ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 198, No. 1, November, pp. 304-313, 1979
Incorporation of Glucose into Lipid-Linked Saccharides in Aorta and Its Inhibition by Amphomycin MOHINDER Department
of Biochemistry,
University
S. KANG of Texas
AND A. D. ELBEIN Health
Science Center,
San Antonio,
Texas
78284
Received March 20, 1979; revised July 20, 1979 The particulate enzyme from pig aorta catalyzed the transfer of glucose from UDPglucose into glucosyl-phosphoryl-dolichol, into lipid-linked oligosaccharides, and into glycoprotein. Radioactive lipid-linked oligosaccharides were prepared by incubating the extracts with GDP-[Wlmannose and UDP-[3H]glucose. When the labeled oligosaccharides were run on Bio-Gel P-4, the two different labels did not exactly coincide; the 3H peak eluted slightly earlier indicating that it was of higher molecular weight than the 14Cmaterial, but there was considerable overlap. The purified oligosaccharide(s) contained glucose, mannose, and N-acetylglucosamine but the ratios of these sugars varied from one enzyme preparation to another, probably depending on the endogenous oligosaccaride-lipids present in the microsomal preparation. Treatment of the [3H]glucose-labeled oligosaccharide with cY-mannosidase gave rise to a 3H-labeled oligosaccharide which moved somewhat faster on Bio-Gel P-4 than the original oligosaccharide, suggesting it had lost one or two sugar residues. These data indicate that mannose and glucose are in the same oligosaccharide. The antibiotic, amphomycin, inhibited the transfer of glucose from UDP-glucose into the lipid-linked saccharides. However the synthesis of glucosyl-phosphoryl-dolichol was much more sensitive then was the synthesis of lipid-linked oligosaccharides. The glucose-labeled oligosaccharide produced in the absence of amphomycin was of high molecular weight based on paper chromatography. But in the presence of partially inhibitory concentrations of antibiotic, the oligosaccharide migrated more rapidly on paper chromatograms. However, amphomycin had no effect on the synthesis of glucosyl-ceramide by the aorta extracts. In fact, the antibiotic may stimulate glucosyl-ceramide by making more of the substrate, UDP-glucose, available for synthesis of this lipid.
Some of the earliest studies on the synthesis of lipid-linked saccharides in eucaryotic cells showed that glucose from UDPglucose (or from glucosyl-phosphoryl-dolichol) was incorporated into lipid-linked oligosaccharides (1). Although it was later shown that mannose from GDP-mannose was also transferred into lipid-linked oligosaccharides by these same membrane fractions (2), it was not really clear whether both of these sugars were transferred to the same oligosaccharide. However, the authors suggested that such was the case (3). More recently, an oligosaccharide was isolated from the lipid-linked oligosaccharides of thyroid and was shown to contain 1 or 2 glucose residues as well as 11 mannose and 2 GlcNAc’ moieties (4). A similar kind of 1 Abbreviations used: GlcNAc, N-acetylglucosamine; PPO, 2&diphenyloxazole; POPOP, 1,4-bis[2-(sphenyl0003-9861/79/130304-10$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
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oligosaccharide was also demonstrated in calf pancreas (5) and oviduct (6). A glucosecontaining oligosaccharide was isolated from the lipid-linked oligosaccharide of mammalian cells infected with vesicular stomatitis virus (‘7) and completely characterized. In addition, biosynthetic studies have shown that the [14C]mannose-labeled oligosaccharide is lengthened when unlabeled UDP-glucose is added to incubation mixtures (8). Thus, these studies show that in a variety of eucaryotic cells, glucose is added to the lipid-linked oligosaccharides near the time of completion of synthesis, and then oxazolyl)]-benzene; NP-40, Nonidet-P40; Solvent A, n-butanol:pyridine:H,O (40~30~40); Solvent B, CHCl,: CH,OH:H,O (65:25:4); Solvent C, CHCl,:CH,OH:HAc: H,O (50:25:7:3); Solvent D, CHC&:CH,OH:NHIOH (75:25:4); TCA, trichloroacetic acid.
