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Biochimica et Biophysica Acta, 4 9 8 ( 1 9 7 7 ) 3 4 1 - - 3 4 8 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 28272

I N C O R P O R A T I O N OF MANNOSE AND GLUCOSE INTO P R E N Y L P H O S P H A T E SUGARS IN ISOLATED HUMAN PLATELET MEMBRANES *

S. DE L U C A **

American National Red Cross, Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, Md. 20014 (U.S.A. (Received February 18th, 1977)

Summary Isolated platelet membranes synthesize mannosylretinyl phosphate and dolichylmannosyl phosphate from GDP-[~4C]mannose, but only dolichylglucosylphosphate is synthesized from UDP- [ 14C ] glucose. Addition of exogenous retinylphosphate specifically stimulates the biosynthesis of mannosylretinylphosphate.

Introduction Prenylphosphate sugar c o m p o u n d s can act as intermediates in the biosynthesis of glycoproteins in a variety of tissues [1--3]. The human platelet is rich in glycoproteins [4,5] and in glycolipids [6,7] and preliminary data indicated that incubation of isolated platelet membranes with UDPG and GDPMan resulted in the incorporation of radioactivity into a lipid-soluble fraction [8] while incubation with UDPGal did not [9]. In this report we have examined the incorporation of mannose and glucose into complex lipids in isolated platelet plasma membranes. Materials and Methods Concentrates of human platelets were provided by the Washington Regional Red Cross Blood Center and prepared from single units (approx. 450 ml) of * Contribution No. 329 from The American National Red Cross. ** Present address: National Institute of Dental Research, Bethesda, Md. 20014, U.S.A. Abbreviations: GDPMan, guanosine diphosphate-mannose; UDPG, uridine diphosphate-glucose; UDPGal, uridine diphosphate-galactose; DMSO, dimethylsulfoxide~ DMP, dolichylmannosylphow phate; MRP, m a n n o s y l r e t i n y l p h o s p h a t e ; RP, retinylphosphate.

342 blood. Platelets were used within 24 h of collection. Platelet plasma membranes were isolated by the glycerol-lysis technique [10] and stored frozen in 20% glycerol. GDP-[~4C]mannose (specific activity 161 C i / m o l ) a n d UDP-[~4C]glucose (specific activity 245 Ci/mol) were purchased from the New England Nuclear Corp., Boston, Mass. DEAE-cellulose was obtained from Eastman Kodak Co., Rochester, N.Y.; Bio-Sil BH (200--325 mesh) from Bio-Rad Laboratories and coated silica gel plates 60F-254 from EM Laboratories, Inc. Synthetic DMP [11] was a generous gift of Dr. Christopher Warren, Carbohydrate Research Laboratory, Massachusetts General Hospital, Boston, Mass. Standard RP [12] and MRP [13] were obtained from Dr. Luigi M. De Luca, National Institutes of Health, Bethesda, Md. Silica gel plates were scraped with an automatic zonal plate scraper from Analabs, Inc. Radioactivity was determined with a Packard Model 3375 liquid scintillation counter and with a Packard TriCarb Sample Oxidizer. The scintillation liquid was made up of toluene (100 ml), 2,5-diphenyloxazone (5 g) and 1,4-bis-[2(5-phenyloxazolyl)] benzene (50 mg). Standard hexoses were detected with AgNO3 [14], sugar phosphates with the molybdate reagent [15]. DMP was visualized employing the anisaldehyde spray [16] and protein was assayed by the method of Lowry et al. [17] using bovine serum albumin as a standard. Glycosylation of gelatin. Preliminary experiments on the biosynthesis of complex glycolipids were made in the system for the glucosylation of acidhydrolyzed collagen [8]. Assay mixtures (0.22 ml) contained 50 pl of collagen acceptor (2.5 mg protein), 0.1 ml platelet membrane fraction (approx. 1.0 mg protein), 15 gl of 0.22 M MnC12, 5 pl of [14C]UDPG (125 nCi, approx. 530 pmol) and 50 pl of 0.05 M maleate buffer (pH 5.7); the reactions were stopped with 1% phosphotungstic acid in 0.5 M HC1 after incubation for 20 min in dim light at 37°C. The precipitate was washed with 4 X 1 ml of 10% trichloroacetic acid and was then extracted with 1 ml of chloroform/methanol (2 : 1, v/v) for 15 min at room temperature. Lipid-bound radioactivity was determined in a liquid scintillation counter using hyamine as solubilizing agent. Biosynthesis of prenylphosphate sugars. GDP-[14C]mannose and UDP-[14C] glucose were used to study the biosynthesis of radioactive prenylphosphate sugars using isolated platelet membranes as a source of enzyme. A typical incubation mixture for the biosynthesis of ['4C]mannolipid contained isolated platelet membranes (1.2 ml; 8 mg protein/ml) and 0.2 ml of each of the following solutions: EDTA, 0.025 M; Tris. HC1, 0.3 M, pH 7.5; MnC12, 0.2 M; ATP, 3 mg/mt; GDP-[14C]mannose 10 pCi (specific activity 160 Ci/M) in a total volume of 2.4 ml. Final pH of the incubation mixture was 7.1. For studies of specific stimulation of MRP synthesis by RP this incubation was scaled down 10-fold. Synthetic RP (20 pg) dissolved in 20 pl of DMSO was added to the incubation mixture, whereas only the solvent DMSO was added to the control incubation. For the biosynthesis of [14C]glucolipid, 3.0 ml of isolated platelet membranes (approx. 10 mg protein/ml), 0.5 ml of each of the solutions described above and 10 ~uCi of UDP-[14C]glucose (specific activity 245 Ci/M) were used.

