r a l of the p e p t i d o g a l a c t o mannans from f u n g i (14,43) c o n t a i n polypeptides with a s i m i l a r amino a c y l composition. We noted that i s o l e u c i n e , l e u c i n e and l y s i n e occurred i n q u a n t i t i e s which are f a r below one residue per mole of polypept i d e (Table I I I ) . T h i s suggests that e i t h e r the molecular weight of peptidophosphogalactomannan i s twice as l a r g e as we c a l c u l a t e d from e q u i l i b r i u m u l t r a c e n t r i f u g a t i o n experiments, or that the polypeptide i s heterogeneous. T h i s heterogeneity could r e s u l t from the p r e p a r a t i o n c o n t a i n i n g polypeptides of s l i g h t l y d i f f e r e n t number of r e s i d u e s , with some species c o n t a i n i n g a h i s - i l e - l e u - l y s or h i s - l e u - i l e - l y s on one end. Carboxy-terminal amino a c i d analys i s of the polypeptide showed that none of the above amino a c i d s was at the C-terminal end. Amino-terminal amino a c i d determinat i o n s were made using dansyl c h l o r i d e as a reagent to d e r i v a t i z e the f r e e amino groups. The d e r i v a t i z e d amino acids were separated on polyamide l a y e r s and s e r i n e , aspartate, glutamate and g l y c i n e residues were shown to be d e r i v a t i z e d (11). A f i f t h f l u o r e s c e n t area was observed which was i d e n t i f i e d as dansyl-ethanolamine. Treatment of peptidophosphogalactomannan with pronase served to modify the polypeptide so that i t contained a s e r i n e residue on both the C-terminal and N-terminal ends, but the number of i s o l e u c y l , l e u c y l and l y s y l residues per mole of polypeptide r e mained considerably l e s s than one. We have extended t h i s work by t r e a t i n g the peptidophosphogalactomannan with anhydrous HF which cleaves O - g l y c o s i d i c a l l y l i n k e d residues at 0°C without c l e a v i n g the peptide bond (27). The mass of t h i s polypeptide p r e p a r a t i o n i s about 3,000 daltons and e l u t e s as one sharp peak from a B i o - G e l column (not shown). The p r e p a r a t i o n was chromâtographed on a weak anion exchange r e s i n and the polypeptide was r e s o l v e d i n t o 4 f r a c t i o n s ( F i g . 7). Attempts at sequencing these i n d i v i d u a l f r a c t i o n s suggest that none are completely homogenous (44). Thus we conclude that the peptidophosphogalactomannan i s derived from at l e a s t 4, and p o s s i b l y from many more, p o l y p e p t i d e s . The i m p l i cations with respect to f u n c t i o n w i l l be discussed i n a l a t e r section. Non-carbohydrate c o n s t i t u e n t s . We have shown that a l l of the phosphorus occurs i n d i e s t e r linkage (10) yet n e i t h e r the methylat i o n a n a l y s i s nor the NMR spectroscopy provided any evidence f o r the occurrence of a g l y c o s y l - l - p h o s p h o r y l r e s i d u e . F i n d i n g dansyl-ethanolamine f o l l o w i n g d e r i v a t i z a t i o n of peptidophosphogalactomannan with dansyl groups, suggested that phosphoethanolamine might be attached to the mannan. However, a q u a n t i t a t i v e determination of ethanolamine revealed only about one r e s i d u e per mole of peptidophosphogalactomannan (11). I t was a l s o


