Biosynthesis of mannosyl- and glucosyl-phosphoryl polyprenols in Mycobacterium smegmatis

ARCt|IVES OF BIOCHEMISTRY AND BIOPHYSICS

160, 311-322 (1974)

Biosynthesis of Mannosyl- and GlucosyI-Phosphoryl Polyprenols in Mycobacterium srnegmatis Evidence for Oligosaccharide-PhosphoryI-Polyprenols J O H N S C H U L T Z AND A L A N D. E L B E I N

Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78284 Received August 17, 1973 A particulate enzyme fraction from Mycobacterium smegmatis catalyzed the incorporation of mannose from GDP-[14C]mannose and glucose from UDP-[14C]glucose into endogenous lipid acceptors. The properties of the isolated [14C]glycolipids are similar to those reported for other glycosyl-phosphoryl-polyprenols. Synthesis of the glucolipid from UDP-[14C]glucose was reversed by the addition of UDP whereas synthesis of the mannolipid from GDP-p~C]mannose was reversed by the addition of GDP. In addition, the pm-ified [14C]mannolipid gave rise to GDP-p~C]mannose when incubated with the particulate enzyme in the presence of GDP and Mg~+. Formation of both glycolipids required Mg2+ with the optimum concentration for GDP-[~4C]mannose incorporation being about 10 mM. The Km for GDP-mannose was estimated to be about 5 X 10-6 Mand the pH optimum was 8.0. The enzyme was depleted of endogenous lipid by treatment with acetone; incorporation of radioactivity from sugar nucleotides into chloroform:methanol with this enzyme preparation was almost totally dependent on the addition of ficaprenyl phosphate. In addition to synthesis of mannosyl-phosphoryl-polyprenols, evidence is presented to show the presence of lipid-linked mannose oligosaccharides in the purified "mannolipid" fraction. Thus, mild acid hydrolysis of purified "mannolipid" gave radioactive compounds which migrated like disaccharides and trisaccharides as well as a radioactive material which remained at the origin of paper chromatograms. The disaccharide was shown to be a mannobiose in which the reducing sugar was not labeled. The role of lipid carriers as intermediates in extracellular polysaccharide synthesis is now well established (1). Thus, in the biosynthesis of peptidoglycan (2, 3), lipopolysaccharide O-antigen (4, 5), capsular polysaccharide (6), and teichoic acid (7), the polysaccharide repeating units are assembled on a polyisoprenol to form an oligosaccharide covalently bound to the lipid through a pyrophosphate linkage. I n addition to these lipid intermediates, Scheret al. (8) demonstrated the synthesis of mannosylmonophosphoryl-polyisoprenol in micrococci, and Lahav, Chin, and Lennarz (9) showed that this compound is the donor of the lateral mannosyl branches in the microCopyright 9 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

coccal mannan. Mannosyl-phosphoryl--decaprenol has also been synthesized in Mycobacterium tuberculosis (10). A glucosylphosphoryl-isoprenol has been synthesized in Salmonella and shown to be involved in lipopolysaccharide biosynthesis (11). Sugar-phosphoryl-p olyisoprenols have also been implicated in plant and animal systems (1). I n animal systems, these compounds appear to be involved in glycoprotein synthesis b u t their role in plants has not been established (see Discussion). The present report demonstrates the incorporation of mannose from GDP-[I~C]mannose and glucose from UDP-[i4C]glucose into m a n n o s y l - a n d glucosyl-phosphoryl 311

312

SCHULTZ AND ELBEIN

lipids by membrane preparations of M. smegmatis. In addition, evidence is presented to indicate that the enzyme preparation also forms mannose o]igosaccharides attached to polyprenyl-phosphate. MATERIALS AND METHODS

Preparation of particulate el,zyme. M. smegmalis was grown in Trypticase Soy B r o t h as previously described (12). Cell-free extracts were p r e p a r e d by sonic oscillation and cell debris was removed b y centrifugation at 10,000g. The s u p e r n a t a n t fraction was centrifuged overnight at 50,000g, and the pellet resulting from this centrifugation was homogenized in 0.01 M Tris buffer, p H 8.0, and used in the following experiments. This enzyme p r e p a r a t i o n generally had 100-150 mg of protein per ml. Assay of the enzyme. I n c u b a t i o n mixtures for assay of glycolipid f o r m a t i o n contained the following components on a final volume of 0.25 ml: GDP-[14C]mannose, 1 X i0 -4 ~moles (25,000 cpm), or UDP-[t4C]glucose, 2 X I0 -4 gmoles (60,000 cpm); MgCl~, 2.5 ~nloles; Tris buffer, pH 7.4, i0 ~moles, and an appropriate amount of enzyme (usually 10--25 /zl or 1-2.5 mg of particles). After incubation for 15 rain at 37~ the reaction was stopped by the addition of 2.5 ml of chloroform: methanol (C:M) (2:1), followed by 0.75 ml of water. The mixture was shaken well and the layers were separated by centrifugation. The lower layer was removed and placed in a clean tube and the upper layer was reextracted with i ml of C:M. The layers were again separated by ccntrifugation and the lower layer was removed and combined with the first extraction. The combined chloroform layers were extracted with C:M:H20 (3:48:47) and the upper layer was discarded. One milliliter of the chloroform layer was placed in a scintillation vial, dried, and counted in toluene scintillator to determine its radioactive content. In the case of mannolipid formation, only one radioactive lipid was found in these incubations (Rf = 0.3, solvent A) so that determination of radioactivity in the chloroform layer was a direct measure of its formation. However, in the case of the glucolipid several radioactive lipids were synthesized. Therefore, it was necessary to chromatograph incubation mixtures on Silica Gel plates to isolate the acidic glycolipid. The radioactive b a n d with an Rs of 0.30-0.4 in solvent A was scraped from the plates and counted.

