Glycoprotein Enzymesin Aspergillus fumigatus - Journal of Bacteriology

Vol. 160, No. 1

JOURNAL OF BACTERIOLOGY, OCt. 1984, p. 67-75

0021-9193/84/100067-09$02.00/0 Copyright © 1984, American Society for Microbiology

Effect of Castanospermine on the Structure and Secretion of Glycoprotein Enzymes in Aspergillus fumigatus ALAN D. ELBEIN,* MICHAEL MITCHELL, AND RUSSELL J. MOLYNEUX Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284; and Natural Products Chemistry Research Unit, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Berkeley, California 94710 Received 1 March 1984/Accepted 4 July 1984

Aspergillusfumigatus secretes a number of glycosidases into the culture medium when the cells are grown in a mineral salts medium containing guar flour (a galactomannan) as the carbon source. At least some of these glycosidases have been reported to be glycoproteins having N-linked oligosaccharides. In this study, we examined the effect of the glycoprotein processing inhibitor, castanospermine, on the structures of the N-linked oligosaccharides and on the secretion of various glycosidases. Cells were grown in the presence of various amounts of castanospermine; at different times of growth, samples of the media were removed for the measurement of enzymatic activity. Of the three glycosidases assayed, I-hexosaminidase was most sensitive to castanospermine; and its activity was depressed 30 to 40% at 100 ,ug of alkaloid per ml and even more at higher alkaloid concentrations. On the other hand, ,I-galactosidase activity was hardly diminished at castanospermine levels of up to 1 mg/ml, but significant inhibition was observed at 2 mg/ml. I-Galactosidase was intermediate in sensitivity. Cells were grown in the presence or absence of castanospermine and labeled with [2-3H]mannose, [6-3H]glucosamine, or [1-3H]galactose to label the sugar portion of the glycoproteins. The secreted glycoproteins were digested with pronase to obtain glycopeptides, and these were identified on Bio-Gel P-4 (Bio-Rad Laboratories). The glycopeptides were then digested with endoglucosaminidase H to release the peptide portion of susceptible structures, and the released oligosaccharides were reisolated and identified on Bio-Gel P-4. The oligosaccharides from control and castanospermine-grown cells were identified by a combination of enzymatic and chemical studies. In control cells, the oligosaccharide appeared to be mostly Man8GlcNAc and Man9GlcNAc, whereas in the presence of alkaloid, the major structures were Glc3Man7GlcNAc and Glc3Man8GlcNAc. These data fit previous observations that castanospermine inhibits glucosidase I. Aspergillus spp. produce a number of extracellular glycosidases, all of which appear to be glycoproteins. Included among these enzymes are oa-mannosidase (39), ,-N-acetylhexosaminidase (25), P-glucosidase (30), a-glucosidase (31), a-galactosidase (2), a-fucosidase (17), 3-galactosidase (1), Imannosidase (9), cellulase (16), and so on. Several of these enzymes have been highly purified, and carbohydrate analysis has demonstrated the presence of mannose and Nacetylglucosamine (GlcNAc) as the major sugars. Since the glycosylation and secretion of several of these glycoproteins were shown to be inhibited by the antibiotic tunicamycin (34), it seems likely that these enzymes contain N-linked high-mannose chains (22). In animal cells, the oligosaccharide chains of the N-linked glycoproteins are biosynthesized via a lipid-mediated pathway whereby the sugars GlcNAc, mannose, and glucose are transferred to dolichyl-phosphate to form a Glc3Man9GlcNAc2-pyrophosphoryl-dolichol (7, 37). This lipid-linked saccharide is the donor of oligosaccharide to protein to form the N-linked glycoprotein, i.e., Glc3Man9GlcNAc2-protein (15). Once this glycoprotein has been formed, the oligosaccharide chain may undergo a number of processing reactions to give rise to either high-mannose, hybrid, or complex types of oligosaccharides (33). The initial processing reactions involve the removal of all three glucose residues. Thus, glucosidase I, a membrane-bound enzyme that is located in the rough endoplasmic reticulum, removes the terminal al,2-linked glucose (3, 4, 10, 26), whereas glucosidase II, another membrane-bound glucosidase that may also be in *

the endoplasmic reticulum, removes the two remaining al, 3-linked glucose units (35, 43, 44). These reactions give rise to a MangGlcNAc2-protein that may be the direct precursor to the high-mannose glycoproteins. Or, this oligosaccharide may be further trimmed by the removal of some mannose residues to give other, shorter high-mannose structures or to eventually give rise to hybrid and complex structures (15). One useful technique to study biosynthesis and function of the oligosaccharide portion of the glycoprotein is through the use of inhibitors that either prevent glycosylation of the protein or modify the structure of the oligosaccharide (8). An example of the latter type of inhibitor is the plant alkaloid castanospermine (14). We have found that this alkaloid is a fairly specific inhibitor of glucosidases (32), and that it inhibits the glycoprotein-processing enzyme glucosidase I (29). Thus, when influenza virus is raised in kidney cells in the presence of castanospermine, the viral glycoproteins contain oligosaccharides of the composition Glc3Man7_9GlcNAc2, ratherthan the typical high-mannose and complex chains found in this hemagglutinin (29). Since castanospermine inhibits normal processing in animal and plant cells (H. Hori, Y. T. Pan, R. J. Molyneux, and A. D. Elbein, Arch. Biochem. Biophys., in press), it was of interest to determine what effect it would have on the oligosacccharide structure of the Aspergillus spp. glycoproteins, and whether it would alter the secretion of these enzymes. In this paper we describe the results of these studies. MATERIALS AND METHODS Materials. p-Nitrophenyl-glycosides were used as substrates for the various glycosidases and were purchased from