66

FUNGAL

POLYSACCHARIDES

demonstrated that r a d i o l a b e l e d ethanolamine i n the growth medium was incorporated i n t o peptidophosphogalactomannan and that t r e a t ment of the polymer r e l e a s e d about 40% of the r a d i o a c t i v i t y with the remainder s t i l l covalent attached to the p o l y s a c c h a r i d e (12). T h i s suggests that ethanolamine or i t s d e r i v a t i v e s occur i n two environments w i t h i n the peptidophosphogalactomannan. However, e s s e n t i a l l y a l l of the ~P f 3 2 p _ i ; L c l peptidophosphogalacto mannan remained a s s o c i a t e d with the phosphogalactomannan. The small amount of 32 which was released was eluted from B i o - G e l P-2 i n f r a c t i o n s (68-73) c o i n c i d e n t with those which a l s o contain *C from ethanolamine ( F i g s . 8,9) as w e l l as carbohydrate. T h i s f r a c t i o n may represent a mannotetraose, or p o s s i b l y a mannopentaose, u n i t c o n t a i n i n g c o v a l e n t l y bound phosphorus and ethanol amine, or a d e r i v a t i v e of ethanolamine. Peptidophosphogalacto mannan c o n t a i n i n g -^C from L-i^C-CH^) methionine was prepared and the polymer was t r e a t e d with a l k a l i . As occurred with the l ^ C ethanolamine-labeled glycopeptide, approximately 40% of the l^C was r e l e a s e d as a low molecular weight substance and the remainder was covalent a s s o c i a t e d with the phosphogalactomannan. Nearly a l l of the which was r e l e a s e d was eluted i n f r a c t i o n s 95-101 from the Bio-Gel P-2 column s i m i l a r to that when the polymer was l a b e l e d with l ^ C from ethanolamine ( F i g . 9) (12). I t was shown (12) that f r a c t i o n s 38-45, 65-75 and 95-105 contained N,N -dimethylaminoethanol with the l a r g e s t quantity i n f r a c t i o n s 65-75. The occurrence of Ν,Ν -dimethylaminoethanol i n the peptidophos phogalactomannan was demonstrated by g a s - l i q u i d chromatography of the h y d r o l y s i s products of the Smith degradation r e a c t i o n s (12). Doubly l a b e l e d peptidophosphogalactomannan c o n t a i n i n g 14 from L - ( C H - C ) m e t h i o n i n e and H from (1- H) ethanolamine was prepared and the polymer was subjected to Smith degradation procedure. T h i s treatment r e s u l t e d i n the formation of two -labeled substances ( F i g . 10). These substances a l s o contained phosphorus as shown by the 32 i n these f r a c t i o n s as demonstrated i n separate experiments with 32 - l a b e l e d peptidophosphogalactomannan (not shown). A c e t o l y s i s of 32p^ 3 _ L - ^ C -methioninel a b e l e d peptidophosphogalactomannan showed that a l l of the r a d i o a c t i v i t y extracted i n t o the aqueous phase. Thus the a c e t o l y s i s procedure cleaved the phosphoethanolamine/phospho-NjN'-dimethylaminoethanol residues from the mannan and n e g l i g i b l e carbohydrate was a s s o c i a t e d w i t h these f r a c t i o n s . These r e s u l t s , when taken c o l l e c t i v e l y , s t r o n g l y suggest that phosphoethanolamine/phosphoN,Ν'-dimethylaminoethanol occurs i n two separate environments i n peptidophosphogalactomannan. The g a l a c t o f u r a n o s y l residues are removed enzymically from the peptidophosphogalactomannan without l o s s of phosphorus or ethanolamine. The methylation data presen ted i n another s e c t i o n suggests that phosphorus may be attached to residues which have f r e eand hydroxy1 groups at carbon atoms 3 and 4. Sandford Matsuda; Fungal Polysaccharides Pphospho-N,N r e l i m i n a r y ACS Symposium Series; American Chemical Society: 1980. i s l o c a t e d i-dimethylaminoethanol 3n1two NMRenvironments; spectroscopy one a ladjacent s with o shows aWashington, phosphoethanolamine-/ to that a f the r eDC, e 3 hydroxy1 1 nucleus 3


Q

aDe

e

P

1Z

?

1

C

14

3

3

3

P

P

H

T

e t l i a n o

L a m i n e >

o

r

4.

Glycopeptide

GANDER E T A L .

Structural

Studies

67

4

Figure 8. Distribution of P following chromatography of the low-molecularweight substances from P-labeled glycopeptide on Bio-Gel Ρ2 (12). The lowmolecular-weight substances from alkalitreated peptidophosphogalactomannan that separated from the high-molecular-weight substances on Sephadex G-50 were chromatographed on a column of Bio-Gel P-2, minus 400 mesh (93 X 2.5 cm) with distilled deionized water as the eluent. Fractions (3.4 mL) were analyzed for P and the data are given as cpm per sample. 32


ε Q. U r-A û.

32

2-

FRACTION

32

Experimental Mycology

Figure 9. Distribution of C (shaded region) and carbohydrate following chromatography of the low-molecular weight substances from alkali-treated peptidophosphogalactomannan from Cethanolamine-labeled peptidophospho galactomannan on Bio-Gel P-2. The low-molecular-weight substances were obtained and treated as described in Figure 13 and (12). 14

14

40

60 80 FRACTION

100

Experimental Mycology

Figure 10. Separation of Smith degra dation products of double-labeled pepti dophosphogalactomannan on Dowex-2formate (12). Double-labeled peptidophos phogalactomannan was prepared containing H and C from (l- H)e hanolamine (shaded region) (500 μα) and L-(CΗ - C^methio nine (100 μΟι) added U a culture (150 mL) 2.5 days after adding approximately 10 P. charlesii spores. The growth conditions and growth media are described elsewhere (12). Ten mg of peptidophosphogalactomannan obtained from this culture filtrate after 9 days were subjected to Smith degradation and the products were fractionated on Dowex-2-formate according to the proce dure of Wells and Dittmer (47).

