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Biochirnica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 4 4 5 - - 4 5 5 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 27847

EVIDENCE F O R MANNOLIPIDS AS I N T E R M E D I A T E S IN MANNOSE T R A N S F E R TO T H Y R O I D R O U G H MICROSOMAL G L Y C O P R O T E I N S

CATHERINE RONIN and SIMONE BOUCHILLOUX

Laboratoire de Biochimie Mddicale, Facultd de Mddicine, Bd. J. Moulin, 13385 Marseille, Cedex 4 (France) (Received August 6th, 1975)

Summary Thyroid rough microsomes catalyzed the transfer of mannose from GDPmannose to endogeneous glycoprotein(s) and to glycolipids comprising a recently described dolichol phosphomannose extractable with usual organic solvents and a material tentatively identified as an oligosaccharide lipid. The labeling of the t w o lipids was consistent with a role in mannose transfer to glycoprotein(s). When partially purified dolichol phospho[14C] mannose was incubated with rough microsomes, a part of the label appeared in the second lipid, suggesting a role as intermediate, and less rapidly in glycoprotein(s). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis did n o t allow to ascertain whether or not the glycoproteins receiving label from these sugar lipids comprised thyroglobulin precursors.

Introduction In a previous study [1--3] rough microsomes from thyroid were incubated, either in conditions that did or did n o t allow peptide synthesis in the presence of [14C] monosaccharides proximal to the peptide b a c k b o n e of thyroglobulin, mannose and N-acetylglucosamine, given in the form of active nucleotides. Thyroglobulin precursors were looked for b y various techniques. Since then, it appeared that glycolipids might constitute an important part of the radioactivity taken up in the particles: when sodium dodecyl sulfate/polyacrylamide gels after electrophoresis were processed for counting w i t h o u t prolonged immersion in dilute acid, they exhibited at the frontal d y e an important radioactive peak more or less coincident with a diffuse periodic acid/Schiff reactive * Abbzeviation: Do1. dolichol.

446

area. Extracting the particles with chloroform-alcohols lead to a substantial decrease of these materials. Several laboratories have recently reported on a family of ~-isoprene-saturated polyprenol phosphates of about 20 units (dolichol phosphate) able to accept mannose from GDP-Man in the microsomes from various mammalian tissues including thyroid [4--8]. That Dol-P-Man might serve as a mannosyl carrier to unidentified glycoproteins has been recently found in liver [5], myeloma t u m o r [6] and oviduct [7]. We report experiments relevant to this problem in thyroid and have also studied the labeling and possible function of another mannolipid distinguished b y its solubility in water-saturated CHC13/CH3OH, possibly a dolichol p y r o p h o s p h o oligosaccharide [9--13]. Experimental procedure GDP[U-14C] mannose (74--154 Ci/mol) was from the Radiochemical Centre, and UDP[6-3H]N-acetylglucosamine (6.6 Ci/mmol) used in a few assays from N.E.N.C. Rough microsomes were prepared as previously described from sheep or occasionally pig thyroid (procedure 1 in ref. 1). They were immediately resuspended for incubation without detergent at a final concentration of 10--15 mg protein per ml of buffer I (20 mM Tris. HC1, pH 7.4, 25 mM KC1, 5 mM MGC12)/0.25 M sucrose supplemented with 5--10 mM MnC12 and 2.5 mM dithiothreitol [1]. When the donor was GDP[14C]Man (2 pCi/ml) and occasionally UDP[3H] GlcNAc, a number of incubations were carried out in a medium allowing peptide synthesis, i.e. comprising 0.5 mM GTP, 1.0 mM ATP, 10 mM phosphoenolpyruvate, pyruvate kinase, a supplement of unlabeled amino acids and a post-microsomal liver supernatant filtered through Sephadex G-25, as described in ref. 1. When the donor was a [~4C] mannose-labeled lipid phase or a biosynthetic DoI-P-[~4C] Man purified up to the first DEAE-cellulose step of Waechter et al. [7] and freed from salt, it was evaporated to dryness and introduced (7 • 104--15 • 104 dpm/ml incubation) as an emulsion in a small volume of buffer I/sucrose with Triton X-100, the final medium (otherwise as above) containing 0.02% of the latter and 0.1 mM GDPMan. It is known that GDPMan counteract a displacement of the label from Dol-P-Man to endogeneous GDP [5,6]. All incubations were at 36°C with shaking, the volumes being from 0.5 ml in pilot assays up to several ml in preparative experiments. For analytical purposes, small aliquots were taken up in duplicate and either subjected to lipid extraction (eventually diluted to 0.2 ml MgC12,4 mM, with 1 mg of carrier immunoglobulin G ) o r to a trichloracetic acid precipitation as in ref. 1. It is worth mentioning that once trichloracetic acid precipitated, thyroglobulin becomes soluble in organic solvents (from which it would be removable by partitioning with water). At preparative scales samples were either extracted for lipids w i t h o u t carrier protein, or respun, washed and stored frozen until a further use such as digitonin extraction for immunological assays. Although several methods for lipid extraction were used at the beginning of this work [5,6], we finally adopted the following procedure (cf. refs. 7, 11): samples either analytical or preparative were treated with 2 vols. of CH3OH and 3 vols. of CHC13 thoroughly mixed and centrifuged. The upper phase was