Corresponding author. 67

68

ELBEIN, MITCHELL, AND MOLYNEUX

Sigma Chemical Co., St. Louis, Mo. [2-3H]Mannose (25 Ci/ mmol) and [1-3H]galactose (12 mCi/mmol) were obtained from Pathfinders Laboratories, St. Louis, Mo., and [63H]glucosamine (19 Ci/mmol) was from New England Nuclear Corp., Boston, Mass. Castanospermine was isolated in 0.3% yield from the seeds of Castanospermum australe and was crystallized from ethanol (14). Bio-Gel P-4 (200 mesh and -400 mesh) were purchased from Bio-Rad Laboratories, Richmond, Calif. Guar flour was obtained from General Mills Chemicals Inc., Minneapolis, Minn. Endo-,BN-acetylglucosaminidase H (Endo H) was from Health Research Inc., Albany, N.Y., and jack bean ac-mannosidase was from Sigma. Growth conditions. Aspergillus niger was grown at 30°C in a liquid medium that has the following composition (in grams per liter): KH2PO4, 2; (NH4)2SO4, 1.4; urea, 0.3; CaCl2, 0.3; MgSO4, 0.3; mannose, 0.1; yeast extract, 0.05; and guar flour, 5. The fungus was maintained on agar slants of the above medium. For innoculation of flasks, a loop of spores was removed from the slant and dispersed in 2 ml of sterile distilled water. Samples of this suspension were pipetted into 125-ml flasks containing 25 ml of the liquid medium. Various amounts of castanospermine, sterilized by filtration (filters from Millipore Corp., Bedford, Mass.), were added to the flasks as indicated below. Radioactive sugars were also added to some flasks to label the glycoproteins. The flasks were placed on a rotary shaker and allowed to incubate for up to 144 h. Samples of the medium were removed at various times and examined for the activity of a number of glycosidases. Assay of glycosidases. Each sample of the medium (i.e., various time points and various castanospermine concentrations) was assayed for the activity of several different glycosidases. The reaction mixtures for these assays contained the following components in a final volume of 0.4 ml: 2 p.mol of the appropriate p-nitrophenyl glycoside, 10 ,umol of sodium acetate buffer (pH 5.0), and various amounts of the medium. Several samples of medium were selected that gave linear responses of activity. Incubation times were usually 30 min at 37°C, but an appropriate time was selected that was in the linear range of enzyme activity. At the end of the incubation, the reaction was stopped by the addition of 2.5 ml of 0.4 M glycine buffer (pH 10.4), and the amount of liberated p-nitrophenol was measured at 410 nm. Preparation of radioactive glycopeptides. As indicated above, flasks containing various amounts of castanospermine and control flasks were innoculated with various radioactive sugars to label the glycoproteins. In these experiments, the entire contents of the flask were removed at the indicated time, usually 120 h, and filtered to remove the cells. The filtrate (about 20 ml) and the cell wash (10 ml) were combined and concentrated in either of the following ways. In some cases, the filtrates were concentrated to about 2 ml with an Amicon filtration apparatus with a UM 10 filter. The 2-ml concentrate was then placed in a tube, and 10 ml of ice cold acetone was added. The mixture was allowed to stand overnight at -20°C, and the precipitate was harvested by filtration, dissolved in 2 ml of water, and dialyzed overnight against several liters of 25 mM Tris buffer (pH 7.5). In the other case, the filtrates were lyophilized. The dried material was dissolved in 2 ml of water and dialyzed against several liters of 25 mM Tris buffer (pH 7.5). This buffer was used for two reasons. First of all, these fungi secrete cellulases, and dialysis against buffers of low pH may result in dissolution of the dialysis bag. Second, the Tris

J. BACTERIOL.

buffer is the appropriate buffer for the next step, which involves the proteolytic enzyme pronase. After dialysis for 24 h, the contents of the dialysis bags were removed and placed in screw-capped tubes. One milliliter of pronase solution (5 mg of enzyme per ml in 50 mM Tris buffer [pH 7.5] containing 5 mM CaCl2) was added to each tube, and the mixtures were incubated for 24 h at 37°C. At the end of that time, another 1 ml of pronase solution was added, and incubations were continued for another 24 h. After the incubation, 2 ml of 25% trichloroacetic acid was added to each tube, and the mixtures were placed in an ice bath for 30 min to precipitate the protein. The protein was removed by centrifugation and discarded, and the supernatants were extracted four or five times with

B - Hexosaminidase

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FIG. 1. Effect of castanospermine on glycosidase activity in media. Cells were grown in various concentrations of alkaloid; at the times shown, samples of the medium were removed, and the activities of the various glycosidases were measured by using the appropriate p-nitrophenyl glycoside.

