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 SCHULTZ AND ALAN 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 in- corporation 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 Mg 2+ 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 M and the pH optimum was 8.0. The enzyme was depleted of en- dogenous 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 pre- sented 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 bio- synthesis of peptidoglycan (2, 3), lipopoly- saccharide O-antigen (4, 5), capsular poly- saccharide (6), and teichoic acid (7), the polysaccharide repeating units are assembled on a polyisoprenol to form an oligosac- charide covalently bound to the lipid through a pyrophosphate linkage. In addi- tion to these lipid intermediates, Scheret al. (8) demonstrated the synthesis of mannosyl- monophosphoryl-polyisoprenol in micro- cocci, and Lahav, Chin, and Lennarz (9) showed that this compound is the donor of the lateral mannosyl branches in the micro-

Copyright �9 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

coccal mannan. Mannosyl-phosphoryl--dec- aprenol has also been synthesized in Myco- bacterium tuberculosis (10). A glucosyl- phosphoryl-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). In animal systems, these com- pounds appear to be involved in glycoprotein synthesis but their role in plants has not been established (see Discussion).

The present report demonstrates the in- corporation 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 E L B E I N

lipids by membrane preparations of M. smegmatis. In addition, evidence is pre- sented to indicate that the enzyme prepa- ration also forms mannose o]igosaccharides attached to polyprenyl-phosphate.

M A T E R I A L S AN D M E T H O D S

Preparation of particulate el,zyme. M. smegmalis was grown in Trypt icase Soy B r o t h as previously described (12). Cell-free ext racts were prepared by sonic oscil lat ion and cell debris was removed by centr i fugat ion at 10,000g. The supe rna tan t f ract ion was centr i fuged overnight at 50,000g, and the pel let resul t ing from this cent r i fugat ion was homogenized in 0.01 M Tris buffer, pH 8.0, and used in the following experiments. This en- zyme prepara t ion generally had 100-150 mg of prote in per ml.

Assay of the enzyme. Incuba t ion mixtures for assay of glycolipid format ion conta ined the fol- lowing 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 ccntrif- ugation 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 radio- active band wi th an Rs of 0.30-0.4 in solvent A was scraped from the plates and counted.

Isolation and purification of radioactive lipids. In order to obta in sufficient amount of radio- active glycolipid for charac ter iza t ion and to use as a subs t ra te in o ther react ions, assay mix- tures were scaled up 50-100 times. Reac t ion mix-

tures were ex t rac ted wi th C : M as described. The radioact ive glycolipids were then purified by chromatography on DEAE-cel lu lose (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 l i ter of C : M (2:1) followed by 500 ml of methanol . In the case of the mannol ipid , less t han 5% of the radioact iv i ty placed on the column was removed in these two washes, whereas wi th the glucolipid the neut ra l [14C]glucolipids were removed in the wash. The columns were then eluted wi th 0.1 • ammonium aceta te in 99% methanol . Fract ions of 15 ml were collected and tubes conta ining rad ioac t iv i ty were pooled, con- cen t ra ted to dryness, t aken up in 40 ml of C :M, and ext rac ted wi th 16 ml of H20 to remove am- monium aceta te (10). The organic layer was re- moved, concent ra ted to dryness, and again ex- t r ac t ed wi th water as described above. N i n e t y to ninety-f ive per cent of the rad ioac t iv i ty in- corpora ted into C : M from GDP-mannose was recovered in the ammonium aceta te fract ion. The glycolipids in the ammonium aceta te f rac t ion were subiccted to saponification in 0.1 N NaOH for 15 min a t 37~ as described (8) and were then rechromatographed on DEAE-cel lulose.

Chromatography. Thin- layer ch romatography for lipids was done on Silica Gel G plates in the following solvent systems: (A) chloroform: me thano l :H20 (65:25:4); (B) ch loroform:metha- nol :acet ic ac id :H20 (25:15:4:2); (C) chloroform: methano l : ammonium hydroxide (75: 25: 4). Lipids were visualized wi th iodine or wi th rhodamine . Sugars were chromatographed on W h a t m a n 3 ~ paper in the following solvent systems: (D) n -p ropano l : e thy l ace t a t e :wa te r (7:1:2) ; (E) n -bu tano l :pyr id ine :0 .1 N HC1 (5:3:2); (F) n -bu ta - no l :py r id ine :H20 (6:4:3). Sugars were located on papers wi th alkaline silver n i t ra te (13). Sugar nucleotides were chromatographed on W h a t m a n 3M~ paper in the following solvents : (G) e thanol : 1 M ammonium acetate , pH 7.4 (7:3) or (H) isobutyric acid: ammonium hydroxide : water (57:4:39). Sugar nucleot]des were located by the i r u l t rav io le t absorpt ion. Radioac t ive compounds were located wi th a Packard Strip Scanner.