Isolation and purification of radioactive lipids. I n order to obtain sufficient a m o u n t of radioactive glycolipid for c h a r a c t e r i z a t i o n and to use as a s u b s t r a t e in o t h e r reactions, assay mixtures were scaled up 50-100 times. R e a c t i o n mix-

tures were e x t r a c t e d w i t h C : M as described. T h e radioactive glycolipids were t h e n purified b y c h r o m a t o g r a p h y on DEAE-cellulose (acetate) as previously described (8). The lipids were placed on the column (2 X 35 cm) in C : M and the column was washed with 1 liter of C : M (2:1) followed b y 500 ml of methanol. I n the case of the mannolipid, less t h a n 5% of the radioactivity placed on t h e column was removed in these two washes, whereas w i t h the glucolipid the neutral [14C]glucolipids were removed in the wash. The columns were t h e n eluted with 0.1 • a m m o n i u m acetate in 99% methanol. Fractions of 15 ml were collected and tubes containing r a d i o a c t i v i t y were pooled, conc e n t r a t e d to dryness, t a k e n up in 40 ml of C : M , a n d extracted w i t h 16 ml of H20 to remove amm o n i u m acetate (10). The organic layer was removed, c o n c e n t r a t e d to dryness, and again ext r a c t e d w i t h w a t e r as described above. N i n e t y to ninety-five per cent of the r a d i o a c t i v i t y incorporated into C : M from G D P - m a n n o s e was recovered in t h e a m m o n i u m acetate fraction. T h e glycolipids in the a m m o n i u m acetate f r a c t i o n were subiccted to saponification in 0.1 N N a O H for 15 m i n a t 37~ as described (8) and were t h e n r e c h r o m a t o g r a p h e d on DEAE-cellulose. Chromatography. Thin-layer c h r o m a t o g r a p h y for lipids was done on Silica Gel G plates in t h e following solvent systems: (A) chloroform: m e t h a n o l : H 2 0 (65:25:4); (B) c h l o r o f o r m : m e t h a nol:acetic a c i d : H 2 0 (25:15:4:2); (C) chloroform: m e t h a n o l : a m m o n i u m hydroxide (75: 25: 4). Lipids were visualized w i t h iodine or w i t h rhodamine. Sugars were c h r o m a t o g r a p h e d on W h a t m a n 3 ~ p a p e r in the following solvent systems: (D) n-propanol:ethyl acetate:water (7:1:2) ; (E) n - b u t a n o l : p y r i d i n e : 0 . 1 N HC1 (5:3:2); (F) n - b u t a n o l : p y r i d i n e : H 2 0 (6:4:3). Sugars were located on papers w i t h alkaline silver n i t r a t e (13). Sugar nucleotides were c h r o m a t o g r a p h e d on W h a t m a n 3M~ p a p e r in the following solvents : (G) e t h a n o l : 1 M a m m o n i u m acetate, p H 7.4 (7:3) or (H) isobutyric acid: a m m o n i u m hydroxide : water (57:4:39). Sugar nucleot]des were located by t h e i r u l t r a v i o l e t absorption. R a d i o a c t i v e compounds were located w i t h a P a c k a r d Strip Scanner. Analytical methods. Hexose was determined b y the a n t h r o n e m e t h o d (14); p h o s p h a t e b y t h e m e t h o d of Chen et al (15); protein b y the Lowry m e t h o d (16).

Hydrolysis and isolation of monosaccharides from lipids. The purified glycolipids were hydrolyzed in the following way: a sample of t h e glycolipid in C : M was p i p e t t e d into a tube and dried u n d e r a s t r e a m of nitrogen. The lipid was suspended in 1 ml of 50% propanol and HC1 was added to the desired concentration (0.001-0.01 s ) . The tube was placed in a boiling water b a t h and

BIOSYNTHESIS OF POLYPRENOLS IN MYCOBACTERIUM SMEGMATIS aliquots (0.1 ml) were removed at the appropriate times and pipetted into tubes containing 1 ml of ice cold NaOH of sufficient concentration to neutralize the added HC1. Two and one-half milliliters of C:M (2:1) were then added, and the lipid was partitioned into the organic phase. The aqueous phase was removed and counted to determine the extent of hydrolysis. The aqueous phase was deionized with mixed-bed ion-exchange resin (Dowex 50-H+ and Dowex 1-COa=) and chromatographed to identify the sugars. Materials. GDP-p4C]mannose (150mCi/mmole) and UDP-[14C]glucosc (227 mCi/mnmle) were purchased from New England Nuclear Company. DEAE-cellulose was obtained from Sigma Chemical Company and was washed and converted to the acetate form as described by Rouser et al. (17). Distilled solvents were used in the final stages of purification. All other chemicals were reagent grade commercial preparations. Ficaprenyl phosphate and mannosyl-phosphorylficaprenol were kindly supplied by Drs. Warren and Jeanloz (18).

