Inhibition of N-linked Complex Oligosaccharide Formation by 1-Deoxynojirimycin, an Inhibitor of Processing Glucosidases*

THE JOURNAL OF BIOI.OGICAL CHEMISTRY Vol. 257, No. 23, Issue of December IO, pp. 14155-14161. 1982 Printed in U.S.A. Inhibition of N-linked Complex Oligosaccharide Formation by 1-Deoxynojirimycin, an Inhibitor of Processing Glucosidases* (Received for publication, March 1, 1982) Brigitte Saunier3, Richard D. Kilker, Jr.8, Jan S. Tkaczq, Andrea Quaronill, and Annette Herscovics**$$ From the Laboratory for Carbohydrate Research. Departments of Biological Chemistry and Medicine, Hartlard Medical School and Massachusetts General Hospital, Boston, Massachusetts 2114, the )I Gastrointestinal Unit, Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 2114, and the **McGill Cancer Centre, McGill Uniztersity, Montreal, Quebec, Canada H3G I Y6 Glucosidase activities which remove glucose residues ent in complex oligosaccharides and rendered high from GlcZMangGlcNAcz and GlclManyGlcNAcz oligosac- mannose oligosaccharides less susceptible the to action charides were obtained in soluble form from Saccha- of a-mannosidase. It is concluded that deoxynojirimyromyces cerevisiae X-218 without detergent. These two cin probably inhibits the formation of complex oligoenzyme activities were clearly separated from the saccharides by interfering with processing glucosi- GlcsMangGlcNAcz oligosaccharide glucosidase which dases, and that it should be useful to modify the strucwas shown previously to remove the terminal glucose ture of oligosaccharides on glycoproteins. residue from GlcsMan9GlcNAcz oligosaccharide (Kilker, R. D., Jr., Saunier, B., Tkacz, J. S., and Herscovics, A. (1981) J. Biol. Chem. 256, 5299-533) and from the a- and b-glucosidases which act on p-nitrophenylgluco- The major pathway for the biosynthesis of the N-linked pyranosides. The activities with both GlcZMan9GlcNAcz carbohydrate chains in eucaryotes involves the transfer of the and GlclMan9GlcNAcz had the same properties and oligosaccharide, Glc.+Man9GlcNAc2, from dolichyl pyrophoswere inhibited to the same extent by glucose and, of phate to protein and its subsequent processing to yield various various a- and /3-linked glucose disaccharides tested, oligosaccharide structures with the same inner core (1-7). In by both nigerose and maltose. In contrast, the animal cells, either high mannose or complex oligosaccharides GlcsMan9GlcNAcz oligosaccharide glucosidase was not affected by glucose, but it was inhibited by kojibiose. are formed, whereas in yeast polymannosyl outer branches are added to the inner core. In both cases, processing of the These results suggest that there are two specific a- oligosaccharide precursor begins with the removal of the glucosidases responsible for oligosaccharide process- glucose residues. At least two distinct glucosidases have been ing in S. cerevisiae. described in animal tissues (8-13), and in previous work, we The glucose analog, 1-deoxynojirimycin, greatly in- reported the partial purification and characterization of a hibited both partially purified oligosaccharide glucosi- soluble yeast glucosidase which releases the terminal glucose dases from S. cerevisiae and the calf pancreas micro- residue from Glc.rMansGlcNAc2 (14). We now report the parsome glucosidases which remove all three glucose restial purification of another glucosidase from Saccharomyces idues. The effect of deoxynojirimycin on intact cells was examined using confluent IEC-6 intestinal epithelial cells after labeling for 24 h with [2- H]mannose. Labeled glycopeptides obtained by exhaustive pronase digestion were fractionated on Bio-Gel P-6, before and after treatment with endo-fl-n-acetylglucosaminidase H to distinguish between high mannose and complex N- linked oligosaccharides. Deoxynojirimycin (5 mm) greatly decreased the proportion of radioactivity pres- * This work was supported by Research Grants AM-3564 and GM-31265 from the National Institutes of Health, United States Public Health Service, by Research Grant PCM-787644 from the National Science Foundation, and by the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisernent in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address, Laboratoire de Biochimie, Unite denseignement et de Recherches St. Louis Lariboisiere, 45 Rue des Saints-Peres, 756 Paris, France. Present address, Chemistry Department, Drew University, Madison, NJ 794. 