343 The total volume of the reaction mixture was 6.0 ml. The mixtures were incubated at 37°C in dim light for 20 min. Purification of prenylphosphate sugars. Unless otherwise stated, the reactions were stopped by the addition of five volumes of chloroform/methanol (2 : 1, v/v) and the washed organic phase, which contained the extracted lipid, was placed on columns of DEAE-cellulose prepared according to Rouser [18] and equilibrated in 99% methanol. Further purification of prenylphosphate sugars was obtained on a column of silicic acid packed in chloroform. The lipid fraction was eluted from the column with a chloroform/methanol gradient. The last step of purification was achieved by thin-layer chromatography. At least two mannolipids are known to be synthesized by rat liver membrane [19,20]; consistent separation of these two compounds has been obtained using thin-layer chromatography on silica gel plates with chloroform/methanol/ water (60 : 25 : 4, v/v) as the eluting solvent. This system was first developed by Tkacz et al. [21]. DMP has Re 0.4--0.5; MRP has RF 0.2--0.25. Results

Incorporation of glucose from UDPG into glucolipid. Under conditions described in Materials and Methods for the glucosylation of the galactosyl moiety in gelatin, about 6% of the total incorporated radioactivity was extracted from the reaction mixture into the organic solvent and the incorporation into the lipid-soluble fraction reached a maximum value at 10 min, whereas incorporation into the protein fraction reached a maximum value at 30 min. In an incubation system using purified platelet membranes and UDPG, [~4C]glucose was incorporated into the lipid phase. Prenylphosphate [14C]glucose was obtained b y chromatography on DEAE-cellulose and silicic acid, as described. By this procedure, neutral and less acidic glycolipids were removed by elution from DEAE-cellulose in 99% methanol and from silicic acid in chloroform/methanol (8 : 1, v/v). In general, purification by DEAE-cellulose was the preferred method. The purified prenylphosphate-[~4C]glucose had an RE value of 0.5, on thinlayer chromatography in chloroform/methanol/water (60 : 25 : 4, v/v) as expected for dolichylglucosylphosphate. The purified glucolipid was recovered unchanged, as judged by thin-layer chromatography (RE 0.5) when subjected to alkaline hydrolysis in 0.1 M NaOH at 37°C for 10 min (Fig. 1), conditions which hydrolyze compounds with allylic phosphates such as RP [12] and MRP [19]. Biosynthesis of mannolipids by platelet plasma membranes. When platelet plasma membranes were incubated with GDP-[14C]mannose there was a rapid uptake of radioactivity both in the crude lipid-soluble fraction and in the phospholipid fraction purified on DEAE-cellulose; maximum incorporation was reached in 20 min. Chromatography of the crude lipid fraction on DEAE-cellulose yielded a single radioactive peak (Fig. 2A) which was further purified on silicic acid to yield another single peak (Fig. 2B). The labeled fraction from silicic acid was further purified by thin-layer chromatography; two peaks of radioactivity were consistently obtained (Fig. 2C), which cochromatographed with standards RP and DMP.