3

14

3

3

14

8

FRACTION Experimental Mycology


68

FUNGAL

POLYSACCHARIDES


group and the other with the phospho-compounds not adjacent to a f r e e hydroxy1 group. Cytoplasmic Peptidophosphogalactomannan. Fungal p e p t i d o galactomannans have been extracted from powdered preparations (43). However, we were unable to o b t a i n phosphogalactomannan from the c e l l w a l l s of 3-day c u l t u r e s of _P. c h a r l e s i i (20). When we examined e x t r a c t s of c h a r l e s i i c e l l s we found galactofurano s y l residues both i n a s o l u b l e cytoplasmic and a membrane-bound cytoplasmic f r a c t i o n . The s o l u b l e cytoplasmic f r a c t i o n was separated from the mem branes by c e n t r i f u g a t i o n at about 100,000 χ g and the glycopept i d e s i s o l a t e d by a procedure described p r e v i o u s l y (10). The galactofuranosy1-containing glycopeptide(s) which were i s o l a t e d had chromatographic p r o p e r t i e s which are s i m i l a r to those of the e x t r a c e l l u l a r peptidophosphogalactomannan except that the " c y t o plasmic" s o l u b l e - g l y c o p e p t i d e may be somewhat l a r g e r . The p o l y peptide was estimated to contain 61-64 amino a c y l residues of which 27 were s e r y l and t h r e o n y l . Treatment of the glycopeptide with a l k a l i r e s u l t e d i n the r e l e a s e of carbohydrate from approxi mately 14 s e r y l and t h r e o n y l residues. The o l i g o s a c c h a r i d e s which were r e l e a s e d were f r a c t i o n a t e d on B i o - G e l P-2 and mannosec o n t a i n i n g saccharides e l u t i n g i n the p o s i t i o n s of mannotriose and mannobiose were obtained. The p o l y s a c c h a r i d e which e l u t e d i n the v o i d volume contained galactose and mannose. Galactose was r e l e a s e d by e i t h e r 0.01 N HC1 or g a l a c t o f u r a n o s i d a s e . The glycopeptide was d e r i v a t i z e d with dansyl c h l o r i d e and the dansyl-glycopeptide was hydrolyzed and three fluorescence areas c o i n c i d e n t with d a n s y l - g l y c i n e , d a n s y l - s e r i n e and d a n s y l ethanolamine were observed on the sheets f o l l o w i n g two dimensional chromatography on sheets of polyamide. The occurrence of ethanolamine was confirmed by i t s p o s i t i o n of e l u t i o n from a column of the amino a c i d analyzer, and i t was q u a n t i f i e d by com p a r i s o n to reference s o l u t i o n of ethanolamine. A 22 hr h y d r o l y sate of glycopeptide i n 6 N HC1 contained about 0.3 moles of ethanolamine per mole of glycopeptide. Our estimate of the molecular weight of the s o l u b l e "cytoplasmic" glycopeptide i s at present based on i t s p o s i t i o n of e l u t i o n from a Sephadex column, and SDS d i s c polyacrylamide g e l e l e c t r o p h o r e s i s , which give a value between 80,000 and 90,000. The glycopeptide was shown to contain phosphorus and when subjected to i s o e l e c t r i c focusing the glycopeptide banded i n the region of pH 2-3. This evidence suggests that the polymer may be a precursor of the e x o c e l l u l a r peptidophosphogalactomannan which has been c h a r a c t e r i z e d more e x t e n s i v e l y . Membrane-bound Peptidophosphogalactomannan. The membranes obtained f o l l o w i n g c e n t r i f u g a t i o n at about 100,000 χ g were f r a c t i o n a t e d by i s o p y c n i c sucrose gradient u l t r a c e n t r i f u g a t i o n . Six



4.

GANDER

ET

AL.

Glycopeptide

Structural

Studies

69

membrane-containing bands were v i s i b l e and each band was removed and the membranes p e l l e t e d . The enzyme a c t i v i t i e s a s s o c i a t e d with each band and the occurrence of g a l a c t o f u r a n o s y l residues based on the r e l e a s e of galactose with galactofuranosidase or with 0.01 N HC1 at 100°C f o r 90 min was determined f o r each band. I t was found that membrane band V (p = 1.18 g/cc) was the only one which contained g a l a c t o f u r a n o s y l r e s i d u e s . T h i s f r a c t i o n was a l s o p a r t i c u l a r l y r i c h i n a c i d phosphatase although i t contained only about 5% of the t o t a l p r o t e i n a p p l i e d to the gradient. The s p e c i f i c a c t i v i t y of a c i d phosphatase was 4-fold greater i n mem brane f r a c t i o n V than that i n the crude membrane p r e p a r a t i o n . In c o n t r a s t , the s p e c i f i c a c t i v i t i e s of Mg-ATPase, Na/K-Mg-ATPase, glucose-6-phosphate phosphatase, 5'-nucleotidase and a l k a l i n e phosphatase i n membrane f r a c t i o n V were e i t h e r equal to that i n the crude membrane p r e p a r a t i o n or s e v e r a l f o l d l e s s than that of the crude membrane p r e p a r a t i o n . Membrane f r a c t i o n V was t r e a t e d with a s e r i e s of detergents, chaotropic agents, t r y p s i n , or a l k a l i i n an attempt to s o l u b i l i z e the galactofuranosy1-containing substances. Although a number of such treatments p a r t i a l l y s o l u b i l i z e d the galactofuranosy1-con t a i n i n g substance(s), the i n s o l u b l e f r a c t i o n a l s o contained g a l a c t o f u r a n o s y l r e s i d u e s . Treatment of f r a c t i o n V with CHCI3:methanol 2:1 (v/v) r e s u l t e d i n concentrating a l l of the g a l a c t o f u r a n o s y l c o n t a i n i n g substances at the i n t e r f a c e . These substances were s o l u b i l i z e d by making the suspension 1 M with KC1 followed by a d j u s t i n g the s o l u t i o n to 2% i n sodium deoxycholate (Table V). Although t h i s treatment s o l u b i l i z e d a l l of the g a l a c t o f u r a n o s y l c o n t a i n i n g substances, i t only s o l u b i l i z e d 11% of the p r o t e i n i n the membrane f r a c t i o n . The galactofuranosy1-containing substances were p u r i f i e d by combining g e l permeation chromatography on B i o - G e l A-5m followed by rechromatography on concanavalin A-Sepharose 4B. The KC1-D0Cs o l u b l e substances were a p p l i e d to Bio-Gel A-5m i n 40 mM T r i s - H C l 40 mM NaCl-0.24% DOC, pH 7.5 b u f f e r (Tris-NaCl-DOC b u f f e r ) and the column i r r i g a t e d with t h i s b u f f e r system. The g a l a c t o f u r a n o s y l c o n t a i n i n g substances were e l u t e d i n f r a c t i o n s 105-115 which con tained l e s s than 10% of the p r o t e i n . These f r a c t i o n s were pooled and a p p l i e d to a concanavalin A-Sepharose 4B column. The adsor bent was washed with the Tris-NaCl-DOC b u f f e r u n t i l the e l u a t e coming through had n e g l i g i b l e absorbance at 280 nm. The adsorbent was then i r r i g a t e d with Tris-NaCl-DOC b u f f e r c o n t a i n i n g 1% methylα-D-mannopyranoside. Galactofuranosy1-containing substances were obtained i n f r a c t i o n s 31-36 ( F i g . 11). The DOC-soluble galactofuranosy1-containing polymer was electrophoresed on SDS d i s c polyacrylamide g e l s . A broad band which was h e a v i l y s t a i n e d with Coomassie blue migrated at an average R of 0.19 (not shown). Carbohydrate c o n t a i n i n g polymers which s t a i n e d with periodate S c h i f f reagent a l s o migrated i n a broad band i n t h i s r e g i o n . Two f a i n t bands which s t a i n e d with Coomassie blue migrated with R s 0.79 and 0.82. A molecular f