Reversal of reaction with purified [~4C]glycolipid and particulate enzyme. The glycolipid to be used as a substrate in these experiments was dried under a stream of nitrogen and taken up in an appropriate amount of methanol so that 10-t~l aliquots would contain sufficient radioactivity for the experiments. The lipid was incubated in 0.01 ~ Tris buffer, ptI 7.5, in the presence of 2 t,moles of MgCl~ and 100 ~l of p~rticulate enzyme in a final volume of 200 td. Nucleoside monophosphate or nueteoside diphosphate (0.4 ~mole) was added as indict.ted in the tables. After incubation for 15 min, 2.5 ml of C:M and 0.75 ml H~O were added, and the mixture was shaken. The layers were separated by centrifugation and the lower layer was removed and saved. The upper layer was extracted with 1 ml of C:M and the lower layer was combined with the first extraction. Radioactivity in the C:M was determined. The aqueous phase was spotted on Whatman 3MM and chromatographed in solvent G for isolation of sugar nucleotides. Sugar nucleotide areas were cut out and the papers were counted in toluene scintillator.

Acetone treatment of the particulate enzyme. In order to remove endogenous lipids from the enzyme preparation, the particulate enzyme was treated with acetone as described by Troy et al. (6). The enzyme was added with stirring to 40 vol of cold acetone (-20~ and was then centrifuged at -20~ The supernatant was removed and the pellet was dried in vacuo to remove any remaining acetone. The pellet was then resuspended in the original volume of 0.01 M Tris, pH 8.0, and gently homogenized. This enzyme prepa-

313

ration was then assayed in the same manner as the original enzyme preparation. However, in order for this acetone treated enzyme to show activity, it was necessary to add a lipid acceptor. Ficaprenyl-phosphate was able to serve as an acceptor of mannose and glucose with this enzyme fraction. Fieaprenyl-phosphate (kindly supplied by Drs. Warren and Jeanloz) was suspended either in 0.1% Triton X-100 or in methanol for addition to the enzyme. Methanol was actually a better solvent since it was not inhibitory as long as its final concentration was less than 10%. Triton X-100, on the other hand, was somewhat inhibitory. RESULTS

Synthesis of Mannolipid and Glucolipid and Reversal of Synthesis by Nucleoside Diphosphates W h e n the p a r t i c u l a t e e n z y m e f r a c t i o n from M. smegmatis was i n c u b a t e d w i t h G D P - [ l t C ] m a n n o s e or UDP-p4C]glucose, rad i o a c t i v i t y was i n c o r p o r a t e d i n t o C : M soluble products. F i g u r e 1 shows t h e effect of t i m e on the i n c o r p o r a t i o n of r a d i o a c t i v i t y from G D P - p 4 C ] m a n n o s e i n t o C : M . T h e r e a c t i o n was n o t s t r i c t l y p r o p o r t i o n a l t o t i m e p r o b a b l y because the synthesis of t h e lipid is reversed b y G D P (see below) a n d also because G D P - m a n n o s e a n d m a n n o lipid are used i n other reactions. I n t h e ease of G D P - m a n n o s e i n c o r p o r a t i o n , o n l y one lipid was formed i n these reactions, a n d this was the acidic " m a n n o l i p i d " described below. However, i n t h e case of t h e glucose incorporation, several r a d i o a c t i v e lipids were formed from UDP-[14C]glucose a n d only a small p r o p o r t i o n ( a b o u t 10%) of the r a d i o a c t i v i t y i n C : M was f o u n d to be i n the acidic glucolipid. Therefore, i n this case m e a s u r e m e n t of r a d i o a c t i v i t y inc o r p o r a t e d i n t o C : M was n o t a good indic a t i o n of acidic glucolipid f o r m a t i o n . React i o n m i x t u r e s h a d to be c h r o m a t o g r a p h e d to s e p a r a t e the glucolipid before d e t e r m i n i n g it's radioactive c o n t e n t . N o t o n l y was r a d i o a c t i v i t y from G D P [l*C]mannose i n c o r p o r a t e d i n t o lipid b u t it was also i n c o r p o r a t e d i n t o m a t e r i a l w h i c h was insoluble i n trichloroacetic acid (Fig. 2). T h e r a t e of i n c o r p o r a t i o n into t h e trichloroacetic, acid-insoluble materiM was m u c h lower t h a n t h a t into lipid a n d a p p e a r e d t a

314

SCHULTZ AND ELBEIN 4000"

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TIME OF INCUBATION ( MINUTES )