1600

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Fig. 1. Alkaline hydrolysis of [ 14C]glucolipid. C*4ClGlucolipid (2700 dpm) prepared as described in Materials and Methods was subjected to mild alkaline hydrolysis following the procedure described in the legend to Fig. 3. The organic phase was examined by thin-layer chromatography in chloroform/methanol/ water (60 : 25 : 4, v/v) on silica gel in comparison with an equal amount of the [ *4C]glucolipid which e-----e, intact glucolfpid; X- - - - - -X, glucolipid after had not been subjected to alkaline hydrolysis. mild alkali hydrolysis. The position of standard DMP is shown.

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Fig. 2. Purification of mannolipids. GDP-[t4C]mannose was incubated with platelet membranes under conditions described in Materials and Methods. The organic extract was purified by sequential chromatography on A, DEAE-cellulose with a linear gradient of W.1 M ammonium acetate; B. sihcic acid with a linear gradient from chloroform/methanol (8 : 1. v/v) to chloroform/methanol (1 : 1. v/v); C, thin-layer chromatography on silica gel in chloroform/methanol/water (60 : 25 : 4. v/v) on 5000 dpm of the purified mannolipid. Radioactivity emerging in the column wash is not shown. The shape of the linear gradients

345

Characterization of mannolipids. The total 14C-labeled mannolipid from silicic acid was subjected to mild basic hydrolysis in 0.1 M NaOH at 37°C for 10 min as a further method of identification; DMP is stable under these conditions. Following such treatment [14C]MRP (R F 0.2) was degraded {Fig. 3B). Under these conditions standard RP is hydrolyzed [12] to anhydroretinol (R). [~C]Mannose and ['4C]mannose phosphate were found in the aqueous phase (Fig. 3C).

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Fig. 3. H y d r o l y s i s of the m a n n o l i p i d s . ( 1 ) M i l d alkaline h y d r o l y s i s . [ 1 4 C ] M a n n o l i p i d ( 1 0 0 0 0 d p m ) , p u r i f i e d t h r o u g h D E A E - c e l l u l o s e , w a s i n c u b a t e d a t 3 7 ° C f o r 10 rain in 0.1 M N a O H in c h l o r o f o r m / m e t h a nol (1 : 4, v / v ) , t h e n b r o u g h t to n e u t r a l i t y w i t h 1 M a c e t i c acid a f t e r c o o l i n g t o 4°C. C h l o r o f o r m (0.8 m l ) , m e t h a n o l (0.2 m l ) , a n d w a t e r ( 0 . 2 ral) w e r e a d d e d a n d t h e a q u e o u s p h a s e w a s a p p l i e d t o W h a t m a n N o 3MM p a p e r a n d e h r o m a t o g r a p h e d f o r 24 h in i s o b u t y r i c a c i d / a m r a o n i a / w a t e r (57 : 4 : 39, v / v ) . T h e c h r o r a a t o g r a m w a s c u t in 1 - c m p i e c e s a n d c o u n t e d in t o l u e n e / P P O / P O P O P . T h e o r g a n i c p h a s e w a s c h r o m a t o g r a p h e d o n l a y e r s of silica gel, d e v e l o p e d in c h l o r o f o r m / r a e t h a n o l / w a t e r ( 6 0 : 25 : 4, v / v ) . DMP a n d RP, w h i c h h a d b e e n s u b j e c t e d to t h e s a m e p r o c e d u r e of m i l d alkaline h y d r o l y s i s , w e r e i n c l u d e d as c o n t r o l s . ( A ) I n t a c t r a a n n o l i p i d b e f o r e m i l d alkali t r e a t m e n t . (B) M a n n o l i p i d a f t e r m i l d alkali t r e a t m e n t . R is t h e h y d r o l y s i s p r o d u c t of RP. (C) P a p e r c h r o m a t o g r a p h y of t h e a q u e o u s p h a s e : M a n a n d Manol-P i n d i c a t e t h e p o s i t i o n of s t a n d a r d m a n n o s e a n d m a n n o s e 1 - p h o s p h a t e . (2) A c i d h y d r o l y s i s . [ 1 4 C ] M a n n o l i p i d ( 3 0 0 0 d p r a ) p u r i f i e d t h r o u g h D E A E - e e l l u l o s e was t r e a t e d w i t h 2 M HCI (1 ral) a t 1 0 0 ° C f o r 2.5 h, in a closed vial, u n d e r n i t r o g e n . A t c o m p l e t i o n o f the h y d r o l y s i s , t h e HCI w a s r e m o v e d b y flash e v a p o r a t i o n . T h e h y d r o l y s a t e w a s dissolved in w a t e r (0.1 m l ) a p p l i e d t o W h a t m a n p a p e r No. 3MM a n d d e v e l o p e d in b u t a n o l / p y r i d i n e / w a t e r (9 : 5 : 4, v / v ) f o r 20 h, w i t h s t a n d a r d m o n o s a e c h a r i d e s . T h e c h r o r a a t o g r a m w a s c u t in 1-cra p i e c e s a n d c o u n t e d . (D) C h r o m a t o g r a r a of acid h y d r o l y s a t e of m a n n o l i p i d w i t h s t a n d a r d glucose a n d r a a n n o s e .