GLUCOSE INCORPORATION
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the mannose- and glucose-containing oligosaccharide is transferred to the protein (6, 9, 10, 11). In this report, we show that extracts of pig aorta tissue also incorporate glucose from UDP-glucose into glucosyl-phosphoryldolichol, into lipid-linked oligosaccharide and into protein. The glucosyl-phosphoryldolichol was partially characterized by thin layer chromatography as well as by its susceptibility to acid hydrolysis, while the lipid-linked oligosaccharide was characterized by isolation of the oligosaccharide portion. Some evidence suggests that the mannose and glucose are in the same oligosaccharide. The antibiotic, amphomycin, inhibits the transfer of glucose from UDP-glucose into glucosyl-phosphoryl-dolicyol, and this antibiotic appears to cause a shift in the size of the glucose-labeled oligosaccharide to a smaller sized oligosaccharide. This antibiotic did not inhibit the transfer of glucose into glucosyl-ceramide. EXPERIMENTAL
PROCEDURES
Materials. GDP-[‘4C]mannose (278 mCiimmol), UDP-[3H]glucose (l-5 Ci/mmol), and UDP-[“Clglucose (150-250 mCi/mmol) were purchased from New England Nuclear. Stachyose, raffinose, and other sugars, as well as dolichyl-phosphate were from Sigma Chemical Company. Bio-Gel P-4 was from Bio-Rad Laboratories and silica gel F-254 thin layer plates were &om Br&nann Instruments. cr-Mannosidase was purified from jack bean mean as described (12) and amphomycin was a generous gift of Mr. W. Minor, Bristol Laboratories, Syracuse, New York. All organic solvents and other chemicals were from commercial sources and were of the highest grade available. Analytical methods. Radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrometer using a Triton/toluene scintillator (500 ml of Triton X-100, 5 g of 2,5-diphenyloxazole (PPO), 0.1 g of 1,4-bis[2-(5-phenyloxazolyl)J-benzene (POPOP) in 1 liter of toluene). Paper chromatograms were scanned with a Packard radiochromatogram scanner, Model 7201. Chemical and chromatographic methods. Lipidlinked oligosaccharides were hydrolyzed in 0.02 N HCl in 2 ml of 50% propanol at 95°C for 20 min. Following this hydrolysis, 1 ml of CHCl, was added to give a CHC1,:propanol:HtO mixture of 1:l:l. After thorough mixing, the layers were separated by centrifugation, and the aqueous phase which contained oligosaccharides was taken to dryness. Polyprenyl-linked monosaccharides were purified on
SACCHARIDES
305
DEAE-cellulose as previously described (13, 14). Lipid-linked oligosaccharides were purified on DEAEcellulose packed, equilibrated, and eluted with CHCl,:CHBOH:H,O (l&10:3). The column was eluted batchwise with increasing concentrations of ammonium formate in this solvent and the lipid-linked oligosaccharides were eluted at 25 mM ammonium formate. Hexoses were quantitated by the anthrone method (15) and hexosamines by the Elson-Morgan procedure (16). Oligosaccharides were incubated with 1 unit of cr-mannosidase (12) in 200 ~1 of 0.01 M acetate buffer, pH 5.0 for 12 h under toluene at 37°C. Descending paper chromatography of radioactive oligosaccharides was done on Whatman 3MM paper in Solvent A, n-butanol:pyridine:HZO (40:30:40). Standard sugars were visualized with the silver nitrate dip (17). Thin layer chromatography of the lipids was done on silica gel F254 plates in Solvent B, CHCl,: CH,OH:HpO (65:25:4); Solvent C, CHCl,:CH,OH: HAc:H,O (50:25:7:3); or Solvent D, CHCla:CHSOH: NH,OH (75:25:4). The location of radioactive lipids was determined by cutting the plates into l-cm sections which were scraped into scintillation vials and counted in toluene scintillation fluid. Dolichyl-phosphate and mannosyl-phosphoryl-dolichol were run as standards and were visualized with iodine vapors or by the determination of radioactivity. Monosaccharides were identified by paper chromatography in Solvent E, n-butano1:pyridine:O.l N HCI (5:3:2) and by gas chromatography on a column