Fio. 1. Effect of time of incubation on the incorporation of radioactivity front GDP-[14C]man, Incubation mixtures were as described in the text and contained 10 td of the particulate enzyme (about 1-2 mg of protein). At the indicated times, samples were removed and lipids were partitioned into C : M as indicated in the text. Radioactivity in lipid was determined. h a v e a lag. T h i s is c o n s i s t e n t w i t h t h e i d e a t h a t t h e l i p i d m i g h t b e a p r e c u r s o r to t h i s insoluble m a t e r i a l . H o w e v e r , t h e r e l a t i o n ship b e t w e e n t h e s e t w o p r o d u c t s has n o t y e t b e e n established, n o r h a s t h e c h e m i c a l n a t u r e of t h e t r i c h l o r o a e e t i c a e i d - i n s o h i b l e m a t e r i a l b e e n established. As s h o w n in T a b l e I, t h e s y n t h e s i s of t h e g l u e o l i p i d could b e r e v e r s e d b y t h e a d d i t i o n of U D P w i t h t h e s u b s e q u e n t f o r m a t i o n of UDP@4C]glucose. In this experiment, the [~C]glueolipid w a s s y n t h e s i z e d f r o m U D P [~4C]glueose a n d t h e p a r t i c u l a t e e n z y m e in t h e first i n c u b a t i o n . T h e n , r e s i d u a l U D P [~4C]glueose was r e m o v e d b y c e n t r i f u g a t i o n a n d r e s u s p e n s i o n of t h e p a r t i c u l a t e e n z y m e w h i c h was i n c u b a t e d a second t i m e e i t h e r w i t h o u t a d d i t i o n or in t h e p r e s e n c e of U M P 'or U D P . I t can b e seen t h a t in t h e p r e s e n c e

of radioactivity

from

ODP-[14C]mannose into chloroform:methanolsoluble and triehloroaeetie acid-insoluble material. Incubations were as described in the text and contained 25 or 50 ~1 of particulate enzyme. Samples were withdrawn at the indicated times and the lipid was isolated by extraction with C:M. After removal of the lower phase, the upper phase was made 10~o with respect to trichloroacetic acid and the tubes were placed in the cold overnight. The precipitate was isolated by eentrifugation, washed several times with 10~0 triehloroaeetie acid, and its radioaetive content was determined. Legend is as follows: Incorporation into lipid with 25 tL1 (O O) and 50 ul ( [ 3 - - [ 3 ) of enzyme; incorporation into triehloroaeetie acid-insoluble material with 25 td (O ..... 9 ) and 50 ;~1 (A ..... A) of enzyme. of U D P , t h e r e was a significant d e c r e a s e in r a d i o a c t i v i t y in t h e g l u c o l i p i d w i t h a corr e s p o n d i n g increase in t h e a m o u n t of UDP-[14C]glucose. T h e U D p @ 4 C ] g l u e o s e in t h e c o n t r o l t u b e s p r o b a b l y r e p r e s e n t s residu a l U D P @ 4 C ] g l u c o s e w h i c h was n o t removed by eentrifugation. S o m e w h a t s i m i l a r results were o b s e r v e d w h e n t h e r e v e r s a l of t h e m a n n o l i p i d was s t u d i e d ( T a b l e I I ) , a l t h o u g h in t h i s case t h e r e a c t i o n was n o t as d e p e n d e n t on a d d i t i o n of nueleotide. I n t h e s e e x p e r i m e n t s t h e purified [14C]mannolipid was i n c u b a t e d w i t h the particulate enzyme either without addit i o n or in t h e p r e s e n c e of G M P or G D P .

315

BIOSYNTHESIS OF POLYPRENOLS IN MYCOBACTERIUM SMEGMATIS

REVERSAL

TABLE I OF GLUCOLIPID ACCUMULATION BY ADDITION OF UDP

Additions to 2nd incubation~

Radioactivity in

TABLE II FORMATION OF G D P - [ 1 4 C ] M A N N O S E FROM [14C]MANNOLIPIDa

Additions to incubation

Glucolipid UDP-[14C]glu

Radioactivity in C: M

GDPiilanilose

Expt 1 None b None UMP UDP Expt 2 None b None UMP UDP

2409 2303 2294 421

987 1006 832 23O8

Not incubated None GMP GDP

3056 2992 3056 1000

2044 2011 2881 3449

Purified [14C]mannolipid was suspended in 0.1% Triton X-100. Aliquots containing 3000 cpm were incubated with enzyme (100 gl), 12.5 gmoles of Tris buffer, pH 7.5, 5.0 gmoles of MAC12, and 1 gmole of GDP or GMP in a final volume of 0.5 ml. At the end of the incubation lipids were partitioned into C:M. Aqueous phase was chromatographed in solvent G to isolate GDP-man. Radioactivity in C:M and in GDP-man was determined.

In the first incubation, particulate enzyme (200 X) was incubated with 0.2 gmoles MgCI~, 7.5 gmoles Tris buffer, pI-I 7.5, 2 X 10-4 gmoles UDP-[14C]glu (60,000 cpm) for 10 min. Particulate enzyme was then isolated by centrifugation, resuspended, and incubated for an additional 10 min with or without additions as shown. After the second incubation, mixtures were extracted with chloroform:methanol to separate lipids. The C:M was chromatographed in solvent A to isolate glucolipid and the aqueous phase in solvent G to isolate UDP-glucose. Radioactivity in these compounds was determined. b Not incubated (i.e., no second incubation).

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3110 1994 1965 1505

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Some reversal (i.e., some G D P - [ l ~ C ] m a n nose) was observed even i n t h e absence of nucleotide, a l t h o u g h G D P a n d to a lesser e x t e n t G M P s t i m u l a t e d this reversal. T h e reversal i n t h e absence of a d d e d n u c l e o t i d e a p p e a r s to be due to t h e presence of endogenous G D P i n t h e p a r t i c u l a t e enzyme. Thus, a compound migrating with authentic G D P i n solvents G a n d H a n d h a v i n g a guanosine ultraviolet absorption spectrum was isolated from t h e p a r t i c u l a t e e n z y m e b y e x t r a c t i o n w i t h 5 % trichloroacetic acid.

