Hydrophobic mannosides act as acceptors for trypanosome a-mannosyltransferases

Glycobiology vol. 7 no. 4 pp. 549-558, 1997 Hydrophobic mannosides act as acceptors for trypanosome a-mannosyltransferases Jillian R.Brownlr2, Maria Lucia S.Giither1, Robert A.Field2 and Michael A J.Ferguson1"3 'Department of Biochemistry, University of Dundee, Dundee DD1 4HN, Scotland and 2Scbool of Chemistry, University of St. Andrews, SL Andrews, Fife KYI6 9ST, Scotland 'T• o whom correspondence should be addressed Key words: dolichol/glycosylphosphatidylinositol/mannosyltransferase/trypanosome Introduction The tsetse fly—transmitted African trypanosomes, which cause human sleeping sickness and a variety of livestock diseases, are able to survive in the mammalian bloodstream by virtue of their dense cell-surface coat. This coat consists of 5 million copies of a homodimer of a 55 kDa GPI-anchored and Nglycosylated glycoprotein called the variant surface glycoprotein (VSG; Cross, 1990). The majority of the VSGs fall into two major types, based on peptide homology towards their COOH-termini (Holder, 1981; Boothroyd, 1985). The type-1 VSGs generally have a single N-glycosylation site about 50 residues from the mature COOH-terminus of the protein that is occupied by oligomannose structures (Zamze et al., 1990). The type-2 VSGs generally have two N-glycosylation sites, one about 50 residues from the COOH-terminus occupied by oligomannose structures and another 5-6 residues from the COOH© Oxford University Press 549 Downloaded from http://glycob.oxfordjournals.org/ by guest on April 20, 2016 A series of hydrophobic mannosides were synthesized and tested for their ability to act as acceptor substrates for mannosyltransferases in a Trypanosoma brucei cell-free system. The thiooctyl a-mannosides and octyl a-mannosides all accepted single mannose residues in a-linkage, as judged by thin layer chromatography of the products before and after jack bean a-mannosidase digestion. The mannosylation reactions were inhibited by amphomycin, suggesting that the immediate donor was dolicholphosphate-mannose (Dol-P-Man) in all cases. The transferred a-mannose residues were shown to be both ocl-2 and a l - 6 linked by Aspergillus phoenicis a-mannosidase and acetolysis treatments, respectively. These data suggest that the compounds can act as acceptor substrates for the DolP-Man dependent otl-2 and al-6 mannosyltransferases of the GPI biosynthetic pathway and/or the dolichol-cyde of protein N-glycosylation. One of the compounds, Manotl6Manal-O-(CH 2 ) 7 CH3, inhibited endogenous GPI biosynthesis in the cell-free system, suggesting that it could be a substrate for the trypanosome Dol-P-Man:Man2GlcN-Pl a 1-2 mannosyltransferase. terminus occupied by complex structures that can include polylactosamine structures (Zamze, 1991; Zamze et al, 1991). The relative abundance of the VSG protein in Trypanosoma brucei has made this organism extremely useful for the study of GPI anchor biosynthesis. The structure of the type-1 VSG GPI anchor is known (Ferguson et al., 1988; Strang et al, 1993), and the principal features of the GPI biosynthetic pathway in trypanosomes were elucidated using a cell-free system based on washed trypanosome membranes (Masterson et al, 1989, 1990; Menon et al., 1990b). The first step in the pathway involves the transfer of GlcNAc from UDP-GlcNAc to endogenous phosphatidylinositol (PI), via a sulfhydryl-dependent GlcNAc-transferase (Milne et al, 1992), to form GlcNAc-PI which is rapidly de-N-acetylated (Doering et al, 1989) to give glucosaminyl-PI (GlcN-PI). Three aMan residues are sequentially transferred onto GlcN-PI from dolichol-phosphate mannose (Dol-P-Man) (Menon et al., 1990a) to produce the intermediate Manal-2Manal-6Manal-4GlcN-PI (Man3GlcNPI). All of the mannosylated intermediates can be found in both inositol-acylated and nonacylated forms, and the Man3GlcN(acyl)PI species appears to be the preferred substrate for ethanolamine phosphate (EtNP) addition (Giither and Ferguson, 1995), the donor for which is phosphatidylethanolamine (Menon et al, 1993), to give rise to glycolipid C (EtNPMan3GlcN-(acyl)PI). Glycolipid C is then deacylated to form glycolipid A' (EtNP-Man3GlcN-PI), that undergoes a series of fatty acid-remodeling reactions (Masterson et al, 1990) in which the fatty acids of the PI moiety are removed and replaced with myristate to yield the mature GPI precursor glycolipid A. This mature precursor is transferred to the VSG polypeptide in the endoplasmic reticulum in exchange for a C-terminal GPI signal peptide, reviewed by Udenfriend and Kodukula (1995). The VSG anchor is subsequently modified by the addition of otGal residues from UDP-Gal (Pingel and Duszenko, 1992), most likely in the Golgi apparatus (Bangs et al, 1988). We have recently described assays for the GlcNAc-PI deN-acetylase (Milne et al., 1994) and for the first a-mannosyltransferase of the GPI biosynthetic pathway in trypanosomes (Smith et al., 1996) based on the use of a chemically synthesized GlcNAc-PI and GlcN-PI substrates (Cottaz et al, 1993). In an attempt to establish assays for other glycosyltransferases of the GPI biosynthetic pathway, we have prepared and tested a series of synthetic hydrophobic thiooctyl mannosides and octyl mannosides. One of these, M a n a l - 6 M a n a l - S (CH2)7CH3, has been shown to act as an acceptor for the trypanosome UDP-Gal:GPI anchor a 1-3 galactosyltransferase that is unique to this organism (Pingel et al, 1995). In this article, we show that the trypanosome cell-free system mannosylates this mannoside as well as three other synthetic mannosides and that at least one of