447 removed, the lower phase and the interphase washed 3 times with theoretical upper phase [11] and the resultant lower phase saved. The material at interphase was dispersed in CHC13/CH~OH/4 mM MgC12 (3 : 2 : 1), centrifuged and the lower phase combined with the previous one, giving phase 1. The interphase dried under a stream of nitrogen was washed 3 times with water, dried again and extracted 3 times with CHC13/CH3OH/H20 (10 : 10 : 3) giving phase 2 and a residual delipidated pellet. Column chromatography on DEAE-cellulose acetate prepared according to Rouser et al. [14] was essentially as described by Waechter et al. [7] in order to analyze and purify the [14C] Man labeled phase 1. After equilibration of the column in CHC13/CI-I3OH (7 : 3), the sample was applied in the same solvent. It was eluted successively with CH3OH and a stepwise gradient of 10, 50 and 300 mM a m m o n i u m acetate in CH3OH/H20 (99 : 1). To analyze the [~4C]Manlabeled phase 2 and comparatively GlcNAc labeled derivatives, which were postulated to be more acidic than Dol-P-Man, small sized columns were equilibrated in CHC13/CH3OH/H20 (10 : 10 : 3) and developed with a stepwise ammonium acetate gradient in the same solvent [9,12,15]. Chromatography on Whatman SG 81 paper was carried o u t in the following solvents: A, n-propanol/H20 (5 : 3), B, n-propanol/H20 (7 : 3) and C, CHC13/ CH3OH/H20 (65 : 25 : 4). Chromatography on Whatman 3 MM paper was in solvent D, isobutyric acid/NH4OH/H20 (59 : 4 : 39). Double antibody immunoprecipitation of digitonin extracts from labeled microsomes was performed after a dialysis as described in ref. 2, except for the presence of 1% Triton X-100 during precipitation and subsequent washings. This has been done with the idea of minimizing unspecific associations to the precipitates. These immunoprecipitates were counted without trichloracetic washing. It was checked that no coprecipitation of mannolipids was occuring. Controls included the use of an excess of carrier sheep thyroglobulin [2]. Results and Comments

Microsomal labeling from GDP[14C] Man and some properties o f the mannolipids The time course of incorporation of [ 14C] mannose from its active nucleotide into the microsomal acceptors of lipid phase 1, lipid phase 2 and residual pellet is shown in Fig. 1. Without peptide synthesis (Fig. 1A), there was a rapid labeling of phases 1 and 2 and then a slow decrease. In the assay containing ATP and GTP necessary for peptide synthesis (Fig. 1B), the time course of labeling of phas e 1 was greatly affected and the incorporation in phase 2 comparatively high. In b o t h systems the labeling of the residual pellet was slow and went on increasing all the time, reaching a b o u t 30% of the total radioactivity incorporated after 90 min. In a " c h a s e " experiment (Fig. 2), a 10-rain incubation mixture w i t h o u t peptide synthesis was rapidly sedimented, the particles resuspended and incubated with an excess of cold GDPMan. This gave, as compared to a control n o t given here, an abrupt instead of a slow decrease of the radioactivity in phase 1 with time, a less pronounced decreasing effect in phase 2, which was even less in the pellet. At 40 rain, the label in each c o m p a r t m e n t has decreased to respectively