Effect of castanospermine on the secretion of enzymes. Aspergillusfumigatus secretes a number of enzymes into the medium when the organism is grown in a mineral salts medium with guar as the carbon source (30, 31). Since a number of these enzymes appear to be glycoproteins having N-linked high-mannose oligosaccharides (34), it was of interest to determine whether the processing inhibitor, castanospermine, would have any effect on the synthesis and secretion of these enzymes. Various amounts of castanospermine, from 10 p.g/ml up to 2 mg/ml, were added to 125-ml flasks containing 25 ml of the guar medium, and the flasks were innoculated with a spore suspension of the organism. The flasks were placed on a rotary shaker at room temperature for up to 144 h; every 24 h, 2 ml of medium was removed and filtered. The filtrate was examined for the presence of a number of glycosidases. Figure 1 presents the results of one such experiment. In this case, we compared the activities of 1-hexosaminidase (1-Nacetylhexosaminidase), ,B-galactosidase, and ot-galactosidase. There was some difference in the amount of castano-

Glc3Man9GlcNAc, Glc2Man9GlcNAc, GlclMan9GlcNAc, MangGlcNAc, Man8GlcNAc, and Man7GlcNAc. Oligosacdigested with a-mannosidase, and the prodon Bio-Gel P-4. Oligosaccharides were also subjected to methylation analysis (12), and the radioactive methylated mannose derivatives were identified by thin-layer chromatography. Enzymatic digestions. Endo H is an enzyme that cleaves some N-linked oligosaccharides between the two internal GlcNAc residues (40). The specificity of this enzyme requires that the mannose residue that is linked al,6 to the ,3linked mannose be substituted with an (x,3-linked mannose were

ucts were rechromatographed

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(21). Thus, this enzyme will act on many high-mannose and hybrid chains, but not on complex structures. Digestions with Endo H were done in 0.2 ml of 50 mM citrate buffer (pH 6.5). Enzyme (10 mU) and a few drops of toluene were added, and the mixtures were incubated for 24 h. At that time, another 10 mU of enzyme was added, and incubations were continued for 24 h. Digestions with jack bean utmannosidase were done in 50 mM sodium acetate buffer (pH 5.0) in a final volume of 0.2 ml. RESULTS

ethyl ether to remove the trichloroacetic acid. The aqueous layers were then concentrated to dryness and separated on Bio-Gel P-4 columns. Gel filtration of radioactive glycopeptides and oligosaccharides. The radioactive glycopeptides were separated on a 1.5- by 150-cm column of Bio-Gel P-4 (200 mesh). The column was calibrated with a variety of standard oligosaccharides, and the radioactive materials were run under the same conditions. Samples were eluted with 0.3% acetic acid, and 1.5-ml fractions were collected. Samples of every other fraction were removed for the determination of radioactivity. The radioactive peaks were pooled and concentrated to a small volume. The peaks were then digested with Endo H (see below), and the products of this reaction were rechromatographed on the same Bio-Gel P-4 column. Partial characterization of glycopeptides and oligosaccharides. Glycopeptides and oligosaccharides were sized on a 1.5- by 200-cm column of Bio-Gel P-4. The column was calibrated with various oligosaccharide standards including charides

~I

GLYCOPROTEIN ENZYMES IN A. FUMIGATUS

VOL. 160, 1984

I

4

4

1

20 30 40 50 60 70 FRACTION NUMBER

FIG. 2. Effect of castanospermine of the structures of the mannose-labeled glycopeptides and oligosaccharides from secreted glycoproteins. Cells were grown in castanospermine and labeled with [2-3H]mannose. The medium was removed at 120 h and concentrated on an Amicon filter, and the protein was precipitated by the addition of 5 volumes of acetone. After standing overnight at -20°C, the protein precipitate was isolated and digested with pronase. The profiles in panel A show the elution patterns of the glycopeptides from control cells (upper), cells in 50 ,ug of alkaloid per ml (middle), or cells in 1 mg of alkaloid per ml (lower). The glycopeptide peaks were pooled, digested with Endo H, and rechromatographed on Bio-Gel P-4 (B). Standards are shown by arrows as follows: G6, Glc3Man9GlcNAc2; M9, Man9GlcNAc; M5, Man5GlcNAc.

70


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FRACTION NUMBER FIG. 3. Determination of size of oligosaccharides from control and castanospermine-grown cells. Oligosaccharides released by Endo H were chromatographed on a calibrated column of Bio-Gel P4. Standards shown are as follows: 12, Glc3Man9GlcNAc; 11, Glc2Man9GlcNAc; 10, Glc1MangGlcNAc; 9, MangGlcNAc; 8, Man8GlcNAc. Samples of each fraction were counted to determine their radioactive content.

spermine required to inhibit the activities of these three compared with the activities in control cells. Thus, the ,-hexosaminidase (upper panel) began to appear in the medium of control cells at about 72 to 96 h of growth, and this activity then increased rapidly over the next 48 h. However, when 100 ,ug of castanospermine per ml was included in the medium, there was a significant decrease in the activity of this enzyme (about 40% of control values), and this decrease in activity was even more pronounced at higher concentrations of castanospermine. On the other hand, the 3-galactosidase and the cx-galactosidase were much less affected by the presence of castanospermine (middle and lower curves), although a decrease in the activities of these enzymes was also observed. Thus, the ,-galactosidase was inhibited about 20 to 25% at 1 mg of castanospermine per ml and about 35 to 40% at 2 mg of this alkaloid per ml. The a-galactosidase was even less susceptible to castanospermine, and levels of this enzyme were almost the same as in control cells at concentrations of alkaloid up to 1 mg/ml. However, at 2 mg/ml, the activity of a-galactosidase was depressed about 25 to 30%. Castanospermine did not inhibit the activities of any of the above glycosidases when added directly to incubation mixtures of enzyme and its p-nitrophenylglycoside substrate. The decrease in activities of these enzymes could be due to an inhibition or slowdown in the secretion of the proteins, or it could be the result of an inhibition in the synthesis of the glycoproteins. It is also possible that the decreased activities could be due to a more rapid turnover of the glycoproteins. Based on the time course studies, there is no reason to enzymes, as

