Biosynthesis of mannan and mannolipids from GDP-Man by membrane fractions of sycamore cell cultures

BIOCHIMIE, 1976, 58, 1195-1211.

Biosynthesis of mannan and mannolipids from GDP-Man by membrane fractions of sycamore cell cultures. M a r g a r e t M e r y l SMITH

(*), Mich61e AXELOS a n d C l a u d e P~AuD-LENO~L ~.

Laboratoire de B i o c h i m i e [onctionnelle des plantes, Ddpartement de Biologie, Centre Universitaire de L u m i n g , 13288 Marseille, Cedex 2, France. (21-6-1976). Summary. - - l~Iembrane f r a c t i o n s h a v e been p r e p a r e d f r o m sycamore (Acer pseudoplatanus) cell suspe.nsion.s grown in .liquid medium. These f r a c t i o n s catalyzed the t r a n s f e r of m a n n o s y l - u n i t s f r o m GDP-[14C] Man into a p o l y m a n n o s i d e , a m a n n o l i p i d and oligosaccharid,e-lipid s. The p o l y m a n n o s i d e was p a r t i a l l y solubilized b y proteolytic digestion or m a c e r a t i o n in sodium dodecyl s u l f a t e - u r e a mixtures. However no evidence is a v a i l a b l e of a covalent linkage between the b i o s y n t h e s i z e d glycan a n d peptides. The s t r u c t u r a l a n a l y s i s of the [14C]mannan showed t h a t the polysaceharide was a h o m o p o l y m e r of [~-(1--~'4) linked m a n n o s e w i t h a few branches. During the i n c u b a t i o n of t h e m e m b r a n e s w i t h t h e substrate, the p o l y m e r chains elongated w i t h a large n u m b e r of sugar u n i t s a n d c o n t a i n e d 25 to 40 hexose residues per non-reducing end m o n o m e r . GDP-Gle was a competitive i n h i b i t o r of the GDP-Man : ~ - m a n n a n m a n n o s y l - t r a n s f e r a s e , whereas GDP-Man activated a GDP-Gle : ~-glucan glucosyl-transferase present in the same m e m b r a n e p r e p a r a t i o n . Two k i n d s of glycolipids were synthesized in the presence of GDP-[14CIMan. The first (I) c o n t a i n e d a p o l a r moi.ety characterized as m a n n o s e - p h o s p h a t e a n d was very s i m i l a r to p o l y p r e n y l - p h o s p h a t e - m a n n o s e identified in p l a n t s b y A]am and Hemming. The other m a n n o H p i d ( I I ) w a s hydrolyzed by mild acid into labeled oligosaccharides of h i g h molecular weight. This m a t e r i a l was separated into two oligosaccharide fractions, the first (II A) of M~V over 500,0; t h e second (II B) of MW a r o u n d 1700. I I B c o n t a i n e d at most two labeled m a n n o s e residues per chain, linked to the n o n - r e d u c i n g end of un.labeled u n i t s w h i c h prob a b l y contained n e u t r a l sugars a n d N-acetyl-osamine(s) n e a r the reducing end. 01igosaceharid.e II A seemed to contain one or several I I B chains.

INTRODUCTION.

d e a l t w i t h t h e g l y c o s y l a t i o n of p l a n t p r o t e i n s . In experiments have shown that exogenous D - g l u c o s a m i n e s u p p l i e d to a v a r i e t y of p l a n t tiss u e s w a s i n c o r p o r a t e d i n t o g l y c o p r o t e i n s [9, 10]. T h e r e is s o m e e v i d e n c e f o r t h e g l y c o s y l t r a n s f e r f r o m n u c l e o t i d e - s u g a r s to p r o t e i n s i n t h e p r e s e n c e of p l a n t e n z y m e p r e p a r a t i o n s [11-14]. W i t h t h e e x c e p t i o n of e x t e n s i n [15], t h e n a t u r e of t h e acc e p t o r p r o t e i n is n o t k n o w n .

vivo

I n t h e c o u r s e of s t u d i e s o n s u g a r t r a n s f e r s b y m e m b r a n e f r a c t i o n s of s y c a m o r e (Acer pseudoplatanus L.) ceils, it w a s o b s e r v e d t h a t m a n y s u g a r s were actively transferred from the nucleotides u g a r s u b s t r a t e to m e m b r a n e - b o u n d m a t e r i a l . M a n nose from GDP-E14C]Man was incorporated into membranes from which radioactive polymer(s) and lipidic compounds could be isolated. The biosynthesis of mannans and glucomannans [1, 2, 3] b y p l a n t e n z y m e p r e p a r a t i o n s h a s b e e n described but the biosynthesis of polymannoside by sycamore cell extracts was quite unexpected s i n c e t h e s e c u l t u r e d c e l l s c o n t a i n v e r y little, i f a n y , m a n n o s e i n t h e i r cell w a l l s [4, 5, 61. T h i s p o l y m a n n o s i d e m i g h t also b e a p a r t of t h e p r o s t h e t i c g r o u p of g l y c o p r o t e i n ( s ) : o l i g o m a n n o s i d e s a r e k n o w n to b e p r e s e n t i n s o m e p l a n t g l y c o p r o t e i n s , f o r i n s t a n c e l e c t i n s [7, 81. F e w s t u d i e s h a v e (*) P r e s e n t address : D e p a r t m e n t of Biology, Carleton University, Ottawa, Canada, KIS 5B6. To w h o m all correspondence should be addressed.

T h e b i o s y n t h e t i c p a t h w a y s of t h e p r o s t h e t i c g r o u p Of a n i m a l a n d m i c r o b i a l g l y c o p r o t e i n s a r e now well documented ; in a number of cases, the c a r r i e r r o l e of p o l y i s o p r e n y l - p h o s p h a t e - o l i g o s a c c h a r i d e s h a s b e e n e l u c i d a t e d [16-20]. I t h a s also b e e n s h o w n t h a t t h e s u g a r s of U D P - G l c , U D P G l c N A c o r G D P - M a n c a n b e t r a n s f e r r e d to l i p i d s and produce lipid-oligosaccharides in the pres e n c e of p l a n t e n z y m e p r e p a r a t i o n s [13, 14, 21]. It w a s t h o u g h t of i n t e r e s t to s t u d y t h e n a t u r e a n d t h e s t r u c t u r e of t h e p o l y m a n n o s y l - c o n t a i n i n g compounds synthesized by the membrane fract i o n s of s y c a m o r e cells, b e a r i n g i n m i n d t h a t t h e

M. M. Sm i t h and coll.

1196

glycan nfight be l i n k e d to a protein. It was also u n d e r t a k e n to characterize the lipids w h i c h were labeled after i n c u b a t i o n of the m e m b r a n e fractions w i t h GDP- [14C] Man. MATE:RIALS AN[D METHODS. CHEMICALS AND ENZYMES. GDP-a-o-mannose, d i s o d i u m salt, was o b t a i n e d from Sigma Chem. Co., CsDP-a-D-glucose from Calb i o c h e m a n d GDP-[U14C]Man, specific activity 80 C i / m o l e from R a d i o c h e m i c a l Centre, Amersham. GDP-[U-I~CIGlc, specific activity 44 Ci/ mole was p r e p a r e d as p r e v i o u s l y described [22]. Samples of (2,3,6-), (2,4,6-), (3,4,6-) a n d (2,3,4-) tri-O-methyl-ctmethyl-D-mannosides were generous gifts of Drs B. F o u r n e t a n d J. Montreuil. The date m a n n a n was f r a c t i o n A o b t a i n e d i n the m a n n e r described by Meier [23], from the 14 per cent potassium h y d r o x i d e extract of date nuts. After t o t a l acid h y d r o l y s i s of this m a n n a n a n d p a p e r c h r o m a t o g r a p h i c analysis, essentially m a n n o s e was detected, with at most 5 per cent of other sugars (Glc > GaD. T r y p s i n (E.C. 3.4.2.1.4.) twice crystallized, protease of Streptomgces griseus (Pronase type VI, E.C. 3.2.1.4.), atkaline phosphatase of E. colt (type I,II, E.C. 3.1.3.1.) a n d m a n n a n a s e , a crude cellulase p r e p a r a t i o n from Aspergillus niger, were commercial samples from Sigma Chem. Co.

e q u i p p e d with an integrator. The radioactivities of i n d i v i d u a l samples were m e a s u r e d w i t h a scintillation c o u n t e r ( I n t e r t e c h n i q u e SL40) i n the mixtures : a) P P 0 5 g/l, POPOP 0.2:5 g/1 i n "toluene, b) Instagel (Packard Inst. Co.), c) n a p h t a l e n e 60 g/l, PPO 4 g/l, POPOP 0.2 g/1 in methanolethylene glycol-dioxane (10/2/88) (*). The radioactivities of samples on glass fibre discs or chrom a t o g r a p h i c papers were c o u n t e d in 5 ml of mixture a ; c h r o m a t o g r a p h i c silica samples a n d polya c r y l a m i d e gel slices 'were counted in 1.5 ml of b ; aqueous samples were c o u n t e d i n 1.5 ml of b or in 10 ml of c. The c o u n t i n g efficiencies were calculated with geometrically i d e n t i c a l controls. CHROMATOGRAPHY AND ELECT'ROPHORE~S!IS. D e s c e n d i n g p a p e r c h r o m a t o g r a m s were on W h a t m a n No 1 or 3MM, washed w i t h 10 g/1 oxalic acid, w a t e r a n d ethanol ; p a p e r electrophoreses were r u n on W h a t m a n No 1 or Schleicher a n d Schfill No 2040 p a p e r strips. Solvent a n d buffer system are listed i n table I. After development of chromatograms, r e d u c i n g sugars were detected w i t h b e n z i d i n e reagent [241 and r e d u c e d products of periodate o x i d a t i o n by alkaline silver nitrate [24]. F o r detection of methyl-sugars and glycitols c h r o i n a t o g r a p h e d i n solvents c o n t a i n i n g b o r i c acid, m e t a p e r i o d a t e a n d b e n z i d i n e sprays were used [24] ; preferably, 1 p e r cent s o d i u m metaperiodate in e t h a n o l - w a t e r ( 1 / I ) was sprayed, the c h r o m a t o g r a m s w e r e kept h u m i d for 15 rain, then dried a n d s p r a y e d w i t h 1 per cent t r i p h e n y l t e t r a -

TABLE I. Nr I

II III IV v vI vii viii IX x xI xII xIII xIv

Solvent mixture or buffer Pyridine-ethyl acetate-water id. 1-propanol-ethyl a c e t a t e - w a t e r id. Butanone-acetic add-water id. 1-butanol-acetic acid-water EthanoM M ammonium acetate id. Chloroform-methanol-water Chloroform-methanol-acetic acid-water Aeetone-heptane 100 mM ammonium acetate, 10 mM EDTA, pH 5.5 2 N acetic acid

RADIOACTIVE MEASUREMENTS.

Radioactive traces a n d relative measures of radioactivities o11 tile c h r o m a t o g r a m s were obtained w i t h a 4 ~ s c a n n e r (Tracerlab I n s t r u m e n t Co.),

BIOCHIMIE, 1976, 58, n ° 11).

Volume ratio 1/2/2 21517 71112 61113 91111 91112 41112 7/3 65135 65/25/4

Observations Upper phase id. Saturated with boric acid to. pH 3.8 id.

251151412 1/1

zolium chloride i n 1 N NaOH-methanol (1/1) : red spots a p p e a r e d b y h e a t i n g 2 rain at 110°C. (*) In tables, figures and text, all solvent mixtures intended by vol,umes.

Mannan

and mannolipid

P L A N T MATERIAL.