1 Present address, Department of Microbiology, Squibb Institute for Medical Research, P.O. Box 4, Princeton, NJ 854. $$To whom correspondence should be addressed at the McGill Cancer Centre, 3655 Drummond Street, Montreal, Quebec, Canada H3G 1Y6. 14155 cereuisiae which removes glucose residues from both GlcsMan9GlcNAc2 and GlclMan,,GlcNAcs. Inhibitors of processing of the oligosaccharide moiety would be useful to modify the structure of the oligosaccharides of glycoproteins in order to study the function of the carbohydrate moiety in the biological activity of glycoproteins. For this reason, we have investigated several glucosidase inhibitors, and now report that the antibiotic, 1-deoxynojirimycin, is a potent inhibitor of the yeast and pancreas glucosidases which release the three glucose residues from Glc.rMan:,- GlcNAcz oligosaccharide. Deoxynojirimycin, produced by Bacillus species, is the reduced form of nojirimycin, an antibiotic synthesized by several strains of Streptomyces. These compounds are glucose analogs with an NH group substituting for the atom in the pyranose ring; they have been shown to inhibit intestinal a-glucosidases and pancreatic a-amylase both in vivo and in uitro (15-18). In the present work we also show that deoxynojirimycin inhibits the formation of N-linked complex oligosaccharides in cells in culture. EXPERIMENTAL PROCEDURES Materials-The source of chemicals has been described in previous publications (14, 19). Bio-Gel P-4 (-4 mesh) and Bio-Gel P-2 (1-2 mesh) were purchased from Bio-Rad Laboratories. Endoa-N-acetylglucosaminidase H, from Miles Laboratories; jack bean w-

~~~ - 14156 Inhibition of N-linked Complex Oligosaccharide Synthesis mannosidase, Pipes', and isomaltose, from Sigma; gentiobiose and cellobiose were from Pfanstiehl Laboratories; maltose, from Eastman Kodak, and concanavalin A-Sepharose was purchased from Pharmacia. Kojibiose was a gift from Dr. K. Matsuda, Tohoku University, Japan: nigerose, from Dr. I. cj. Goldstein, University of Michigan, and sophorose from Dr. H. Sandermann, cjr., University of Freiburg, Federal Pepublic of Germany. 1-Deoxynojirimycin (BAY h5595) was obtained from Drs. E. Truscheit and D. Schmidt, Bayer Research Laboratories, Wuppertal, West Germany. Preparation of Oligosaccharide Substrates-The glucose-labeled oligosaccharides, Glcl, Glc2, and GlcI. were prepared by incubation of chicken liver microsomes with UDP-[6-"HJglucose, or of calf pancreas microsomes with UDI"["C]glucose, as described previously (14). For endo-8-n-acetylglucosaminidase H treatment, each labeled oligosaccharide (Glcl, Glc2, and G1c.J was incubated for 16 h at 37 "C in 1 pl of.4 M citrate-phosphate buffer, ph 5.5, containing 2 milliunits of enzyme, and a drop of toluene. The reaction was stopped by boiling for 2 min. For wmannosidase digestion, each labeled oligosaccharide was incubated fur 72 h in 5 pl of.2 M sodium acetate buffer, ph 4.5, containing.2 mg/ml of bovine serum albumin, 2 mm zinc sulfate, and I drop of toluene. Three units of a-rnannosidase were added at the beginning, and 1.5 units after 24 and 48 h of incubation. The reaction was stopped by boiling for 2 min. The u-mannosidase-treated oligosaccharides were chromatographed on a column of Bio-Gel P-4 (-4 mesh, 1 X 22 cm) in.1 M pyridine acetate, ph 5.5, containing.5 mm sodium azide. Fractions of.5 ml were collected; bovine serum albumin was used to determine the void volume. A sample of the original oligosaccharides was chromatographed on the same column for comparison. scraped from the dishes in phosphate-buffered saline, and extracted Purification of Yeast Oligosaccharide Glucosidases-The proce- three times with chloroform/methanol/water (1:13) (v/v) to redure used was a modification of that described previously (14). A move any lipid-bound oligosaccharides. This material was used as a crude extract obtained from S. cerecisiae X-218-1B a was subjected source of cell pellet glycopeptides. to precipitation between 2 and 6% saturated ammonium sulfate. Cell surface and cell pellet glycopeptides were obtained by exhaus- The subsequent chromatography on DEAE-Sephadex was performed tive pronase digestion as