these compounds, Manal6Manal-O-(CH2)7CH3, appears to act as an acceptor for the Dol-P-Man:Man2GlcN-PI ot-mannosyltransferase of the GPI biosynthetic pathway. J.RBrown et al excised bands. Representative results using the Manal6Manal-S-C 8 and Manal-6Manal-O-C 8 compounds are shown in (Figure 1, lanes 3-15), where the [3H]mannosylated products of these acceptors migrate close to a tri-mannoside standard. For the four active acceptors, the recovered counts increased with acceptor concentration up to a maximum at about 1, 2, 0.5, and 2 raM for Manal-S-C 8 , Manal-O-Cg, Manal-6Manal-S-C 8 , and Manal-6Manotl-O-C 8 , respectively, but then decreased above 2 mM. The reason for the decrease in acceptor activity is most likely due to the detergentlike properties of the acceptors. Consistent with this view, the inclusion of detergents such as Triton X-100 (0.4 mM) or n-octyl-fJ-D-glucoside (10 mM) also significantly reduced the [3H]mannosylation of the acceptors (data not shown). The sensitivity of the trypanosome cell-free system to detergents, both stimulatory and inhibitory, has been previously noted (Smith et al, 1996). The efficiencies of the different acceptors at their optimal acceptor concentrations, relative to the maximum rate of transfer measured using 2 mM Manod-6Manal-0-C 8 (i.e., 20 pmol/h/109 cell equivalents of membrane), are shown in Table I. The [3H]mannosylation of Manal-6Manal-O-C 8 (Figure 1, lane 14) is of a similar order of magnitude to the [3H]mannosylation of synthetic GlcNAc-PI (Figure 1, lane 1), the most potent compound for priming the GPI biosynthetic pathway in the trypanosome cell-free system (Smith et al, 1996), suggesting that Manotl-6Manal-O-C 8 is also a good acceptor for trypanosome mannosyltransferase(s). Characterization of the [3H]mannosylated products In all cases, the major radioactive [3H]mannosylated products had Rf values consistent with the addition of a single [3H]Man residue to the acceptors (see for example Figure 1). The Front• >• • DPM GPI - —»*<- DPM -<- M 2 OC 8 I — <• • M 3 OC 8 origin • GlcNAc-PI + Man2SC8(mM) Man ? 0C 8 (mM) - 2 3 4 5 6 7 8 9 - 0.1 0.3 0.5 1.0 1.5 2.0 4.0 10 11 12 13 14 15 0.1 0.3 0.5 1.0 1.5 4.0 Fig. 1. Synthetic mannosides act as acceptors for trypanosome mannosyltransferases. Washed trypanosome membranes were incubated with GDP[3H]Man and various acceptor compounds. The extracted glycolipids were analysed by HPTLC using solvent system A and fluorography. In the absence of exogenous acceptor, Dol-P-Man (DPM) is the only major product (lane 2). The bands running below Dol-P-Man m lane 1 are [%]Man-labeled GPI intermediates, formed by the processing of the exogenous synthetic GlcNAc-PI acceptor (Smith et aL, 1996), indicating that the trypanosome GPI-pathway a-mannosyltransferases are functional. Lanes 3-9 and lanes 10-15 show the [3H]mannosylated products of the Manal-6Manal-S-C 8 (Man^SCg) and Manal-6Manal-O-Cg (MarijOCj) acceptors, respectively, wben incubated with the washed membranes al the concentrations indicated. The positions of tbe nonradioactive standards of Manor l-6Manal-S-C, (ManjSC,), Manal-fiManorl-O-Cj (ManjOC^, and Manal-6(Manal-3)Manal-O-C, (Man3OC») are indicated on the left and the right of the chromatogram. The [3H]Man-labeled bands marked as GPI in lane 1 are the GPI intennedifltes formed in the control incubation with exogenous GlcNAc-PL 550 Downloaded from http://glycob.oxfordjournals.org/ by guest on April 20, 2016 Results Hydrophobic mannosides act as mannose acceptors in the trypanosome cell-free system The compounds Manpi-S-C 8 , Manotl-S-Cg, Manal-O-C 8 , Manal-6Manal-S-C 8 , Manal-6Manal-O-C 8 , and Glcal6Glcal-S-Cg (where Cg denotes the -(CH2) 7 CH 3 alkyl chain) were tested for their abilities to act as acceptor substrates for mannosyltransferases present in washed trypanosome membranes. The compounds were added at various concentrations to the membranes together with GDP-[3H]Man and in the presence of N-ethylmaleimide and tunicamycin. The products were recovered by solvent extraction, partitioned between water and butan-l-ol, and the butan-1-ol phases were analyzed by HPTLC. Under these conditions, Dol-P-Man is the only major labelled product (Figure 1, lane 2) because the synthesis of endogenous GPI intermediates is substantially suppressed by the inhibition of the UDP-GlcNAc:PI aGlcNAc transferase of the GPI pathway by N-ethylmaleimide (Milne et al, 1992) and the synthesis of dolichol-cycle intermediates is substantially suppressed by tunicamycin. However, if exogenous synthetic GlcNAc-PI is added a range of radiolabeled GPI intermediates from Man,GlcN-PI to EtNP-Man3GlcN-PI are formed (Smith et al, 1996) (Figure 1, lane 1), showing that the GPI pathway ot-mannosyltransferases are active in this preparation. In this assay system, the Glcal-6Glcal-S-Cg compound showed no mannose-acceptor activity and the Man|31-S-C8 compound showed negligible acceptor activity (data not shown). In contrast, the other four compounds produced significant radiolabeled bands when the products were analyzed by HPTLC. The radioactivity associated with the [3H]mannosylated products was estimated by scintillation counting of the Trypanosome mannosyltransferase acceptor substrates Table L Characterization of the [3H]mannosylated products that result from transfer of [3H]Man to the synthetic glycoside acceptors [3H]Man linkage (%) Relative efficiency1" (%) Compound (optimum cone.