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Fig. 1. T i m e c o u r s e o f [ 14C] M a n t r a n s f e r f r o m G D P - [ 14C] M a n i n t o r o u g h m i c r o s o m e s f r o m s h e e p thy= r o i d ( d p m / m g m i c r o s o m a l p r o t e i n ) . ( A ) w i t h o u t , a n d (B) w i t h , p e p t i d e s y n t h e s i s . L i p i d p h a s e 1, lipid p h a s e 2 a n d r e s i d u a l p e l l e t w e r e o b t a i n e d as d e s c r i b e d in E x p e r i m e n t a l P r o c e d u r e . T h e s a m e p a r t i c l e s w e r e u s e d in (A) a n d (B). M e a n o f 2 i n d e p e n d e n t e x p e r i m e n t s w i t h d e t e r m i n a t i o n s in d u p l i c a t e .

35, 69 and 80% of the values in this control. Even so the radioactivity in the pellet kept gradually increasing and represented up to 66% of the total incorporated after 90 min {Fig. 2). Altogether these results suggest a precursor product relationship between the two organic phases and the residual sediment expected to comprise glycoproteins. Optimal conditions for the incorporation of mannose into phase 1 were the presence of MnC12, 2.5--7.5 mM, (in addition to 5 mM MgC12 in buffer I/ sucrose} and a pH of approx. 6.0. EDTA was strongly inhibitory. Triton X-100

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Fig. 2. T i m e c o u r s e o f [ 1 4 C ] M a n t r a n s f e r i n t o r o u g h m i c r o s o m e s f r o m s h e e p t h y r o i d ( d p m / m g m i c r o s o m a ) p r o t e i n ) w h e n a 1 0 - r a i n p u l s e l a b e l i n g w i t h G D P - [ 1 4 C ] M a n (assay w i t h o u t p e p t i d e s y n t h e s i s ) w a s f o l l o w e d ( a r r o w ) b y a " c h a s e " i n c u b a t i o n o f t h e r e s p u n p a r t i c l e s ( 1 0 m i n a t 1 5 0 0 0 r e v . / m i n ) , in t h e p r e s e n c e o f 0.1 m M G D P M a n a d d e d t o t h e u s u a l i n c u b a t i o n m e d i u m .

449

enhanced the labeling in a 0.05--0.2% range, although it lowered the incorporation into the terminal pellet. The mannose label in phase 1 was found to be associated almost exclusively with a single c o m p o n e n t which had, as reported by Waechter et al. [7], the properties of a dolichol phosphomannose (Dol-P-Man). It was labile to dilute acid (0.01 M HC1 at 100°C for 10 min, giving a 98% conversion to hydromethanolic soluble radioactivity) and stable to alkaline treatment (0.1 M NaOH at 37°C for 10 rain allowing a 96% recovery as such). All the radioactivity applied in CHC13/CH3OH (7 : 3) to a DEAE-cellulose column was retained and 95% of it eluted as a single symetrical peak with 10 mM a m m o n i u m acetate (Fig. 3). Continuing with 50 and 300 mM a m m o n i u m acetate, the latter removed a tiny peak or more acidic radioactivity in a region where others have situated a c o m p o u n d postulated to be a Dol-P-P-[GlcNAc] 2-Man, on the basis of an in vitro labeling of l y m p h o c y t e membranes with GDp[t4C] Man in the presence of a synthetic DoI-P-P-[GlcNAc] 2 (ref. 16). When analyzed by paper chromatography, the mannose-labeled phase 1 revealed a single c o m p o n e n t with Rr values of 0.85, 0.75, 0.65 and 0.96 in solvents A, B, C and D. In contrast, the mannose label in phase 2 appeared somewhat heterogeneous (and/or unstable) in spite of its distinctive insolubility in CHC13/CH3OH and solubility in water-saturated CHC13/CH3OH, suggestive of an oligosaccharide lipid [10--13]. Small amounts of contaminating Dol-P-[14C]Man were occasionally present. When analyzed on DEAE-cellulose using a water-saturated solvent, most of the retained radioactivity eluted with 10 mM a m m o n i u m acetate (Fig. 4, b o t t o m ) , as one might expect under these conditions of a dolichol