described previously (19). They were then as described previously, except that the size of the column was 3.5 X chromatographed on a column of Bio-Gel P-6, before and after 22 cm. and that gradient elution was performed with 1 liter of.1 to treatment with endo-p-n-acetylglucosaminidase H to distinguish be- 1. M potassium phosphate buffer, ph 6.8. Fractions of 8.5 ml were tween high mannose and complex N-linked oligosaccharides as decollected. The fractions containing Glc? and Glc: oligosaccharide scribed previously (19). glucosidase activity were combined and lyophilized as were those Treatment of High Mannose Oligosaccharides with u-mannosicontaining Glc I oligosaccharide glucosidase activity. These prepara- dase-to determine the proportion of radioactivity released bv (ttions were used for all experiments unless otherwise indicated. mannosidase, high mannose oligosaccharide fractions obtained from In some cases, the oligosaccharide glucosidases obtained from Bio-Gel P-6 after treatment with endo-ib-n-acetylglucosaminidase H DEAE-Sephadex were subjected to gel filtration on a column of Bio- Gel P-2 (1.5 x 85 cm) previously equilibrated with 2 mm potassium were lyophilized and then incubated for 18 h at 37 "C in 1 pl of 12.5 mm sodium acetate, ph 5., containing 25 mm sodium chloride,.25 phosphate, ph 6.8. containing.2% Hibitane. The column was mm ZnSOI, 25 pg of chloramphenicol, 25 pg of cycloheximide, 5 pg of eluted with the same buffer, and fractions of I ml were collected. bovine serum albumin, and 1.4 units of jack bean a-mannosiclase Yeast Oligosaccharide Glucosidase Assays-For Glct and Glc. (Boehringer Mannheim). After incubation, the samples were boiled oligosaccharide glucosidases, enzyme samples (5-1 pl) were incu- for 3 min and passed through a small column (1 ml) of concanavalin bated for 6 min at 37 "C in 4 pl of either 6 mm sodium maleate A-Sepharose in a Pasteur pipette. The released labeled mannose was buffer, ph 6., or 5 mm potassium phosphate buffer, ph 6.8, contain- eluted with 4 ml of a solution containing 1 mm each MnCL, MgCI,, ing 2 cpm of [ 'H]glucose-labeled Glcl or Glc? oligosaccharides and hovine serum albumin (1 mg/ml). For Glc.{ oligosaccharide glucosidase, enzyme samples (1 pl) were incubated for 6 min at 37 "C in 4 pi of 2 nm potassium phosphate, ph 6.8, containing 2 cpm of either ['Hlglucose- or ["C]glucoselabeled Glcl oligosaccharide and bovine serum albumin (1 mg/ml). and CaCL. The labeled oligosaccharide adsorbed to the gel was then eluted with 4 ml of 1 mm a-methyl-o-mannopyranoside in the same solution. Control incubations without u-mannosidase were processed in the same way. The per cent of the total radioactivity released as mannose was calculated. Analytical Methods-Thin layer chromatography was performed The reactions were stopped by boiling for 2 min. The labeled on precoated plates of silica gel G (.25 mm thick, E. Merck A. G., glucose released was determined using either of two methods. In method A, the samples were chromatographed on paper as described previously (14); in method B, the samples were passed through a column of concanavalin A-Sepharose (.5 X 2 cm) equilibrated with a solution containing 5 nm each of MgCL, MnCL, and CaCL, and.5 mm NaN,. The labeled glucose was eluted with 3 ml of the same solution. Oligosaccharides in the sample mixtures were retained on the column. The same column could be used repeatedly about 1 times. This method was not appropriate for experiments with ctmannosidase-treated oligosaccharide substrates. Oligosucchande Glucosidase Assays in Ctrlf Pancreas MLrrosones-Calf pancreas microsomes were prepared as described previously (2), except that the membranes were washed with 1 mm phosphate buffer, ph 6.8, before storage at -8 "C since l'ris buffer is inhibitory to the glucosidases. Microsomal pellets were homogenized in 1 rnm potassium phosphate buffer, ph 6.8, just before use. ~~ ~ ' The abbreviations used are; Pipes, 1,4-piperazinediethanesulfonic acid Glc,, Glc.rMan~3GlcNAcL; Glcr, GlcnMansGlcNAcr; Glc:, GlclMan&lcNAc2. All sugars are of the D configuration. Incubation of pancreas microsomes was done at 37 C with 2 cpm of either [ 'HIGlc-labeled Glc.:, Glc?, or Glcl oligosaccharides in 5 pl of 6 mm potassium phosphate buffer, ph 6.8, containing.8c:; soybean