*) Manal-S^CH^CHj (1 mM) Manal-O-{CH2>7CH3 (2 mM) Mana 1 -6Mana 1 -S^CH^CHj (0.5 mM) Mana 1 -6Mana 1 -CMCH^CHj (2mM) al-2 al-6 69 60 48 24 52 76 47 31 69 100 44 56 *The concentration of acceptor that gave the maximum yield of [3H]mannosylated product Th • e acceptor efficiencies are expressed relative to the yield of the [3rf)mannosylated product of Mana 1-6Mana 1-O-(CH 2 )TCH3 (20 pmol/h/109 cell equivalents of washed membranes). Front - Man2OC8->. -Man2SC8 OriQin " JBAM Acceptor 5 [Std] 6 Man 2 SC 8 Man 2 OC 8 7 8 9 10 ManSC8 ManOC 8 Fig. 2. Characterization of the [3H]mannosylated products of the synthetic mannosides. HPLC-purified [3H]mannosylated products of the Manal-S-Cg (ManSC,), Manal-O-C, (ManOCg), Manal-6Manal-S-Cg (MaiVjSCj), and Manal-6Manal-O-C, (MarijOQ) mannosides and a [3H]myristate labeled Man3GlcN-PI standard (Std) were incubated with and without jack bean a-mannosidase (JBAM), as indicated. The products were partitioned between water and butan-1-ol and the butan-1-ol phases were analyzed by HPTLC using solvent system A and fluorography. The conversion of Man3GlcN-PI to GlcN-PI (lanes 1 and 2) serves as a positive control for the a-mannosidase. The standards indicated on the left and right of the chromatogram are as described in the Figure 1 caption. The analyses summarised in Table I show that for all of the acceptors both al-2 and al-6 (but not a 1-3/4) linked products were detected suggesting that at least two different ot-mannosyltransferases present in the trypanosome membranes were utilising the hydrophobic mannosides as acceptor substrates. The nature of the a-mannosyltransferase activities The trypanosome cell-free system contains Dol-P-Man synthetase and therefore converts some of the added GDP-[3H]Man to Dol-P-[3H]Man (see Figure 1). In order to assess whether the a-mannosyltransferases responsible for the [3H]mannosylation of the acceptor compounds were GDP-Man- or Dol-P-Mandependent, the enzymic reactions were performed in the presence and absence of amphomycin and calcium. In the presence of Ca2+ this antibiotic is a potent inhibitor of trypanosome Dol-P-Man synthetase (Prado-Figueroa et ai, 1994) and should therefore inhibit Dol-P-Man-dependent, but not GDPMan-dependent, aMan transfer. The results (Figure 4A,B) show that in all cases preincubation of the cell free system with amphomycin and Ca abrogated the formation of both DolP-[ H]Man and the [3H]mannosylated products of the acceptor compounds. Thus, the a-mannosyltransferases detected with the hydrophobic mannoside acceptors all appear to be Dol-PMan-dependent enzymes. The substantial (but incomplete) inhibition observed with amphomycin in the absence of added Ca2+ (Figure 4A, lanes 3 and 6; Figure 4B, lanes 11 and 15) is presumably due to the presence of trace amounts of Ca2+ in the cell-free system and/or the amphomycin preparation. The trypanosome cell-free system contains enzymes of the GPI biosynthetic pathway and of the dolichol-cycle of protein 551. Downloaded from http://glycob.oxfordjournals.org/ by guest on April 20, 2016 [3H]mannosylated products were purified from radiolabeled Dol-P-Man by reverse phase HPLC, subjected to digestion with jack bean a-mannosidase and repartitioned between water and butan-1-ol (Figure 2). In each case the enzyme quantitatively removed the [3H]Man residue attached to the acceptor, resulting in the loss of the labelled band upon HPTLC analysis. These results show that the compounds can act as acceptor substrates for ot-mannosyltransferase(s) present in the trypanosome membranes. In order to assess the nature of the glycosidic linkage(s) between the transferred [3H]Man residues and the acceptor molecules, each HPLC-purified product was subjected to exhaustive digestion with Manal-2Man-specific A.phoenicis a-mannosidase followed by acetolysis. In this way the relative proportions of al-2-, al-6-, and al-3/4-linked [3H]Man could be ascertained. The results of one representative analysis, for Manal^Manal-O-Cg, are shown in Figure 3 and the results for the four compounds are shown in Table I. The first digestion of the [ 3 H]mannosylated M a n a l - 6Manal-O-C 8 product with A.phoenicis a-mannosidase released a significant amount of radioactivity (56%) that was recovered in the aqueous phase of a water/butan-1-ol partition and that comigrated with free mannose by HPTLC (Figure 3A, lane 4). Subsequent digestion of the material that remained in the butan-1-ol phase with more A.phoenicis mannosidase did not result in any further release of free [3H]Man (Figure 3B, lane 2), showing that the mannosidase digestion was exhaustive. A small amount of the undigested material appears in the aqueous phase of the control (Figure 3A, lane 2) and in both A.phoenicis a-mannosidase digests (Figure 3A, lane 4; Figure 3B lane 2). This is accounted for in the figures shown in Table I. Acetolysis of the standards Manal-6Manal-0-Cg, Manal6Manal-S-C 8 , and Manal-3Manal-O-Cg showed that the procedure gave quantitative cleavage of Manal-6Man glycosidic bonds and only partial cleavage of Manal-3Man glycosidic bonds, as expected (compare Figure 3C lanes, 2, 4, and 6). The results also showed that both the O and S glycosidic bonds to the C 8 aglycone were quantitatively cleaved by this procedure, producing exclusively free Man from Manal6Manal-O-C 8 and Manal-6Manal-S-C 8 and a mixture of Manal-3Man disaccharide and free Man from M a n a l 3Manal-O-C 8 . Thus, the exclusive release of free [3H]Man from a radiolabeled glycoside by acetolysis is consistent with an al-6 linkage between the [3H]Man residue and the acceptor whereas the release of a radiolabeled disaccharide would be consistent with the presence of some a 1-3/4 linkage. The result shown in Figure 3D (lane 1), which shows the liberation of only free [3H]Man, indicates that all of the labeled product remaining from the exhaustive