T

f


FUNGAL

70

POLYSACCHARIDES

Table V S o l u b i l i z a t i o n of Galactofuranosy1-containing Substance(s) from I n t e r f a c e Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on February 28, 2018 | https://pubs.acs.org Publication Date: June 10, 1980 | doi: 10.1021/bk-1980-0126.ch004

3

G a l a c t o f u r a n o s y l Residues Soluble Insoluble d

Treatment

1 M KC1 1 M TCA, pH 7.2 1 mM phosphotungstic 2% DOC 1 M KC1 + 2% DOC

a) b) c) d)

a c i d , pH 6.8

+ + + +

Membranes were t r e a t e d with 10 volumes CHCl :MeOH, 2:1 (v/v) and the m a t e r i a l accumulating a t the i n t e r f a c e was c o l l e c t e d . Treated f o r two hours at 4°C. Measured with galactose oxidase a f t e r treatment with g a l a c tofuranosidase. Soluble a f t e r c e n t r i f u g a t i o n a t 100,000 χ g f o r 1 hour. 3

Figure 11. Fractionation of polymer on a concanavalin A-Sepharose 4B column. Galactofuranosyl-containing fractions from a Bio-Gel A-5m column were pooled and applied to a column of concanavalin ASepharose 4B. The adsorbent was washed successively with 40mM Tris-HCl-40mM NaCl-0.24% deoxycholate, pH 7.5, fol lowed by the same buffer containing 1% methyl-a-O-mannopyranoside starting with Fraction 27. Fractions containing 100 drops were collected and 0.5 mL samples were assayed for galactofuranosyl residues (21). Fractions containing galactofuranosyl resi dues are indicated with a bar.

ο

10

20

30

FRACTION NUMBER


40


4.

GANDER

ET

AL.

Glycopeptide

Structural

Studies

71

weight of 105,000 was estimated f o r the m a t e r i a l at Rf 0.19. However, i t i s apparent that the polymer i s very heterogenous. Amino a c i d analyses were performed on the glycopeptide pre p a r a t i o n f o l l o w i n g h y d r o l y s i s of the polymer(s) i n 6 N HC1 f o r 22 hr. Approximately 38% of the amino acids were represented by s e r y l and threonyl residues (Table V I ) . In a d d i t i o n , the amino a c i d composition i s s i m i l a r to that obtained f o r both the e x t r a c e l l u l a r and s o l u b l e "cytoplasmic" peptidophosphogalactomannans. The polypeptides contained only t r a c e q u a n t i t i e s of aromatic amino a c y l r e s i d u e s , no detectable q u a n t i t i e s of s u l f u r - c o n t a i n i n g amino a c y l r e s i d u e s , and were r i c h i n s e r i n e , threonine and a l a n i n e . The percentage of p r o l i n e decreased as the glycopeptide s i z e i n creased. Thus, the average number of amino a c y l residues i s c a l c u l a t e d on the b a s i s of a polymer c o n t a i n i n g two p r o l y l r e s i dues. Treatment of the galactofuranosy1-containing polymer(s) with a l k a l i r e s u l t e d i n a decrease of 11 s e r y l and 9 t h r e o n y l r e s i d u e s , based on a polypeptide of 110 amino a c y l r e s i d u e s . Mannobiose was the major low molecular weight o l i g o s a c c h a r i d e released by a l k a l i (not shown). However, only 49% of the carbohydrate a p p l i e d to the column was e l u t e d . Experiments were conducted to determine the f r e e amino groups a v a i l a b l e f o r r e a c t i o n with dansyl c h l o r i d e . Only one f a i n t f l u o r e s c e n t area i n the r e g i o n of d a n s y l - l y s i n e was observed. No dansyl-ethanolamine was observed. However, i f the polymer was f i r s t t r e a t e d with 6 N HC1 to hydrolyze the amino a c i d s , dansyl amino a c i d s were obtained. Q u a n t i t a t i v e a n a l y s i s f o r ethanolamine from the amino a c i d analyzer gave 1.3 ethanolamine residues f o r 2 p r o l i n e r e s i d u e s . The low s o l u b i l i t y of the polymer i n aqueous s o l u t i o n may have been the primary f a c t o r l e a d i n g to lack of d e r i v a t i z a t i o n of the i n t a c t glycopeptide. The composition of peptidophosphogalactomannan from 2-day c u l t u r e f i l t r a t e s was compared to that of the cytoplasmic s o l u b l e - and membrane-bound galactofuranosy1-containing substances (Table V I ) . Galactose released by d i l u t e a c i d accounts f o r 6065% of the t o t a l carbohydrate. These data when coupled with that obtained with galactofuranosidase, suggests that the three g l y c o peptides contain about the same percentage of g a l a c t o f u r a n o s y l r e s i d u e s . The DOC-soluble polymer appeared to contain about 26% p r o t e i n as compared to about 15% polypeptide i n the s o l u b l e g l y c o peptides. The r a t i o of hexose:Ρ of approximately 15:1 i s con s i d e r a b l y l e s s than the 30:1 value which we r o u t i n e l y obtain f o r e x t r a c e l l u l a r peptidophosphogalactomannan c o n t a i n i n g a l a r g e per centage of g a l a c t o f u r a n o s y l r e s i d u e s . At present we have no explanation f o r t h i s observation. -Table VII shows that 2-day c u l t u r e s have produced i n a l l three forms of the peptidophosphogalactomannan a q u a n t i t y e q u i v a l e n t to 11 ymoles of hexose/100 ml of c u l t u r e . The average germination time i s 36 hr. We c a l c u l a t e that a quantity of peptidophosphogalactomannan equivalent to 0.9 pmole h r ~ l 100 ml"-*-