346

All of the radioactivity in the mannolipid fraction was in [:4C]mannose, as shown by acid hydrolysis and paper chromatography (Fig. 3D}. pH optimum for prenylphosphate mannose, [J4C]MRP and [~4C]DMP were separated by thin-layer chromatography as in Fig. 3A, from incubations run at different pH values. It was found that the ratio of the two components varied at different pH values. MRP constituted about 10% of the total mannolipid at pH 6 and about 18% at pH 7 (Fig. 4). The amounts of retinyl derivatives at more extreme pH values were negligible, although there was still considerable synthesis of the dolichyl compound. Metal ion requirement. The following ions were tested to determine their requirement in the biosynthesis of the total mannolipid fraction isolated following chromatography on DEAE-cellulose: K +, Na +, Mn 2+, Co 2+, Cd 2+, Ca 2+, Mg 2+, and Zn2+(Table I). Maximum activity was obtained with 15 mM Mn2÷and slightly lesser activity with Co 2+. Stimulation of MRP synthesis by RP. The addition of 20 pg of RP to the platelet membrane system specifically stimulated [I~C]MRP synthesis from 1010

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347 TABLE I M E T A L I O N R E Q U I R E M E N T F O R S Y N T H E S I S OF M A N N O L I P I D S BY P L A T E L E T P L A S M A MEMBRANES T h e s a m e i n c u b a t i o n m i x t u r e d e s c r i b e d in Materials a n d M e t h o d s was sealed d o w n to a final v o l u m e of 0 . 0 6 0 m l w i t h d i f f e r e n t c a t i o n s r e p l a c i n g Mn 2+. 0 . 0 3 0 m l of p l a t e l e t m e m b r a n e s u s p e n s i o n (8 m g p r o t e i n / m l ) a n d 0.2 /aCi of G D P - [ 1 4 C ] m a n n o s e w a s u s e d in e a c h i n c u b a t i o n . T h e r e a c t i o n p r o c e e d e d f o r 20 m i n , 3 7 ° C , in d i m light. T h e c h l o r o f o r m / m e t h a n o l e x t r a c t w a s p u r i f i e d t h r o u g h D E A E - c e l l u l o s e as d e s c r i b e d . System

DMP/incubation

Complete Minus Mn 2+ Mn 2+ r e p l a c e d b y : Co 2+ Mg 2+ Ca 2+ Cd 2+ Zn 2+ Na+ K+