A.phoenicis a-mannosidase digestions must have contained exclusively a l - 6 linked [3H]Man. JJCBrown et aL B Frant From- Z - 8 Man 3 O C 8 Origin- - Aq Control • BuOH - Origin- Downloaded from http://glycob.oxfordjournals.org/ by guest on April 20, 2016 BuOH - ^ Aq APAM-1 APAM-2 D Front Front - • - iiii.Miiiii-iimrHil~aMin • -* ' »« • • ^t i •* I D-Man - > • Manl-3Man OrtginOriB 1 " ' Acetolysis Mana1-6Man al-SC e + Mana1-6Man a 1 - 0 C 8 Mana1-3Man a l - O C 8 - ~- + + - - • - + + - + - - " + ~ 7 8 9 O-Man H1-3M M1-6M 1 Acetolysis + Fig. 3. Analysis of the linkage composition of the [ 3 H]Man-(Manal-6Manal-O-C,) product by exhaustive exoglycosidase digestion and partial acetolysis. (A) The HPLC-purified [3H]mannosylated product of the Manal-6Manal-O-C, acceptor was digested with (APAM-1) and without (Control) A.phoaucis cr-mannosidase, and the products were partitioned between water (Aq) and butan-1-ol (BuOH), as indicated. Aliquots of the products were analysed by HPTLC using solvent system A and fluorography. (B) The butan-1-ol phase material from the APAM-1 digest was redigested with A.phoenicis a-mannosidase (APAM-2), and the products were partitioned between water (Aq) and butan-1-ol (BuOH), as indicated. Aliquots of the products were analyzed by HPTLC using solvent system A and fluorography. (C) Mannoside standards (Manal-6Manal-S-C,, lanes 1 and 2; Manal^5Manal-O-Cj, lanes 3 and 4 and Manor l-SManal-O-Cg, lanes 5 and 6) were peracetylated and subjected to acetolysis (+) or to control incubations (-) and de-O-acetylated prior to HPTLC analysis using solvent system B and orcinol-staining. Standards of free mannose (D-Man), Manal-3Man (M1-3M), and Manal-6Man (M1-6M) were chromatographed on the same HPTLC plate and stained with orcinol (lanes 7, 8, and 9, respectively). Note: the minor bands seen running ahead of the major products in lanes 2, 4 and 6 are due to incomplete de-O-acetylation, as judged by electrospray-mass spectrometry (data not shown). (D) the butan-1-ol phase material from the APAM-2 digest was subjected to acetolysis and the products were analysed by HPTLC using solvent system B and fluorography. The standards indicated on the sides of (A) and (B) are as described in the Figure 1 caption. In (D), D-Man indicates the position free mannose and Manl-3Man indicates the position of Manor 1 -3Man disaccharide. 552 Trypanosome mannosyttransferase acceptor substrates B Front - r ManOC 8 /ManSC 8 sx tl DPM ' r Man 2 OCg/ Man 2 SC g - Man 3 OC 8 • originStd Acceptor - *" * "••• 2 + 3 + 4 + - Man2SCg 5 + " - Man 6 7 8 + + - + + 2 OCg 9T 1^" 11 12 Man1SC 8 13 1 4 1 5 1 6 Man 10Cg Fig. 4. Amphomycin inhibits the transfer of [3H]Man to the mannoside acceptors. The formation of Dol-P-[3H]Man (DPM) and the [3H]mannosylated products of the di-mannosides (A) and the mono-mannosides (B) was measured using the standard assay in the presence and absence of amphomycin and Ca2+, as indicated. The glycolipid extracts were analyzed by HPTLC using solvent system A and fluorography. In (A), lane 1 (Std) shows trypanosome Dol-P-[3H]Man formed by the standard assay in the absence of acceptor substrate. The standards and acceptors indicated at the sides of and below the cbromalograms are as described in the Figure 1 caption. N-glycosylation (Low et al., 1991). Both of these pathways contain distinct Dol-P-Man dependent a 1-2 and a 1-6 mannosyltransferases. In order to assess whether the hydrophobic mannosides could act as acceptors for the GPI pathway enzymes, the compounds were preincubated with the trypanosome cell-free system which was subsequently labeled with UDP-[3H]GlcNAc in the presence of excess (0.4 mM) GDPMan and in the absence of N-ethylmaleimide. Under these conditions, the cell-free system produces a whole range of labeled GPI intermediates, from GlcNAc-PI to glycolipid A' (EtNP-Man3GlcN-PI) and its lyso-form, glycolipid 0 (Masterson et al., 1989; Giither and Ferguson, 1995); see Figure 5A. In previous experiments using the trypanosome cell-free system pre-loaded with and without the Dol-P-Man:Man2GlcN-PI a 1-2 mannosyltransferase inhibitor ManNH 2 al-6Manotl4GlcN-PI we observed that this inhibitor does not cause an accumulation of the GPI intermediate proceeding the metabolic block (i.e., Man2GlcN-PI) but rather exhibits its effects by reducing the formation of the downstream-components glycolipids A' and 9 (Ralton et al., 1993). Thus, the addition of compounds to the cell-free system that inhibit, or compete with the endogenous substrates for, GPI pathway a-mannosyltransferases should have little effect on the labeling of the earlier intermediates but should result in a reduction in the combined amount of label in the final products, namely glycolipids A' and 0. Of the four compounds tested, only Manal-6ManalO-C8 clearly produced this effect (Figure 5B.C). As a control, the hydrophobic diglucoside Glcotl-4Glcpl-O-Cg was also tested. In this case, the formation of glycolipids A' and 0 were stimulated (Figure 5D), suggesting that the inhibition seen with the dimannoside was a specific effect and not simply due to the detergent-like properties of the compound. The possibility that the observed inhibition might be due to the production of GDP (due to the turnover of GDP-Man during the mannosylation of the Manal-6Manal-O-Cg acceptor) can be ruled out since the addition of up to 1 mM GDP to the cell free system had no effect on the labelling of GPI intermediates (data not shown). Similarly, the results shown in Figure 1 suggest that the dimannoside has no effect on the trypanosome Dol-P-Man synthetase activity required to produce the Dol-P-Man donor. Discussion