trypsin inhibitor, 15 mm EDTA,.5%. Triton X-1, and 1 mg/ml of bovine serum albumin. The amount of microsomal protein was 1 pg,.6 pg, and 1 pg for Glc,, Glc?, and Glci oligosaccharide glucosidases, respectively. The incubation time was 3 min for Glcl and 2 min for Glcl and Glc.3 oligosaccharide glucosidases. The con- ditions of each assay were established to be linear with respect to time, substrate, and enzyme concentrations. Method B was used to determine the labeled glucose released. Glycoprotein Biosynthesis in Intestinal Epithelial Cells in Chlture-Rat small intestinal epithelial cells (IEC-6 cells) were cultured to confluence in 1-mm diameter dishes as described previously (19). After 3-4 days of confluence, the cells were labeled for 24 h with I)- [2-'H]mannose(5 pci/ml) in 1 ml oflow glucose (1 g/liter) Ihlbecco's modified essential medium supplemented with 5c; dialyzed fetal calf serum, 5 units/ml of penicillin, 5 pg/ml of streptomycin, 1 pg/ml of insulin, and 4 mm L-glutamine. The medium also contained different concentrations of deoxynojirimycin during the entire labeling period. After 24 h the cells were washed three times with 1 ml of phosphate-buffered saline and incubated for 1 min at room temperature with 4 ml of phosphate-buffered saline containing.1? trypsin and.5% EDTA. The extracted cell surface material was removed with a Pasteur pipette and the cells were washed with 2 nd of phosphate-buffered saline, ph 7.9. The solutions were combined and centrifuged to remove any detached cells. The resulting supernatant was used as source of cell surface glycopeptides. The cells were Darmstadt, Germany) in chloroform/methanol/water (1:1:3)(v/v). Sugars were detected with the anisaldehyde reagent (21). Assays for glycosidases using p-nitrophenylglycosides and protein determinations were done as described previously (14). Radioactivity was determined in a Packard liquid scintillation spectrometer, model 3255. using Hydrofluor (National Diagnostics, Somerville, NJ). RESULTS Partial Purification of Glc, and GLc2 Oligosaccharide Glucosidases In previous work we showed that a significant part of the oligosaccharide glucosidase from S. cerevisiae using Glc:, as substrate was recovered in the supernatant (27, X g) obtained from the yeast homogenate without using detergent. Similarly, when assays for oligosaccharide glucosidase activity were done using Glc? and Glc, oligosaccharides as substrates, about 754 of the total activity was recovered in the superna-

Inhibition of N-linked Complex Oligosaccharide Synthesis 14157 tant (27, X g). The enzyme activity with these two substrates was not affected by Triton X-1. The Glcl and Glcz oligosaccharide glucosidase activities were quantitatively precipitated between 2 and 6% saturated ammonium sulfate, resulting in a 2-fold increase in specific activity. After dialysis against 1 mm potassium phosphate buffer, ph 6.8, and subsequent lyophilization, the activities were stable for at least 3 months at -2 C. When this preparation was chromatographed on DEAE- Sephadex A-25, the Glc, and Glc2 oligosaccharide glucosidase activities were eluted together at a lower phosphate concentration than the Glc:, oligosaccharide glucosidase (Fig. 1A). The elution profile was identical for both Glcp and Glcl substrates. The Oligosaccharide glucosidases obtained from DEAE-Sephadex still contained,&glucosidase and a-mannosidase acting on p-nitrophenylglycosides (Fig. 1B). They were free of a-glucosidase acting on p-nitrophenyl a-d-glucopyranoside which was shown previously not to bind to the resin (14). The recovery of Glc, and Glcl oligosaccharide glucosidase activities from DEAE-Sephadex was about 35%) and the overall purification was 5-fold. After lyophilization, the preparation was stable for at least 3 months at -2 C. The fractions obtained from DEAE-Sephadex containing Glc, and Glcn oligosaccharide glucosidase activities, and those containing Glc., oligosaccharide glucosidase activity were each chromatographed on Bio-Gel P-2 (Fig. 2). Both oligosaccharide glucosidase preparations were excluded from the gel, I* 6-5 - 4-3 - 2-1 - Oo r 4 i= b T 2 o pnph-a-o-mon 5 6 7 8 9 IO 11 12 FRACTION NUMBER f 8.5 ml I FIG. 1. DEAE-Sephadex A-25 column chromatography. A preparation (about 1.4 g of protein) obtained after ammonium