The s y c a m o r e cell line o r i g i n a t e d from Lainport's culture [25]. Cells ~ e r e g r o w n i n l i q u i d susp e n s i o n as described by Lescure [26]. 450 m l of early s t a t i o n a r y phase m o t h e r culture were transferred into 6 1 flasks c o n t a i n i n g 1.2 1 of m e d i u m . After a weekly i n c u b a t i o n (2 to 3 cell generations), cells were collected a n d r a p i d l y washed w i t h 3 1 w a t e r at 2°C on a s i n t e r e d glass filter (120 g fresh, 10 g d r y material). The cells were used i m m e d i a tely or frozen i n l i q u i d n i t r o g e n a n d stored u n d e r n i t r o g e n at - - 2 5 ° C . MEMBRANE FRACTIONS.

100 g of cells (fresh) were crushed w i t h m o r t a r a n d pestle i n l i q u i d nitrogen. All the s u b s e q u e n t operations were p e r f o r m e d at 2°-4°C. The homogenate was s u s p e n d e d i n 225 ml of the m i x t u r e : 10 mM Tris-C1, pH 7.5, 5 mM 2-mercaptoethanol (TBIS-ME buffer), c o n t a i n i n g 7.5 g/l p o l y v i n y l p y r r o t i d o n e (MW 40,000), 5 HaM EDTA a n d 240 g/1 sucrose. The s u s p e n s i o n was squeezed t h r o u g h two layers of gauze ; the residue was r i n s e d with 75 m l of the same m i x t u r e but c o n t a i n i n g 180 g / l sucrose a n d was filtered again. The pooled filtrates were freed from cell debris by c e n t r i f u g a t i o n 5 rain at 2,500 × g a n d the s u p e r n a t a n t was centrifuged 30 rain at 143,000 × g (35,000 rpm, rotor 35 Beckman). The pellet was h o m o g e n i z e d i n 1.2 ml of TRIS-ME buffer c o n t a i n i n g 180 g/1 sucrose w i t h a Potter-Elvejhem h o m o g e n i z e r a n d a Teflon plunger, deposited at the top of l a y e r e d 250 a n d 450 g/1 sucrose solutions i n TRIS-ME a n d centrifuged at 113,000 × g (25,000 rpm, rotor SW27 Beckman) for 2 h. The selected active fraction b a n d e d at the interface of sucrose layers w h e r e the d e n s i t y was a r o u n d 1.13. This fraction m a y be referred to as m i c r o s o m e s i n the sense given b y Fleischer a n d K e r v i n a [27J. The p r o t e i n content of the m i c r o s o m a l fractions was m e a s u r e d a c c o r d i n g to Itzhaki a n d Gill [28] after p r e c i p i t a t i o n by 10 per cent TCA a n d delip i d a t i o n b y acetone washing. Alternatively, protein was m e a s u r e d by the m e t h o d of Lees a n d P a x m a n [29] adapted to automatic titration. F o r both methods, the control was b o v i n e s e r u m albumin. I N C U B A T I O N C O N D I T I O N S AND R E C O V E R Y OF I~ADIOACTIVE P R O D U C T S .

S t a n d a r d samples were as follows : conical vials c o n t a i n e d an aliquot of the microsome susp e n s i o n (3 to 12 mg p r o t e i n / m I ) i n the m i x t u r e : TRIS-ME) 350 g/1 sucrose, 20 mM MgC12 a n d the BIOCHIMIE, 1976, 58, n o 10.

1197

biosynthesis.

substrate 30 ~M G,DP-[U-I~C]Man i n a total volume 120 ~l. After i n c u b a t i o n 30 m i n at 18°C, samples were f r a c t i o n a t e d a c c o r d i n g to m e t h o d A : the m i x t u r e was t r a n s f e r r e d onto a GF/B W h a t m a n glass fibre disc on a s i n t e r e d glass filter. The l i q u i d phase was w a s h e d off with s u c t i o n b y 5 × 1 m l of TRIS-ME and 2 × 1 ml water. The disc was then washed by 3 × 1 ml ethanol-water (7/3) a n d 5 × 1 ml c h l o r o f o r m - m e t h a n o l (2/1). The dried disc was m o i s t e n e d with 100 mM TrisC1 buffer, pH 7.5, c o n t a i n i n g 1 mg t r y p s i n a n d incubated 25 m i n at 25°C. The disc was then washed w i t h water, ethanol and c h l o r o f o r m - m e t h a n o l (2/1). The r a d i o a c t i v e p o l y m e r r e m a i n i n g on the disc was used as described i n the e x p e r i m e n t a l part. A 5 per cent TCA t r e a t m e n t p r i o r to t r y p s i n digestion did not change the results. W h e n larger scale i n c u b a t i o n m i x t u r e s were used, they were i n a c t i v a t e d i n a 100°C water bath for 3 rain a n d stored at - - 2 5 ° C u n t i l f r a c t i o n a t i o n b y m e t h o d B : 10 volumes of w a t e r were added to the i n a c t i v a t e d s u s p e n s i o n and the i n s o l u b l e material was s e d i m e n t e d i n a conical tube 3 rain at 1,500 × g. The pellet was w a s h e d stepwise with 3 i n i t i a l volumes of water, 6 vol. of ethanol-water (7/3), twice w i t h 6 vol. chloroform-Inethanol (2/1), 6 vol. c h l o r o f o r m - m e t h a n o l - w a t e r (10/10/3) a n d 6 vol. ethanol-water (7/3). The pellet c o n t a i n i n g the radioactive p o l y n i e r ~'as kept at - - 2 5 ° C i n ethanol. METHODS

FOR

CHEMICAL ANALYSIS' OF

MANNANS.

H y d r o l y s i s . - - The date m a n n a n and the [14C] p o l y m e r w e r e submitted to total acid h y d r o l y s i s by a h y d r o c h l o r i c acid solution saturated w i t h HC1 gas at - - 1 6 ° C , in sealed vials for 24 h at 25°C. Partial h y d r o l y s e s were p e r f o r m e d in the same way but the h y d r o c h l o r i c solution was saturated at 0°C a n d the r e a c t i o n time r e d u c e d to 1.5 h. After h y d r o l y s i s , acid was r e m o v e d b y lyop h i l i s a t i o n . The radioactive p o l y m e r was hydrolyzed directly on the glass fibre disc a n d the solubilized m a t e r i a l was w a s h e d off w i t h w a t e r after the h y d r o l y s i s step. P e r i o d a t e o x i d a t i o n . - - Date m a n n a n was dissolved i n 2 N s o d i u m h y d r o x i d e u n d e r n i t r o g e n a n d the pH adjusted to 5.1 w i t h acetic acid. Sodium periodate was then added to a final conc e n t r a t i o n 20 mM. To m o n i t o r potential overoxidation, periodate c o n s u m e d was m e a s u r e d d u r i n g the oxidation p e r i o d by the thiosulfate method, versus a m a n n a n - f r e e control. F r o m the periodate oxidized material, the derived polyalcohol was p r e p a r e d a c c o r d i n g to Goldstein et al. [30], h y d r o -

1198

M. M. S m i t h a n d coll.

lyzed i n 0.5 N h y d r o c h l o r i c acid, 29 h at 25°C, deionized on ion exchange resins a n d d r i e d u n d e r v a c u u m for c h r o m a t o g r a p h i c analysis. Total hydrolysis of the polyalcohol was p e r f o r m e d i n 2 N h y d r o c h l o r i c acid, 2 h at 100°C. The h y d r o l y s a t e was n e u t r a l i z e d a n d the glycitol acetates were p r e p a r e d b y b o r o h y d r i d e red u c t i o n and acetylation [.31]. Glycitol acetates were f r a c t i o n a t e d by GLC i n a stainless steel c o l u m n (1.8 m × 3 mm), filled w i t h 1.3 per cent EGS, 1.7 per cent silicone GE XF 1150 on GasChrom Q 100-2c00 mesh (Applied Science Inc.), with a l i n e a r t e m p e r a t u r e g r a d i e n t 150-190°C (2°C/rain). The vector gas was N 2 (2,0 m l / m i n ) ; the peaks were detected b y a flame i o n i z a t i o n detector at 220°C a n d the i n j e c t i o n t e m p e r a t u r e was 210°C. Periodate oxidation of the [14C]mannose polym e r was p e r f o r m e d as above w i t h a sufficient a m o u n t of date m a n n a n a d d e d as carrier. The oxidized m a t e r i a l was r e d u c e d b y s o d i u m boroh y d r i d e , p a r t i a l l y or totally h y d r o l y z e d a n d analyzed b y TLC or p a p e r c h r o m a t o g r a p h y . Methylation. - - Date m a n n a n was m e t h y l a t e d twice by dimethylsulfate a n d solid s o d i u m hydroxide in a n h y d r o u s t e t r a h y d r o f u r a n [32]. An aliquot of the p r o d u c t was then p e r m e t h y l a t e d twice by the m e t h o d of Hakomori, a c c o r d i n g to L i n d b e r g [33]. After each m e t h y l a t i o n step, the r e a c t i o n m i x t u r e was diluted w i t h water, extensively dialyzed and dried u n d e r vacuum. At the end, the i n f r a - r e d s p e c t r u m of the p e r m e t h y l a t e d m a n n a n showed no h y d r o x y l a b s o r p t i o n band. The c o m p o u n d was t h e n m e t h a n o l y z e d b y 7 per cent (weight/volume) HC1 in m e t h a n o l , in a sealed viai, 40 h at 100°C. The acid was r e m o v e d w i t h silver carbonate a n d the solution was c o n c e n t r a ted for GDC [.34]. The analysis was p e r f o r m e d i n a stainless steel c o l u m n (3 m × 3 ram) loaded w i t h Chromosorb W I~MDS 60-80 mesh, c o n t a i n ing 3 p e r cent C a r b o w a x 6 000 (Appl. Sc.). The vector gas was H 2 (45 m l / m i n ) ; the peaks were detected by c a t h a r o m e t r y w i t h the t e m p e r a t u r e p r o g r a m of figure 4 ; i n j e c t o r a n d detector were at 210°C. The [~4C]polymer, to w h i c h date m a n n a n was a d d e d as carrier, ~vas m e t h y l a t e d a n d p e r m e t h y lated on its glass fibre support. The m a t e r i a l was m e t h a n o l y z e d as above. The m o n o m e r s were analyzed by TLC a n d GLC, as described i n text.

w a t e r (l/l), at 50°C for 20 rain. F o r TLC analysis, the r e a c t i o n m i x t u r e s were deposited a n d dried directly on silica plates. Alkaline h y d r o l y s i s was p e r f o r m e d i n 1 ml of 0.3 N s o d i u m h y d r o x i d e i n 1-propanol-water (15/20), 2 h at 64°C. Dowex 50 (H +) was then a d d e d to remove Na ÷ ions. The n e u t r a l i z e d solution was centrifuged and the resin pellet was washed with water and chloroform-methanol (2/1). The w a s h i n g s were m i x e d w i t h the i n i t i a l s u p e r n a t a n t a n d the m i x t u r e was shaken vigorously. The aqueous a n d o r g a n i c phases were then separated b y c e n t r i f u g a t i o n a n d the aqueous phase, c o n c e n t r a t e d u n d e r vacuum, was analyzed b y p a p e r electrophoresis. H y d r a z i n o l y s i s of the b o r o h y d r i d e r e d u c e d oligosaccharides derived from l i p i d l I was performed in 200 ~1 of a n h y d r o u s h y d r a z i n e , 40 h at 100°C, i n sealed tubes, u n d e r n i t r o g e n [35]. H y d r a z i n e was r e m o v e d b y e v a p o r a t i o n u n d e r v a c u u m a n d the samples analyzed by p a p e r electrophoresis. Methanolysis of r e d u c e d di- a n d t r i s a c c h a r i d e s derived from l i p i d II was p e r f o r m e d as described for the m e t h y l a t e d [14C]mannan. POLYACRYLAMIDE GEL E L E C T R O P H O R E S I S

OF

THE

RADIOACTIVE POLYMER.