of 3% OV-275. Columns of Bio-Gel P-4 were equilibrated and run in 0.15% acetic acid. Columns were standardized with blue dextran, ovalbumin glycopeptide (or oligosaccharide), stachyose, raffinose, maltose, and mannose. Preparation and assay of particulate enzyme. The intimal layer of fresh pig aorta was stripped and homogenized in 50 mM Tris-HCI buffer, pH 7.5, as described previously (14). The homogenate was filtered through cheesecloth and centrifuged at 50009 for 10 min. The pellet was discarded and the supernatant liquid was centrifuged at 100,OOOgfor 90 min. The high speed pellet was resuspended in 50 mM Tris buffer (approximately 1.5 ml for every 20 ml of supernatant liquid) by gentle homogenization. This particulate fraction served as the enzyme source. Incubation mixtures contained the following components in a final volume of 0.4 ml: 100 ~1 of 50 mM Tris buffer, pH 7.5; 2 mM MnCl,; GDP-[‘4C]mannose or UDP-[3H or 14C]glucose (60,000 cpm); and 100 ~1 of enzyme (usually 1 or 2 mg of protein). Following an incubation for varying time periods at 37”C, the reaction was stopped by the addition of CHC13:CH,0H: H,O (1:1:0.6) to make a mixture of 1:l:l and the various lipids were extracted by the multiple extraction procedure described previously (13, 14). Amphomycin was generally added, in the amounts indicated in the figures, before the addition of enzyme. However, in time course experiments, antibiotic and enzyme were pre-
KANG AND ELBEIN
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incubated for 2 min before the addition radioactive substrate.
of the
Preparation of assay of soluble enzyme. The particulate enzyme was solubiliied by vortexing with 0.5% NP-40 for about 2 or 3 min (about 30 s, five or six times with cooling in between) as described (18, 19). The mixture was centrifuged at 100,OOOgfor 45 min and the supernatant liquid was used as the enzyme source. Assay mixtures were essentially as indicated for the particulate enzyme except that dolichyl-phosphate had to be added as the glycosyl acceptor. Solvent was removed with a stream of N, and lipid was resuspended in 0.5% NP-40. Substrates, MnCl,, and antibiotic were added, followed by 100 ~1 of enzyme (about 500 pg of protein). Lipids were extracted by the multiple extraction method used for the particulate enzyme.
RESULTS
Incorporation of Glucose into the LipidLinked Saccharides and Glycoprotein When UDP-[3H] or [14C]glucose was incubated with the particulate fraction from pig aorta, radioactivity was incorporated into the various lipid-linked saccharide intermediates and into glycoprotein. In Fig. 1, the transfer of radioactivity from GDP-[14C]mannose into lipid and protein is compared to the transfer of radioactivity from UDP-[14C]glucose by the particulate
enzyme preparation. Thus, the curves on the left of Fig. 1A show the incorporation of these sugars into glucosyl-phosphoryldolichol and mannosyl-phoshoryl-dolichol as a function of time and incubation. Radioactivity in both of these monosaccharidelipids reached a maximum within a few minutes and then declined, probably due in part to the transfer of radioactivity to lipid-linked oligosaccharides and partly to breakdown during the incubation. The glucose-lipid was isolated from large scale incubations and compared to mannosylphosphoryl-dolichol, previously characterized from aorta (14). Both lipids had the same Rf values upon thin layer chromatography in an acidic, a basic, and a neutral solvent (Solvents B, C, D). Furthermore, the glucose-lipid bound to DEAE-cellulose and was eluted with ammonium acetate in the same area as mannosyl-phosphoryl-dolichol, and also showed the same lability to mild acid as did mannosyl-phosphoryl-dolichol (data not shown). Thus, the glucoselipid appears to be glucosyl-phosphoryldolichol. However, as indicated below, about lo-20% of the radioactivity in the glucolipid behaved as a neutral lipid and had the same properties as glucosylceramide.