244 36 211 168 156 115 50 16 16

cpm per mg of platelet membrane per 30 min to 3080 cpm. No effect of RP on DMP synthesis was found. Discussion These experiments show that isolated platelet plasma membranes are active in synthesizing two different prenylphosphate-mannoses from GDPMan. Chromatographic and hydrolytic data indicate that these are dolichylmannosylphosphate and mannosylretinylphosphate. Different pH optima for the formation of the two products, as had also been found in rat liver system [20] suggests the presence of two different mannosyltransferases specific for each product. The addition of synthetic retinylphosphate to the reaction mixture specifically stimulates the biosynthesis of MRP. Derivatives of retinol (C20) are less hydrophobic than their dolichol (C100) counterparts; it has recently been reported that 70% of MRP is recovered from the upper phase of a solvent extraction procedure while DMP is exclusively found in the lower phase [13]. This less hydrophobic character of MRP supports the idea of a different functional site in the membrane for the two classes of compounds. Platelet membranes are also active in synthesizing glucosyldolichylphosphate, b u t no glucosyl derivative of retinylphosphate was found. The possible role of the mannolipids and the glucolipid in platelet metabolism and function remains to be elucidated. Acknowledgement Supported, in part, by U.S.P.H.S. grants No. HL 14697 and No. AI 09017. References 1 H e m m i n g , F.W. ( 1 9 7 4 ) in B i o c h e m i s t r y o f L i p i d s ( G o o d w l n , T.W., ed.), Vol. 4, pp. 3 9 - - 9 8 , U n i v e r s i t y P a r k Press, B a l t i m o r e , Md.

348 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Waeehter, C.J. and Lennarz, W.J. (1976) Annu. Rev. Biochem. 45, 95--112 Maestri, N. and De Luca, L.M. (1973) Biochem. Biophys. Res. Commun. 53, 1344--1349 Pepper, D.S. and Jamieson, G.A. (1968) Nature 219, 1252--1253 Pepper, D.S. and Jamieson, G.A. (1969) Biochemistry 8, 3362--3369 Marcus, A.J., Ullman, H.L. and Saffier, L.B. (1969) J. Lipid Res. 10, 108--114 Marcus, A.J., Ullman, H.L. and Safier, L.B. (1972) J. Clin. Invest. 51, 2602--2612 Barber, A.J. and Jamieson, G.A. (1971) Biochim. Biophys. Acta 252, 533--545 Barber, A.J. and Jamieson, G.A. (1971) Biochim. Biophys. Acta 252, 546--552 Barber, A.J. and Jamieson, G.A. (1970) J. Biol. Chem. 245, 6 3 5 7 - - 6 3 6 5 Warren, C.D. and Jeanloz, R.W. (1973) Biochemistry 12, 5038--5045 Frot-Coutaz, J.P., Silverman-Jones, C.S. and De Luca, L.M. (1976) J. Lipid Res. 17, 220--230 Silverman-Jones, C.S., Frot-Coutaz, J.P. and De Luca, L.M. (1976) Anal. Biochem. 75, 664--667 Trevelyan, W.E., Procter, D.P. and Harrison, J.S. (1950) Nature 1 6 6 , 4 4 4 - - 4 4 5 Dittmer, J.C. and Lester, R.L. (1964) J. Lipid Res. 5, 126--127 Dunph y, P.J., Kerr, J.D., Pennock, J.F., Whittle, K.J. and Fenney, J. (1967) Biochim. Biophys. Acta 136, 136--147 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 Rouser, G., Kritehevsky, G., Heller, D. and Lieber, E. (1963) J. Am. Oil Chem. Soe. 40, 425--454 Barr, R.M. and De Luca, L.M. (1974) Biochem. Biophys. Res. C ommun. 60, 355--363 Rosso, G.C., De Luca, L.M., Warren, C.D. and Wolf, G. (1975) J. Lipid Res. 16, 235--243 Tkacz, J.S., Herscovics, A., Warren, C.D. and Jeanloz, R.W. (1974) J. Biol. Chem. 249, 6372--6381