J. BACTERIOL.

believe that the alkaloid is affecting the turnover of the glycosidases or increasing the degradation. Alterations in structure of mannose-labeled glycopeptides induced by castanospermine. To examine the effect of castanospermine on oligosaccharide structure, cells were grown in alkaloid and labeled with [2-3H]mannose. After growth in the label for 48 to 72 h, the medium was collected and concentrated to 2 ml on an Amicon filter. The concentrate was cooled, and 5 volumes of acetone, cooled to -20°C, was added to precipitate the protein. After standing for 24 h -20°C, the protein was isolated by centrifugation. The precipitate was digested exhaustively with pronase, and the glycopeptides were examined on Bio-Gel P-4. Figure 2A shows the elution profiles of the mannose-labeled glycopeptides from control cells and from cells grown in several concentrations of castanospermine. Two peaks of radioactivity were seen in control and castanospermine-grown cells, but there were some significant differences between these various cells. Thus, in each case (control and castanospermine treated), a major peak of radioactivity eluted at fractions 38 through 46 (i.e., near the void volume), and this peak appeared to be similar in control and castanosperminegrown cells. However, the second, smaller peak was clearly different in the presence of alkaloid. In control cells, this second peak was rather broad, eluting in fractions 54 through 64, whereas at 1 mg of castanospermine per ml, this peak eluted earlier (fractions 48 through 56), indicating that it was larger in size. This peak was near the Glc3Man9GlcNAc2 standard. These data indicated that this alkaloid was causing changes in the structure of the oligosaccharide chains. The glycopeptides from control and castanosperminetreated cells were treated with Endo H and rechromatographed on the Bio-Gel P-4 column. This enzyme cleaves high-mannose oligosaccharides and glycopeptides between the two internal GlcNAc residues, but does not act on complex chains or on certain types of high-mannose structures (21). The elution profiles of the Endo H-digested samples are shown in Fig. 2B. In each case, the first large peak did not shift after this enzyme treatment. However, since this peak elutes near the void volume, it may be too large to be able to detect the small shift that would be caused by Endo H digestion (i.e., loss of GlcNAc-peptide). Or this peak may be resistant to this enzyme. This peak was also resistant to a-mannosidase, indicating that the mannose residues were not a linked or were blocked with other sugars. Because of its large size it seems likely that this peak 1 represents the cell wall mannan or polymannan-protein aggregates. It is not known whether this structure is part of the glycosidases or is secreted as a separate polymer. However, its content of radioactivity was unaffected by the

alkaloid. On the other hand, peak 2 was susceptible to Endo H in both control and treated cells. However, the new peak resulting from Endo H was different in control cells as compared with treated cells (Fig. 2B). In control cells, the new peak eluted with or just after the MangGlcNAc standard. On the other hand, the peak in castanospermine-grown cells eluted earlier and was only slightly smaller than the Glc3Man9GlcNAc standard. Thus, the alterations caused by castanospermine must be in the oligosaccharide rather than the peptide portion of the glycoprotein. Characterization of mannose-labeled oligosaccharides from control and castanospermine-treated cells. The mannoselabeled oligosaccharides released by Endo H from control and castanospermine-treated cells were chromatographed on a long calibrated column of Bio-Gel P-4 to determine their size. Figure 3 shows the elution profiles of these oligosac-


VOL. 160, 1984

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(N

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0

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U

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FRACTION NUMBER

FRACTION NUMBER

FIG. 4. Effect of (x-mannosidase digestion on the structures of control and castanospermine-induced oligosaccharides or glycopeptides. (B) control glycopeptides before (upper) and after (lower) Q-mannosidase digestion. (A) Castanospermine-induced oligosaccharides before (upper) and after (lower) ox-mannosidase digestion. In each case, the mannose-labeled glycopeptide or oligosaccharide was chromatographed on the Bio-Gel P-4 column and then treated with a-mannosidase and reexamined on Bio-Gel P-4. Standards shown by the arrows are as follows: G3, Glc3MangGlcNAc; Mg, MangGlcNAc; M5, Man5GlcNAc; S, stachyose; M, mannose. Samples of each fraction were counted to determine their radioactive content.

charides. In both the control cells and the treated cells, the oligosaccharides were not homogeneous, but represented a spectrum of sizes. That is probably not surprising since these oligosaccharides were derived from cell-secreted glycoproteins. Nevertheless, the oligosaccharides from control cells were clearly of lower molecular weight than those from castanospermine-grown cells. The major peak in control cells eluted near the hexose8GlcNAc areas. On the other hand, the major oligosaccharide from castanosperminegrown cells eluted near the hexoseI0GlcNAc standard with a second peak in the hexosegGlcNAc area. These data support the idea that castanospermine inhibits processing and thus prevents the trimming of sugars from the oligosaccharides. To learn more about the structures of the oligosaccharides from these cells, they were treated with oL-mannosidase to determine how many mannose residues could be released. If castanospermine prevented the removal of glucose residues from the Glc3Man9GlcNAc2-protein, this glycopeptide (or oligosaccharide) should be only partly susceptible to cxmannosidase since the glucoses cap some of the mannose chains. Figure 4 shows the results of these digestions. In Fig. 4A, the elution profile of the castanospermine-derived oligosaccharides is shown before and after the cx-mannosidase treatments. In this experiment, the oligosaccharides were run on a shorter Bio-Gel P-4 column, and thus the resolution of species is not as good as that in Fig. 3. However, the untreated oligosaccharides eluted in a broad peak, indicating a heterogeneous mixture from hexose12GlcNAc (G3) to hexose10GlcNAc (mostly hexose1lGlcNAc, upper profile). This oligosaccharide was only partly susceptible to a-mannosidase digestion with the release of about 20% of the radioactivity as free mannose (lower profile). Also, after