(6)
TIME
(mid
FIG. 1. Incorporation of glucose and mannose into lipid-linked saccharides and glycoproteins as a function of time. Reaction mixtures containing either GDP-[Wlmannose or UDP-[3H]glucose were as described in the text with the aorta particulate enzyme. At the indicated times, a sample was removed and the individual lipids were separated by solvent extraction. The remaining pellet was washed with TCA and water before being digested with Pronase. (A) Incorporation into lipid-linked saccharides. Curves on left show synthesis of mannosyl-phosphoryl-dolichol and glucosyl-phosphoryldolichol. Curves on right show formation of glucose-labeled and mannose-labeled oligosaccharide-lipids. (B) Incorporation of glucose and mannose into glycoprotein.
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(10:10:3), and then declined with time of incubation. Some of this decline was due to transfer of oligosaccharide to protein, but some was also due to degradation of these lipids during incubation. Whether this degradation is due to hydrolysis by glycosidases involved in processing (22-24), or is due to other hydrolytic enzymes is not known. Partial characterization of the glucose-labeled oligosaccharide-lipid is discussed below. Figure 1B shows the incorporation of glucose and mannose into the glycoprotein as a function of time of incubation. In this case, the pellet after lipid extraction was washed with 5% TCA and then several times with water. The remaining pellet was digested with Pronase and the radioactivity released was determined as a function of time. It can be seen that the radioactivity in glucose decreased with incubation of 15 min or longer and by 6 h only about 60% of the radioactivity remained in the glycoprotein. Mannose, on the other hand, only decreased slightly with long incubations. When the entire pellet (i.e., before Pronase digestion) was examined as a function of time, the curves resembled those shown here with glucose radioactivity decreasing with long incubations, except that the radioactivity in the pellet was higher. The loss of glucose from the glycoproteins as a function of time may be due to the action of glycosidases involved in processing. Kinetics of Labeling of Glucose-Containing Oligosaccharide-Lipid
NUMBER
FIG. 2. Gel filtration profiles of glucose-labeled and mannose-labeled oligosaccharides formed at various times of incubation. Incubation mixtures were as described in Fig. 1. Lipid-linked oligosaccharides were isolated and hydrolyzed to liberate the oligosaccharides. The oligosaccharides were chromatographed on a 100~cm column of Bio-Gel P-4. Aliquots of each tube were assayed for radioactivity.