sulfate precipitation, dialysis, and lyophilization was redissolved to yield 1 mm potassium phosphate buffer, ph 6.8, containing.2% Hibitane. The sample was then applied to a column of DEAE-Sephadex A-25 (3.5 x 22 cm). The column was first eluted with 5 ml of the same buffer and then with a linear gradient of.1 to 1. M potassium phosphate, as indicated by the arrow. The glucose released from Glc,, Glcn, and Glc:~ oligosaccharides was measured using 1 p1 of each fraction (A). The total P-glucosidase and u-mannosidase activities using the appropriatep-nitrophenylglycosides was also measured (B). No activity with Glc:l was detected in fractions 6-8. Method B was used for assay. pnph-p-~ -Glc, p-nitrophenyl P-D-glucopyranoside; pnph-u-d-glc, p-nitrophenyl a-d-ghcopyranoside. 4 3 2 1 3 4 5 6 7 fr4cji ON NUM3.M FIG. 2. Gel filtration of oligosaccharide glucosidases. A, a lyophilized sample containing Glc oligosaccharide glucosidase activity was obtained after DEAE-Sephadex chromatography. It was redissolved in its original volume of water (4 ml) and was dialyzed against 2 mm potassium phosphate buffer, ph 6.8. It was then lyophilized and redissolved in 1 ml of water and chromatographed on Bio-Gel P-2 as described under Experimental Procedures. B, a lyophilized sample containing Glc2/Glcl oligosaccharide glucosidase activity was obtained after DEAE-Sephadex chromatography. It was dissolved in one-quarter of the original volume of water. dialyzed against 2 mm potassium phosphate buffer, ph 6.8, and then chromatographed on Bio-Gel 1-2 as described under Experimental Procedures. The fractions were assayed for the oligosaccharide glucosidases using method B and for /?-glucosidase using p-nitrophenyl /?-D-ghcopyranoside (pnph-8-d-gzc). There was no /?-glucosidase activity detectable in fractions 3-5. whereas P-glucosidase acting on p-nitrophenyl p-d-glucopyranoside was included. a-mannosidase activity towardp-nitrophenyl a-o-rnannopyranoside was not detectable. The elution profiles obtained for the oligosaccharide glucosidases demonstrated heterogeneity. The columns in Fig. 2 were eluted with 2 mm phosphate buffer. When Glc:, oligosaccharide glucosidase was chromatographed on Bio-Gel P-2 in 25 mm potassium phosphate, it was included in the gel, showing that this enzyme undergoes aggregation at low phosphate concentrations as previously described for the corresponding rat liver enzyme (8). The preparation containing Glc2 and Glcl oligosaccharide glucosidase activities could not be chromatographed in 25 mm potassium phosphate buffer because phosphate was inhibitory at that high concentration. The enzyme could not be stored at this stage without significant loss of activity. Properties of GZcl and GZQ Oligosaccharide Glucosidases-The enzyme preparation obtained from DEAE-Sephadex which contained Glcl and Glc? oligosaccharide glucosidase activities (Fig. 1A) showed a parallel time-dependent release of glucose with Glc, and Glc2 as substrates, but no significant release with Glc:~ (Fig. 3). The specific activity (counts/min of glucose released/mg of protein) with Glc: as substrate was much higher (2-3 times) than that using Glc,, but this is partly due to the fact that thenzyme preparation removes both glucose residues from Glc.? and that both are labeled. Periodate oxidation showed that 6% of the radioactivity in Glce was in the terminal glucose residue. The release of glucose was dependent on protein concentration up to at least 1.2 mg/ml using Glc, as substrate, and up to.6 mg/ml

14158 Inhibition of N-linked Complex Oligosaccharide Synthesis o 2 4 6 eo too 12 TfME lminuted FIG. 3. Rate of glucose release from Glc,, Glcn, and GlcB oligosaccharides. Samples obtained from the DEAE-Sephadex column (fractions 7-8, Fig. 1A) were incubated for various periods of time with each of the glucose-labeled oligosaccharides (2 cpm) in 4 pl of potassium phosphate buffer, ph 6.8, containing 1 mg/ml of bovine serum albumin. The assay was performed using method B. 15 pg of enzyme protein was used with Glc2 and 3 pg of enzyme protein was used with Glc, and Glc.{ oligosaccharides. TABLE I Effect of glucose and disaccharides on. oligosaccharide glucosidases Enzyme fractions obtained after Bio-Gel P-2 chromatography were assayed under standard conditions using Method B, in the presence of various concentrations of disaccharides. Concentration rnm Glucose 1. 