mannosidase treatment, the larger oligosaccharide became much more homogeneous and mostly migrated in the hexoseloGlcNAc area. This suggests that most of the mannose residues that were released were derived from the hexose12GlcNAc and hexose1 1GlcNAc species (i.e., Glc3Man9GlcNAc and Glc2Man9GlcNAc). In other studies, we have found that the Glc3Man9GlcNAc is relatively resistant to (x-mannosidase, and the release of mannose from that oligosaccharide is very slow (29; Hori et al., in press). The effect of ox-mannosidase was also determined on the mannose-labeled structures from control cells. However, in this study we used the control cell glycopeptides rather than the oligosaccharides. Figure 4B compares the profiles of these glycopeptides before and after Q-mannosidase digestion. In this case, the untreated glycopeptide migrated in a rather broad area, emerging before, with, and after the Glc3Man9GlcNAc standard. However, after treatment with a-mannosidase, the large-molecular-weight radioactive peak completely disappeared and was replaced by a radioactive peak in the mannose area as well as one or two radioactive peaks eluting near the Man5GluNAc standard. At least one of these peaks is probably the Man1GlcNAcGlcNAc-peptide, since a GlcNAc residue migrates like 2.1 hexoses on Bio-Gel P-4, and the peptide would probably be equal to 1 or more hexoses. Thus, it seems likely that the control cell glycopeptides are almost completely susceptible to x-mannosidase and probably have little, if any, glucose. Further characterization of the mannose-labeled oligosaccharides was done by methylation analysis. Both the Endo H-released oligosaccharides from control cells and from castanospermine-treated cells were subjected to complete methylation. After complete acid hydrolysis, the methylated

72


STANDARDS I

2A

3,4,6 I

2

J. BACTERIOL.

-

.12.3Ao6

R 15 CONTROL OLIGO. ,}

10

were identified. Three radioactive peaks were observed corresponding to 2,3,4,6-tetramethylglucose, 2,4,6-trimethylglucose, and 3,4,6-trimethylglucose (data not shown). Since three glucose derivatives were detected, the oligosaccharide must be a Glc3Man7_9GlcNAc. Effect of castanospermine on glucosamine-labeled glycopeptides. Since the N-linked oligosaccharides also contain glucosamine, we examined the effect of castanospermine on the incorporation of [3H]glucosamine into the secreted glycoproteins. Cells were grown in several concentrations of the alkaloid and labeled with [3H]glucosamine. The secreted

proteins

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DISTANCE FROM ORIGIN (cm) FIG. 5. Methylation analysis of control and castanospermine oligosaccharides. Oligosaccharides were methylated by the 14akomori procedure (12); after complete methylation and isolation, the

oligosaccharides were subjected to complete acid hydrolysis. The radioactive methylated mannose derivatives were identified by thinlayer chromatography in benzene-acetone-water-ammonium hydroxide (10:200:3:1.5). Standards were prepared by methylation of yeast mannan and ovalbumin and are as follows: 2,4, 2,4-dimethylmannose; 3,4,6, 3,4,6-trimethylmannose; 2,4,6, 2,4,6-trimethylmannose; 2,3,4,6, 2,3,4,6-tetramethylmannose. mannoses were identified by thin-layer Figure 5 compares the radioactive

chromatography.

methylated mannoses of control cells with those of castanospermine-grown cells. In the control cells (upper profile), the expected methylated mannoses, i.e., 2,3,4,6-tetramethylmannose, 3,4,6-trimethylmannose, and 2,4-dimethylmannose, were observed, indicating the presence of terminal mannose, 2-substituted mannose, and 3,6-substituted mannose. The approximate ratio of radioactivity in these three species was 1:0.47:0.76. This is close to the expected for a Man78GlcNAc oligosaccharide. Since we cannot be certain that all of the mannose residues are equally labeled, one might expect some departure from the theoretical value. Onrthe other hand, the lower profile of Fig. 5 shows the identification of methylated mannoses in the alkaloid-derived oligosaccharide. The distinguishing characteristic in this case was the presence of a small radioactive peak of 2,4,6-trimethylmannose that was absent from control oligosaccharides. A mannose substituted in the 3 position is strongly suggestive of 9ligosaccharides contain-

ing glucose. Thus, as expected, this oligosaccharide also contained terminal, 3,6-substituted, 2-substituted, ahd 3substituted mannose residues in the approximate ratio of 1.0:0.83:0.44:0.37. The lower than expected radioactive conmannose may be due to unequal labeling

tent in 2-substituted

in the mannose residues. To be certain that the castanospermine-induced oligosaccharide contained three glucose residues, A. fumigatus was grown in castanospermine (1 mg/ml) and labeled with [13H]galactose. The glycopeptides were isolated, digested with Endo H, and reisolated on Bio-Gel P-4. The oligosaccharide was then methylated, and the methylated glucoses