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FIG. 3. Effect of Mg ~+ concentration on the incorporation of radioactivity into C:M. Reactions were as described in the text except that Mg ~+ was varied.

T h e i n c o r p o r a t i o n of m a n n o s e i n t o t h e m a n n o l i p i d was s t r o n g l y d e p e n d e n t on t h e presence of M g 2+ as s h o w n i n Fig. 3. T h e o p t i m u m c o n c e n t r a t i o n of M g 2+ was a r o u n d 10 m~r. T h e effect of G D P - m a n n o s e conc e n t r a t i o n is s h o w n i n Fig. 4. T h e a p p a r e n t Km for G D P - m a n n o s e was a b o u t 5 • 10 -6

M. T h e p H o p t i m u m for m a n n o s e i n c o r p o r a t i o n i n t o C : M was a b o u t 8.0 i n T r i s m a l e a t e buffer (Fig. 5). W e h a v e n o t b e e n able to do these e x p e r i m e n t s w i t h glucolipid s y n t h e s i s because of the fact t h a t more t h a n 90 % of t h e a c t i v i t y i n c o r p o r a t e d i n t o C : M

Properties

316

SCHULTZ AND ELBEIN from UDP-[14C]glucose is into other lipids. However, formation of the acidic glucolipid was dependent on Mg 2+ and had a p H optimum of around 8.0.

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Effect of the Addition of Ficaprenyl-Phosphate

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FIG. 4. Effect of GDP-mannose concentration on the incorporation of radioactivity into C:M. Reactions were as described in the text except that GDP-mannose was varied.

6000-

When the particulate enzyme was treated with acetone as described b y T r o y et al. (6), it was no longer able to catalyze the incorporation of radioactivity from G D P [14C]mannose into C : M . As shown in Fig. 6, the addition of ficaprenyl-phosphate to the enzyme preparation resulted in the restoration in the incorporation of radioactivity into C : M . T r e a t m e n t of the enzyme with acetone resulted in considerable loss of enzymatic activity which could not be totally restored even by the addition of ficaprenyl-phosphate. However, this enzyme preparation was useful for demonstrating the requirement for ficaprenylphosphate as a mannose acceptor. Ficaprenyl-phosphate also stimulated the incorporation of radioactivity from U D P [~4C]glucose into the phospholipid fraction. On the other hand, dolichyl-phosphate did not work as an acceptor lipid with the acetone-treated enzyme. The radioactive product formed from GDP-[~C]mannose and ficaprenyl-phosphate was extracted with C : M and purified on DEAE-cellulose. This radioactive lipid had identical properties to the mannolipid formed from GDPmannose with endogenous lipid acceptors.

4000-

Isolation and Characterization of Glycolipids

2000-

FIG. 5. Effect of pH oi1 the incorporation of radioactivity from GDP-mannose into C:M. Reactions were as described in the text except that pH was varied as indicated. Tris buffer was used in all cases.

The mannolipid formed in a large-scale incubation with GDP-[14C]mannose (4 X 106 epm) and the particulate enzyme or t h e glucolipid formed from UDP-[l~C]glucose (2 • 106 cpm) were purified as described b y Scher et al. (20). This involved a preliminary separation on DEAE-cellulose, followed b y saponification and a second chromatography on DEAE-cellulose. In some cases the second DEAE-cellulose column was eluted with a linear gradient of ammonium acetate in 99 % methanol whereas in other cases it was eluted with 0.1 ~ ammonium acetate in 99 % methanol. As shown in Fig. 7 for the mannolipid, the

BIOSYNTHESIS OF POLYPRENOLS IN MYCOBACTERIUM SMEGMATIS

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FIG. 6. Effect of the addition of ficaprenyl-P on the incorporation of mannose by the acetone-treated enzyme. Ficaprenyl-P, suspended in 0.1% Triton X-100, was added to the enzyme as indicated. Incubation mixtures were as indicated in the text and contained GDP-[14C]man. After 15-rain incubation, incorporation of radioactivity into C:M was determined.

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ELUATE

FIG. 7. Purification of mannolipid on DEAE-cellulose cohmm. Conditions were as described in the text. After washing the column with C:M and methanol, the lipid was eluted with 0.1 • ammonium acetate in 99% methanol. Fractions of 15 ml were collected, and radioactivity in an aliquot of each fraction was determined. g l y e d i p i d s e l u t e d in a f a i r l y s y m m e t r i c a l p e a k w h e n e l u t e d w i t h 0.1 ~, a m m o n i u m a c e t a t e . I n t h e ease of t h e m a n n o l i p i d , a p p r o x i m a t e l y 1.5 X 106 c p m were incorp o r a t e d i n t o C : M f r o m 4 X 10 G e p m of GDP-[t4C]mannose, a n d 1.2 X 106 e p m were r e c o v e r e d in t h i s p e a k off of D E A E cellulose. I n t h e ease of t h e glueolipid,

a b o u t 1 X 10 ~ c p m were i n c o r p o r a t e d i n t o C : M f r o m 2 X 106 c p m of U D P - [ I 4 C ] g l u eose a n d 10,000 e p m were f o u n d in t h e purified p e a k f r o m D E A E - e e l l u l o s e . T h e m a n n o l i p i d a n d t h e glueolipid h a d m o b i l i t i e s on t h i n - l a y e r c h r o m a t o g r a p h y s i m i l a r to t h o s e p r e v i o u s l y r e p o r t e d (8, 19, 20) for 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 s