72

FUNGAL

POLYSACCHARIDES

Table VI


Amino A c i d Composition

Amino A c i d

of Peptidophosphogalactomannans

Exocellular

Serine Threonine Alanine Glycine Valine Proline Aspartate/Asparagine Glutamate/Glutamine Histidine Isoleucine Leucine Lysine Arginine Phenylalanine Tyrosine Methionine

Peptidophosphogalactomannans Soluble-cytoplasmic Mem brane bound

9 6 4 3 2 2 1 1 1 0.5 0.5 0.4 Τ Τ Τ Τ

16 11 7 8 4 2 4 5 1-2 1 1-2 1 Τ Τ Τ

24 19 14 13 8 2 10 8 5 5 5 3-4 Τ Τ Τ

-

-

The number of moles of each amino a c i d per mole of peptidophosphogalactomannan i s based on 2 p r o l y l residues per mole.

Table V I I Chemical C h a r a c t e r i z a t i o n of Glycopeptides Source of Glycopeptide Growth medium Supernatant DOC-soluble

pmoles An-Hex: flask 2.5 10.4 4.0

a

An-•Hex phosphate

An -Hex protein

Galactose^ An-Hex

16 15 12

6.7 7.0 3.8

0.66 0.64 0.60

a

a

a

Anhydrohexose. Polymer t r e a t e d with 0.05 N HC1 f o r 90 minutes at 110°C. Galac tose r e l e a s e d measured by the coupled g a l a c t o s e oxidase-horser a d i s h peroxidase assay.



4.

GANDER E T A L .

Glycopeptide

Structurai

73

Studies

of c u l t u r e i s formed. Therefore, 8-day c u l t u r e s should produce a q u a n t i t y o f peptidophosphogalactomannan equivalent to about 140 pmoles of hexose. F i g . 2 shows that c u l t u r e f i l t r a t e s from 8-day c u l t u r e s contain approximately t h i s quantity of pep tidophosphogalactomannan. The p h y s i c a l p r o p e r t i e s of membrane-bound peptidophospho galactomannan suggest that i t i s amphipathic which i s i n contrast to those o f s o l u b l e peptidophosphogalactomannans. Furthermore, only a p o r t i o n of the phosphogalactomannan region was s o l u b i l i z e d by t r e a t i n g membrane-bound peptidophosphogalactomannan with alkali. This suggests that the phosphogalactomannan contains hydrophobic substances attached by covalent linkages which are s t a b l e to 0.5 Ν NaOH. No evidence was obtained f o r f a t t y a c y l residues attached t o the mannan. However, membrane-bound peptido phosphogalactomannan contained approximately 13-fold more 1 4 per mole of hexosyl residues than s o l u b l e cytoplasmic peptidophospho galactomannan when the peptidophosphogalactomannans were i s o l a t e d from 48-hr c u l t u r e s to which ( l - ^ C ) acetate had been added a f t e r 24 hr (Table V I I I ) . E s s e n t i a l l y a l l of the r a d i o a c t i v i t y was removed from the growth medium w i t h i n 6 h r a f t e r the a d d i t i o n o f ( l - 1 4 c ) a c e t a t e (not shown). E s s e n t i a l l y none of the C was i n corporated i n t o e i t h e r the g a l a c t o f u r a n o s y l or mannopyranosyl r e s i d u e s . A small amount of the l^C incorporated i n t o the amino a c i d s . H y d r o l y s i s of the -^C-labeled peptidophosphogalacto mannan i n 2 N HC1 f o r 4 h r a t 110°C followed by t h i n l a y e r chromatography i n appropriate solvents showed that the l^C s not incorporated p r i m a r i l y i n t o f a t t y a c i d s or c h o l e s t e r o l . Treatment of -^C-labeled peptidophosphogalactomannan with 4 Ν KOH f o r 5 h r at 110°C released -^C (13%) which was s o l u b l e i n CHCI3: methanol 2:1 ( v / v ) . The r a d i o a c t i v i t y i n the organic solvent migrated c o i n c i d e n t with sphingosine i n two solvent systems ( F i g s . 12,13) but somewhat d i f f e r e n t than sphingosine i n two other s o l vent systems ( F i g . 14,15). An a l i q u o t of the organic e x t r a c t was treated with I0^~ (45) and the r e a c t i o n mixture was extracted with methylene c h l o r i d e . The methylene c h l o r i d e e x t r a c t was chromatographed. Periodate treatment caused about 40% of the r a d i o a c t i v i t y to be d i s p l a c e d from the o r i g i n where sphingosine i s l o c a t e d , to an Rf of 0.5 ( F i g . 16). S i m i l a r treatment of sphingosine r e s u l t e d i n a product which had an Rf of 0.61. These experiments show that membrane-bound peptidophospho galactomannan contains s e v e r a l f o l d more 14 derived from acetate than does the s o l u b l e peptidophosphogalactomannans. This suggests that the amphipathic nature of the membrane-bound peptidophospho galactomannan may be a r e s u l t of hydrophobic substance(s) derived from acetate which are attached to the phosphogalactomannan. No evidence was obtained f o r f a t t y a c y l residues or s t e r o l s being attached by e s t e r linkage to the phosphogalactomannan. Some of the 1 C was r e l e a s e d by 4 Ν NaOH and the substance(s) released has p r o p e r t i e s s i m i l a r to a sphingosine d e r i v a t i v e . The 4 Ν a l k a l i should have r e l e a s e d any f a t t y a c y l residues i n amide linkage C