5. 1. Kojibiose 2. 1. Nigerose 2.5 5. 1. Maltose 2.5 5. 1. I ND, not determined. Inhibition Glc, Glc, Glc, - 45 75 85 18 41 48 37 77 7 5 75 9 16 36 4 33 56 64 1 ND with Glca. Both Glc, and Glcy oligosaccharide glucosidase activities responded in parallel to changes in ph, and had a broad ph optimum between 5.8 and 6.8. The Glcl and Glca oligosaccharide glucosidase activities do not require divalent cations since 1 mm of either MgCla, MnC12, or EDTA had no effect, but they were inhibited 6% by 5 mm Tris. Similar activities were obtained using 5 mm phosphate, maleate, or Pipes buffers at ph 6.8. However, higher phosphate concentrations of 1-2 mm caused an inhibition of 2-556, respectively. Effect of a-mannosidase and Endo-P-N-acetylglucosaminidase H Treatment of Oligosaccharides-The three oligosaccharide substrates were treated with a-mannosidase; the prod- ucts were chromatographed on a high resolution column of Bio-Gel P-4 and then used as substrates for the oligosaccharide glucosidases. The number of mannose residues removed was estimated from the relative values as described by Hubbard and Robbins (22). The removal of3-4 mannose residues from Glc:{ oligosaccharide did not inhibit the Glc:c oligosaccharide glucosidase activity; on the contrary, a stimulation of about 3% was observed. In contrast, the Glcl and Glca oligosaccharide glucosidase activities were completely inhibited by removal of a similar number of mannose residues. Treatment of the oligosaccharides with endo-p-n-acetylglucosaminidase H had no effect on the oligosaccharide glucosidases. Effect of Monosaccharides and Disaccharides on Oligosaccharide Glucosidases-Various monosaccharides and glucose-containing disaccharides were tested as potential inhibitors of the glucosidases. As previously shown for Glc:] oligosaccharide glucosidase, mannose, galactose, N-acetylglucosamine, glucose 6-phosphate, and 2-deoxyglucose had no effect on the Glc, and Glc? oligosaccharide glucosidases. Both activities, however, were inhibited to the same extent by glucose which had no effect on Glc:{ oligosaccharide glucosidase (Table I). The effect of disaccharides was studied using the enzyme preparations obtained after Bio-Gel P-2 chromatography. Thin layer chromatography at the end of the incubation showed that no degradation of any of the disaccharides had occurred. Glc:, oligosaccharide glucosidase was completely inhibited by 2 mm kojibiose which contains an a-1,2 linkage, whereas Glc, and Glcs oligosaccharide glucosidases were inhibited to the same extent by both nigerose (a-1,3) and maltose (a-1,4)(table I). No effect on the glucosidases was DEOX YNOJlRfA-4 YCfN @MI FIG. 4. Effect of deoxynojirimycin concentration on yeast oligosaccharide glucosidases. Partially purified (DEAE-Sephadex) Gkl oligosaccharide glucosidase obtained from S. cerecisiae was incubated with [ H]glucose-labeled Glc:,, and partially purified Glcd Glc, oligosaccharide glucosidase was incubated with [ H]glucose-labeled Glc, and Glc, oligosaccharides in the presence of different concentrations of deoxynojirimycin as described under Experimental Procedures. observed with 2-1 mm isomaltose (a-1,6), sophorose (p-1,2), gentiobiose (/3-1,6), or cellobiose (/3-1,4). Effect of Deoxynojirimycin on Glucosidases The antibiotic deoxynojiromycin inhibits both partially purified yeast glucosidases at very low concentrations (Fig. 4). A 5% inhibition was observed at about 2 p~ for Glc:{ oligosaccharide glucosidase and at about 2 p~ for Glcl and Glcr oligosaccharide glucosidase activities. Deoxynojirimycin also inhibits the glucosidases present in calf pancreas microsomes using all three substrates, a 5% inhibition being observed at about 2 p~ (Fig. 5). Since the pancreas glucosidases were not separated, and since all of the glucose residues of the substrates are labeled, it is not possible to compare the sensitivity of the different pancreatic glucosidases as was done for the yeast enzymes. In this case, Triton X-1 was required in the assay because the glucosidases were membrane-bound; EDTA and soybean trypsin inhibitor were found to stimulate the pancreas glucosidase activities, probably by protecting th enzymes against proteolytic degradation.