318

SCHULTZ AND ELBEIN

from other sources. Thus, as shown in Table III, both the glucolipid and the mannolipid migrated rapidly in an acidic solvent (R/ = 0.76), slowly in a basic solvent (RI --- 0.11), and had intermediate mobility in a neutral solvent (RI = 0.31). Synthetic mannosyl - phosphoryl - ficaprenol, kindly supplied b y Drs. Warren and Jeanloz (18) showed identical mobilities in these three solvents to the biosynthesized mannolipid and glucolipid. Ficaprenyl-P (also supplied b y Drs. Warren and Jeanloz) migrated the same as the other lipids in the neutral solvent but had a faster mobility in the acidic solvent and a slower mobility in the basic solvent (Table III). Some differences in mobility of these lipids was observed from time to time suggesting that migration m a y depend on concentration of lipid, TABLE III THIN-LAYER

CHROMATOGRAPHIC VARIOUS

MOBILITIES

OF

LIPIDS

Lipid A [14C]Mannolipid [14C]Glucolipid Synthetic mannosyl phosphoryl-ficaprenol Fic aprenyl-P

R/in solvent B C

0.31 0.29 0.31

0.76 O.77 0.75 .: 0.91

0.38

0.11 0.09 0.10 0.02

time allowed for equilibration of solvent, a n d / o r amount of salt present in the sample. However, in general, mobilities were as shown in Table I I I and in all cases the three glycolipids ran together. The sugar portions of both mannolipid and glucolipid were very susceptible to acid hydrolysis as shown in Fig. 8. Thus, in 0.01 N HC1 at 100~ 50 % of the glucolipid was hydrolyzed in 1 rain whereas at the lowest acid strength tested (0.001 N) complete hydrolysis occurred in 7 min at 100~ Similar results were seen for the mannolipid and have previously been reported for mannosyl-phosphoryl-decaprenol from M. tuberculosis (10). The sugars released from these lipids were identified by paper chromatography in solvents D, E, and F; the major radioactive sugar in the mannolipid was mannose, but smaller amounts of larger oligosaccharides were also found (see below). The only radioactive sugar detected in the glucolipid was glucose but since considerably less radioactive glucolipid was available for hydrolysis, oligosaccharides m a y not have been detected even if present.

Evidence for Oligosaccharide-PhosphorylPolyprenols Although the major radioactive sugar released from the mannolipid b y mild acid hydrolysis was mannose, smaller

150

I00-

f

50-

o

i

~

t

3

g

~

i

6

~

n

6

t

9

,'o

TIME ( M i n u t e s )

FIG. 8. Acid hydrolysis of glucolipid. Lipid was placed in 0.01 • HC1 at 100~ Samples were removed at the indicated times and neutralized, and lipids were partitioned into the C:M phase. Radioactivity in the aqueous phase was determined.

BIOSYNTHESIS OF POLYPRENOLS IN MYCOBACTERIUM SMEGMATIS amounts of other radioactive peaks which migrated like oligosaccharides were also observed on paper chromatograms as shown in Fig. 9. Tracing A shows the water-soluble components released from the purified lipid fraction by mild acid hydrolysis. The peak which migrated near trehalose and was presumably a disaecharide contained as much as 15 % of the total radioactivity in the purified lipid whereas the slower peak (migrating like a trisaceharide) contained 1-2 % of the total activity. Small amounts of radioactivity (0.5 % of the total) were also observed at or near the origin of the ehromatograms. The relative proportions of radioactivity in these various compounds varied greatly from one lipid preparation to another and apparently depended on the condition used in the synthesis of the mannolipid (i.e., length of incubation, amount of enzyme, etc.). This is shown in Fig. 9, tracings B and C, where it can be seen that when the purified mannolipid preparation (Fig.

9A) was incubated with the particulate enzyme, radioactivity disappeared from each of the oligosaccharide areas suggesting that it might be incorporated into polymer. The transfer of radioactivity from lipid to polymer has not yet been demonstrated and is only speculative. Degradation of the lipid or reversibility of synthesis could also account for this decline in activity. In fact, as shown in Fig. 10 the radioactivity in the lipid-linked disaccharide was apparently more labile or turned over faster than the radioactivity in the mannosyl-phosphorylpolyprenol. In this experiment, "mannolipid" was incubated with the particulate enzyme and at various times samples were withdrawn and the lipid was extracted with chloroform:methanol, subjected to mild acid hydrolysis to release the sugars and paper chromatography of the aqueous phase to isolate the sugars. Radioactivity in each of the oligosaccharides was detected with a Packard strip scanner and quantitated in a liquid scintillation counter. Radioactivity in the larger oligosaccharides

3,500

z0 25,000

~900

AO00

700.