Analytical methods. Hexose was determined by the an throne method (14); phospha te by the me thod of Chen et al (15); prote in by the Lowry method (16).

Hydrolysis and isolation of monosaccharides from lipids. The purified glycolipids were hy- drolyzed in the following way: a sample of the glycolipid in C : M was p ipe t ted into a tube and dried under a s t ream of ni trogen. The l ipid was suspended in 1 ml of 50% propanol and HC1 was added to the desired concent ra t ion (0.001-0.01 s ) . The tube was placed in a boil ing water b a t h and

BIOSYNTHESIS OF POLYPRENOLS IN MYCOBACTERIUM SMEGMATIS 313

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 de- termine 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 (150 mCi/mmole) and UDP-[14C]glucosc (227 mCi/mnmle) were purchased from New England Nuclear Company. DEAE-cellulose was obtained from Sigma Chemi- cal 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. Fica- prenyl phosphate and mannosyl-phosphoryl- ficaprenol 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 monophos- phate 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 centri- fuged at -20~ The supernatant was removed and the pellet was dried in vacuo to remove any remaining acetone. The pellet was then resus- pended in the original volume of 0.01 M Tris, pH 8.0, and gently homogenized. This enzyme prepa-

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 inhibi- tory.

RESULTS

Synthesis of Mannolipid and Glucolipid and Reversal of Synthesis by Nucleoside Di-

phosphates

W h e n the par t icu la te enzyme f rac t ion from M. smegmatis was incuba t ed wi th GDP-[ l tC]mannose or UDP-p4C]glucose, ra- d ioac t iv i ty was incorpora ted in to C : M soluble products . F igure 1 shows the effect of t ime on the incorpora t ion of rad ioac t iv i ty f rom GDP-p4C]mannose in to C : M . The react ion was no t s t r ic t ly propor t ional to t ime p robab ly because the synthesis of the l ipid is reversed b y G D P (see below) a nd also because G D P - m a n n o s e and m a n n o - l ipid are used in other reactions. I n the ease of G D P - m a n n o s e incorporat ion, on ly one lipid was formed in these react ions, and this was the acidic " m a n n o l i p i d " de- scribed below. However, in the case of the glucose incorporat ion, several radioact ive lipids were formed from UDP-[14C]glucose and only a small p ropor t ion (about 10%) of the rad ioac t iv i ty in C : M was found to be in the acidic glucolipid. Therefore, in this case measu remen t of rad ioac t iv i ty in- corporated in to C : M was no t a good indi- ca t ion of acidic glucolipid format ion. Reac- t ion mixtures had to be chromatographed to separate the glucolipid before de te rmin ing i t ' s radioact ive content .

N o t only was rad ioac t iv i ty f rom G D P - [l*C]mannose incorporated in to lipid b u t i t was also incorporated in to mater ia l which was insoluble in tr ichloroacetic acid (Fig. 2). The ra te of incorpora t ion into the tr ichloro- acetic, acid-insoluble materiM was m u c h lower t h a n t h a t into l ipid and appeared t a

314 SCHULTZ AND ELBEIN

4 0 0 0 "

~ 3 0 0 0 - o

o z

cr o 2000 -

>

c~

I 000 -

5 1 0 115 210

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 indi- cated times, samples were removed and lipids were partitioned into C:M as indicated in the text. Radioactivity in lipid was determined.

have a lag. Th is is cons i s ten t w i th t he idea t h a t t he l ip id m i g h t be a p recursor to th is insoluble mate r i a l . However , t he re la t ion- ship be tween these two p roduc t s has no t y e t been es tabl ished, nor has t he chemica l n a t u r e of t he t r i ch lo roaee t i c ae id- insohib le m a t e r i a l been es tabl ished.