were

isolated and digested with

pronase,

and the

liberated glycopeptides were chromatographed on Bio-Gel P-4. Figure 6A shows the profiles obtained from control cells (upper) and cells grown in 1 mg of castanospermine per ml. More of the radioactive glucosamine was found in the second peak, although peak 1 was still labeled. This is additional suggestive evidence that peak 1 represents mannans with a much higher mannose content (relative to glucosamine) than the typical N-linked oligosaccharides of the glycoproteins (as seen in peak 2). Figure 6A also demonstrates that peak 2 in the control cells was of lower molecular weight than that seen in castanospermine-grown cells, since it eluted in later fractions. This is shown more clearly in Fig. 613, where the glycopeptides have been digested with Endb H and then rechromatographed on the Bio-Gel P-4 columns. As in the case of mannose-labeled glycopeptides, peak 2 was susceptible to Endo H as shown by the change in migration after treatment. Thus, the oligosaccharide released from control cells migrated near the MangGlcNAc standard, whereas that from castanospermine-treated cells was larger and migrated near the hexose1lGlcNAc standard. The glucosamine-labeled oligosaccharide from control cells was mostly susceptible to a-mannosidase digestion as demonstrated by the appearance of most of the radioactivity in a peak migrating like Man-GlcNAc. The oligosaccharide from castanospermine-grown cells, on the other hand, was only slightly susceptible to a-mannosidase, and its migration was only altered by a few fractions, indicating the removal of only one or two mannose residues (data not shown). These results are similar to those observed with the mannose-labeled glycopeptides and oligosaccharides. DISCUSSION The results reported in this paper show that the tetrahydroxyoctahydroindolizine castanospermine inhibits the processing of the oligosaccharide chains of the glycoprotein enzymes secreted by A. fumigatus. Thus, cells grown in the presence of this alkaloid produce N-linked oligosaccharides that are larger than the oligosaccharides of normal cell glycoproteins. Based on our partial characterization and the reported structures for the various processing intermediates, the oligosaccharide(s) produced in the presence of castanospermine appear to be mostly Glc3Man1wGlcNAc2 structures, whereas those found in control cell glycoproteins are mostly Manv9GlcNAc2 structures. Thus, the oligosaccharides from normal cells were almost completely susceptible to a-mannosidase digestion and released most of the radioactivity as free mannose indicating the absence of blocking glucose residues. On the other hand, the oligosaccharides from castanospermine-treated cells only released one or two mannose residues by a-mannosidase treatment, suggesting that some of the branches were capped by glucose residues. These data were also confirmed by methylation analysis of

the [3H]mannose-labeled and [3H]glucose-labeled oligosaccharides.


VOL. 160, 1984

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FIG. 6. Effect of castanospermine on the structures of the glucosamine-labeled glycopeptides and oligosaccharides from secreted glycopeptides. Protocol for this experiment was as described in the legend to Fig. 2, except that [6-3H]glucosamine was used as the label. (A) Glycopeptides from control and castanospermine (1 mg/ml)-grown cells. The glycopeptides were digested with Endo H and rechromatographed to give the profiles in B.

The glycoproteins produced in the presence of castanospermine were still secreted from the cells, as shown by the presence of various enzymatic activities in the culture medium (Fig. 1). However, some inhibition in secretion was observed, since the activities in the media were decreased with increased castanospermine concentration. Interestingly enough, the various glycosidases did not show the same sensitivity to the alkaloid. Thus, the P-hexosaminidase was the most sensitive of the glycosidases tested, and its activity was depressed by 30 to 40% at 100 ,ug of alkaloid per ml. On the other hand, a-galactosidase activity was not greatly affected at alkaloid concentrations of up to 1 mg/ml, and decreases in activity were only seen at 2 mg/ml or higher concentrations. We tested the effect of castanospermine directly on the enzymatic activity of the ,-hexosaminidase, the a-galactosidase, and the ,-galactosidase by adding various amounts of alkaloid to the assay mixtures. No inhibition of enzymatic activity was observed. Thus, the decrease in activity in the culture media must be attributed to lower amounts of the specific enzymes, or to less active enzyme in the media. There are several possible explanations to account for the inhibition of secretion observed in these studies. Since castanospermine inhibits the processing glucosidases and prevents the removal of glucose residues fron the N-linked glycoproteins (28), the glucose-containing glycoprotein may be recognized only poorly by the secretory mechanism of the cell. In fact, there is some precedence for this idea. A recent study by Lodish and Kong (24) compared the effects of several processing inhibitors on the secretion of a number of glycoproteins by human hepatoma HepG2 cells. Deoxynojirimycin, also an inhibitor of glucosidase I (20), reduced the rate of secretion of the glycoproteins al-antitrypsin and cx1-