Hydrophobic glycosides have proved to be extremely useful reagents and are often used as acceptor substrates for the assay and characterisation of glycosyltransferases, see for example (Lowary et al., 1994; Pohlentz et al., 1995; Strangier et al., 1995). Hydrophobic monosaccharide glycosides have also been shown to prime the formation of mucin, glycosaminoglycan, glycolipid, and polylactosamine oligosaccharide chains when added to cultured cells and, in some cases, to inhibit the synthesis of endogenous glycoconjugates (Kuan et al., 1989; Freeze et al., 1993; Fritz et al., 1994; Neville et al., 1995, and references therein). The 3-xyloside naroparcil, which has antithrombotic activity, has been shown to prime glycosaminoglycan synthesis in vivo (Masson et al., 1995). Higher glycosides have been also shown to be taken up and metabolised by cells when they are methylated and/or acetylated (Sarkar et al., 1995). Such observations prompted us to assess the usefulness of a series of hydrophobic mannosides as substrates for trypanosome glycosyltransferases in vitro. The data presented here show that all of the synthetic hydrophobic a-monomannosides and a-dimannosides, but not the p-monomannoside or the a-diglucoside, act as acceptors for trypanosome a-mannosyltransferase activities. It is interesting to note that all of these glycosides were substrates for 553 Downloaded from http://glycob.oxfordjournals.org/ by guest on April 20, 2016 Amphomycin CaCI 5 ~ 1 Trypanosome mannosyttransferase acceptor substrates B From- -<• Man 2 OCg/ Man 2 ^ 8 t * origin1 Amphomycin CaO 2 Acceptor - ^ 2 3 4 + + + + Man 2 SC 8 5 6 - + + <<- Man3OC8 7 - 8 9 1*0 11 12 13 14 15 16 + + Man2OC8 Man 10C g Fig. 4. Amphomycin inhibits the transfer of [3H]Man to the mannoside acceptors. The formation of Dol-P-[3H]Man (DPM) and the [3H]mannosylated products of the di-mannosides (A) and the mono-mannosides (B) was measured using the standard assay in the presence and absence of amphomycin and Ca2+, as indicated. The glycolipid extracts were analyzed by HPTLC using solvent system A and fluorography. In (A), lane 1 (Std) shows trypanosome Dol-P-[3H]Man formed by the standard assay in the absence of acceptor substrate. The standards and acceptors indicated at the sides of and below the cbromalograms are as described in the Figure 1 caption. N-glycosylation (Low et al., 1991). Both of these pathways contain distinct Dol-P-Man dependent a 1-2 and a 1-6 mannosyltransferases. In order to assess whether the hydrophobic mannosides could act as acceptors for the GPI pathway enzymes, the compounds were preincubated with the trypanosome cell-free system which was subsequently labeled with UDP-[3H]GlcNAc in the presence of excess (0.4 mM) GDPMan and in the absence of N-ethylmaleimide. Under these conditions, the cell-free system produces a whole range of labeled GPI intermediates, from GlcNAc-PI to glycolipid A' (EtNP-Man3GlcN-PI) and its lyso-form, glycolipid 0 (Masterson et al., 1989; Giither and Ferguson, 1995); see Figure 5A. In previous experiments using the trypanosome cell-free system pre-loaded with and without the Dol-P-Man:Man2GlcN-PI a 1-2 mannosyltransferase inhibitor ManNH 2 al-6Manotl4GlcN-PI we observed that this inhibitor does not cause an accumulation of the GPI intermediate proceeding the metabolic block (i.e., Man2GlcN-PI) but rather exhibits its effects by reducing the formation of the downstream-components glycolipids A' and 9 (Ralton et al., 1993). Thus, the addition of compounds to the cell-free system that inhibit, or compete with the endogenous substrates for, GPI pathway a-mannosyltransferases should have little effect on the labeling of the earlier intermediates but should result in a reduction in the combined amount of label in the final products, namely glycolipids A' and 0. Of the four compounds tested, only Manal-6ManalO-C8 clearly produced this effect (Figure 5B.C). As a control, the hydrophobic diglucoside Glcotl-4Glcpl-O-Cg was also tested. In this case, the formation of glycolipids A' and 0 were stimulated (Figure 5D), suggesting that the inhibition seen with the dimannoside was a specific effect and not simply due to the detergent-like properties of the compound. The possibility that the observed inhibition might be due to the production of GDP (due to the turnover of GDP-Man during the mannosylation of the Manal-6Manal-O-Cg acceptor) can be ruled out since the addition of up to 1 mM GDP to the cell free system had no effect on the labelling of GPI intermediates (data not shown). Similarly, the results shown in Figure 1 suggest that the dimannoside has no effect on the trypanosome Dol-P-Man synthetase activity required to produce the Dol-P-Man donor. Discussion Hydrophobic glycosides have proved to be extremely useful reagents and are often used as acceptor substrates for the assay and characterisation of glycosyltransferases, see for example (Lowary et al., 1994; Pohlentz et al., 1995; Strangier et al., 1995). Hydrophobic monosaccharide glycosides have also been shown to prime the formation of mucin, glycosaminoglycan, glycolipid, and polylactosamine oligosaccharide chains when added to cultured cells and, in some cases, to inhibit the synthesis of endogenous glycoconjugates (Kuan et al., 1989; Freeze et al., 1993; Fritz et al., 1994; Neville et al., 1995, and references therein). The 3-xyloside naroparcil, which has antithrombotic activity, has been shown to prime glycosaminoglycan synthesis in vivo (Masson et al., 1995). Higher glycosides have been also shown to be taken up and metabolised by cells when they are methylated and/or acetylated (Sarkar et al., 1995). Such