The curves on the right of Fig. 1A show the incorporation of mannose and glucose into the lipid-linked oligosaccharides as a function of time of incubation. Again, radioactivity was rapidly incorporated into lipids that were soluble in CHCls:CH30H:H20
In order to study the kinetics of formation of the glucose-containing oligosaccharidelipid, incubation mixtures were prepared which contained GDP-[14C]mannose and UDP-[3H]glucose. At various times, the reactions were harvested and the lipidlinked oligosaccharides were isolated by solvent extraction. The oligosaccharides were released by mild acid hydrolysis and were separated on a 100~cm column of BioGel P-4 as shown in Fig. 2. In very early times, the glucose was seen in two rather broad peaks which emerged after the blue dextran marker, and these two peaks merged into one as the time of the
308
KANG AND ELBEIN
incubation was increased. The final peak (i.e., 15 or 240 min) was of high molecular weight based on the position of its elution from P-4. The peaks seen in Fig. 2, labeled with glucose, are rather broad suggesting that they are probably heterogenous in size. In the experiments on mannose incorporation into oligosaccharides, one rather broad peak was seen in early times which coincided with the smaller sized glucoselabeled oligosaccharide. However, as the time of the incubation increased, the mannose-labeled oligosaccharide became slightly larger in size and also became much sharper, indicating that the oligosacchar-ides were becoming more homogeneous. However, even in very long incubations, the glucose and mannose peaks did not coincide completely. That is, although there was considerable overlap, the peak of glucose radioactivity was slightly larger in size than that of the mannose-labeled oligosaccharide. This is probably due to the fact that the oligosaccharides labeled only with mannose remain more heterogenous in size. But it seems likely that the large oligosaccharide contains both mannose and glucose. Partial Churacterixation of Glucose-Labeled Oligosaccharide-Lipid
A large-scale incubation mixture was prepared with UDP-[3H]glucose and the particulate enzyme, and the lipid-linked oligosaccharides were isolated by solvent extraction. The radioactive lipid bound to a DE AE-cellulose column equilibrated with CHCI,:CH,OH:H,O (10: 10:3) and was eluted with 25 mM ammonium formate. The oligosaccharide was released from this lipid by mild acid hydrolysis and was purified by chromatography on Bio-Gel P-4. The elution pattern of this oligosaccharide on a standardized Bio-Gel P-4 column indicated a molecular weight of about 2500-3000. This oligosaccharide was hydrolyzed in strong acid and the monosaccharides were reduced with NaBH,, acetylated with acetic anhydride, and identified and quantitated by gas liquid chromatography. Two neutral sugars were detected by gas chromatography on a column of 3% OV 275
-.-.-. So
loo FRACTION
NUMBER
150
FIG. 3. Gel filtration of glucose-labeled oligosaccharide before and after treatment with a-mannosidase. Oligosaccharide was run on a 200-cm column of Bio-Gel P-4 (200-400 mesh). Oligosaccharide was treated with cY-mannosidase for 24 h and the digest was treated with TCA to remove protein and then run on this column. Standards shown are stachyose (S), raffinose (R), maltose (M) and glucose (G).
which corresponded to mannose and glucose. A small amount of galactose was also seen. However, the ratio of glucose to mannose varied depending on the enzyme preparation. This variation is probably due to differences in the amount and size of the endogenous lipid-linked oligosaccharides in the various aorta preparations. Thus no attempt was made to determine the actual amounts of sugars present. The individual sugars were also identified by paper chromatographic methods and in some cases by standard calorimetric procedures (15, 16). Thus, the sugars present in the oligosaccharide were mannose, glucose, and glucosamine (i.e., GlcNAc). The oligosaccharide was run on a Bio-Gel P-4 (200 cm) column as shown in Fig. 3. It eluted in a sharp peak after the blue dextran marker. This oligosaccharide ‘was treated with 1 unit of a-mannosidase overnight and the treated glucose-labeled oligosaccharide was rerun on this column. It eluted slightly later than the untreated compound indicating that it was smaller in size. The difference in the elution pattern is indicative of the loss of about two mannose residues. These data indicate that both the glucose and the mannose are in the same oligosaccharide. Acetolysis of the glucose-labeled oligosaccharide did not release any 3H in small molecular weight oligosaccharides (i.e, tetrasaccharides or
GLUCOSE
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INCORPORATION
I 100
I 200 AMPHOMYCIN
INTO LIPID-LINKED
SACCHARIDES
309
I-----300 CONCENTRATION(W)
FIG. 4. Effect of amphomycin on the incorporation of glucose into lipid-linked saccharides and glycoprotein. Incubations were as described in the text with particulate enzyme (200 ~1) and UDP-[3H]glucose. Amphomycin was added as shown in the figure and preincubations were done as described. Incubations were for 20 min at 37°C. The various lipid-linked saccharides and glycoprotein were isolated as indicated under Experimental Procedures.