1 4

w

a

s

w

c


a

74

FUNGAL

Table 1

^C from

POLYSACCHARIDES

VIII

1

[ l - ^ C ] A c e t a t e Incorporated

i n t o DOC-soluble Glycopep


tide 14 Source of

C/ymole Anhydro-

Gly cop ep t i d e

a

hexos e -3 cpm χ 10

Growth medium Supernatant DOC-soluble

a)

1.9 2.7 35.3

Glycopeptides i s o l a t e d and p u r i f i e d from c u l t u r e s grown i n f o u r f l a s k s under standard c o n d i t i o n s . Twenty f o u r hours a f t e r i n o c u l a t i o n 1 mCi ( 1 - C ) a c e t i c a c i d (58 mCi/mmole) was added to each of two f l a s k s . 1 4

b CD

300

200r-

100

1 FROM ORIGIN

Figure 12. Thin layer chromatography of base-hydrolyzed C-laheled deoxycholate-soluhle peptidophosphogalactomannan. C-Labeled deoxycholate-soluble peptidophosphogalactomannan was treated with 4N KOH for 5 hr at 110° C in a sealed evacuated tube. The solution was extracted with CHCl :methanol, 2:1 (v/v). Fifty sample was applied to a silica-gel, thin layer plate and chromato graphed in benzene .di ethyl ether .ethyl acetate-.acetic acid, 80:10:10:2 (v/v/vjv). Reference compounds lysine, ethanolamine, monomethylethanolamine, dimethylethanolamine, and choline were chromatographed in a similar manner. The mobility of sphingosine is depicted in the upper lane. 14

14

3



GANDER

ET AL.

Glycopeptide

Structural

Studies

5 10 CM FROM ORIGIN

Figure 13. Thin layer chromatography of base-hydrolyzed C4abeled deoxycholate-soluble peptidophosphogalactomannan. The C-labeled peptidophosphoga lactomannan was treated as described in Figure 12. A 50-μΖ, sample was applied to a thin layer of silica gel and chromât ο graphed in CHCl :methanol:H 0, 100:42:6 (v/v/v). The mobility of sphingosine is depicted in the upper lane. 14

14

3

2

200

100

5 10 CM FROM ORIGIN

Figure 14. Thin layer chromatography of base-hydrolyzed C-labeled deoxycholate-soluble peptidophosphogalactomannan. The C-labeled peptidophosphoga lactomannan was treated as described in Figure 12. A 50-/xL sample was applied to a thin layer of silica gel and chromatographed in CHCl :methanol:2N ΝΗβΗ, 80:20:2 (v/v/v). The mobility of sphingosine is depicted in the upper lane. 14

14

3


FUNGAL


*

POLYSACCHARIDES

Ο

2005

ο. ο 100-

_ι

Ο

ι

5

1 10

ΐ-

15

CM FROM ORIGIN

Figure 15. Thin layer chromatography of base-hydrolyzed C-labeled deoxycholate-soluble peptidophosphogalactomannan. The C-hbeled lipo-^peptidophosphogalactomannan was treated as described in Figure 12. A 50-sample was applied to a thin layer of silica gel and chromatographed in CHCl :methanol:H 0, 49:4:2 (v/v/v). The mobility of sphingosine is depicted in the upper hne. 14

14

3

2

Figure 16. Thin layer chromatography of periodate-treated base-hydrolyzed C-labeled lipo-peptidophosphogalactomannan. The C-hbeled lipo-peptidophosphogalactomannan was treated as described in Figure 12. Α 50-μΕ sample was applied to a thin layer of silicic acid and chromatographed in hexane.diethylether, 9:1 (v/v). The C remined at the origin as shown in the cross hatched area. Α 100-μΊ-, sample was treated with periodate according to the procedure of Sweeley and Moscatelli (45), and the sample applied to a thin layer silica gel plate and chromatographd in hexane-.diethylether. Sphingosine remained at the origin, and sphingosine treated with periodate mi grated with an R of 0.61. 14

14

14

;



4.