observations prompted us to assess the usefulness of a series of hydrophobic mannosides as substrates for trypanosome glycosyltransferases in vitro. The data presented here show that all of the synthetic hydrophobic a-monomannosides and a-dimannosides, but not the (3-monomannoside or the a-diglucoside, act as acceptors for trypanosome a-mannosyltransferase activities. It is interesting to note that all of these glycosides were substrates for 553 Downloaded from http://glycob.oxfordjournals.org/ by guest on April 20, 2016 Std • * J JLBrown et at Man3GlcN-PI 300 M«n3GlcN-acy/PI GlcN-PI 100 GlcNAc-PI B Man3GlcN-PI 300 Man3GlcN-acy/P <D GlcN-PI 100 GlcNAc-PI CD Man3GlcN-PI 300 13 O ManjGlcN-acy/PI Man2G!cN-PI O GlcN-PI 100 GlcNAc-PI Man3GlcN-PI 300 Man^lcN-acy/PI 100 GlcNAc-PI The ability of all of the hydrophobic glycosides to act as acceptors for at least two different trypanosome a-mannosyltransferases limits their usefulness as substrates for mannosyltransferase purification in the early stages of membrane solubilization and fractionation. However, once an activity such as the GPI pathway a 1-2 mannosyltransferase has been partially purified, perhaps using more complex and specific substrates such as Man2GlcN-PI, the simpler and synthetically more accessible Mana 1-6Mana 1-O-C8 analog should be extremely useful as an acceptor substrate for monitoring column fractions and for enzyme characterisation. Finally, the ability of Manotl-6Manal-0-C 8 to inhibit the GPI pathway (Figure 5) suggests that this relatively small and simple structure could be used as the starting point for the synthesis of a series of analogs that might be more potent GPI pathway inhibitors. Given the dependence of African trypanosomes, and related trypanosomatid parasites, on their highly abundant GPI-anchored surface glycoconjugates (McConville and Ferguson, 1993), this pathway represents a suitable target for the development of novel chemotherapeutic agents. Materials and methods Materials 0 8 cm Fig. 5. The dimannoside Manal-6Manal-O-C, inhibits the GPI-palhway in the trypanosome cell-free system. Washed trypanosome membranes were incubated with UDP-[3H]GlcNAc and GDP-Man (A) and in the presence of 0.1 mM (B) and 0.2 mM ( Q Manal-6Manal-O-C, or 0.2 mM Glcal-6Glcal-O-C 8 (D). The glycolipid extracts were analysed by HPTLC using solvent system A and a linear analyzer TLC-scanneT. The identities of the peaks are as described in the Introduction. 554 GDP-[3H]Man (9.8 Ci/mmol), UDP-[3H]GlcNAc (48.8. Ci/mmol), and En3Hance were purchased from Dupont NEN. Jack bean a-mannosidase was from Boehringer-Mannheim and Aspergillus phoenicis a-mannosidase was from Oxford GlycoSystems. Octyl maltoside (Glcal^GlcBl-CXCH^CHj) was purchased from Calbiochem. The [3H]myristate-labeled glycolipid standard Mana l-2Manal-6Manal-4GIcNal-6?I (Man3GlcN-PI) was prepared as described previously (Milne et aL, 1992). Synthetic GlcNAc-PI was prepared according to (Cottaz et aL, 1993). Manal-3Man and Manal-6Man disaccharides were obtained from Dextra-Laboratories. All solvents and general reagents were from BDH-Merck or from Sigma. Downloaded from http://glycob.oxfordjournals.org/ by guest on April 20, 2016 A' Dol-P-Man-dependent, rather than GDP-Man-dependent, a-mannosyltransferases. Of the compounds tested, only Manet l-6Manal-O-C 8 clearly showed an ability to inhibit the GPI pathway, presumably by competing with endogenous GPI intermediates for GPI pathway a-mannosyltransferase(s). Thus, it is likely that this compound acts as an acceptor for the trypanosome GPI pathway a 1-2 and/or a 1-6 mannosyltransferase. It is not possible to define unambiguously which of the two enzymes act on this substrate, but it seems likely that it is the Dol-P-Man: Man2GlcN-PI a 1-2 mannosyltransferase since (1) Manal6Manal-0-C g is a close analogue of the natural acceptor, namely, Manal-6Manal-4GlcNod-6PI (Figure 6) and (2) the effects of the dimannoside on the cell-free system (Figure 5) were similar to those previously observed for a specific inhibitor of the aforementioned enzyme (Ralton et aL, 1993). Whether the Dol-P-Man:Man2GlcN-PI a 1-2 mannosyltransferase activity in the trypanosome cell-free system would account for all of the a 1-2 transferase activity observed when Manal6Manal-O-C 8 is used as an acceptor, or whether some is due to the dolichol-cycle Dol-P-Man-dependent a 1-2 mannosyltransferase(s) (the natural substrate for which is Man7GlcNAc2-PP-Dol; see Figure 6), has not been resolved. However, the a 1-6 transferase activity detected using the dimannoside acceptor is more likely to be of the dolichol-cycle than of the GPI pathway since Manal-6Manal-O-C 8 is a better analog of the dolichol-cycle acceptor (Ma^GlcNAc^PPDol; see Fig. 6) than the GPI acceptor (i.e., Manal-4GlcNal6PI). Trypanosome maimosyltransferasc acceptor substrates Synthesis of thiooctyl glycosides and octyl glycosides Preparation of trypanosomes and the trypanosome cell-free system Trypanosoma brucei (variant MTTatl .4) bloodstream forms were isolated from infected rats and cell lysates were prepared as described by (Masterson et aL, 1989) with the modifications described in (Masterson and Ferguson, 1991). Mannosyltransferase assay Membranes for the mannosyltransferase assays were prepared by thawing an aliquot of lysate (1 ml containing 5 x 10s cell equivalents) and washing the membranes twice in 50 mM sodium-Hepes buffer, pH 7.4, containing 25 mM KC1, 5 mM MgCl2, 0.1 mM N"" p-tosyl-L-lysine chloromethyl ketone (TLCK) and 2 jig/ml leupeptin. The membranes were finally resuspended at 3 x 10^ cell equivalents/ml in 2x concentrated incorporation buffer (100 mM