smaller) or monosaccharides. However, it did result in a shift from the single large 3H-labeled oligosaccharide to several somewhat smaller ones suggesting that some unlabeled mannose was released (data not shown). The amount of radioactivity in these compounds was too low for further identification. Effect of Amphomycin on the Incorporation of Glucose into the Lipids The effect of amphomycin on the transfer of radioactivity from UDP-[3H]glucose into the various lipids and into pellet was examined with the aorta particulate enzyme as shown in Fig. 4. It can be seen that glucose incorporation into the monosaccharide-lipids (i.e., 2:l lipids) was strongly inhibited, such that 50% inhibition occurred at about 100 pg of amphomycin per incubation mixture. Amphomycin was previously shown to inhibit the transfer of GlcNAc from UDP-GlcNAc into GlcNAcpyrophosphoryl-dolichol and the transfer of mannose from GDP-mannose into mannosylphosphoryl-dolichol(16). While the transfer of glucose into lipid-linked oligosaccharides and into insoluble pellet (Fig. 4) was also inhibited by amphomycin, these reactions were much less sensitive to antibiotic than was the formation of glucosyl-phosphoryl-dolichol.
In order to examine the amphomycin inhibition in more detail, the incorporation of glucose into the lipids as a function of time of incubation was studied as demonstrated in Fig. 5. It can be seen from the curves on the left that radioactivity into glucosyl-phosphoryl-dolichol was inhibited more than 75% at all times by 50 ,ug of amphomycin and almost complete inhibition occurred at 100 pg of antibiotic or higher. However, as shown by the curves on the right, incorporation of glucose from UDPglucose into the lipid-linked oligosaccharides was much less sensitive to antibiotic, such that only about 20% inhibition occurred at 50 pg of amphomycin. From these data it appears that some of the glucose residues may be directly transfered from UDPglucose into the lipid-linked oligosaccharides. These results are reminiscent of studies with GDP-mannose where amphomycin also inhibited the formation of mannosyl-phosphoryl-dolichol but allowed a direct transfer of mannose to lipid-linked oligosaccharides (27). In this regard, we have tried to study the direct transfer of glucose from glucosyl-phosphoryl-dolichol to lipid-linked oligosaccharides but have had only limited success in demonstrating any transfer of glucose. Thus, we do not know the effect of amphomycin on this specific transfer.
310
KANG AND ELBEIN
P
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FIG. 5. Time course of formation of glucosyl-phosphoryl-dolichol and lipid-linked oligosaccharides in the presence of amphomycin. Incubations with particulate enzyme (100 gl) were as described in Fig. 4 but samples were removed at various times as shown in the figure. Lipids were isolated as described.
Nature of the Lipids Formed Presence of Amphomycin
in the
In order to determine whether this antibiotic caused any alterations in the nature of the lipid-linked saccharides, large-scale incubations were done in the absence or in the presence of several concentrations of amphomycin. The radioactive glycolipids were isolated by extraction with chloroform and identified by thin layer chromatography as shown in Fig. 6. In the absence of antibiotic, most of the radioactivity was in a lipid which had the same mobility as mannosyl-phosphoryldolichol with smaller amounts in a second peak with the same mobility as galactosylceramide. Based on their mobilities in a basic and neutral solvent (not shown), these two compounds are glucosyl-phosphoryl-dolichol and glucosyl-ceramide. The identify of the former compound was supported by its acid lability and alkaline stability and by the fact that it bound to DEAE-cellulose columns. The latter compound, on the other hand, did not bind to DEAE-cellulose and was stable to mild acid and alkaline treatments. Figure 6 shows that with increasing amphomycin concentrations there was a progressive decrease in the radioactivity in the first peak (glucosyl-phosphoryl-dolichol) but the radioactivity in the second peak (glucosylceramide) did not change. In fact in some experiments, the radioactivity in,this peak
increased to some extent, probably as a result of more UDP-glucose being available because of decreased glucosyl-phosphoryldolichol synthesis. Thus, amphomycin has no effect on formation of glucosyl-ceramide.