Gels were p r e p a r e d a c c o r d i n g to L a e m m l i [36], i n tubes (6 m m × 100 ram). The s e p a r a t i n g gels (7.5 per cent a c r y l a m i d e a n d 0.2 per cent N,N'm e t h y l e n e - b i s - a c r y l a m i d e ) were p o l y m e r i z e d i n 375 mM Tris-C1 buffer, pH 8.8. The stacking gels (3 per cent a c r y l a m i d e a n d 0.08 p e r cent bis-acrylamide were in 12,5 mM Tris-C1 buffer, p H 6.8. Both gels c o n t a i n e d 0.1 per cent SDS (*) a n d 0.5 M urea. A c r y l a m i d e and b i s - a c r y l a m i d e were recrystallized before use. Electrode buffer was 25 mM Tris, 19.2 mM glycine, 0.1 per cent SDS, pH 8.3. Samples were solubilized in 60 mM Tris-C1 buffer, pH 6.8, 1 p e r cent SDS, 143 mM 2-nlercaptoethanol, 6 M urea, 2 per cent sucrose and 0.01 p e r cent b r o m o p h e n o l blue. After electrophoresis, 2 to 3 h at 6 mA per gel, one of two d u p l i c a t e d samples was stained w i t h R 250 Coomassie blue a n d the other was sliced i n 2 m m sections for radioactivity measurements. RESULTS. F E A C T I O N A T I O N OF T H E INCUBATED MICROSOMES.

M E T H O D S FOR ANALYS~S OF M A N N O L I P I D S .

The n l a n n o l i p i d s were s u b m i t t e d t o , m i l d acid h y d r o l y s i s i n 50 rd of 0.1 N HC1 i n 1-propanolBIOCHIMIE, 19'76, 58, n ° 10..

A n l i c r o s o m e p r e p a r a t i o n c o n t a i n i n g 284 ~g p r o t e i n was i n c u b a t e d i n the s t a n d a r d conditions, (*) Abbreviation : SDS : sodium dodecyl sulfate.

Mannan and mannolipid biosynthesis.

1199

TABLE II.

Fractionation o[ the radioactive incttbation m i x t u r e s (*). Radioaetivities (dpm} Fractionation steps

W a t e r extraction . . . . . . . . . . . . . . . . . . . . Organic solvents extraction . . . . . . . . . . . i Trypsin digestion . . . . . . . . . . . . . . . . . . . . Pronase digestion . . . . . . . . . . . . . . . . . . . . 0.1 N sodium hydroxide extraction . . . . Addition of 4 vol. ethanol to 0.1 N NaOH-soluble fraction . . . . . . . . . . . . .

in the soluble frdction

in the insoluble fraction

604,700 1,850 580 730 6,350

27,350 26,470 25,660 24,930 18,260

200

5,525

(*) The i n c u b a t e d microsolnes were deposited on a glass fibre disc. The insoluble f r a c t i o n r e t a i n e d on the disc a f t e r each step was s u b m i t t e d to the next fractionation, except t h e last for w h i c h the soluble m a t e r i a l was used.

as d e s c r i b e d i n m e t h o d s , w i t h G D P - [ 1 4 C ] M a n (650,000 d p m ) . A f t e r i n c u b a t i o n , t h e r a d i o a c t i v e m i x t u r e , d e p o s i t e d o n a glass f i b r e d i s c w a s f r a c t i o n a t e d s t e p w i s e a c c o r d i n g to m e t h o d A : t h e r e s u l t s a r e l i s t e d i n t a b l e II. T h e f i r s t g r o u p of p r o d u c t s , s o l u b l e i n w a t e r , contained unreacted GDP-Man, mannose-phosp h a t e ( s ) a n d f r e e m a n n o s e , as i n d i c a t e d b y T L C o n c e l l u l o s e i n s o l v e n t IX ; i t w a s n o t f u r t h e r a n a lyzed. The second group was extracted from the r e s i d u e b y e t h a n o l a n d c h l o r o f o r m e - m e t h a n o l : it will be further described in the last paragraphs. A polymeric fraction remained on fhe disc: its radioactivity was contained in mannosyl residues, as s h o w n i n t h e f o l l o w i n g p a g e s . T h e s t e p w i s e p r o t e o l y t i c t r e a t m e n t of t h e p o l y m e r w i t h t r y p s i n and pronase solubilized little radioactivity. After t h e s e t r e a t m e n t s , t h e d i s c w a s s o a k e d 24 h i n 0.1 s o d i u m h y d r o x i d e , at 26°C u n d e r n i t r o g e n , t h e n w a s h e d w i t h w a t e r . T h e r a d i o a c t i v i t i e s of t h e r e s i d u e o n t h e d i s c a n d of t h e p o o l e d a l k a l i n e extracts and washings were measured. The cond i t i o n s of t h i s a l k a l i n e t r e a t m e n t w e r e c h o s e n f o r B - e l i m i n a t i o n of a n y O - I n a n n o s y l - h y d r o x y a l n i n o a c y l r e s i d u e . 25 p e r c e n t of t h e r a d i o a c t i v i t y o n t h e d i s c w a s f o u n d to b e s o l u b i l i z e d b y t h i s t r e a t m e n t . F i v e m g of d a t e m a n n a n , d i s s o l v e d i n 1 N s o d i u m h y d r o x i d e , w e r e a d d e d to t h e a l k a l i n e extract obtained in the experiment described by t a b l e II. T h e m i x t u r e w a s n e u t r a l i z e d b y a c e t i c a c i d a n d t h e p o l y m e r s p r e c i p i t a t e d b y a d d i t i o n of 4 vol. e t h a n o l . A l m o s t all t h e r a d i o a c t i v i t y w a s recovered in the precipitate, showing that the radioactive material soluble in sodium hydroxide did not contain short chain oligosaccharides. The p r o p e r t i e s of t h e s o d i u m h y d r o x i d e s o l u b l e o r

BIOCHIMIE, 19.76, 58, n ° 10.

insoluble polymeric fractions correspond of e - l i n k e d m a n n a n s .

to t h o s e

TABLE I I I .

Solubilization of the radioactive p o l y m e r s by SDS and urea. Radioactivity Fraction dpm Deposited on glass fibre disc . . . . . 32,800 Solubilized in 30 rain by 1 per cent SDS-buffer (*) . . . . . . . . . . . . . . . I 310 Solubilized in 30 rain by 6 M urea- , 1 per cent SDS-buffer(*) . . . . . 13,600 Additional 13 h t r e a t m e n t by the same solvent . . . . . . . . . . . . . . . 1,100 Recovered on filter . . . . . . . . . . . . . 17,800

per cent 100 1

41.5 3.4 54

(*) Buffer : 60 mM Tris-C1, 142 mM m e r c a p t o e t h a n o l , pH 6.8.

ASSOCIATION OF [14~]POLYMER WITH PROTEINS.

I n t h e a b o v e e x p e r i m e n t , it w a s o b s e r v e d t h a t the radioactive polymer was partially soluble in mild alkali. This observation raised the question of a l i n k a g e b e t w e e n t h e p o l y m a n n o s i d e a n d a peptide chain. To investigate this problem, a mic r o s o m e p r e p a r a t i o n (2.3 m g p r o t e i n ) w a s i n c u b a t e d ~vith G D P - [ 1 4 C ] M a n (2,200,000 d p m ) i n a t o t a l vol. 400 ~l, u n d e r t h e s t a n d a r d c o n d i t i o n s . T h e s u s p e n s i o n w a s t h e n f r a c t i o n a t e d b y method B. A f t e r d e l i p i d a t i o n , t h e i n s o l u b l e m a t e r i a l c o n t a i n e d 26.2,000 d p m . A n a l i q u o t w a s d e p o s i t e d o n a g l a s s f i b r e d i s c a n d s o a k e d i n e a c h of t h e d i s s o l v i n g n l i x t u r e s s p e c i f i e d b y t a b l e III.

1200

M. M. Smith

Almost no r a d i o a c t i v i t y w a s e x t r a c t e d by buffer e d SDS, w h e r e a s 6 M urea plus SDS w a s effective : 45 p e r cent of the r a d i o a c t i v e p o l y m e r w a s dissolved. An a t t e m p t to f r a e t i o n a t e the soluble m a t e r i a l on a S e p h a d e x G 150 c o l u m n w a s unsuccessful : 96 p e r cent of the r a d i o a c t i v i t y deposited at the top of the c o l u m n r e m a i n e d strongly a d s o r b e d on the gel and could not be eluted by the urea-SDS b u f f e r ; 4 p e r cent w e r e found in the e x c l u s i o n volume.

v~ i

4 ¢o

'9 × 3 E D_ 2 >

._o

o,

and coll.

thesis, such peptides should m i g r a t e by electrophoresis. Aliquots of the r a d i o a c t i v e p o l y m e r s u s p e n d e d in 6 M urea-1 p e r cent SDS b u f f e r e d solution w e r e l a y e r e d on stacking gels for p o l y a c r y l a m i d e elect r o p h o r e s i s , as d e s c r i b e d in methods. E a c h l a y e r c o n t a i n e d an average of 100 ~g protein. After e l e c t r o p h o r e s i s , s t a i n i n g of the gels w i t h Cooniassie blue e x h i b i t e d a r e p r o d u c i b l e series of peptide b a n d s but none was r a d i o a c t i v e . T h e radioa c t i v i t y was r e c o v e r e d at the top of the stacking gels. Since the S'D,S-peptide b i n d i n g m i g h t be i m p o s s i b l e on a c c o u n t of steric h i n d r a n c e due to p o l y s a c c h a r i d e chains, the [14C]polymer was partially h y d r o l y z e d by m a n n a n a s e of A s p e r g i l l u s n i g e r , as d e s c r i b e d in the next p a r a g r a p h : no r a d i o a c t i v e g l y c o p e p t i d e was detected by electrophoresis. A p r e t r e a t m e n t of the p o l y m e r w i t h pronase o b l i t e r a t e d all the p e p t i d e bands o b s e r v e d on the gels but t h e beh.aviour of the r a d i o a c t i v e material w a s not m o d i f i e d . These results suggest that the [14C]polymannoside is strongly a d s o r b e d but not c o v a l e n t l y linked to m e m b r a n e proteins.

1

-O O iv,

0

#

0

10

~'""i':i,,,m:~ . . . . . . . . . . . . . . . . . . . . . . 20 30 40

Tube number FIG. 1. - - Fractionation on a Biogel PIO column of the [l~C]mannosc polymer. Column : 0.5 cm × 17.5 cm ; [14C] polymer 5.0 i!~1containing 17,z~0,0dpm ; t00 p,1 fractions eluted by 60 mM Tris-C1, 142 mM mercaptoethano1, 1 per cent SDS, 6M ure.a, pH 6.8 buffer ; Ve : exclusion volume, determined bv dextran 200.0 blue elution ; circles : native [14C]i)olylner ; squares : [14C]polymer kept 15 h at 22°C ; triangles [14G]polymcr treated by 500 ~!xgpronase in 5'0 !~tl of the sam.e buffer, 16 h at 30°C.