Inhibition of N-linked Complex Oligosaccharide Synthesis 14159 I I I I I 2 4 6 8 IO DEOXYNOJiRiM YCIN (p MI FIG. 5. Effect of deoxynojirimycin concentration on calf pancreas oligosaccharide glucosidases. Calf pancreas microsomes were incubated with [ HJglucose-labeled Glc!, Glc2, and Glc, substrates as described under Experimental Procedures in the presence of different concentrations of deoxynojirimycin. labeled high mannose oligosaccharides (Figs. 6, A and D, and 7, A and D). Labeling of the cells in the presence of deoxynojirimycin greatly decreased the proportion of cell surface glycopeptides containing labeled complex oligosaccharides from about 76% to about 16% (Fig. 6, Table 11). It also greatly decreased the proportion of labeled complex oligosaccharides in the cell pellet glycopeptides from about 22% to about 5% (Fig. 7, Table 11). The effect of deoxynojirimycin was concentration-dependent, up to about 5 mm. At this concentration, deoxynojirimycin did not significantly affect the total incorporation of radioactivity into the cells although it inhibited labeling of cell surface components about 3%. At a concentration of 1 mm, deoxynojirimycin inhibited the overall incorporation of [2- H]mannose about 2596, but the pattern of glycopeptides was similar to that obtained with 5 mm deoxynojirimycin. E 3 LA Vo MAN I V, MAN It t i t + 111111111111 2 7 7 2 FRACTfONNUMBER C Vo F MAN + t u 2 7 FIG. 6. Effect of deoxynojirimycin on cell surface glycopeptides of IEC-6 cells. Bio-Gel P-6 chromatography of cell surface glycopeptides obtained after a 24-h incubation with [2- H]mannose. A-C, glycopeptides before treatment with endo-p-n-acetylglucosaminidase H; -F, fractions 3-5 from A-C were pooled, lyophilized, digested with endo-/-n-acetylglucosaminidase H, and then chromatographed on the same column of Bio-Gel P-6. A and D were controls without deoxynojirimycin. Band E contained 1 mm deoxynojirimycin; and C and F contained 5 mm deoxynojirimycin. In A-C the fractions with a peak at tubes 35-37 had a K,, =.3, those with a peak at tube 41 had a K$,, =.4, and those with a peak at tubes 49-51 had a Ku,, =.6. t L ul LLL 2 7 2 7 2 7 FRACTION NUMBER F u FIG. 7. Effect of deoxynojirimycin on cell pellet glycopeptides of IEC-6 cells. Bio-Gel P-6 chromatography of cell pellet glycopeptides corresponding to the experiment in Fig. 6. A-C, glycopeptides before treatment with endo-p-n-acetylglucosaminidase H; D-F, fractions 3-55 from A-C were pooled, lyophilized, digested with endo-/-n-acetylglucosaminidase H, and then chromatographed on the same column of Bio-Gel P-6. A and D were controls without deoxynojirimycin; B and E contained 1 mm deoxynojirimycin; C and Fcontained 5 mm deoxynojirimycin. The V,, and Man elution volumes shifted significantly between A and D, and B and E. In A, the fraction with a peak at tube 37 had a Kt,, =.3; in A-C, the fractions with a peak at tubes 41-45 had a K,i, =.4; in D-F, the fractions with a peak at tubes 34-35 had a K,3v =.3, and those with a peak at tubes 46-47 had a K,, =.6. TABLE I1 Effect of Deoxynojirimycin on IEC-6 Cells in Culture Effect of deoxynojirimycin on glycoprotein biosynthesis in ZEC-6 To determine whether deoxynojirimycin affects glycosyla- cells tion in intact cells, we examined the incorporation of [2-, H] Cell surface and cell pellet glycopeptides from IEC-6 cells labeled mannose in IEC-6 rat intestinal epithelial cells in culture. The for 24 h with [2- H]mannose in the presence of different concentralabeled glycopeptides obtained from these cells by exhaustive tions of deoxynojirimycin were chromatographed on Bio-Gel P-6 (Figs. 6 and 7). The total radioactivity in fractions 3-55 of Figs. 6, A- pronase digestion were fractionated by gel filtration before C, and 7, A-C, is given for cell surface and cell pellet, respectively. and after treatment with endo-p-n-acet,ylglucosaminidase H. The per cent of radioactivity recovered in complex and high mannose For both cell surface (Fig. 6) and cell pellet (Fig. 7), the elution oligosaccharides was determined from Figs. 6, D-F, and 7, D-F, after of the larger glycopeptides (k, =.3) was not affected by the treatment with endo-/j-n-acetylglucosarninidase H. endoglycosidase, indicating that these mannose-labeled gly- Radioactivity ~- copeptides contained N-linked complex oligosaccharides. In Cell