5,000 t O

Fro. 9. Radioactive scans of the sugars present in the mannolipid fraction after incubation with the particulate enzyme. "Mannolipid" (50,000 cpm) was suspended in 0.025 ml methanol and incubated with 100 ~,1 of particulate enzyme in 200 ~1 of 0.05 g Tris buffer, pH 7.0. At 0 time (scan A), 15 min (scan B), or 30 min (scan C) each reaction mixture was extracted with chloroform:methanol to reisolate the lipid. The lipid was then subjected to mild acid hydrolysis and the sugars chromatographed in solvent D.

319

15

"L. 50

45

60

.

75

90

TIME(MINUTES)

Fro. tO. "Mannolipid" (50,000 cpm) was incubated with 100 ~1 of particulate enzyme as

described in Fig. 9. At the times indicated the lipid was extracted with chloroform:methanol and the radioactive sugars were released by mild acid hydrolysis and isolated by paper chromatography. Papers were scanned with a Packard strip scanner and radioactivity in the various oligosaccharides was quantitated by liquid scintillation counting.

320

SCHULTZ AND ELBEIN

appeared to disappear at approximately the same rate as that in the disaceharide, b u t since these compounds had much lower activity to begin with, the data must be considered to be less reliable. Although it was not possible to obtain sufficient amounts of the larger lipid-linked oligosaccharides for complete characterization, the disaccharide was obtained in sufficient amounts for partial eharacterization. The disaceharide was eluted from the paper and rechromatographed in solvents E and F. I t was tentatively characterized as a mannobiose on the basis of the following evidence. As shown in Fig. 11, complete acid hydrolysis in 2 N HC1 at 100~ for 2 hr gave rise to one radioactive and one silver-staining compound which migrated with authentic mannose. Even after NaBH4 reduction of the disaceharide and complete acid hydrolysis, only radioactive mannose

was observed (Fig. 11), indicating that the reducing end of the disaccharide was either not labeled or contained much less radioactivity. This suggests that the disaccharide is formed by transfer of mannose from GDP[14C]mannose to endogenous mannosylphosphoryl-polyprenol. The mannose and mannitol areas of the chromatogram were eluted, acetylated (after NaBH4 reduction of the mannose area), and quantitated by gas-liquid chromatography. Approximately equal amounts of mannose and mannitol were detected. Finally, the hexose (by anthrone using a mannose standard) to reducing sugar ratio in the intact disaeeharide was approximately 2:1. DISCUSSION

Glycolipids of the type described in this report have been synthesized in a number of tissues including bacteria, mammalian cells, and plants. In general, sugar-phosphorylpolyprenols appear to be involved either in the formation of lateral branches on polysaccharide chains or in glycoprotein biosynthesis. Thus, Lahav et al. (9) and Scher and Lennarz (20) showed that in microcoeci mannosyl-phosphoryl-undecaprenol transferred mannose units only to the nonreducing termini of endogenous mannan so t h a t this intermediate appeared to be involved in completion of mannan synthesis. Wright (11) demonstrated a similar function for glueosyl-phosphoryl-undecaprenol in the synthesis of Salmonella lipopolysaccharide. The lipid intermediate was found to be involved in the formation of branches in this polymer. Behrens and Leloir (21) showed t h a t glucosyl-phosphoryI-doliehol 0 84 O ........ was a precursor for a glucoprotein in r a t liver microsomes. Baynes and H e a t h (19) found a similar function for a mannosyli ; ~ i ~ ; :~I~ phosphoryl-doliehol synthesized in rat liver. FIG. 11. Radioactive scans of the sugars re- Retinol monophosphate galactose has also leased from the purified disaccharide by various been shown to serve as an intermediate in chemical treatments. Scan A, untreated disac- glycoprotein synthesis in mouse mastoeycharide; scan B, disaccharide treated with 2 N toma particulate preparations b y Alam tiC1 at 100~ for 2 hr; scan C, disaccharide treated with NaBH4 and then hydrolyzed in 2 N HC1 at et al. (22). Various other workers have demonstrated 100~ for 2 hr. In this solvent, glucose migrated slightly behind mannitol (R mannitol = 0.97) the synthesis of glycosyl-phosphoryl-polywhereas sorbitol ran slower than glucose (R glu = prenols in other organisms, but in these 0.93). cases a function for the lipid has not yet