An aliquot of the r a d i o a c t i v e p o l y m e r was then s u s p e n d e d in urea-SDS buffer a n d l a y e r e d on the top of a Biogel P 10 c o l u n m (fig. 1). The c o l m n n w a s w a s h e d w i t h the urea-SDS b u f f e r : 52 p e r cent of the r a d i o a c t i v i t y was eluted in the exclusion v o l u m e and 37 p e r cent r e m a i n e d at the top. H o w e v e r , the urea-SDS soluble m a t e r i a l was f o u n d to p r e c i p i t a t e on storage, w h e n kept in this buffer for 15 h. W h e n the p o l y m e r was digested w i t h p r o n a s e p r i o r to gel c h r o m a t o g r a p h y , no r a d i o a c t i v e m a t e r i a l of l o w m o l e c u l a r w e i g h t was o b s e r v e d on the elution p r o f i l e (fig. 1). The p a r t i a l solubility of the native p o l y m e r in urea-SDS m a y m e a n e i t h e r that the m a n n a n w a s s t r o n g l y adsorbed on p r o t e i n s or that the p o l y s a e c h a r i d e was c o v a l e n t l y l i n k e d to some protein(s) and that the digestion by p r o n a s e l i b e r a t e d high m o l e c u l a r w e i g h t glycopeptides. A c c o r d i n g to the last h y p o BIOCH1MIE, 1976, 58, n ° 1O.

HYDROLYSIS ANALYSIS

OF OF

THE

RADIOACTIVE

POLYMER

AND

OLIGOSACCHARIDES.

The [ l ~ C j p o l y m e r collected on a glass fibre disc from a s t a n d a r d i n c u b a t i o n m i x t u r e was p u r i f i e d by m e t h o d A and totally h y d r o l y z e d by h y d r o c h l o r i c a c i d . W h a t l n a n 3,M~M p a p e r c h r o m a t o g r a p h y of the p r o d u c t s in solvent I s h o w e d only one r a d i o a c t i v e spot w h i c h c o - c h r o m a t o g r a p h e d w i t h m a n n o s e (Rot e = 1.1). The analysis of the products of p a r t i a l e n z y m a t i c and acid h y d r o l y s i s , as w e l l as the analysis of the m e t h y l a t e d d e r i v a t i v e s w i l l f u r t h e r d e m o n s t r a t e that the only radioactive m o n o m e r was mannose. P a r t i a l acid h y d r o l y s i s of a n o t h e r s a m p l e of the [ l ¢ C j p o l y m e r (56,000 dpm), f o l l o w e d by p a p e r c h r o m a t o g r a p h y of the h y d r o l y z e d m i x t u r e revealed a series of r a d i o a c t i v e spots w h i c h co-chrom a t o g r a p h e d w i t h the series of o l i g o s a c c h a r i d e s o b t a i n e d f r o m a p a r a l l e l p a r t i a l h y d r o l y s i s of date m a n n a n (fig. 2A). A c o m m e r c i a l h y d r o l a s e m i x t u r e of A. n i g e r w a s f o u n d to be able to h y d r o l y s e date m a n n a n . W h e n this n t a n n a n was used as a substrate, oligom a n n o s i d e s ~,ere r e c o v e r e d as t r a n s i e n t p r o d u c t s and nlannose was the only ultimate p r o d u c t . T h e r e f o r e this m i x t u r e contains both ~-mannan h y d r o l a s e and ,~-mannosidase activities. The radio~ctive p o l y m e r s y n t h e s i z e d and p u r i f i e d as pre-

M a n n a n and m a n n o l i p i d biosynthesis. viously d e s c r i b e d (67,000 dpm), was s u s p e n d e d in 10 mM acetate buffer, pH 4.5, c o n t a i n i n g 100 t~g of A. niger h y d r o l a s e . After i n c u b a t i o n 30 rain at 45°C, o l i g o s a c c h a r i d e s w e r e s e p a r a t e d by extraction w i t h e t h a n o l - w a t e r (8/2). T h e ethanol-'water

A

~

0

M4

M3

.4

"¢'a3

10 Distance

Ga[ Olc

M2

~M2

Gal

--

GI¢

--

- - M--

.a

20 from origin (cm)

30

Mcm

FIG. 2. - - Tracings of radioactivity on paper chromatograms of partial acid hydrolysis (A) and enzymatic hydrolysis (B) products of [l$C]mannan. A : chroma-

tography on Whatman 3MM paper, solvent I, development 15.5 h ; B : chromatography on Whatman No 1 paper, solvent I, development 15 h ; Man, M~, Ms, M~ : positions of the products of hydrolysis of the carrier date mannan ; Gal, Glc : control spots.

1201

r a d i o a c t i v e m a t e r i a l submitted to the e n z y m a t i c digestion was still ethanol-insoluble. F o r c o m p l e t e splitting of the p o l y m e r into e t h a n o l - w a t e r (8/2) soluble products, the r a d i o a c t i v e p o l y m e r should be dissolved in s o d i m n h y d r o x i d e and n e u t r a l i z e d before the e n z y m a t i c attack (fig. 6). In o r d e r to d e t e r m i n e w h e t h e r only one or several nlannose units w e r e t r a n s f e r r e d p e r p o l y m e r chain, the r a d i o a c t i v e d i s a c c h a r i d e and t r i s a c c h a r i d e areas w e r e eluted f r o m the c h r o m a t o g r a m s of figures 2A and 2B. The d i s a c c h a r i d e was reduced by s o d i u m b o r o h y d r i d e [30] and m e t h a n o l y zed as d e s c r i b e d in m e t h o d s for the i n e t h y l a t e d m a n n a n s . The T L C on cellulose in solvent 171 of the p r o d u c t s s h o w e d that the m e t h y l - m a n n o s i d e and the m a n n i t o l spots w e r e almost equally labeled (table IV). If only one or a few [14CJmannose units had been t r a n s f e r r e d to the p r i m e r p o l y m e r , one w o u l d expect a s e l e c l i v e labeling e i t h e r of the m e t h y l - m a n n o s i d e if tile m a n n o s e t r a n s f e r takes place at the n o n - r e d u c i n g end or a l a b e l i n g of the m a n n i t o l if the t r a n s f e r takes place at the reducing end of the p r i m e r . In a p r e l i m i n a r y e x p e r i m e n t , w e f o u n d that the

A. niger h y d r o l a s e s p e c i f i c a l l y splitted m a n n o s y l insoluble f r a c t i o n was s u b m i t t e d t w i c e m o r e to the same e n z y m e treatment. The e t h a n o l i c extracts w e r e pooled, c o n c e n t r a t e d u n d e r r e d u c e d pressure and c h r o m a t o g r a p h e d on paper. F i g u r e 2B shows that a series of r a d i o a c t i v e spots co-chrom a t o g r a p h e d w i t h the p r o d u c t s of e n z y m a t i c hydrolysis of date m a n n a n . Mannose was the only d e t e c t e d r a d i o a c t i v e m o n o s a c c h a r i d e ; no h e t e r o d i s a c c h a r i d e a p p e a r e d on this c h r o l n a t o g r a m or on a n o t h e r one r u n in solvent 111. This i n d i c a t e d

m a n n o s y l linkages w h e r e a s a m a n n o s y l - m a n n i t o l linkage was left u n b r o k e n . T h e r e f o r e , the radioactive t r i s a c c h a r i d e , r e d u c e d by b o r o h y d r i d e was s u b m i t t e d to a digestion by this e n z y m e d u r i n g 48 h. R a d i o a c t i v e mannose, m a n n o s y l - n l a n n i t o l and a small a m o u n t of m a n n i t o l w e r e r e c o v e r e d on t h e c h r o m a t o g r a m (table IV). T h e r a n d o m labeling of the d i s a c c h a r i d e and of the t r i s a c c h a r i d e i n d i c a t e that the c h a i n of the o r i g i n a l FHC] p o l y m e r w a s long and r a n d o m l y labeled.

TABLE IV. Analysis of rednced di- and trisaccharides. Compounds analyzed

Treatment

Residues released

Radioactivity (dpm)

Disaccharide

Methanolysis

Mannitol Methyl-mannoside

2,180 1,950

Trisaccharide

Enzymatic hydrolysis

Mannitol

320 1,920 2,510

that the s y c a m o r e cell m i c r o s o m e s h a v e little or no GDP~Man : GDP-Glc e p i m e r a s e activity, s u c h as d e s c r i b e d for Phaseolus m e m b r a n e f r a c t i o n s [37]. In this p a r t i a l e n z y m a t i c h y d r o l y s i s , v e r y little r a d i o a c t i v i t y was f o u n d in o l i g o s a c c h a r i d e s of DP greater t h a n 3 although 50 p e r cent of the

BIOCHIMIE, 1976, 58, n ° 10.

Mannose Mannosyl-mannitol

PERIODATE THE

OXIDATION

RADIOACTIVE

OF

DATE

MANNAN

AND

OF

POLYMER.

To c h a r a c t e r i z e the linkage of the m a n n o s y l residues, the p o l y m e r s w e r e s u b m i t t e d to a controlled d e g r a d a t i o n by p e r i o d a t e oxidation, b o r o h y -

M. M. S m i t h a n d coll.

1202

dride r e d u c t i o n of the p o l y a l d e h y d e s a n d m i l d acid h y d r o l y s i s a c c o r d i n g to the Smith m e t h o d [30]. TABLE V.

Analysis of acetylated m o n o m e r s p r e p a r e d front periodic oxidation products of date m a n n a n . Relative ]molar ratio (" retention__time per cent

Derivative

Glycerol triacetate . . . . . . . Erythritol tetraacetate , . . C (unknown) . . . . . . . . . . . . . Mannitol h e x a a c e t a t e . . . . .

1.0 3.24 3.57 10.9

/ I

10.2 73.4 2.1 14.3

(*) Calculated from the integrated peak areas, corrected for the detector efficiency.

Date m a n n a n was oxidized by periodate as a c a r r i e r control for the study of the r a d i o a c t i v e polymer. After 2,63 h, 1.094 mole p e r i o d a t e per mole of a n h y d r o h e x o s e h a d been reduced. The oxidized p o l y s a c c h a r i d e was r e d u c e d by s o d i u m b o r o h y d r i d e ; b y mild acid h y d r o l y s i s of the polyalcohol, only glycerol, e r y t h r i t o l a n d m a n n o s y l e r y t h r i t o l were detected. The polyalcohol was then totally h y d r o l y z e d by acid ; the alditol acetates were p r e p a r e d a n d analyzed b y GLC, as described in methods. As s h o w n by table V, the presence of a m a j o r peak of e r y t h r i t o l tetraacetate s u b s t a n t i a t e d the (1 --> 4) linkage of the polys a c c h a r i d e p r e p a r e d from date nuts [23]. The size of the glycerol triacetate peak i n d i c a t e d 10 per

cent m a n n o s y l residues p r e s e n t as n o n - r e d u c i n g t e r m i n a l m o n o m e r s w h e r e a s the presence of m a n nitol hexaacetate m a y be due to b r a n c h i n g points or (1 - ~ 3) linkages i n the chains. The [14C]polymer was o b t a i n e d in the convent i o n a l w a y (microsomes 2.8 mg protein, GDP[14C]Man 1,925,000 dpm, total vol. 350 ,.1) a n d was p u r i f i e d by m e t h o d B. The p o l y m e r was digested b y pronase, 1 h at 37°C (600 ~g protease i n 1 ml 10 mM Tris-C1 buffer, pH 7.5). After this 'time, 600 lxg protease were again added and the digest i o n extended b y 30 nfin. The digest was centrifuged at 1,500 x g for 3 rain. The r a d i o a c t i v i t y was r e c o v e r e d in the s u p e r n a t a n t fraction F 1 (104,000 dpm) a n d i n the pellet f r a c t i o n F 2 (101,000 dpm). The r a d i o a c t i v i t y of F 1 was s e d i m e n t e d by the a d d i t i o n of 200 ~xg of c a r r i e r m a n n a n a n d 4 vol. ethanol. It was u n d e r t a k e n to compare the s t r u c t u r e of the r a d i o a c t i v e p o l y m a n n o s i d e s c o n t a i n e d i n the fractions F~ a n d F2 w h i c h were s u b m i t t e d separately to the Smith degradation, as above. The m i l d acid h y d r o l y s i s p r o d u c t s of the polyalcohols were water-soluble. Aliquots of the deionized fractions were c h r o m a t o g r a p h e d i n solvent VII on cellulose layers. Figure 3 shows that the radioactive tracings of F~ a n d F 2 were c o i n c i d e n t a n d that the peaks c o - c h r o m a t o g r a p h e d with glycerol, erythritol, m a n n o s e a n d m a n n o s y l - e r y t h r i t o l controls. The fastest m i g r a t i n g peak was p r o b a b l y glycolaldehyde. No l r i s a c c h a r i d e or higher DP oligos a c c h a r i d e was detected. The slower m i g r a t i n g

Man Ery Gly M2.E M.E

TABLE VI.