GANDER E T

AL.

Glycopeptide

Structural

Studies

77

with the amino group of the s p h i n g o s i n e - l i k e p o r t i o n of the molec u l e . No C-labeled f a t t y acids were found. Treatment of the methylene c h l o r i d e - s o l u b l e substance(s) w i t h p e r i o d a t e r e l e a s e d a substance that was only s l i g h t l y more p o l a r than the f a t t y a l d e hyde r e l e a s e d when sphingosine i s t r e a t e d with p e r i o d a t e . P r e l i m i n a r y gas chromâtography-mass spectrometer of the substance s o l u b i l i z e d by treatment with 4 Ν a l k a l i suggests that the long chain base i s dehydrosphingosine. Phytospingosine and dehydrophytosphingosine are the predominant long chain bases present i n f u n g i and p l a n t s . Dihydrosphingosine composes 3% of the s p h i n g o l i p i d bases i n yeast (45). T r i a c y l d i h y d r o s p h i n g o s i n e has been obtained from the growth medium of Hansenula c i f e r r i i (46). We t e n t a t i v e l y conclude that the membrane-bound amphipathic peptidophosphogalactomannan contains one or more l i p o p h i l i c r e s i d u e s attached to the mannan. The amphipathic polymer w i l l be designated lipo-peptidophosphogalactomannan to d i s t i n g u i s h i t from the s o l u b l e peptidophosphogalactomannans. I t was noted that storage of membrane f r a c t i o n V even at -20°C r e s u l t e d i n a decrease and sometimes a complete l o s s i n lipo-peptidophosphogalactomannan. This membrane f r a c t i o n may contain enzymes which convert lipo-peptidophosphogalactomannan to peptidophosphogalactomannan. Summary I n v e s t i g a t i o n s on the s t r u c t u r e of a peptidophosphogalacto mannan of Pénicillium c h a r l e s i i has been reviewed and new work presented. Carbon-13 NMR spectroscopy has been used to confirm e a r l i e r s t r u c t u r a l s t u d i e s . S t r u c t u r a l s t u d i e s on the p o l y p e p t i d e show that the p o l y p e p t i d e i s derived from s e v e r a l species of prot e i n s . We a l s o r e p o r t the occurrence i n P. c h a r l e s i i e x t r a c t s of a s o l u b l e " c y t o p l a s m i c " peptidophosphogalactomannan and a membrane-bound species which because of a hydrophobic region we have termed lipo-peptidophosphogalactomannan. The two new g a l a c t o mannan d e r i v a t i v e s have been p a r t i a l l y c h a r a c t e r i z e d and have been shown to have amino a c i d composition which i s s i m i l a r to that of the e x t r a c e l l u l a r peptidophosphogalactomannan except that the polypeptide p o r t i o n s are considerably g r e a t e r . The hydrophobic region, u n l i k e the galactomannan, d e r i v e s i t s carbons from acet a t e . P r e l i m i n a r y r e s u l t s suggest that the hydrophobic character i s given by a long chain base with p r o p e r t i e s s i m i l a r to sphingos i n e and dehydrosphingosine. T h i s lipo-peptidophosphogalactomannan has been i s o l a t e d and p u r i f i e d by gel-permeation and a f f i n i t y chromatography. Acknowledgement The work described h e r e i n o r i g i n a t i n g i n my l a b o r a t o r y has been supported by the N a t i o n a l Science Foundation (Research Grant GB 21261), by the General M e d i c a l Sciences d i v i s i o n of the



78

FUNGAL

POLYSACCHARIDES

N a t i o n a l I n s t i t u t e s of Health, United States P u b l i c Health Ser v i c e (Research Grants GM 19978 and GM 20441), by the United States Army Research O f f i c e (Research Grant GM DAHCO4-75-6-0179) and the gas chromatography-mass spectrometry l a b o r a t o r y supported by the U n i v e r s i t y of Minnesota A g r i c u l t u r a l Experiment S t a t i o n , S c i e n t i f i c J o u r n a l S e r i e s No. 10,738, A g r i c u l t u r a l Experiment S t a t i o n , U n i v e r s i t y of Minnesota, S t . P a u l , Minn. 55108. We express our a p p r e c i a t i o n to c o l l a b o r a t o r s , most of whom have been acknowledged i n the r e f e r e n c e s , and a l s o to the many who have made i n d i r e c t c o n t r i b u t i o n s to the advances i n under standing the research d e s c r i b e d . The i n v e s t i g a t i o n s of the i s o l a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of lipo-peptidophosphogalactomannan was conducted by Dr. Beachy i n p a r t i a l f u l f i l l m e n t of the requirements f o r the Ph.D. degree and the ^ C-NMR was conducted by C J . Unkefer i n p a r t i a l f u l f i l l m e n t of the requirements f o r the M.S. degree. 3