No Ampho.
75ug
Ampho.
--.--15Oyg
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DISTANCE
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ORIGIN
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FIG. 6. Thin layer chromatography of glucoselabeled lipids. The glucose-labeled lipids that were extracted with CHCls:CH30H:H20 (1:l:l) were isolated by solvent extraction and streaked on silica gel plates. Solvent B was used. Plates were cut into l-cm sections and the location of radioactivity was determined by scintillation counting. Incubations contained no amphomycin (upper), 75 pg of amphomycin (middle), or 150 pg of antibiotic (lower).
GLUCOSE
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that is, whether the smaller peak is a precursor to the larger one. Thus far, we have not been able to obtain sufficient amounts of the smaller oligosaccharide for characterization. We also do not know whether this effect of amphomycin is directly on the transfer of glucose to lipid-linked oligosaccharides or is a result of antibiotic effect on the endogenous lipid-linked oligosaccharides.
No Ampho.
DISTANCE FROM ORIGINkm)
DISCUSSION
FIG. 7. Paper chromatographic identification of glucose-labeled oligosaccharide formed in the absence or presence of amphomycin. Incubations were as described in preceding figures either without or with (100 and 200 pg) antibiotic. In this experiment UDP-[%]glucose was used as substrate. Lipidlinked oligosaccharides were isolated by solvent extraction and the oligosaccharides were released from the lipids by mild acid hydrolysis and chromatographed on Whatman 3MM paper in Solvent A. Papers were cut into l-cm strips and counted in the scintillation counter. Standard shown is stachyose (S).
The lipid-linked oligosaccharides formed in the presence and absence of amphomycin were also isolated and the oligosaccharides were released by mild acid hydrolysis. Figure 7 shows the radioactive scans of these oligosaccharides when they were chromatographed on Whatman 3MM paper in Solvent A. In the absence of amphomycin (upper scan) only one radioactive peak was detected and this peak remained fairly close to the origin, indicating that it was of high molecular weight. This is the same oligosaccharide observed by gel filtration (Figs. 2 and 3). The migration rate of this oligosaccharide relative to stachyose was 0.24. The middle scan shows that when 100 Fg of amphomycin was added to incubation mixtures, there was a decrease in the radioactivity in this large oligosaccharide while a new faster-migrating peak appeared = 0.52). The lower scan shows that at 200 pg of amphomycin most of the radioactivity in the lipid-linked oligosaccharides was in that oligosaccharide with an of 0.52 and only about 25-30% was in the larger sized oligosaccharide. We do not know whether these two radioactive peaks are related to each other in any way, (R,
Rf
The studies described in this paper show that glucose from UDP-glucose is transformed to lipid-linked oligosaccharides by the particulate enzyme from pig aorta. The glucose incorporated into this oligosaccharide-lipid reached a maximum within the first 15 min of incubation and then declined upon. longer incubations. These results suggested either of the following possibilities: (i) that the glucose-containing oligosaccharide lipid was breaking down during the incubation as a result of instability, or (ii) that the glucose was being removed during the incubation by a specific glycosidase. The latter explanation seems to apply here since in preliminary experiments we examined the aqueous phases during various times of incubation and found a large increase in free glucose as time progressed. Therefore, it seems likely that the glucose-containing oligosaccharide is undergoing trimming during the incubation. The glucose-containing oligosaccharidelipid was larger in size and more homogeneous than were the oligosaccharidelipids which contained only mannose and GlcNAc. Other workers have found that the [ 14C]mannose-oligosaccharide becomes larger in size when incubation mixtures were chased with unlabeled UDP-glucose (8). Thus, mannose and glucose appear to be in the same oligosaccharide as was shown by characterization of several oligosaccharides from different tissues (4-6). Glucose and mannose are also in the same oligosaccharide in oviduct tissue and in the oligosaccharide described here, since in both eases treated with a-mannosidase results in a decrease in the size of the glucose-