Total acid h y d r o l y s i s of the poIyaIcohols derived f r o m periodate oxidation of [l',C]mannan fractions F 1 and F e (*).

Radioactive area

Radioactivity I Molar ratio lepta) per cent F1

09 O~

Glycerol. . . . . . . . . . . . . . . . . Erythritol . . . . . . . . . . . . . . . Mannose . . . . . . . . . . . . . . . . 5

10

Distance from origin

(cm)

FI6. 3. Tracings of chromatograms of the products obtained by mild acid hydrolysis of the [25C] mannan, degraded by the Smith method. Fractions F~ and F._, : see text. TLC on cellulose in solvent VII, development 5.5 h ; control spots : Gly, glycerol ; Ery, erythritol ; Man, mannose ; M.E., mannosyl-erythritol ; M:.E, mannobiosyl-erythritol. -

-

BIOCHIMIE, 1976, 58, n ° 10.

Ft

F~

] 178 [ 253 [ 4.6 [ 4 3 9 3 0 6 4 5 7 76.1 77.5 [ 1491 [ 2310 / 19.3 ] 18.5

(*) Fractions F1 and F~ : see text.

i

0

F~

spot area was eluted from each c h r o m a t o g r a m a n d h y d r o l y z e d w i t h the A. niger h y d r o l a s e p r e p a r a tion. P a p e r c h r o m a t o g r a p h y of the p r o d u c t s i n solvent system H detected m a n n o s e and e r y t h r i t o l of relative r a d i o a c t i v i t y 6 to 4.

Mannan and mannolipid biosynthesis. Total a c i d h y d r o l y s i s of an aliquot of the r a d i o active p o l y a l c o h o l s F~ a n d F2 a n d p a p e r c h r o m a t o g r a p h y of the p r o d u c t s gave the results of Table VI. The m a j o r p r o p o r t i o n of e r y t h r i t o l i n d i c a t e d the m a i n (1 - ~ 4) l i n k a g e of the native p o l y s a c c h a r i d e s , t h e p r e s e n c e of u n a l t e r e d m a n nose suggesting either some b r a n c h i n g p o i n t s or (1 - ~ 3) linkages, p r o b a b l y both. The small r a t i o of g l y c e r o l r a d i o a c t i v i t y to the total i n d i c a ted f e w e r n o n - r e d u c i n g ends t h a n in the date mannan. I

l

II

)

Ill

IV V

i

I

II

1203

the polymer by pronase, have very similar if not identical structures.

METIfYLATION OF THE DATE MANNANAND OF THE [14C] MANNAN. Methylation of the date m a n n a n and s u b s e q u e n t m e t h a n o l y s i s p r o d u c e d the c a r r i e r m a t e r i a l necess a r y for the analysis of the m e t h y l a t e d r a d i o active p o l y m e r . Date m a n n a n (94 rag) was m e t h y lated a n d p c r m e t h y l a t e d as d e s c r i b e d in methods. The d r y p e r m e t h y l a t e d c o m p o u n d (73 rag) was re-

Vl . . . .

'

,,o c3 ¢N

170 ° 130°

................ "......................

g o.

G

8

F

E

DQe

~ e

D C A.B

•

2,3,6

,

\

~

2,4,fl

¢-4 11

3,4,6t 2,3,4t 0

Distance from origin (cm}

)

0

10 20 Retention time (min) Fro. 4. - - GLC tracing of m e t h a n o l y z e d permeH~ylated date mannan, as carrier of m e t h a n o l y z e d permethglated [l~Clmannan. Solid line : m c l h a n o l y z e d mixture ; dashed line : m e t h a n o l y z e d date m a n n a n mixed w i t h the four authentic t r i - O - m e t h y l - a - m e t h y l - D - m a n nosides ; dotted line • t e m p e r a t u r e program ; r o m a n figures : radioactive fractions collected.

FIG. 5. - - TLC and radioactive tracing of m e t h g l a t e d derivatives of [~:,C]mannan and carrier date mannan. L a w e r p a n e l : positions of m e t h y l a t e d derivatives of date m a n n a n (M) and of the four tri-O-methyl-ct-met h y l - v - m a n n o s i d e s ; c h r o m a t o g r a p h y on silica, so.lvent XII, developed 45 min. Compounds A to F were identified to the compounds indicated by the same letter on the GLC profile of figure 4:. Compounds G correspond to dimethyl-derivatives, not shown on fig. 4.

TABLE VII. GLC o f O - m e t h y l Fraction collected

I II

III IV V VI

[I:,C]m e t h y l m a n n o s i d e s .

a - a n d ~-2, 3, 4, 6 - t e t r a - O - m e t h y l unidentified ~-2, 3, 4 - t r i - O - m e t h y l a-3, 4, 6 - t r i - O - m e t h y l -

Molar ratio (per cent)

89 27

2.5 0.7

151

4.1

3.012 215

82.6 5.9 4.2

i

~-2, 4, 6-tri-O-methyla-2, 3, 6-tri-O-methyl~-2, 3, 6-tri-O-methyl-

i

d i - O - m e i h y l - (mixture)

:

Figure 3 and table ¥I underline the fact that f r a c t i o n s F 1 a n d F2, r e s p e c t i v e l y d e r i v e d f r o m t h e s u p e r n a t a n t a n d the s e d i m e n t after digestion of

BIOCHIMIE, 1976, 58, n ° 1().

Radioactivity (dpm)

Carrier derivative of methylmannoside

153

covered and methanolyzed; the products were i d e n t i f i e d b y GLC [341, u s i n g t h e a u t h e n t i c t r i m e t h y l - d e r i v a t i v e s o f c t - m e t h y l - D - m a n n o s i d e as 83

1204

31. M. S m i t h

controls (fig. 4). Peak A was i d e n t i f i e d as 2,3,4,6tetra-0-methyl-c~-incthyl-D-mannoside by its retention time in the GLC system of F o u r n e t a n d Montreuil [34]. Peak B was p r o b a b l y the ~-anomer of the same c o m p o u n d . T h e i r integrated areas a m o u n t e d to 10 per cent of the total surface of peaks detected on the chromatogram. Peak E comigrated with 2,3,6-tri-O-niethyl-a-methyl-D-nmnnoside, peak F p r o b a b l y b e i n g due to the ~-anom e t (pooled surface 85 per cent of the total). Peak C was u n i d e n t i f i e d ; Peak D did not m a t c h a n y t r i m e t h y l - d e r i v a t i v e of a-methyl-D-mannoside. Small peaks due to c o m p o u n d s m i g r a t i n g w i t h the r e t e n t i o n times of 2,4-, 3,6- a n d 4,6-di-O-methyl-a-methyl-D-mannosides [.34] were d e t e c t e d ; their pooled areas did not exceed 2.5 per cent of the total. These results c o n f i r m e d [233 that date m a n n a n c o n t a i n e d essentially (1 - > 4) l i n k e d m a n n o s y l c h a i n s w i t h a few b r a n c h i n g points and 10 per cent of n o n - r e d u c i n g end monomers. Radioactive m a n n a n was p r e p a r e d by the stand a r d method ; 500 .ug of date m a n n a n was added as c a r r i e r and the p o l y m e r s were p u r i f i e d by m e t h o d A. The material on tlIe glass fibre disc (63,000 dpm) was subniitted to Inethylation, as described above ; 33,000 d p m were recovered and methanolyzed. TLC of an aliquot gave a profile of c o m p o u n d s c o r r e s p o n d i n g to the m e t h y l a t e d derivatives of date m a n n a n (fig. 5). A n o t h e r aliquot of the n i e t h y l a t e d a n d methanolyzed [14C]mannan was q u a n t i t a t i v e l y analyzed b y GLC, m o n i t o r e d b y the m e t h y l a t e d derivatives of c a r r i e r date m a n n a n . The r a d i o a c t i v e comp o u n d s eluted w i t h each f r a c t i o n (see fig. 4) were collected at the c o l u m n outlet a n d c o u n t e d in toluene (table VII). The q u a n t i t a t i v e recovery of these c o m p o u n d s was checked w i t h a c o n t r o l GLC of nIethylated date m a n n a n . F r o m the results of table VII, the structure of the radioactive polys a c e h a r i d e parallels the s t r u c t u r e of date m a n n a n with fewer end m o n o m e r s a n d some more dimethyl-derivatives. In this case, n o n statement is possible about the significance of peak D, since a b r o a d fraction i n c l u d e d this peak d u r i n g the collection a n d does not locate the peak accurately.

A N O M E R I C STRUCTURE SYNTHESIZED MANNAN.

OF

THE

ENZYMATICALLY

The structure ~5 of date n I a n n a n has been estab l i s h e d [23, 38]. It was used as a control for the i d e n t i f i c a t i o n of the a n o m e r i c s t r u c t u r e of the m a n n o s y l linkage i n the p o l y s a c c h a r i d e p r e p a r e d w i t h the m i c r o s o m a l fraction. BIOCHIMIE, 1976, 58, n ° 10.

a n d coll.

The parallel c h r o m a t o g r a p h i c m i g r a t i o n of the oligosaecharides resulting from the p a r t i a l h y d r o lysis of the [a4C]mannan a n d the c a r r i e r date i n a n n a n (fig. 2) first p r o v i d e d evidence for the same structure of the a n o m e r i c linkage in both compounds. It has also been m e n t i o n e d that ihe [14C]mannan was soluble o n l y i n strong alkali a n d that its acid h y d r o l y s i s was sh)w : these observations are in favor of a ~ c o n f i g u r a t i o n of the a n o m e r i c linkage.

100 0

-~o~ D

-' :~

so

/

/

~.o L.