antichymotrypsin, but had only marginal effects on the secretion of other glycoproteins. Equilibrium density gradient centrifugation indicated that this al-antitrypsin and (xlantichymotrypsin accumulated in the rough endoplasmic reticulum in the presence of deoxynojirimycin. The authors suggested that the movement of the protein from the rough endoplasmic reticulum to the Golgi required that the Nlinked oligosaccharides be processed to at least MangGlcNAc2 and that glucose residues on these oligosaccharides might retard or prevent their movement. Thus, the results with these hepatoma cells are quite analogous to those described here with Aspergillus sp., and the explanation for the reduction in glycosidase activities in the media could be a reduction in the rate of secretion. There are several other compounds that have also been reported to retard or inhibit the intracellular transport of newly synthesized glycoproteins. For example, monensin is a carboxylic acid ionophore that collapses the proton gradient by the electroneutral exchange of a proton for a monovalent cation (preferably sodium) across a membrane. Thus, monensin causes a rapid dilation of the Golgi elements and blocks the transport of secreted proteins to the extracellular space (18, 36, 41, 43). Chloroquine and ammonium chloride are weak bases that become protonated after entering the intralysosomal space. A primary consequence of this action is the ability of either agent to raise the intralysosomal pH and disrupt the targeting of newly synthesized lysosomal enzymes to the lysosome (13, 42). Since castanospermine and deoxynojirimycin are also weak bases, it is possible that they could also act as lysosomotropic drugs and alter intracellular pH. It is clear from a number of studies that the carbohydrate is not always necessary for protein secretion. For example,

74


in mouse myeloma tumor cells, 2-deoxyglucose prevented the incorporation of glucosamine, mannose, and galactose into secreted protein while allowing the incorporation of leucine to proceed at 40% of the normal rate. The protein that was secreted under these conditions was shown to be the nonglycosylated form of K46. Thus, in this case, glycosylation was not necessary for secretion, although the absence of carbohydrate did appear to retard intracellular transport and export from the cell (6). A number of studies have also been done with tunicamycin, an antibiotic that prevents N-glycosylation of proteins (8, 27, 37). In several of these studies, the nonglycosylated proteins were still secreted from the cells or functioned normally (19, 28, 38), whereas in other cases secretion did not occur (5, 11). A plausible explanation for these variations and one for which some evidence has been gathered suggests that at least one role for the carbohydrate is to help to determine or maintain the conformation of the protein (23). Since the carbohydrate is added during polypeptide synthesis, it may have a great influence on the protein conformation, depending, of course, on the amino acid composition of the protein. That is to say, carbohydrate may be essential in influencing the conformation of some proteins, but not of others. Since castanospermine apparently does not inhibit glycosylation, but causes alterations in the final oligosaccharide structure, it may be possible to correlate subtle changes in structure with alterations in function. The changes in secretion observed with the glycosidases may be an example that needs further examination. ACKNOWLEDGMENT This study was supported by Public Health Service research grant HL-17783 from the National Institutes of Health. LITERATURE CITED 1. Akasaki, M., M. Suzuki, K. Funakoshi, and I. Yamashina. 1976. Characterization of P-galactosidase from a special strain of Aspergillus oryzae. J. Biochem. (Tokyo) 80:1195-1200. 2. Ayda, S., and A. D. Elbein. 1977. Glycoprotein enzymes secreted by Aspergillus niger: purification and properties of a-galactosidase. J. Bacterol. 129:850-856. 3. Burns, D. M., and 0. Touster. 1982. Purification and characterization of glucosidase II, an endoplasmic reticulum hydrolase involved in glycoprotein biosynthesis. J. Biol. Chem. 257:999110,000. 4. Chen, W. W., and W. J. Lennarz. 1978. Enzymatic excision of glucosyl units linked to the oligosaccharide chains of glycoproteins. J. Biol. Chem. 253:5780-5785. 5. Cox, G. S. 1981. Synthesis of the glycoprotein hormone asubunit and plancental alkaline phosphatase by Hela cells: effects of tunicamycin, 2-deoxyglucose and sodium butyrate. Biochemistry 20:4893-4900. 6. Eagon, P. C., and E. C. Heath. 1977. Glycoprotein biosynthesis in myeloma cells. Characterization of nonglycosylated immunoglobulin light chain secreted in the presence of 2-deoxy-Dglucose. J. Biol. Chem. 252:2372-2383. 7. Elbein, A. D. 1979. The role of lipd-linked saccharides in the biosynthesis of complex carbohydrates. Annu. Rev. Plant Physiol. 30:239-272. 8. Elbein, A. D. 1984. Inhibitors of the biosynthesis and processing of N-linked oligosaccharides. Crit. Rev. Biochem. 16:21-49. 9. Elbein, A. D., S. Ayda, and Y. C. Lee. 1977. Purification and properties of a 3-mannosidase from Aspergillus niger. J. Biol. Chem. 252:2026-2031. 10. Grinna, L. S., and P. W. Robbins. 1980. Substrate specificities of rat liver microsomal glucosidases which process glycoproteins. J. Biol. Chem. 255:2255-2258. 11. Hackman, S., A. Kulczycki, Jr., R. G. Lynch, and S. Kornfeld. 1977. Studies on the mechanism of tunicamycin inhibition of IgA and IgE secretion by plasma cells. J. Biol. Chem. 252:4402-