sodiumHepes, pH 7.4, 50 mM KC1, 10 mM MgCl2, 10 mM MnCl2, 20% (v/v) glycerol, 2.0 u,g/ml leupeptin, 2.5 u-g/ml tunicamycin, and 0.2 mM TLCK). Unless otherwise stated, the 2x concentrated incorporation buffer was supplemented with freshly prepared 0.2 mM W-ethylmaleimide to suppress the synthesis of endogenous GPI intermediates (Milne et aL, 1992). For each assay 0.4 u,Ci of GDP-[3H]Man, and various amounts of the synthetic glycosides, were dried in an Eppendorf tube and redissolved in 20 u.1 water. The reactions were initiated by the addition of 20 \x\ of washed membranes (6 x 107 cell equivalents) in 2x incorporation buffer. For the experiments with amphomycin, the washed membranes were preincubated with and without 1 mg/ml amphomycin and/or 10 raM CaCl2 for 15 min on ice prior to starting the reactions. After incubation for 1 h at 30°C the reactions were stopped by the addition of 267 uJ of chloroform/methanol (1:1, v/v) and the lipids were extracted for 16 h at 4"C. After centrifugation, the lipid extract was dried under a stream of N 2 , dissolved in 100 uJ of water-saturated butan-1-ol, and partitioned with 100 u.1 of water saturated with butan-1-ol. AfteT vortexing and centrifugation, the [3H]mannosylated products were recovered in the upper butan-1-ol phase. The aqueous phase was then re-extracted three times with butan-1-ol and the combined butan-1-ol extracts were back-extracted twice with 100 u.1 of water saturated butan-1-ol, dried, and analyzed by HPTLC. Generation of the [3H]mannosylated products of the hydrophobia glycosides for structural characterization Large-scale mannosyltransferase reactions were performed to generate sufficient material for structural characterization. The reaction conditions were similar to those described above except that 5 x 1 0 * cell equivalents of washed membranes were incubated with 4 p.Ci of GDP-[3H]Man in 400 u.1 lx incor- poration buffer. The final concentrations of tbe synthetic glycoside acceptors were 1, 2, 0.5, and 2 mM for Mano 1 -S-Q, Manal-O-C,, Manal-6ManalS-C,, and Manal-6Manal-O-Cg, respectively. After incubation for 1 h at 30°C, reactions were stopped by the addition of 2.6 ml of chloroform/methanol (1:1, v/v) and the lipids were extracted for 16 h at 4°C. After centrifugation, the lipid extracts were dried under a stream of Nj, dissolved in 500 uJ of wateTsaturated butan-1-ol, and partitioned with 500 pJ of water saturated with butan1-ol. After vortexing and centrifugation, the [3H]mannosylated products were recovered in the upper butan-1-ol phase. The aqueous phase was then reextracted three times with 500 u.1 butan-1-ol, and the combined butan-1-ol extracts were then back-extracted twice with 500 uJ of water-saturated butanol-ol. Aliquots of the final butan-1-ol phase were dried and analyzed by HPTLC. The remaining material was dried; redissolved in 100 mM ammonium acetate, 5% acetonitrile; and subjected to reverse phase HPLC. Reverse-phase HPLC The [3H]mannosylated products of the synthetic glycoside acceptors were purified from Dol-P-[3H]Man by reverse phase HPLC on a C, 8 reverse-phase column (Hichrom KR100-5 C18, 25 x 0.46 cm). The column was equilibrated in 100 mM ammonium acetate, 5% acetonitrile. The elution program was as follows: 100 mM ammonium acetate, 5% acetonitrile for 5 min then a linear gradient to 5% acetonitrile over 5 min followed by a linear gradient to 100% acetonitrile over 50 min (held at 100% acetonitrile for 10 min). The flow rate was 1 ml/min. Fractions (1 ml) were collected, and 100 uJ of each fraction was taken for scintillation counting. The [3H]mannosylated products of the synthetic glycoside acceptors all eluted at about 25% acetonitrile. High performance thin layer chromatography (HPTLC) Samples were applied to silica gel-60 aluminum-backed HPTLC plates (Merck). Unless otherwise stated, the plates were developed for 10 cm with solvent system A: one development with chloroform/methanol/1 M ammonium acetate/13 M ammonia/water (180:140:9:9:23, v/v) or with solvent system B: one development with propan-1 -ol/acetone/water (5:4:1, v/v) followed by one development with butan-1-ol/acetone/water (5:3.5:1.5, v/v). The plates were scanned with Raytest Rita linear analyzer and subsequently sprayed with En3Hance and exposed to Kodak XAR-5 film at -70°C for fluorography. The lanes containing nonradioactive compounds (5—10 u,g) were cut out after development of HPTLC, sprayed with orcinol reagent (20 mg/ml orcinol monohydrate in ethanol/conc. HjSO^/water (75:10:5, v/v), and heated for 5 min at 110-C. Exoglycosidase digestions [3H]Mannosylated products were dried and redissolved in 0.1 M sodium acetate buffer, pH 5.0, containing 0.1% (w/v) sodium taurodeoxycholate and incubated for 16 h at 37°C with and without 0.75 U of jack bean a-mannosidase (30 u.1 final volume) or with and without 5 u,U of A.phoenicis ot-mannosidase (10 (il final volume). After incubation the digests were extracted twice with 50 yd of butan-1-ol saturated with wateT, and the combined butan1-ol extracts were back-extracted twice with 50 (xl of water-saturated butan1-ol. The butan-1-ol phases were dried and analyzed by HPTLC. Selective cleavage by partial acetolysis [3H]Mannosylated products were dried in a 1 ml glass vial and acetylated in 40 u,l pyridine/acetic anhydride (1:1, v/v) for 30 min at 100°C. The products were dried in a Speed Vac and residual acetic acid was removed by coevaporation with toluene (2 x 50 \xi toluene). The peracetylated products were dissolved in 30 u.1 acetic anhydride/acetic acid/sulfuric acid (10:10:1, v/v), and acetolysis was performed for 8 h at 37°C. The reaction was quenched by adding 