. / ' / pmJ'

Incubation time (rain) FI(L 6. Kinetics of enzymatic hydrolysis oJ [z,~C] mannan, by itself (1) or in the presence 'of 800 ~tg of date mannan (2). Incubation conditions : [14Clma]lnan 20,000 dpm, A. niger mannanase 40~0 l~tg in 40 mM sodium acetate buffer, pH 4.5, in a total rot. 400 ~tl ; temperature 45°C. Radioactive measurements on 50 ~tl samples. -

-

An i n d e p e n d e n t evidence was p r o v i d e d by the kinetics of enzyulatic h y d r o l y s i s : both p o l y m e r s were dissolved i n 1 N sodiunl h y d r o x i d e a n d the solutions b r o u g h t to pH 4.5 w i t h acetic acid. The radioactive m a n n a n alone (fig. 6-1) or m i x e d with date m a n n a n (fig. 6-2) was h y d r o l y z e d by A. n i g e r u l a n n a n a s e : ethanol-soluble oligosaccharides appeared. D u r i n g the i n c u b a t i o n s , ali(iuots were collected a n d heated 5 rain at 100°C to stop the reaction. 100 ,ttg of c a r r i e r date m a n n a n a n d 4 vol. ethanol were a d d e d to each sample and the r a d i o a c t i v i t y c o n t a i n e d i n the s u p e r n a t a n t s was measured. This e x p e r i m e n t showed that the h y d r o l y s i s of the [14C~ m a n n a n was strongly delayed by the isotopic d i l u t i o n w i t h the B-linked date i u a n n a n . The m o u l d enzyme p r e p a r a t i o n m a y c o n t a i n a - m a n n a n a s e ; if such an enzyme was effective to split the radioactive p o l y s a c c h a r i d e , no c o m p e t i t i o n should be observed w h e n date n i a n n a n was added. Considered together, these results strongly support the c o n t e n t i o n that the [14C]mannan is ~-linked.

Mannan and mannolipid biosynthesis. KINETIC

PROPERTIES

OF

GDP-MAN

THE

: MANNO-

SYL-TRANSFERASE.

T h e time course of m a n n o s e i n c o r p o r a t i o n f r o m GDP-Man was s t u d i e d : f r o m an i n c u b a t i o n mix-

0

E

Q. "-0

2.5

.(2_

"-0

O.

0

t

i

|

10

20

30

Incubation time

FIG. 7.

- -

cu b at ed in the s t a n d a r d c o n d i t i o n s w i t h each r a d i o a c t i v e n u c l e o t i d e - s u g a r by itself or in the p r e s e n c e of an equal c o n c e n t r a t i o n or a ten fold excess of the o t h er unlabeled nucleotide (table VIII). The p o l y n i e r i c f r a c t i o n was isolated by m e t h o d A. It can be seen f r o m the results that the i n c o r p o r a t i o n of m a n n o s e was i n h i b i t e d by GDP-GIc, even at an equal c o n c e n t r a t i o n , w h e r e a s GDP-Man m a r k e d l y stimulated the i n c o r p o r a t i o n of glucose f r o m GDP-Glc w h e n c o n c e n t r a t i o n s of tile t w o nucleotides w e r e equal. W i t h a ten fold excess of GDP-Man, the i n c o r p o r a t i o n of glucose is stinmlated to a lesser extent. The observed results are in a g r e e m e n t w i t h the c o m p e t i t i o n curves p u b l i sh ed by E l b e i n [1] and Villemez [3i. INCORPORATION

(min)

!14~]~IAN

Kinetics of GDP-Man : mannan mannosyltransferase of microsomes.

ture p r e p a r e d as d e s c r i b e d in m e t h o d s (microsomes : 0.76 mg protein, GDp-[14C]mannose :

1205

INTO

OF

THE

[14C]MANN()SE fROM GDPLIPIDIC

~'RACTION

OF

MICRO-

SOMES.

As s h o w n by table II, w h e n the m i c r o s o n i e s w e r e i n c u b a t e d in the p r e s e n c e of GDP-!a4C!Man, some r a d i o a c t i v i t y was r e c o v e r e d in the l i p i d i c f r act i o n . The nature of the r a d i o a c t i v e c o m p o -

TABLE VIII.

Incorporation of m a n n o s e f r o m GDP-rlJC ' ]ManL or incorporation of gh:cose f r o m GDP-[~3C]Glc, in the p r e s e n c e of the other nucleotidesugar. Nucleotide-sugar concentration (uM) GDP-GIc

GDP-p~,C]Man

0 34 340

34 34 34

GI)P-[t'CIGIc

GDP-Man

37 37 37

Radioactivily (dpm)

Incorporation of [J~'C]hexose (pmoles/mg prolcin/30 min)

5,500 919 354

45l 75 29

433 2,415 1,000

40 220 92

0 36 357

825,000 d p m in 150 ~,1), 10 ~1 aliquots w e r e remove d after v a r i o u s p e r i o d s and the r a d i o a c t i v e m a n n a n was p u r i f i e d by m e t h o d A. A l i n e a r synthesis of m a n n a n , w i t h o u t lag, w a s o b s e r v e d (fig. 7). Th e s y n t h e t i c a c t i v i t y m e a s u r e d in s e v e r a l e x p e r i n l e n t s v a r i e d f r o m 0.7 to 1.4 ~mole m a n n o syl t r a n s f e r r e d p e r h o u r p e r m g protein. The m i c r o s o m a l p r e p a r a t i o n was also able to i n c o r p o r a t e the glucosyl residues f r o m G,DP-[14C] Glc into a ~-glucan (*). The Inicrosonms w e r e in(*) Unpublished results.

BIOCHIMIE, 1976, 58, n ° 10.

nents of this f r a c t i o n was i n v e s t i g a t e d in p a r t i c u l ar e x p e r i m e n t s . Microsomes w e r e i n c u b a t e d in s t a n d a r d conditions (1.85 mg protein, GDP-E14CIMan 2,200,000 dpm, total vol. 400 ~1). After i n c u b a t i o n , the mixture was f r a c t i o n a t e d by m e t h o d B. The results are listed in table IX. After w a t e r ex t r act i o n , the pellet was w a s h e d w i t h e t h a n o l - w a t e r (7/3) and e x t r a c t e d stepwise by c h l o r o f o r m - m e t h a n o l (2/1) (fraction A) and c h l o r o f o r m - m e t h a n o l - w a t e r (10/10/3) ( f r act i o n B).

M. M. S m i t h a n d coll.

1206

E t h a n o l w a s h i n g s w e r e f o u n d to c o n t a i n e s s e n tially unmodified or hydrolyzed substrate and were not further analyzed. Fractions A and B w e r e a n a l y z e d i n p a r a l l e l b y 3"LC ( t a b l e X). F r a c tion A exhibited a major well defined radioactive p e a k I a n d a m i n o r p e a k II, w h e r e a s p e a k I I w a s t h e o n l y r a d i o a c t i v e c o m p o n e n t o f f r a c t i o n B.

those previously reported by other authors for mannosyl-phosphorylpolyisoprenols [20, 39i. 0.1 N h y d r o c h l o r i c acid in 1-propanol hydrol y z e d 86 p e r c e n t of t h i s l i p i d i n 20 m i n at 50°C ; a f t e r t h i s , t h e r a d i o a c t i v i t y w a s f o u n d to m i g r a t e with mannose and unaltered material on TLC (fig. 9). P a p e r c h r o m a t o g r a p h y i n s o l v e n t I w h i c h

TABLE IX.

[ ~ t C ] - l a b e l e d m a n n o l i p i d fraclionation from microsomes. Radioactivity (dpm)

Fraction Water-soluble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . First ethanol-water (7/3) extract . . . . . . . . . . . . . . . . . . . . . . . . . Chloroform-methanol (2/1) extract (A) . . . . . . . . . . . . . . . . . . . Chloroform-methanol-water ( 1 0 / I 0 / 3 ) extract (B) . . . . . . . . . . Second ethanol-water (7/3) extract . . . . . . . . . . . . . . . . . . . . . . Residue (mannan) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

F o r a p r e p a r a t i v e p u r i f i c a t i o n of l i p i d s I a n d II, m i c r o s o m e s w e r e i n c u b a t e d as u s u a l ; 'the m i x t u r e was extracted with water and ethanol-water (7/3). T h e s o l i d r e s i d u e w a s d e p o s i t e d o n a glass f i b r e d i s c at t h e t o p of a s i l i c i c a c i d c o l u m n . T h e r a d i o "active m a t e r i a l w a s e l u t e d b y c h l o r o f o r m - m e t h a n o l (2/1) : l i p i d I e m e r g e d f r e e of l i p i d II. A c h l o roform-methanol-water (10/10/3) eluent was then a p p l i e d a n d a n a d d i t i o n a l a m o u n t of l i p i d (I') a p p e a r e d at t h e f r o n t of t h i s s o l v e n t . L i p i d 1I w a s e l u t e d i n t h e n e x t f r a c t i o n s (fig. 8). T h e r a d i o a c t i v e f r a c t i o n s w e r e k e p t at - - 2 5 ° C i n t h e i r elution solvents and analyzed shortly after isolation. TABLE X.

,~ 15

2,000,000 21,000 33,000 26,500 900 It4,0O0

CM

'~1

× E

CMW

11"

._~

~ °o

25 Tube number

50

g

TLC of mannolipids on silica. Rf on solvent system : o

Fraction

tPeak I ....... A i P e a k II B ...............

IV

X

XI

0.69 0.2 to 0.5 0.2 to 0 . 5

0.35 0

0.74 0

0

0

£3

0

5 10 Distance from origin (cm)

15

Fro. 8 . - Silica column chromatography and TLC of [~C]mannolipids. Upper panel : elution profile of a According to its c h r o m a t o g r a p h i c properties, l i p i d I ' of f i g u r e 8 s e e m s to b e i d e n t i c a l to l i p i d I ; it has not been further analyzed.

column (1 >< 21.5 era)' c h r o m a t o g r a p h y of m a n n o l i p i d s on silicic acid (Mallinekrodt, 10~0 mesh) ; e]uents : CM, c h l o r o f o r m - m e t h a n o l (2/1) ; CMW, c h l o r o f o r m - m e t h a n o l - w a t e r (10/10/3) ; v o l u m e of f r a c t i o n s : 1 m l ; lower panel : tracing of TLC of m a n u o l i p i d s on silica, solvent IV, d e v e l o p m e n t 165 rain. ; roman figures : aliquot samples of c o m p o u n d s f r a c t i o n a t e d on t h e silieie column.

ANALY'SIS OF L I P I D ~.

The chromatographic c o n s t a n t s of l i p i d I a r e s h o w n o n t a b l e X ; t h e Rf v a l u e s a r e s i m i l a r to

BIOCHIMIE, 1976, 58, n ° 10.

separates aldoses showed that the radioactive s u g a r w a s i n d e e d m a n n o s e . T h e r e l e a s e of m a n -

1207

Mannan and mannolipid biosynthesis. nose from lipid I by mild acid hydrolysis suggest e d t h e p r e s e n c e of a l a b i l e l i n k e d p h o s p h a t e .

Man__

n e i g h b o u r i n g c o m p o u n d s of w h i c h c o m p l e t e r e s o lution in the native state was unsuccessful. When l i p i d II w a s s u b j e c t e d to h y d r o l y s i s b y 0.1 N h y d r o c h l o r i c a c i d at 50°C f o r 20 m i n , t h e r a d i o a c t i v e m a t e r i a l b e c a m e w a t e r - s o l u b l e a n d its m i g r a t i o n i n s o l v e n t t V w a s r e d u c e d a l m o s t to nought. This material was hydrolyzed by 2 N hyd r o c h l o r i c a c i d at 100°C f o r 3 h : t h e o n l y r a d i o a c t i v e p r o d u c t w a s f o u n d to b e m a n n o s e b y p a p e r c h r o m a t o g r a p h y i n s o l v e n t I.

c o Q_

0

5

10

15

M4

o

ITA

M~3

M2

Olc Man

An

2

~D

~ 0 M6P 0

10 Distance from origin

20 (crn)

FIG. 9. - - Radioactive traces of mild acid hydrolysis (upper panel) and alkaline hydrolysis products (lower panel) of lipid I. Tracing A : TLC on silica of n a t i v e lipid I, solvent IV, d e v e l o p m e n t 3 h ; tracing B : same e x p e r i m e n t w i t h lipid I h y d r o l y z e d b y dilute acid ; tracing C : electrophoresis of a l k a l i n e hydrolysi~ product of lipid I on W h a t m a n No 1 p~.per in buffer XIII, 1.5 h at 45 v o l t / e r a ; tracing D : the same product a f t e r a l k a l i n e p h o s p h a t a s e digestion ; controls : Man, m a n n o s e ; MIP, a - v - m a n n o s e - l - p h o s p h a t e ; M6P, a-D-mannose-6-phosphate.