As shown in T a b l e I , t he syn thes i s of t he g lueol ip id could be reve r sed b y the a d d i t i o n of U D P wi th t he subsequen t f o r m a t i o n of UDP@4C]glucose . I n th is exper iment , t he [~C]glueolipid was syn thes ized f rom U D P - [~4C]glueose a n d the p a r t i c u l a t e enzyme in t he first i ncuba t ion . Then , res idua l U D P - [~4C]glueose was r e m o v e d b y cen t r i fuga t ion and resuspens ion of t he p a r t i c u l a t e enzyme which was i n c u b a t e d a second t ime e i ther w i t h o u t a d d i t i o n or in t h e presence of U M P 'or U D P . I t can be seen t h a t in t h e presence

o

2 0 0 0 / , ~ '''~ o 50X

- - ~ d f t >" /

"- j j

/ / 25,X ~ / "

:~ lO00 ~ d.'" I~ / s.

/ / �9 ~J

,o ~'o 3 ' ~ 5'0 do T I M E (min)

FIG. 2. Incorporat ion of rad ioact iv i ty f rom ODP-[14C]mannose into chloroform:methanol- soluble and triehloroaeetie acid-insoluble ma- terial. 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 trichloro- acetic acid and the tubes were placed in the cold overnight. The precipitate was isolated by eentrif- ugation, washed several times with 10~0 tri- ehloroaeetie 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 , the re was a s ignif icant decrease in r a d i o a c t i v i t y in the g lucol ip id w i t h a cor- r e spond ing increase in the a m o u n t of UDP-[14C]glucose. T h e UDp@4C]g lueose in t h e cont ro l tubes p r o b a b l y represen t s res id- ua l UDP@4C]g lucose which was no t re- m o v e d b y een t r i fuga t ion .

S o m e w h a t s imi lar resul ts were obse rved when the reversa l of t he ma nno l ip id was s t ud i e d (Tab le I I ) , a l t hough in th is case t he r eac t ion was no t as d e p e n d e n t on add i - t i on of nueleot ide . I n these exper imen t s t h e pur i f ied [14C]mannolipid was i n c u b a t e d w i th t h e p a r t i c u l a t e enzyme e i ther w i thou t add i - t i on or in t h e presence of G M P or G D P .

BIOSYNTHESIS OF POLYPRENOLS IN MYCOBACTERIUM SMEGMATIS 315

REVERSAL

TABLE I

OF GLUCOLIPID ACCUMULATION BY ADDITION OF UDP

TABLE II

FORMATION OF GDP-[14C]MANNOSE FROM

[14C]MANNOLIPIDa

Additions to 2nd Radioactivity in Additions to incubation Radioactivity in incubation ~

Glucolipid UDP-[14C]glu

Expt 1 None b 2409 987 None 2303 1006 UMP 2294 832 UDP 421 23O8

Expt 2 None b 3056 2044 None 2992 2011 UMP 3056 2881 UDP 1000 3449

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, re- suspended, 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 com- pounds was determined.

b Not incubated (i.e., no second incubation).

Some reversal (i.e., some GDP-[ l~C]man - nose) was observed even in the absence of nucleotide, a l though G D P and to a lesser ex tent G M P s t imula ted this reversal. The reversal in the absence of added nucleot ide appears to be due to the presence of en- dogenous G D P in the par t icu la te enzyme. Thus , a compound migra t ing wi th au then- tic G D P in solvents G and H and hav ing a guanos ine u l t rav io le t absorp t ion spec t rum was isolated from the par t i cu la te enzyme b y ext rac t ion wi th 5 % trichloroacetic acid.

Properties of the Particulate Mannosyl Transferase

The incorpora t ion of mannose in to the manno l ip id was s t rongly dependen t on the presence of Mg 2+ as shown in Fig. 3. The o p t i m u m concen t ra t ion of Mg 2+ was a round 10 m~r. The effect of G D P - m a n n o s e con- cen t r a t ion is shown in Fig. 4. The a p p a r e n t Km for G D P - m a n n o s e was abou t 5 • 10 -6

C: M GDP- iilanilose

Not incubated 3110 23 None 1994 592 GMP 1965 742 GDP 1505 971

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 chro- matographed in solvent G to isolate GDP-man. Radioactivity in C:M and in GDP-man was determined.

o 600C o

m

~_ �9

N e

2000-

D

o ~ ~ ,~

Mg ~§ CONCENTRATION ( mM )