10 uJ pyridine and 500 u.1 water. After 1 h the peracetylated products were recovered by extraction into 250 \t\ chloroform. The chloroform phase was washed three times with 500 pJ water and dried under a stream of N 2 . The products were de-O-acetylated with 200 uJ concentrated ammonia/methanol (1:1,v/v) for 60 h at 37°C. The resulting products were dried, redissolved in 40% propan-l-ol, and analyzed by HPTLC using solvent system B. GPI pathway competition assay Washed trypanosome membranes were preincubated with and without synthetic glycosides for 5 min at 30°C in 2x concentrated incorporation buffer (without N-ethylmaleimide and supplemented with 4 mM dithiothreitol). Aliquots of 20 u.1 (4 x 107 cell equivalents) were mixed with 20 uJ 0.8 mM GDP-Man containing lu,Ci UDP-[3H]GlcNAc and incubated for 20 min at 555 Downloaded from http://glycob.oxfordjournals.org/ by guest on April 20, 2016 All synthetic and associated analytical procedures were carried out using established methods, as described previously (Field et aL, 1995). Where appropriate, deprotected products were dissolved in water, washed with diethyl ether, and purified by gel filtration on a Bio-Gel P4 column. NMR spectra were recorded on a Varian Unity plus spectrometer ('H, 500 MHz) or a Varian Gemini 2000 ('H, 300MHz; I3 C, 75.4MHz). Electrospray mass spectra were recorded on an Micromass-Quattro triple-quadrupole instrument (Micromass, UK). Thiooctyl a-D-mannopyranoside [Manal-S-CJ and thiooctyl a-l,6-Dmannopyranosyl-a-D-mannopyranoside [Manal-6Manal-S-Cd were prepared as reported previously (Pingel et aL, 1995). Octyl a-D-marmopyranoside [Manal-O^CJ and octyl al,6-D-mannopyranosyl-<l,3-D-mannopyranosyl)-aD-mannopyranoside [Manotl-oXManal-SVManal-O-Cj] were prepared according to published procedures (Oscarson and Tiden, 1993). Octyl a-l,6-Dmannopyranosyl-a-D-mannopyranoside [Manal-6Manal-O-Cg] was prepared by selective mono-glycosylation of octyl 2,4-di-O-benzoyl-a-Dmannopyranoside (Oscarson and Tiden, 1993) with benzobromomannose (Nessetal., 1950) using mercury cyanide as a promoter (Lemieuxeral, 1975). Octyl al,3-D-mannopyranosyl-a-D-mannopyranoside [Manal-3Manal-O-C,] was prepared by selective mono-O-benzoylation of the primary alcohol of octyl 2,4-di-O-benzoyl-a-D-mannopyranoside (Oscarson and Tiden, 1993) using benzoyl cyanide (Paulsen and Bunsch, 1982), followed by glycosylation with benzobromomannose (Ness et aL, 1950) using silver triflate as a promoter (Oscarson and Tiden, 1993). All compounds that were subjected to biological testing gave positive and/or negative ion electrospray mass spectra consistent with their proposed structures (Pingel et aL, 1995). In negative ion mode ions corresponding to [M-Hp were observed whereas in positive ion mode ions corresponding to [M + H p and [M + Nap1" were observed. Full experimental details of the synthetic procedures and analytical data for the compounds can be found elsewhere (Brown, 1997). o o-p-cr i GlycoIipidA (mature GPI precursor) 2&ian-a-L • OMan-a-1 (A) Man2-GlcNHrPI (natural substrate for GPI pathway Dol-P-Man dependent al-2 mannosyltransferase) Man2OCg (synthetic hydropbobic glycoside) (C) MansGlcNAcrPP-DoI (natural substrate for the doUchol cyde Dol-P-Man dependent ctl-6 mannosyltransferase) Man-aMan-a-l-»lan-al-2MaiHa I (D) Man7GIcNAcrPP-Dol (natural substrate for the doUchol cyde Dol-P-Man dependent al-2 mannosyltransferase) Man-o-k "Mm^a-K' ^Man-P-l-4GlcNAc-P-l^iGlcNAc-PP-D<rf Man-a-l-2Man-al-2Man-ai (E) 556 Downloaded from http://glycob.oxfordjournals.org/ by guest on April 20, 2016 (B) Trypanosome mannosyltransferase acceptor substrates 30°C. Glycolipids were extracted and processed for HPTLC as described above. Acknowledgments This work was supported by a program grant from The Wellcome Trust to M.A.J.F., a Wellcome Trust equipment grant to Prof. D. Gani, University of St. Andrews, and a Zeneca Strategic Research Fund grant to R.A.F. J.R.B. thanks The Chemicals and Pharmaceuticals Directorate of the Biotechnology and Biological Sciences Research Council for providing a research studentship. We thank Terry Smith, John Brimacombe, and Ravi Kartha for valuable discussions and advice and Trevor Rutherford for help with some of the NMR analyses. tvLAJJ. is a Howard Hughes Medical Institute International Research Scholar. Abbreviations Dol-P-Man, dolichol-phosphate-mannose; VSG, variant surface glycoprotein; GPI, glycosylphospharidylinositol; PI, pbosphatidylinositol; Cg, Fig. 6. Homologies between the Manal-6Manal-O-C, glycoside and the acceptor substrates for GPI and dolichol-cycle a-mannosyltransferases. The structures of the mature GPI precursor, glycolipid A (A), and the Man2GlcN-PI intermediate (B), that is the natural acceptor substrate for the Dol-P-Man: Man2GlcN-PI a l - 2 mannosyltransferase of the GPI pathway, are compared with the synthetic Manal^Manal-O-Cg acceptor (C). The structures of the natural acceptors of the DoI-P-ManMan^lcNACj-PP-Dol a l - 6 mannosyltransferase (D) and the Dol-P-ManManrGlcNACj-PP-Dol a l - 2 mannosyltransferase (E) are shown underneath for comparison. The boxed areas indicate the sites of structural homology. 557 Downloaded from http://glycob.oxfordjournals.org/ by guest on April 20, 2016 References BangsJ.D., Doering.T.L., Englund,P T. and Hart,G.W. (1988) Biosynthesis of a variant surface glycoprotein of Trypanosoma brucci. J. BioL Chem., 263, 17697-17705. BoothroydJ.C. (1985) Antigenic variation in African trypanosomes. Annu. Rev. MicrobioL, 39, 475-502. BrownJ.R. (1997) Carbohydrate derivatives as substrates for parasite glycosyltransferases. 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