L i p i d I w a s t h e n s u b m i t t e d to a l k a l i n e h y d r o lysis by NaOH in the conditions described in met h o d s ; 30 p e r c e n t of t h e r a d i o a c t i v i t y b e c a m e w a t e r - s o l u b l e a n d 70 p e r c e n t w e r e r e c o v e r e d i n the chloroform-methanol phase. Paper electrophor e s t s of t h e w a t e r - s o l u b l e p r o d u c t s h o w e d t h a t it was negatively charged with a mobility matching mannose-phosphates (fig. 9). T r e a t m e n t of t h i s c o m p o u n d w i t h a l k a l i n e p h o s p h a t a s e (0.3 E.U., 1 h at 37°C i n 50 m M Tris-C1 b u f f e r , p H 8.0) r e s u l ted in a total conversion into mannose. Although the attack by alkali was not complete, the results suggested that lipid I was a mannosyl-phosphoryllipid and probably a polyisoprenol derivative. ANALYSIS OF LIPID II. T h e c h r o m a t o g r a p h i c m i g r a t i o n of l i p i d II o n s i l i c a i n s o l v e n t I V (fig. 8) s u g g e s t e d a m i x t u r e of

BIOCHIMIE, 197,6, 58, n ° l(t.

10

~MIP

20 Dislonce from origin (cm)

30

Fro. 10. - - Radiochromalograms of enzymatic hydrolysis products of mannolipid It (upper tracing) and partial acid hydrolysis of fractions I1 A and 11 B of the same mannolipid (lower tracings), W h a t m a n No 1 p a p e r c h r o m a t o g r a p h y , 16 h in solvent I ; control spots : see Fig. 2 ; pet" cent of radioactivity in each peak integrated f r o m the tracing.

]IA TrB

.:

\]

r ;

"i Elution volume (m 0

Sephadex G 2S column chromatography of mild acid hydrolysis products of lipid II. Column : 0.4 X 110 cm ; solid line : r a d i o a c t i v i t y of f r a c t i o n s ; dotted line : e]ntion of d e x t r a n blue 2,000 ; dashed line : elution of control i n u l i n e oligofructosides of M W a : 180, b : 504, c : 1152, d : 1638. Fro.

11. --

L i p i d I I w a s s u b j e c t e d to a l k a l i n e h y d r o l y s i s and paper electrophoresis by the method used for l i p i d I. Most of t h e r a d i o a c t i v i t y w a s r e c o v e r e d o n t h e s t a r t i n g s t r e a k of t h e e l e c t r o p h o r e s i s p a p e r

1208

M. M. S m i t h a n d coll.

and no c o n c l u s i o n c o u l d be d r a w n f r o m this experiinent. T h e w a t e r - s o l u b l e p r o d u c t of nlild acid h y d r o lysis of l i p i d I~l was then subjected to e n z y m a t i c h y d r o l y s i s by the A. niger h y d r o l a s e (enzyule 200 ~g in a m u I o n i u m acetate buffer, pH 4.8, 45°C). After an i n c u b a t i o n p e r i o d of 45 rain u n d e r condit i o n s r e q u i r e d for the ~41 ->- 4) m a n n a n to be split into short o l i g o s a c c h a r i d e s (fig. 2 and 6), only m a n n o s e and a b r o a d peak of large o l i g o s a c c h a r i des w e r e detected on the c h r o m a t o g r a m (fig. 10). An exhaustive i n c u b a t i o n p e r i o d of 17 h w i t h the same enzynie gave a s i m i l a r c h r o m a t o g r a p h i c patt e r n a n d 35 p e r cent m a n n o s e was liberated. Tbese results suggested several types of m a n n o s y l linkages in l i p i d II, of w h i c h at least one was not sensitive to ~-mannanase. To f r a c t i o n a t e the w a t e r - s o l u b l e moieties of l i p i d II and estimate the MW of the fractions, the p r o d u c t of m i l d acid h y d r o l y s i s d u r i n g 90 rain w a s n e u t r a l i z e d by s o d i u m h y d r o x i d e , b r o u g h t to dryness, dissolved in w a t e r a n d l a y e r e d at the too of a S e p h a d e x G25 (,fine grade) column. It was eluted by 10 mM a m m o n i u m acetate pH 4.8 buffer (fig. 11). I n u l i n e oligofructosides w e r e r u n on the same c o l u m n as MVV niarkers. The r a d i o a c t i v i t y was eluted in t w o distinct peaks II A and I I B ; II A was eluted in the e x c l u s i o n v o l u m e : its M~V w a s o v e r 5,000, w h e r e a s H B w a s s o m e w h a t polydisperse w i t h a m a x i m m n at MW 1,700-1,800. ANALYSIS OF FRACTIONS A AND B OF LIPID I I .

In o r d e r to investigate the p r e s e n c e of N-acetylo s a m i n e residues in o l i g o s a c c h a r i d e s A2, A3, An and Bn, the r a d i o a c t i v e peaks w e r e eluted f r o m

Man

GIc_N_{ol)

An

Bn

5

Distance

15

I0

from orlgin

@m)

Fro. 12. - - Etectrophoretic traces of hydrazinolgsis products of A,, and B~. Electrophoresis on Schleicher and Schiill paper in buffer XIV, 1 h at 62 volt/cm ; controls : Man, mannose and Glc-N-(ol), glucosaminito].

papers, r e d u c e d w i t h s o d i u m b o r o h y d r i d e , deionized and h y d r a z i n o l y z e d as i n d i c a t e d in methods. Tlae p r o d u c t s of h y d r a z i n o l y s i s w e r e b r o u g h t to dryness and tested by p a p e r e l e c t r o p h o r e s i s . The snlall o l i g o s a c c h a r i d e s A2 and A3 w e r e found to be n e u t r a l w h e r e a s the o l i g o s a c c h a r i d e s An or Bn e x h i b i t e d a d i s t i n c t p a t t e r n w i t h about 40 p e r cent of the r a d i o a c t i v i t y in the n e u t r a l area and 60 p e r cent in the p o s i t i v e l y c h a r g e d m o l e c u l e s (fig. 12). These results suggested that, besides r a d i o a c t i v e mannose, the w a t e r - s o l u b l e nloieties of l i p i d II c o n t a i n e d u n l a b e l e d n e u t r a l sugars and p r o b a b l y also acetyl-osamines.

F r a c t i o n s II A and II B w e r e c o n c e n t r a t e d to dryness u n d e r v a c u u m ; a m m o n i u m acetate was e l i m i n a t e d by h i g h v a c u u m and each f r a c t i o n was p a r t i a l l y h y d r o l y z e d in h y d r o c h l o r i c acid saturated w i t h HC1 gas at 0°C, d u r i n g 4.5 h at 22°C in sealed vials. T h e p r o d u c t s of these h y d r o l y s e s w e r e c h r o m a t o g r a p h e d on p a p e r (fig. 10). The c h r o m a t o g r a p h i c p a t t e r n s of f r a c t i o n s I1 A and I I B w e r e s t r i k i n g l y similar. The o l i g o s a c c h a r i d e s A2 and A3 or B2 a n d B3 did not m a t c h a n y di- or t r i s a c c h a r i d e o b t a i n e d f r o m date ~-(1->- 4) m a n nan.

After GDP-Man was i n c u b a t e d w i t h the sycam o r e cell m i c r o s o m e s , t h r e e m a i n r e a c t i o n products w e r e isolated fronl the w a t e r - i n s o l u b l e fraction. T h e first c o m p o u n d has been s h o w n to be a p o l y m a n n o s i d e , the t w o others w e r e lipids.

O l i g o s a c c h a r i d e s B2 and B3 w e r e eluted f r o m paper, r e d u c e d w i t h s o d i u m b o r o h y d r i d e , deionized on A m b e r l i t e IR 45 (OH-) a n d D o w e x 50 (H ~) m i x e d beds and m e t h a n o l y z e d as d e s c r i b e d in methods. The p r o d u c t s w e r e c h r o m a t o g r a p h e d by 'PLC on silica in s o l v e n t VI w i t h a-methyl-9-mannoside and m a n n i t o l as controls. T h e r a d i o a c t i v i t y migrated quantitatively with methyl-niannoside : t h e r e f o r e these o l i g o s a c e h a r i d e s c o n t a i n e d only one r a d i o a c t i v e m a n n o s e l o c a t e d on the n o n - r e d u cing end.

The p o l y m a n n o s i d e w a s only p a r t i a l l y soluble in s o d i u m h y d r o x i d e solutions or buffered SDSurea m i x t u r e s : this situation m a d e it difficult to estimate the m o l e c u l a r w e i g h t of this c o m p o u n d . T h e question w a s r a i s e d to k n o w if the r a d i o a c tive m a n n o s i d e w a s b o u n d to an a c c e p t o r peptide. In f a v o r of this v i e w it should be r e c a l l e d that the p o l y m e r w a s p a r t l y solubilized by m i l d alkali : this w o u l d suggest that the p o l y m a n n o s i d e is b o u n d t h r o u g h an O-glycosidic linkage to the h y d r o x y l group of amino-acids. On the o t h e r

BIOCHIMIE, 1976, 58, n ° 10'.

DISCUSSION.

M a n n a n a n d m a n n o l i p i d biosynthesis. h a n d , about half the r a d i o a c t i v i t y of the p o l y m e r did not s e d i m e n t after protease digestion. The [14C]polymannoside was also p a r t i a l l y soluble i n the m i x t u r e SDS-urea, a solvent k n o w n as a dissociating agent of proteins. These features do not demonstrate the b i o s y n t h e s i s of glycoproteins. Especially, no radioactive glycopeptide was characterized b y electrophoresis. Such glycopeptides have been detected after i n c o r p o r a t i o n in vivo of u-glucosamine in p l a n t tissues [9, 10]. Other authors have p r e s e n t e d evidence for the synthesis of glycoproteins from nucleotide-sugars by p l a n t enzyme p r e p a r a t i o n s [11-141. It seems to us that this evidence still requires f u r t h e r data. F r o m our e x p e r i m e n t s the p o l y m a n n o s i d e appears to be a h o m o p o l y m e r strongly b o u n d to m e m b r a n e proteins but not to be the prosthetic group of a glycoprotein. The study by chemical a n d enzymatic m e a n s of the linkages b e t w e e n m a n n o s y l u n i t s has s h o w n that this m a n n a n was essentially a 6-(1 -~- 4) linked chain. F r o m the periodate oxidation and the m e t h y l a t i o n results, the part of the molecule synthesized d u r i n g the i n c u b a t i o n w i t h GDP-[14C] Man seems to consist of chains c o n t a i n i n g 25 to 40 m a n n o s y l residues. F r o m the a m o u n t of detected d i m e t h y l - m a n n o s i d e s , a weak b r a n c h i n g of these chains can be expected (one or two b r a n c h e s for 40 m a n n o s y l units). The a m o u n t of m a n n o s y l e r y t h r i t o l detected after periodate oxidation of the [ l ~ C ] m a n n a n is h i g h e r t h a n this model w o u l d assume. However, m a n n o s y l - e r y t h r i t o l may originate both from b r a n c h i n g points a n d (1 - ~ 3) linkages scattered i n the molecule. It is also likely that the i n s o l u b i l i t y of the p o l y s a c c h a r i d e p r e v e n ted its total o x i d a t i o n b y periodate and t h e r e b y accounts for the p r e s e n c e of a slight excess of u n a t t a c k e d m a n n o s e monomers. I n agreement w i t h the findings of other authors [38], the same s t r u c t u r a l model holds for date nut m a n n a n A, although this p o l y s a c c h a r i d e has shorter chains a n d more b r a n c h i n g points t h a n the radioactive polymer. The second a n d t h i r d classes of radioactive c o m p o u n d s synthesized from GDP-E14C]Man were solubilized b y c h l o r o f o r m - m e t h a n o l or chlorof o r m - m e t h a n o l - w a t e r solvents. T h e i r chromatog r a p h i c f r a c t i o n a t i o n a n d analysis have first s h o w n that n o n e of these lipids was a m a n n o s y l steride or a m a n n o s y l - g l y c e r i d e . This is i n contrast with the results o b t a i n e d b y i n c u b a t i o n of the microsomes w i t h UDP-Glc [40] or UDP-Xyl (*), w h i c h p r o d u c e d steryl-sugars or w i t h UDPGal (*) w h i c h p r o d u c e d galactosyl-glycerides. The (*) Unpublished results.