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pyrophosphate derivative [10,12,15]. The peak unretained in that case may correspond to contaminating Dol-P-Man. We have analyzed with an identical column a CHC13/CH3OH extract (i.e. a phase 1) obtained from thyroid rough microsomes previously incubated with UDP[3H] GlcNAc. Its label emerged in a single peak again at 10 mM ammonium acetate (Fig. 4, top}. The c o m p o u n d s Dol-P-P-GlcNAc and DoI-P-P-[GIcNAc] 2, likely to be present, have been recently identified in a few mamalian membranes on the basis of in vitro labeling experiments in the presence of authentic chemically synthesized acceptors [15, 16,17]: a c o m m o n relatively acidic behaviour on DEAE-cellulose has been reported. Upon paper chromatography, the mannose label in phase 2 revealed some heterogeneity and variability. Rf values for the major species were 0.63, 0.55, 0.03 and 0.70 in solvents A, B, C, D respectively, and for an accompanying minor species approximately 0.2 in solvents A, B and D.

451

It was interesting to get some information about the configuration of the linkages established: using a purified a-mannosidase (from Boehringer) essentially as described by others [18] we have obtained (single experiment, assays and appropriate controls without enzyme in duplicate) a 98% disappearance of the mannose label of a phase 2 (rendered water-soluble), whereas the label associated with a phase I was unmodified. It is m o s t probable that the latter corresponds to Dol-P-/3-Man, as in pancreas microsomes and human lymphocytes

[18]. Microsomal labeling from exogeneous [~4C] mannolipids As shown in Fig. 5, partially purified Dol-p-[14C]Man or a [14C] mannoselabeled phase 1, when incubated in the presence of rough microsomes, acted as mannose donors to acceptors in phase 2 and residual pellet. A precursor-product relationship between these last t w o was suggested, in Fig. 5A, as in other assays with Dol-P-[14C] Man and varying the conditions for membrane disruption. In all cases the label in the terminal pellet after 60 min did not exceed a few percent of the supply. Breakdown to hydro-methanolic soluble products (upper phase) was generally less than in Fig. 5A. Interestingly enough, a preliminary gel filtration analysis on Biogel P4 indicated that this water-soluble material was mainly accounted for by oligosaccharidic c o m p o u n d s rather than by free mannose: consequently it might derive from a degradation of the mannosylated oligosaccharide lipid, postulated in phase 2. Incubating rough microsomes with exogeneous [14C] mannose-labeled phase 2 again lead (2 preliminary assays, not shown) to an incorporation of label, which was very rapid in this case into the residual pellet (analyzed later on, as will be seen, by sodium dodecyl sulfate polyacrylamide gel). These results support the idea that an oligosaccharide lipid receiving some mannose from Dol-P-Man might be involved in the transfer of a pre-assembled

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452

saccharide chain to polymeric glycoconjugates. Convincing experimental evidence has been recently obtained in other mammalian systems that strongly suggest such a mechanism for the synthesis of the "core" portion of certain glycoproteins [11,12,19].

Mannosylated glycoproteins In digitonin extracts of GDP[ ~4C] Man labeled microsomes (without peptide synthesis), the proportion of radioactivity found to be immunoreactive through the double antibody technique in the presence of Triton X-100, was close to 12% of the total trichloracetic acid insoluble material (mean of 2 independent experiments with assays in duplicate). This would correspond to 27% of the non-lipid material in this case. When examined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis such extracts exhibited complex radioactivity profiles, as it was the case for unextracted labeled microsomes (the latter shown in Fig. 6A). Some label