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Haworth, W. N . ; Raistrick, H . ; Stacey, M. Biochem. J. 1937, 31, 640. Clutterbuck, P . ; Haworth, W. N . ; Raistrick, H . ; Smith, G . ; Stacey, M. Biochem. J . 1934, 28, 94. Haworth, W. N . ; Raistrick, H. K . ; Stacey, M. Biochem. J. 1935, 29, 2668. Gorin, P. A. J.; Spencer, J. F. T. Can. J. Chem. 1959, 37, 499. Hough, L.; Perry, M. B. Chem. Ind. (London) 1956, p. 768. Hough, L.; Perry, M. B. J. Chem. Soc. (London) 1962, p. 2801. Preston, J . F . ; Gander, J. E . Arch. Biochem. Biophys. 1968, 124, 504. Preston, J . F . ; Lapis, E.; Gander, J. E.; Westerhouse, S. Arch. Biochem. Biophys. 1969, 134, 316. Preston, J . F . ; Lapis, E.; Gander, J. E . Arch. Biochem. Biophys. 1969, 134, 324. Gander, J. E.; Jentoft, Ν. H . ; Drewes, L . R.; Rick, P. D. J. B i o l . Chem. 1974, 249, 2063. Rick, P. D.; Drewes, L . R.; Gander, J. E . J. B i o l . Chem. 1974, 249, 2073. Gander, J. E . Exper. Mycol. 1977, 1, 1. Preston, J . F.; Lapis, E.; Gander, J. E . Can. J. Microbiol. 1970, 16, 687. Lloyd, K. O. Biochemistry 1970, 9, 3446. Lloyd, K. O. Biochemistry 1972, 11, 3884. Sakaguchi, O.; Yokota, K . ; Suzuki, M. Yakugaku Zasshi 1967, 87, 1268. Sakaguchi, O.; Yokota, K . ; Suzuki, M. Japan. J. Microbiol. 1969, 13, 1. Azuma, I . ; Kimura, H . ; Hirao, F.; Tsubura, E.; Yamamura, Y . ; Misaki, A. Japan. J. Microbiol. 1971, 15, 237.


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22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

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79

Bulman, R. Α . ; Chittenden, G. F. Biochim. Biophys. Acta 1976, 444, 202. Gander, J. E.; Fang, F. Biochem. Biophys. Res. Commun. 1976, 71, 719. Rietschel-Berst, M . ; Jentoft, N. H.; Rick, P. D . ; Pletcher, C. P . ; Fang, F.; Gander, J. E . J. B i o l . Chem. 1977, 252, 3219. Gander, J. E.; Fang, F. Biochem. Biophys. Res. Commun. 1974, 58, 368. Drewes, L . R.; Rick, P. D.; Gander, J. E . Arch. Microbiol. 1975, 104, 101. Jordan, J. M . ; Gander, J. E . Biochem. J. 1966, 100, 694. Gander, J. E.; Drewes, L . R.; Fang, F.; L u i , A. J . B i o l . Chem. 1977, 252, 2187. Porath, J.; Axen, R.; Ernback, S. Nature 1967, 215, 1491. Mort, Α . ; Lamport, D. Anal. Biochem. 1977, 82, 289. Fischer, W.; Zapf, J. Z. Physiol. Chem. 1964, 337, 186. Dubois, M . ; G i l l e s , Κ. Α . ; Hamilton, J. K . ; Rebers, P. Α . ; Smith, F. Anal. Chem. 1956, 28, 350. Ames, B. N . ; Dubin, D. J. B i o l . Chem. 1960, 235, 769. Parvin, R.; Smith, R. A. Anal. Biochem. 1969, 27, 65. Lowry, O. H . ; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J . B i o l . Chem. 1951, 193, 265. Gros, C.; Labouesse, B. Eur. J . Biochem. 1969, 7, 463. Klatt, K. P . ; Gander, J. E . Can. J. Microbiol. 1968, 14, 579. Staub, A. M . , in "Methods in Carbohydrate Chemistry" (Whistler, R. L.; Wolfrom, M. L . eds.) V o l . V, p. 5; Academic Press: New York, 1965. Gander, J. E . unpublished. Lee, Y.-C.; Ballou, C. E . Biochemistry 1965, 4, 257. Price, V. E.; Greenstein, J. P. Arch. Biochem. 1948, 18, 383. Kessler, G . ; Nickerson, W. J. J. B i o l . Chem. 1959, 234, 2281. O'Connor, J. V . ; Nunez, H. Α . ; Barker, R. Biochemistry 1979, 18, 500. Stewart, T. S.; Mendershausen, P. B . ; Ballou, C. E . Biochem istry 1968, 7, 1843. Stewart, T. S.; Ballou, C. E . Biochemistry 1968, 7, 1855. Barker, S. Α . ; Basarab, O.; Cruickshank, C. N. D. Carbohyd. Res. 1967, 3, 325. Tonn, S. J.; Gander, J. E . unpublished. Sweeley, C. C . ; Moscatelli, E . A. J. Lipid Research 1959, 1, 40. Stodola, F. H . ; Wickerham, L . J.; Scholfield, C. R.; Dutton, H. J. Arch. Biochem. Biophys. 1962, 98, 176. W i l l s , M. Α.; Dittmer, J . C. Biochemistry 1966, 5, 3405.

RECEIVED August 13, 1979.


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