BIOCHIMIE, 1976, 58, n ° 10.

1209

m e m b r a n e fractions exhibit a specific glycolipid b i o s y n t h e t i c p a t t e r n a c c o r d i n g to the n a t u r e of the nucleotide-sugar substrate. M a n n o l i p i d I was first s h o w n to be sensitive to m i l d acid h y d r o l y s i s at 50°C a n d p r o d u c e d o n l y free mannose. The r a p i d rate of h y d r o l y s i s suggested that m a n n o s y l was b o u n d to phosphate or pyrophosphate. This fact j o i n e d to the detection of m a n n o s e - p h o s p h a t e after alkaline h y d r o l y s i s suggested, a c c o r d i n g to Herscovics et al [41], that the a n o m e r i c configuration of the m a n n o s e - l - p h o s phate in lipid I was 8. A c c o r d i n g to these findings a n d to the study of the solubility a n d chromatographic properties of l i p i d I, it seems very similar if not i d e n t i c a l to m a n n o s y l - p h o s p h o r y l - p o l y p r e n o l w h i c h was characterized in plants by Alam a n d H e m m i n g [42]. This l i p i d has been described as a possible i n t e r m e d i a t e for m a n n o s e t r a n s f e r from GDP-Man to p l a n t p o l y m a n n o s i d e s although it was not clear w h e t h e r m a n n o s y l - p h o s p h o r y l p o l y p r e n o l is a necessary i n t e r m e d i a t e or a storage c o m p o u n d w h i c h could regenerate GDP-Man from GDP [11, 43]. An alternative suggestion w o u l d be that l i p i d I is synthesized b y a memb r a n e fraction w h i c h catalyses m a n n o s e t r a n s f e r into oligosaccharide-lipids such as lipid II a n d p e r h a p s into glycoproteins [18] while the synthesis of the p o l y s a c c h a r i d e is catalyzed b y a n o l h e r m e m b r a n e fraction, w i t h o u t r e q u i r e m e n t for l i p i d interlnediates. P h y s i c a l a n d chemical properties of l i p i d II also recall the properties of p o l y p r e n o l - p h o s p h a t e derivatives, p a r t i c u l a r l y b y the ease of its m i l d acid h y d r o l y s i s at moderate t e m p e r a t u r e [16]. The identification of the l i p i d b o u n d to sugar moiety obviously requires more i n f o r m a t i o n : for u n k n o w n reasons l i p i d II did not p r o v i d e evidence for the presence of a negatively charged c o m p o u n d after alkaline hydrolysis. The more i n t e r e s t i n g feature of this l i p i d is that, after mild acid h y d r o lysis, l i p i d II was f r a c t i o n a t e d i n two compounds. One of them (II B) h a d an estimated MW n e a r 1,700 and the second (II A) was over 5000 : both c o m p o u n d s gave exactly the same chromatograp h i c p a t t e r n after p a r t i a l acid h y d r o l y s i s (fig. 10). Consequently, II A may be a p o l y m e r of I I B or, alternatively, II A m a y c o n t a i n a I I B c h a i n b o u n d by an acid-labile linkage to an u n k n o w n core molecule. The results of e n z y m a t i c a n d p a r t i a l acid h y d r o l y s i s have s h o w n that every I I B c h a i n accepted either one or two m a n n o s y l units b o u n d to the n o n - r e d u c i n g end of an endogenous acceptor. E n z y m a t i c h y d r o l y s i s easily l i b e r a t e d the last radioactive m a n n o s e from the chains while the next radioactive m a n n o s e was stable. F r o m the h y d r a -

1210

M. M. Smith

z i n o l y s i s of t h e di- a n d t r i s a c c h a r i d e ( s ) o b t a i n e d f r o m II A, it c o u l d b e s e e n t h a t t h e s e c o m p o u n d s w e r e n o t c h a r g e d : t h e s u g a r s n e x t to r a d i o a c t i v e m a n n o s e u n i t s n m s t b e n e u t r a l . H y d r a z i n o l y s i s of t h e a c i d - r e s i s t a n t c o r e of t h e w a t e r - s o l u b l e m o i e t i e s of l i p i d II (An a n d B n ) a n d e l e c t r o p h o r e s i s have shown that positively charged groups were present in the higher oligosaccharides. The specif i c i t y of t h i s m e t h o d [25] s u g g e s t e d t h a t t h e n a t i v e oligosaccharides contained N-acetyl osamine(s) near their reducing ends. From the electrophoretic p a t t e r n s (fig. 12), it a p p e a r s t h a t m o r e t h a n o n e positively charged unit per chain should be present. A t e n t a t i v e m o d e l of t h e b a s i c u n i t of l i p i d I [ would be : I

2

[Ma n] --~-I-'~an] -->- ['qeutral stl~a r]n --~" IAcetyl-osa mine]n' w h e r e n ~ 3, n ' _~ 2, n q- n ' = 8-10 a n d l i n k a g e 1 is l a b i l e . Such a structure recalls the structure of the oligosaccharides linked to plant glycoproteins, the s o y a b e a n l e c t i n f o r i n s t a n c e [7]. I f t h e d e s c r i b e d lipids are intermediates in plant glycoprotein bios y n t h e s i s , t h e l a c k of a c c e p t o r p r o t e i n i n t h e m i c r o s o m e system m a y e x p l a i n w h y t h e o l i g o s a c c b a r i d e - l i p i d s a r e t e r m i n a l p r o d u c t s of t h e m a n n o s e transfer from GDP-Man. Nothing in the m o i e t y of l i p i d ~-(1 - > 4 ) m a n n a n active mannose into each chain. lipid II may act mannan.

s t r u c t u r e of t h e o l i g o s a c c h a r i d e I I r e c a l l s t h e s t r u c t u r e of t h e w h e r e a l a r g e n u m b e r of r a d i o residues have been transferred T h e r e f o r e , it is u n l i k e l y t h a t as a p r e c u r s o r m o l e c u l e f o r t h e

As m e n t i o n e d i n t h e i n t r o d u c t i o n , it w a s s u r p r i s i n g t h a t t h e s y c a m o r e m i c r o s o m e s w e r e a b l e to s y n t h e s i z e a m a n n a n w h i c h is a b s e n t f r o m t h e cell w a l l s . A n e x p l a n a t i o n is t h a t t h i s p o l y s a c c h a r i d e n m y b e a p a r t of a p e p t i d o g l y c a n i n t h e i n t a c t cell. An alternative explanation would he that the enzyme system physiologically operates with GDPGlc as s u b s t r a t e . E v e n i f ~ D P ~ M a n is p r e s e n t , it w o u l d f a v o r t h e i n c o r p o r a t i o n of g l u c o s e f r o n l G D P - G l c i n t o a g l u e a n , as s h o w n i n t e x t . I f t h i s e x p l a n a t i o n is v a l i d , t h e p h y s i o l o g i c a l r e a c t i o n is t h e b i o s y n t h e s i s of a ~ - g l u c a n , p e r h a p s of a g l u c o mannan containing little mannose. Acknowledgements. This work has been supported h y t h e Centre N a t i o n a l de la Recherche Scientifiq~e (ER 10~), the Ddldgation Gdndrale & la Recherche S c i e n t i f i q u e et Technique (Contrat N ° 72-7-0178 ; Action eoncertde : Membranes Biologiques, Structure et Fonctions) a n d the Uniuersit5 d'A ix-MarseiIle lI. BIOCHIMIE, 1976, 58~ n ° 10.

and coll. One of us (M.M.S.) is grateful to these o r g a n i z a t i o n s and the Centre National des (Eaw'cs Universitaires for post-doctorate rcsearch grants. R~suM~. Des fractions m e l n b r a n a i r e s ont 6t6 prdpar4cs h part i r de suspensions cellulaires d'6rable (Acer pseadoplatanus) cultiv~es en milieu ]iquide. Ces fractions catalysent le t r a n s f c r t d'unit6s m a n n o s y l du GDP-Man [14C] dans un~ m a n n a n e , u n m a n n o l i p i d e et des oligosaccharides-lipides. Le p o l y m a n n o s i d e est p a r t i c l l c m e n t soluble apr~s digestion prot6olytique ou m a c 6 r a t i o n dens lc melange dodecyl sulfate-nrde. Cependant il n ' y a pas de preuve d'une liaison covalente entre la glyeane et u n peptide accepteur. Le polysaccharide se pr6sente comme u n h o m o p o l y m 6 r e dont les u n i t e s m a n n o s y l sont lides p r i n c i p a l e m e n t en ~(1 -->- 4), avec un t a n x de b r a n c h e m e n t faible. Au cours de l ' i n c u b a t i o n des f r a c t i o n s m e m b r a n a i r e s en pr6sence de substrat, chaque chalne est allongdc d'un g r a n d h o m b r e de restes m a n n o s e : 25 h 40 p a r extrdmit6 n o n rdductrice. Le GDP-Glc est u n i n h i b i t e u r compdtitif de la m a n n o s y l - t r a n s f d r a s e a~ors que ]e GDP-Man active ]e GDP-Glc : [3-glucane glucosyl-transfdrase contenue dans la m~me p r d p a r a tion m e m b r a n a i r e . Deux glycolipides sont f o r i n t s h partir de GDP-Man[14C]. Le p r e m i e r (I), c o n t e n a n t u n e p a r t i e h y d r o s o l u b l e identifiable au m a n n o s e - p h o s p h a t e , posshde les propridtEs des pol.yprenol-phosphate-mannose identifies, n o t a m m e n t d a n s ]es plantes, p a r M a i n et Hemming. Le second lipide (II) f o u r n i t p a r h y d r o l y s e acide faihle des o]igosaccharides de poids molEculaire Elevd. Deux f r a c t i o n s ¢,ligosaccharidiques out 6t6 isoIdes, l ' u n e de PM 1700 environ (IIB), l ' a u t r e de PM supdrieur h 50.00 (II A). I I B contient au m a x i m u m deux restes m a n n o s e marquEs p a r chaine, lies h Uextrdmit6 non rEductrice d'une moldcule qui p o u r r a i t e o n t e n i r des oses n e u t r e s n o n m a r q u 4 s et des restes N-ac6tyl-osamine h l'extrdmit6 rEductrice. L'oligosaccharide II A semble contenir une ou p l u s i e u r s chaines I I B . REFERENCES. 1. 2. 3. 4. 5. 6. 7.

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