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Fig. 6. R a d i o a c t i v i t y p r o f i l e s in s o d i u m d o d e c y ] s u l f a t e / p o l y a c r y l a m i d e gels a f t e r e l e c t r o p h o r e s i s . R o u g h m i c r o s o m c s f r o m s h e e p t h y r o i d : ( A ) l a b e l e d f r o m G D P - [ 1 4 C ] Man ( r e s p u n particles); (B) the s a m e a f t e r d e l i p i d a t i n n ( r e s i d u a l p e l l e t ) ; (C) l a b e l e d f r o m e x o g e n e o u d D o I - p - [ 1 4 C ] M a n a n d (D) f r o m e x o g c n e o u s [ 1 4 C ] M a n - p h a s e 2 ( t o t a l i n c u b a t i o n w i t h o u t d e l i p i d a t i o n ) . S a m p l e s A , C and D w e r e e q u i l i b r a t e d f o r 1 h in 1% s o d i u m d o d e c y l s u l f a t e 0 . 1 M T r i s / g l y c i n e b u f f e r , p H 8 . 2 ; s a m p l e B, d i g e s t e d o v e r n i g h t at 50°C in the s a m e b u f f e r p l u s 1% ~ - m e r c a p t o e t h a n o l . O t h e r c o n d i t i o n s essentially as in r e f . 3 e x c e p t f o r 5% a c r y l a m i d e , 0 . 1 1 6 % m e t h y l e n e - b i s a c r y l a m i d e and s o a k i n g t h e gels f o r 2 h in 1 0 % t r i c h l o r o a c e t i c acid. D o t t e d line ahead o f t h e f r o n t a l b r o m o p h e n o l b l u e . A m o n g t h e m a r k e r s w a s a f u l l y i o d i n a t e d t h y r o g l o b u l i n , partly d i s s o c i a t e d i n t o subunits.

453 might represent a half-sized thyroglobulin subunit, an apparent mol.wt, close to 300 000 being expected for a partially glycosylated uniodinated subunit; smaller sized thyroglobulin chains or related species, still largely hypothetical (cf. refs. 3, 20, 21) might also be present together with other unrelated glycoproteins. After lipid extraction, the denatured residue still revealed several glycoproteins, and the frontal label had vanished (Fig. 6B). As seen in Fig. 6C and D, unextracted microsomal suspensions labeled from either Dol-P-['4C] Man or from a ['4C] mannose labeled phase 2 did exhibit a partly comparable profile, in that heterogeneously sized glycoproteins became radioactive: whether or not thyroglobulin precursors had been labeled is not clear. It is worth mentioning that a control using Dol-P-[14C] Man with sodium dodecyl sulfate-treated rough microsomes failed to exhibit any labeling except for the frontal region. General discussion Attachment of peripheral sugars to the complex carbohydrate units of "serum t y p e " glycoproteins occurs one by one and is, for the most, catalyzed by Golgi-located glycosyltransferases. Using Golgi-enriched particles from severai tissues including thyroid [22], a transfer of sialic acid, galactose and Nacetylglucosamine is observable from their active nucleotides to specifically deglycosylated glycoproteins. Membrane integrity does n o t seem essential and some of the transferases lend themselves to partial purification. On the contrary, the process responsible for attachment of more internal sugars, i.e. biosynthesis of the saccharide " c o r e s " containing several mannose and two N-acetylglucosamine molecules linked to asparagine, has remained poorly understood. That it occurs in rough membranes and begins soon after, or at the end of, peptide synthesis, has been suggested in numerous studies (see ref. 23). But many difficulties were encountered in the search and use of efficient exogeneous acceptors [ 13]. The possibility of using a liver microsomal extract and UDPGlcNAc to obtain a transfer of GlcNAc to ribonuclease A remains controversial [24,25]. In the meanwhile a possible role of membranous lipids for the glycosylation of some glycoproteins was suggested [26,27]. During the last years it became well known that a family of dolichol phosphate, and pyrophosphate saccharide derivatives, although present at very low concentrations in eukaryotic membranes, were nevertheless identifiable by labeling techniques [5--9,13,15--17, 28]. Increasing evidence appeared, showing that some of them might ultimately act as sugar donors to polymers such as glycoproteins [5--7,10--12,19]. Compounds such as Dol-P-Man or Dol-P-P-GlcNAc might simply represent carriers of monosaccharides across membranes. As regards p y r o p h o s p h o derivatives of oligosaccharides, inasmuch as their sugar moities would contain sequences resembling the internal " c o r e " of numerous glycoproteins, i.e. [GlcNAc]:13-Man-[a-Man]n, as reported for liver [11], m y e l o m a t u m o r [12] and oviduct [19], they would be good condidates for a preassembly and subsequent transfer of these portions. It would of course be important to determine the exact nature of the final acceptor(s). With some of these views in mind, we studied further the endogeneous labels (lipid and non-lipid labels) obtained by incubating thyroid rough microsomes