J . BACTERIOL . 4408. 12. Hakomori, S. 1964. A rapid permethylation of glycolipid and polysaccharide catalyzed by methyl sulfinyl carbanion in dimethyl sulfoxide. J. Biochem. (Tokyo) 55:205-208. 13. Hasilik, A., and E. F. Neufeld. 1980. Biosynthesis of lysosomal enzymes in fibroblasts. J. Biol. Chem. 259:4937-4945. 14. Hohenschutz, L. D., E. A. Bell, P. J. Jewess, D. P. Leworthy, R. J. Pryce, E. A. Arnold, and J. Clardy. 1981. Castanospermine, a 1,6,7,8-tetrahydroxyoctahydroindolizine alkaloid from the seeds of Castanospermum australe. Phytochemistry 20:811814. 15. Hubbard, S. C., and R. Ivatt. 1981. Synthesis and processing of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 50:555-583. 16. Hurst, P. L., J. Neilsen, P. A. Sullivan, and M. G. Shepherd. 1977. Purification and properties of a cellulase from Aspergillus niger. Biochem. J. 165:33-41. 17. Iwashita, S., and F. Egami. 1973. a-D-fucosidase from Aspergillus oryzae: characterization of a-D-fucosidase with a-D-galactosidase activity. J. Biochem. (Tokyo) 73:1217-1222. 18. Johnson, D. C., and M. J. Schlesinger. 1980. Vesicular stomatitis virus and Sindbis virus glycoprotein transport to the cell surface is inhibited by ionophores. Virology 103:407-424. 19. Keller, R. K., and G. D. Swank. 1978. Tunicamycin does not block ovalbumin secretion in the oviduct. Biochem. Biophys. Res. Commun. 85:762-768. 20. Kilker, R. D., Jr., B. Saunier, J. S. Tkacz, and A. Hercovics. 1981. Partial purification from Saccharomyces cerevesiae of a soluble glucosidase which removes the terminal glucose from the oligosaccharide Glc3Man9GlcNAc2. J. Biol. Chem. 256:5299-5303. 21. Kobata, A. 1979. Use of endo and exoglycosidases for structural studies of glycoconjugates. Anal. Biochem. 100:1-14. 22. Kornfeld, R., and S. Kornfeld. 1976. Comparative aspects of glycoprotein structure. Annu. Rev. Biochem. 45:217-237. 23. Leavitt, R., S. Schlesinger, and S. Kornfeld. 1977. Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of VSV and Sindbis virus. J. Biol. Chem. 252:9018-9023. 24. Lodish, H. F., and N. Kong. 1984. Glucose removal from Nlinked oligosaccharides is required for efficient maturation of certain secretary glycoproteins from the rough endoplasmic reticulum to the golgi complex. J. Cell Biol. 98:1720-1729. 25. Mega, T., T. Ikenaka, and Y. Matsushima. 1972. Studies on Nacetyl-D-glucosaminidase of Aspergillus oryzae. Il. Substrate specificity of the enzyme. J. Biochem. (Tokyo) 71:107-114. 26. Michael, J. M., and S. Kornfeld. 1980. Partial purification and characterization of the glucosidases involved in the processing of asparagine-linked oligosaccharides. Arch. Biochem.

Biophys. 199:249-258.

27. Mozraki, A., J. A. O'Malley, W. A. Carter, A. Takatsuki, G. Tamura, and E. Sulkowski. 1978. Glycosylation of interferons. Effects of tunicamycin on human immune interferon. J. Biol. Chem. 253:7612-7615. 28. Olden, K., R. M. Pratt, and K. M. Yamada. 1978. Role of carbohydrates in protein secretion and turnover: effects of tunicamycin on the major cell surface glycoprotein of chick embryo fibroblasts. Cell 13:461-473. 29. Pan, Y. T., H. Hori, R. G. Saul, B. A. Sanford, R. J. Molyneux, and A. D. Elbein. 1983. Castanospermine inhibits the processing of the oligosaccharide portion of the influenza viral hemagglutinin. Biochemistry 22:3975-3984. 30. Rudick, M., and A. D. Elbein. 1973. Glycoprotein enzymes secreted by Aspergillus fumigatus. Purification and properties of ,-glucosidase. J. Biol. Chem. 248:6506-6513. 31. Rudick, M. J., and A. D. Elbein. 1974. Glycoprotein enzymes secreted by Aspergillus fumigatus. Purification and properties of a-glucosidase. Arch. Biochem. Biophys. 161:281-290. 32. Saul, R., J. P. Chambers, R. J. Molyneux, and A. D. Elbein. 1983. Castanospermine, a tetrahydroxylated alkaloid that inhibits 3-glucosidase and ,-glucocerebrosidase. Arch. Biochem. Biophys. 221:593-597. 33. Schachter, H., S. Narasimhan, P. Gleeson, and G. Vella. 1983.

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Control of branching during biosynthesis of asparagine-linked oligosaccharides. Can. J. Biochem. Cell. Biol. 61:1049-1066. Speake, B. K., D. J. Mailey, and F. W. Hemming. 1981. The effect of tunicamycin on secreted glycosidases of Aspergillus niger. Arch. Biochem. Biophys. 210:110-117. Spiro, R. J., M. J. Spiro, and V. D. Bhoyroo. 1979. Processing of carbohydrate units of glycoproteins. Characterization of a thyroid glucosidase. J. Biol. Chem. 254:7659-7667. Srinivas, R. V., L. R. Melsen, and R. W. Compans. 1982. Effects of monensin on morphogenesis and infectivity of Friend murine leukemia virus. J. Virol. 42:1067-1075. Struck, D. K., and W. J. Lennarz. 1981. The function of saccharide-lipids in synthesis of glycoproteins, p. 35-73. In W. J. Lennarz (ed.), The biochemistry of glycoproteins and proteoglycans. Plenum Publishing Corp., New York.

38. Struck, D. K., P. B. Siuta, M. D. Lane, and W. J. Lennarz. 1978. Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. J. Biol. Chem. 253:5332-5337. 39. Swaminathan, N., K. L. Matta, L. A. Donoso, and 0. P. Bahl.


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