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

M. The p H o p t i m u m for mannose incorpora- t i on in to C : M was abou t 8.0 in T r i s - malea te buffer (Fig. 5). We have no t been able to do these exper iments wi th glucolipid synthesis because of the fact t h a t more t h a n 90 % of the ac t iv i ty incorpora ted in to C : M

316 SCHULTZ AND ELBEIN

~000-

(a

6 o o o - z

~ 4000 -

~ 2 D 0 0 -

GDP -- MANNOS I: CONClVNTRATION ( . M }

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-

4000-

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.

from UDP-[14C]glucose is into other lipids. However, formation of the acidic glucolipid was dependent on Mg 2+ and had a pH opti- mum of around 8.0.

Effect of the Addition of Ficaprenyl-Phos- phate

When the particulate enzyme was t reated with acetone as described by Troy 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 radio- act ivi ty into C :M. Trea tment of the en- zyme with acetone resulted in considerable loss of enzymatic activity which could not be totally restored even by the addition of ficaprenyl-phosphate. However, this en- zyme preparation was useful for demon- strating the requirement for ficaprenyl- phosphate as a mannose acceptor. Fica- prenyl-phosphate also stimulated the in- corporation 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 proper- ties to the mannolipid formed from GDP- mannose with endogenous lipid acceptors.

Isolation and Characterization of Glycolipids

The mannolipid formed in a large-scale incubation with GDP-[14C]mannose (4 X 106 epm) and the particulate enzyme or the glucolipid formed from UDP-[l~C]glucose (2 • 106 cpm) were purified as described by Scher et al. (20). This involved a pre- liminary separation on DEAE-cellulose, followed by 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 317

z

,.'R,

l l : o l lc

(b

I O 0 0 -

500 -

~ ,.8. m

F iCAF 'RENYL- P ( n mo les )

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 de- termined.

,0ooJ y'

I 0oo -

5o0 -

I

o �84 1 i 150 200 0 I00 250

ml OF 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 lu ted in a f a i r ly s y m m e t r i c a l p e a k when e lu ted w i th 0.1 ~, a m m o n i u m ace ta te . I n the ease of t he manno l ip id , a p p r o x i m a t e l y 1.5 X 106 c p m were incor- p o r a t e d in to C : M f rom 4 X 10 G e p m of GDP-[ t4C]mannose , and 1.2 X 106 epm were recovered in th is peak off of D E A E - cellulose. I n t he ease of t he glueol ipid ,

a b o u t 1 X 10 ~ cpm were i nco rpo ra t ed in to C : M f rom 2 X 106 c p m of UDP-[ I4C]glu - eose and 10,000 epm were found in the pur i f ied p e a k f rom D E A E - e e l l u l o s e .

T h e ma nno l ip id and the g lueol ip id h a d mobi l i t ies on t h in - l a ye r c h r o m a t o g r a p h y s imi lar to those p rev ious ly 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 by Drs. Warren and Jeanloz (18) showed identical mobilities in these three solvents to the biosynthesized mannolipid and glucolipid. Ficaprenyl-P (also supplied by 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 may depend on concentration of lipid,

TABLE III T H I N - L A Y E R C H R O M A T O G R A P H I C M O B I L I T I E S O F

V A R I O U S L I P I D S

Lipid R/in solvent

A B C

[14C]Mannolipid 0.31 0.76 0.11 [14C]Glucolipid 0.29 O. 77 0.09 Synthetic mannosyl 0.31 0.75 0.10

phosphoryl-ficaprenol .: Fic aprenyl-P 0.38 0.91 0.02

time allowed for equilibration of solvent, and /or 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) com- plete 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 chroma- tography 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 may not have been detected even if present.

Evidence for Oligosaccharide-Phosphoryl- Polyprenols

Although the major radioactive sugar released from the mannolipid by mild acid hydrolysis was mannose, smaller

150

I00-

5 0 -

f

t i n t o i ~ 3 g ~ 6 ~ 6 9 ,'o

TIME (Minu tes)

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 319

amounts of other radioactive peaks which migrated like oligosaccharides were also ob- served 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 man- nolipid (i.e., length of incubation, amount of enzyme, etc.). This is shown in Fig. 9, trac- ings B and C, where it can be seen that when the purified mannolipid preparation (Fig.

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 chloro- form:methanol to reisolate the lipid. The lipid was then subjected to mild acid hydrolysis and the sugars chromatographed in solvent D.