312
KANG AND ELBEIN
labeled oligosaccharide. However, in the aorta oligosaccharide, only one or two mannose residues could be removed by a-mannosidase digestion. It is not clear whether the inability of a-mannosidase to remove more mannose residues is due to protection of additional mannose residues by terminal glucoses or whether the a-mannosidase only hydrolyzes certain glycosidic linkages. In addition to the oligosaccharide-lipid, glucose from UDP-glucose was also transferred to glucosyl-phosphoryl-dolichol and to glucosyl-ceramide. Attempts to show the transfer of glucose from glucosyl-phoshoryldolichol to the lipid-linked oligosaccharides have not been very successful with aorta extracts although some such transfers have been reported in liver (20) and other tissues (21). Radioactivity was also incorporated in our studies into insoluble material, at least part of which is glycoprotein. The radioactive glucose in the TCA-washed pellet declined with time of incubation (as did the radioactivity in the lipid-linked oligosaccharides) suggesting that it was removed by enzymatic cleavage. In several tissues, a glucosidase has been identified in the particulate fraction which apparently selectively removes glucose from the glucosylated protein (22, 23). In addition time course studies in vivo have shown that three glucose residues are removed from the protein during a period of 4-8 h after transfer (24). A mannosidase was previously identified in the Golgi (25), and this enzyme may be involved in glycoprotein processing. In the case of the glucose-labeled pellet described here, at least part of this material is probably glycoprotein since some of the radioactivity is solubilized by Pronase digestion. Several laboratories have shown that the glucose-labeled oligosaccharidelipid is transferred to protein (6, 10, 11) and some studies suggest that this oligosaccharide-lipid is a better substrate for transfer to protein than is the glucosefree, oligosaccharide-lipid (10, 11). In the aorta system, we are not certain as to the role of the glucose-containing oligosaccharidelipid since we have not yet compared its transfer to that of the mannose-labeled oligosaccharide-lipid.
Studies described here show that the antibiotic amphomycin inhibits the transfer of glucose from UDP-glucose into glucosylphosphoryl-dolichol and that this transfer was much more sensitive to antibiotic than was the synthesis of oligosaccharide-lipid. These findings are reminiscent of previous studies on the effect of amphomycin on mannose incorporation (2’7). In that study amphomycin blocked the synthesis of mannosyl-phosphoryl-dolichol much more effectively than the formation of oligosaccharide-lipid. Thus, when mannosyl-phosphoryl-dolichol formation was completely inhibited with amphomycin, mannose was still transferred from GDP-mannose to lipid-linked oligosaccharides. The radioactivity in this lipid-linked oligosaccharide appeared to be exclusively in one oligosaccharide with the mobility of a heptasaccharide. In the case of the glucose-labeled oligosaccharide lipids, the radioactivity was in one large oligosaccharide(s) which remained near the origin upon paper chromatography. But in the presence of amphomycin, the glucose-labeled material migrated faster upon paper chromatography indicating that is was smaller in size. This oligosaccharide has not been characterized yet because of limitations in the amount of radioactive material available. This effect of amphomycin could reflect a direct inhibition of glucose incorporation into lipid-linked oligosaccharides or it could be the result of amphomycin effect on the nature of the endogenous lipid acceptors. Thus in the presence of antibiotic, the oligosaccharide-lipids could be of smaller size resulting in the smallersized [3H]G1c-oligosaccharide. Interestingly, while amphomycin inhibited the formation of glucosyl-phosphoryl-dolichol, it has no effect on the transfer of glucose from UDP-glucose to ceramide to form glucosyl-ceramide. In fact, in some experiments this reaction was stimulated by amphomycin probably because more UDP-glucose was available as a result of inhibition of glucosyl-phosphoryl-dolichol. ACKNOWLEDGMENTS This research was supported by a grant from the National Institutes of Health (HL17783) and a grant from the Robert A. Welch Foundation (AQ-366).
GLUCOSE
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