321

BIOSYNTHESIS OF POLYPRENOLS IN MYCOBACTERIUM SMEGMATIS

been demonstrated. Takayama and Goldman (10) showed the synthesis of mannosyl-phosphoryl-undecaprenol by extracts of M. tuberculosis. A mannolipid with similar properties was synthesized in mung bean seedlings by Villemez and Clark (23), Villamez (24), and Kauss (25). In these cases the lipid moiety was not identified. However, these lipids are probably polyprenols since Alan and Hemming (26) showed that the addition of betulaprenolphosphate to extracts of mung beans stimulated the incorporation of mannose from GDP-mannose into chloroform:methanol. Forsee and Elbein (27) demonstrated the synthesis of glucosyl and mannosyl-phosphoryl-polyprenols in extracts of cotton fibers. Ficaprenyl-phosphate was able to serve as a glycosyl acceptor in this system (28). Sentandreu and Lampen have recently reported the synthesis of mannosylphosphoryl-polyprenol in yeast (29). It seems likely that these glycolipids will also be found to be involved in polymer synthesis but in what capacity remains to be established. In addition to monosaccharide-phosphoryl-polyprenols, the particulate enzyme from M. smegmatis also forms oligosaccharides containing mannose which are linked to the lipid. Since the mannose moiety at the reducing end of the disaccharide is apparently unlabeled, these oligosaccharides appear to arise by transfer of mannose from GDP-mannose to endogenous mannosyl-phosphoryl-polyprenol. Parodi et al. (30) have shown that rat liver microsomes catalyze the synthesis of oligosaccharides of glucose attached to dolichyl-phosphate or dolichyl pyrophosphate apparently from [14C]ghicosyl--phosphoryl-dolichyl. In this case, reduction of the labeled oligosaccharide with NaBH4 followed by acid hydrolysis gave rise to radioactive glucose but no sorbitol indicating that the labeled glucose is not incorporated at the reducing end of the oligosaccharide. Although the role of the mannosyl lipids of M. smegmatis is not known at this time, it should be pointed out that mycobacteria produce a number of cell wall polysaccharides, one of which is an arabinomannan.

Thus, these lipids could be precursors for this polysaccharide. ACKNOWLEDGMENT This work was supported by Grant AI09402 from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. LENNARZ, W. J., AND SCHER, M. Biochim. Biophys. Acta 265,417.

K.

(1972)

2. ANDERSON, J. S., MATSUKASHI,M., HASKIN~ M. i . , AND STROMINGER,J. L. (1965) Proc. Nat. Acad. Sci. U S A 53, 881. 3. ANDERSON, J. S., MATSUKASHI,M., I~ASKIN, M. A., AND S~IROMINGER, ft. L. (1967) 3". Biol. Chem. 242, 3180. 4. WEINnR, I. M., HIGVCm, T., ROTHFIELD, L., SALTMARSH-ANDRE~V,M., OSBORN, M. J., AND HORECKER, B. L. (1965) Proc. Nat. Aead. Sci. U S A 54, 228. 5. WRIGHT, A., DANKERT, M., AND ROBBINS, P. W. (1965) Proe. Nat. Acad. Sci. U S A 54, 235. 6. TROY, F. A., FREEMAN, F. E., AND HEATH, E. C. (1971) J. Biol. Chem. 246, 118. 7. HussEu H., ANDBADDILEY,J. (1972) Biochem. J. 127, 39. 8. SCHER, M., LENNARZ, W. J., AND SWEELEY, C. C. (1968) Proc. Nat. Acad. Sci. U S A 59, 1313. 9. LAHAV,M., CHIU, W. H., AND LENNARZ,W. J. (1969) J. Biol. Chem. 244, 5890. 1O. TAKAYAMA,K. AND GOLDMAN, D. S. (1970) J. Biol. Chem. 245, 6251. 11. WRIGHT,A. (1971) J. Bacteriol. 105,927. 12. LAPp, D. F., PAT'rERSON,B. W., AND ELREIN, A. D, (1971) J. B w l . Chem. 246, 4567. 13. TREVELYAN, W. E., PROCTER, D. P., AND HARRISON, J. S. (1950) Nature (London) 166, 444. 14. LoEwus, F. (1952) Anal. Chem. 24, 219. 15. CHEN, P. S., TORIBARA,T. Y., AND WARNER, H. (1956) Anal. Chem. 28, 1756. 16. LowRY, D. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. 17. ROUSER, G., KRITCHEVSKY, G., HELI~ER, D., AND LEIBER, E. (1963) J. A m er. Oil Chem. Soc. 40, 425. 18. WARREN, C. D., AND JEANLOZ, R. W. (1972) Biochemistry 11, 2565. 19. BAYNES, J. W., AND HEATH, E. C. (1972) Fed. Proc. 31, 1239. 20. SCHER, M., AND LENNARZ, W. J. (1969) J. Biol. Chem. 244, 2777.

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21. BEHRENS, N. H., AND LELOIR, L. H. (1970) Proe. Nat. Acad. Sci. 66, 153. 22. ALAM, S. S., BARS, M., RICHARDS, J. B., AND HEMMING, •. W. (1971) Biochem. J. 121, 198. 23. V1LLAMaZ, C. L., AND CLANK, A. F. (1969) Biochem. Biophys. Res. Commun. 36, 57. 24. V1LLAMEZ, C. L. (1970) Biochem. Biophys. Res. Commun. 40, 636. 25. KAr~ss, H. (1969) Fed. Eur. Biochem. Soc. Left. 5, 81.

26. ALAM, S. S., AND HEMMING, F. W. (1971) Fed. Eur. Biochem. Soc. Lett. 19, 60. 27. FORSEE, W. T., AND ELBEIN, A. D. (1972) Biochem. Biophys. Res. Commun. 49, 930. 28. Fons~E, W. T., AND ELBEIN, A. D. (1973) J. Biol. Chem. 248, in press. 29. SENTANDREU, •., AND LAMPEN, J. O. (1972) Fed. Eur. Biochem. Soc. Lett. 27, 331. 30. PARODI, A. J., STANELONI, R., CANTARELLA, A. I., LELOI~, L. F., BE~nENS, N. H., CARMINATTI, H., AND LEVY, J. R. (1973) Carbohydrate Res. 26, 393.