454 with GDP-[14C] Man (or UDP[3H] GlcNAc, in a few experiments): it has been shown in previous work [1--3], that digitonin extracts from such particles exhibit significant immunoreactivities in the presence of an antiserum raised against homologous thyroglobulin, although electrophoresis on sodium dodecyl sulfate/polyacrylamide gels revealed mainly heterogeneously sized labeled species smaller than half-sized thyroglobulin. The results reported here confirm that in thyroid {rough} microsomes, a compound having several properties of Dol-P-Man is rapidly labeled from GDPMan, as first demonstrated by Waechter et al. [7], and that it represents almost exclusively the radioactivity extractable with CHC13/CH3OH {phase 1). That a ~-Man link is formed, as deduced from stability to a specific glycosidase, has been also very recently reported for thyroid from a chemical study [29]. The time courses of mannose incorporation into this Dol-P-Man (phase 1) and a more acidic (postulated pyrophospho) oligo saccharide derivative extractable with CHC13/CH3OH/H20 (10 : 10 : 3} {phase 2} were compatible with both being precursors for the glycosylation of non-lipid polymeric residual material (Figs. 1 and 2). This does not exclude some degradative processes by endogenenous exoglycosidases and phosphatases, which probably are enhanced as the membranes disrupt, either during prolonged incubations or with Triton X-100. Whether or not the nucleotides included in the assays with peptide synthesis have an inhibitory effect on the breakdown of the label in phase 2 {Fig. 1B as compared to 1A) is unknown at present. More direct arguments for a lipid-mediated process of glycosylation in thyroid rough microsomes are provided by the kind of experiments reported in Fig. 5, where previously labeled mannolipids, especially a partially purified DolP-[~4C]Man, are used. An intermediary role for the mannosylated oligosaccharide lipid appearing in phase 2 is suggested. But it appears that this compound (probably a pyrophosphate) is very unstable in the conditions of incubations used so far. The nature of the polymeric acceptors, especially when mannolipids were the donors, remains uncertain, except for being largely accounted for by glycoproteins: whether or not thyroglobulin-related species have been labeled cannot be concluded from the sodium dodecyl sulfate gels data, although this is a possiblity. In any case, even after the labeling with GDP-mannose, the immunoreactivity has been modest. Other approaches for specific identification are currently under study. References 1 Bouchilloux, S., Chabaud, O. and Ronin, C. (1973) Biochim. Biophys. Acta 322, 401---420 2 Torresani, J., Chabaud, O., Ronin, C., Bouchinoux, S. and Lissitzky, S. (1973) Bioehim. Biophys Acta 322, 421--436 3 Roques, M.0 Torresani, J., Bouchilloux, S. and Lissitzky, S. (1973) Biochim. Biophys. Acta 3 2 2 437---447 4 Evans° P.J. and Hemming, F.W. (1973) FEBS Lett. 31,335---338 5 Richards, J.B. and Hemming, F.W. (1972) Biochem. J. 130, 77--93 6 Baynes, J.W., Hsu, A.F. and Heath, E.C. (1973) J. Biol. Chem. 248, 5 6 9 3 - - 5 7 0 4 7 Waechter, C.J., Lucas, J.J. and Lennarz, W.J. (1973) J. Biol. Chem. 248, 7 5 7 0 - - 7 5 7 9 8 Tkacz, J.S., Herscovics, A.0 Warren, C.D. and Jeanloz° R.W. (1974) J. Biol. Chem. 249, 6372---6381 9 Behrens, N.H.° Parodi, A~I. and Leloir, L.F. (1971) Proc. Natl. Acad. Sei. U.S. 68, 2 8 5 7 - - 2 8 6 0

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