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-phosphoryl- polyprenol. In this experiment, "manno- lipid" 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 de- tected with a Packard strip scanner and quanti tated in a liquid scintillation counter. Radioactivity in the larger oligosaccharides

3,500 z 0 25,000

~900 AO00

t " L . .

700. 5,000

O 15 50 45 60 75 90

TIME (MINUTES)

Fro. tO. "Mannol ipid" (50,000 cpm) was in- cubated 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 chroma- tography. Papers were scanned with a Packard strip scanner and radioactivity in the various oligosaccharides was quantitated by liquid scintil- lation counting.

320 SCHULTZ AND ELBEIN

appeared to disappear at approximately the same rate as tha t in the disaceharide, bu t since these compounds had much lower act ivi ty 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 character- ization, the disaccharide was obtained in sufficient amounts for partial eharacteriza- tion. The disaceharide was eluted from the paper and rechromatographed in solvents E and F. I t was tentat ively characterized as a mannobiose on the basis of the follow- ing 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

........ 0 �84 O

i ; ~ i ~ ; :~I~

FIG. 11. Radioactive scans of the sugars re- leased from the purified disaccharide by various chemical treatments. Scan A, untreated disac- charide; scan B, disaccharide treated with 2 N tiC1 at 100~ for 2 hr; scan C, disaccharide treated with NaBH4 and then hydrolyzed in 2 N HC1 at 100~ for 2 hr. In this solvent, glucose migrated slightly behind mannitol (R mannitol = 0.97) whereas sorbitol ran slower than glucose (R glu = 0.93).

was observed (Fig. 11), indicating that the reducing end of the disaccharide was either not labeled or contained much less radio- activity. This suggests tha t the disaccharide is formed by transfer of mannose from GDP- [14C]mannose to endogenous mannosyl- phosphoryl-polyprenol. The mannose and mannitol areas of the chromatogram were eluted, acetylated (after NaBH4 reduction of the mannose area), and quanti tated by gas-liquid chromatography. Approxi- mately equal amounts of mannose and mannitol were detected. Finally, the hexose (by anthrone using a mannose standard) to reducing sugar ratio in the intact disae- eharide 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-phosphoryl- polyprenols appear to be involved either in the formation of lateral branches on poly- saccharide chains or in glycoprotein bio- synthesis. Thus, Lahav et al. (9) and Scher and Lennarz (20) showed that in microcoeci mannosyl-phosphoryl-undecaprenol trans- ferred mannose units only to the nonreduc- ing termini of endogenous mannan so tha 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 tha t glucosyl-phosphoryI-doliehol was a precursor for a glucoprotein in ra t liver microsomes. Baynes and Heath (19) found a similar function for a mannosyl- phosphoryl-doliehol synthesized in rat liver. Retinol monophosphate galactose has also been shown to serve as an intermediate in glycoprotein synthesis in mouse mastoey- toma particulate preparations by Alam et al. (22).

Various other workers have demonstrated the synthesis of glycosyl-phosphoryl-poly- prenols in other organisms, but in these cases a function for the lipid has not yet

BIOSYNTHESIS OF POLYPRENOLS IN MYCOBACTERIUM SMEGMATIS 321

been demonstrated. Takayama and Gold- man (10) showed the synthesis of man- nosyl-phosphoryl-undecaprenol by extracts of M. tuberculosis. A mannolipid with simi- lar 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 poly- prenols since Alan and Hemming (26) showed that the addition of betulaprenol- phosphate to extracts of mung beans stimu- lated the incorporation of mannose from GDP-mannose into chloroform:methanol. Forsee and Elbein (27) demonstrated the synthesis of glucosyl and mannosyl-phos- phoryl-polyprenols in extracts of cotton fibers. Ficaprenyl-phosphate was able to serve as a glycosyl acceptor in this system (28). Sentandreu and Lampen have re- cently reported the synthesis of mannosyl- phosphoryl-polyprenol in yeast (29). I t seems likely that these glycolipids will also be found to be involved in polymer synthe- sis but in what capacity remains to be es- tablished.

In addition to monosaccharide-phos- phoryl-polyprenols, the particulate enzyme from M. smegmatis also forms oligosac- charides 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 man- nosyl-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 polysaccha- rides, 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 In- fectious Diseases.

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