surface Cell pellet contrast, the smaller glycopeptides (ICab =.4) yielded smaller Deoxynoji- ~- rimycin Total fragments (Kav =.6) after treatment with endo-p-n-acetyl- C$I;- High man- Total Corn- High mannose plex nose glucosaminidase H, thereby demonstrating that they contain mm vm X cpm X N-linked high mannose oligosaccharides. As we have shown 1- a 1 previously (19), after 24 h of labeling, the cell surface glyco- 21 76 24 15 22 78 peptides of these cells were greatly enriched in N-linked 461 18 54 12 15 85 labeled complex oligosaccharides, particularly in confluent 5 84 14 16 13 5 95 cells, whereas the cell pellet glycopeptides contained mostly IO 12 21 79 83 2 98 I

1416 Inhibition N-linked Complex of Oligosaccharide Synthesis 4 TABLE I11 Effect of a-mannosidase on high mannose oligosaccharides High mannose oligosaccharides obtained after endo-b-n-acetylglucosaminidase H treatment (from Figs. 6, D-F, and 7, D-F) were treated with a-rnannosidase and the proportion of mannose released by a-mannosidase was determined using concanavalin A-Sepharose as described under Experimental Procedures. Labeled mannose released Deoxynojirimycin _ ~ Cell surface Cell pellet f71 M 84 85 1 4 63 5 32 When high mannose oligosaccharides obtained after endo- P-N-acetylglucosaminidase H digestion were treated with a- mannosidase, a large proportion (85%) of the radioactivity was released from oligosaccharides derived from control cells (Table 111). The proportion of radioactivity released by a-mannosidase from oligosaccharides derived from deoxynojirimycin-treated cells was much smaller, about 32 and 4% for cell surface and cell pellet oligosaccharides, respectively. This decrease suggests, that in the presence of deoxynojirimycin, the high mannose oligosaccharides contain glucose residues which render some of the mannose residues inaccessible to tr-mannosidase. Assuming that all the mannose residues of these oligosaccharides are uniformly labeled and that the oligosaccharides are derived from a Glc:IMan&lcNAc2 precursor with the structure proposed by Li et al. (23), then a maximum of 89 and 56% of the radioactivity would be released by a-mannosidase from the glucose-free and glucose-containing oligosaccharides, respectively. DISCUSSION We have partially purified a glucosidase from S. cereuisiae which removes glucose residues from both Glc2 and Glcl oligosaccharides. The evidence available so far indicates that the same enzyme is probably acting on both substrates. The activities with Glce and Glcl exhibit the same elution profile during ion exchange chromatography, the same kinetics of inactivation at 37 C (data not shown), the same response to ph changes, and a similar inhibition by glucose, disaccharides, and deoxynojirimycin. The Glcs/GlcI oligosaccharide glucosidase is distinct from the Glc,, oligosaccharide glucosidase which we have described previously (14), and both oligosaccharide glucosidases can be separated from a- and,!?-glucosidases which utilize p-nitrophenylglucopyranosides as substrates. These studies indicate that there two are glucosidases involved in processing of oligosaccharides in S. cereuisiae as was previously shown in liver (8-1). The two oligosaccharide glucosidases respond differently to removal of mannose residues from their substrates; whereas Glc:( oligosaccharide glucosidase is slightly stimulated by removal of 3-4 mannose residues from the pentamannosyl branch of the oligosaccharide, Glce/Glcl oligosaccharide glucosidase is completely inhibited by removal of mannose residues. The behavior of the yeast Glc.j oligosaccharide glucosidase is different in this respect from that of the corresponding liver enzyme (24). Although in liver the Glc:] oligosaccharide glucosidase was not as sensitive to removal of mannose residues as the Glce/Glc, oligosaccharide glucosidase, an increasing inhibition occurred with the removal of an increasing number of mannose residues. The yeast GlcJGlcI oligosaccharide glucosidase is as sensitive as the liver enzyme to alterations of the pentamannosyl branch of the oligosaccharide substrates (1, 24). The effect of various disaccharides on the oligosaccharide glucosidases was studied using enzyme preparations obtained after gel filtration because at this stage of purification the oligosaccharide glucosidases did not act on either a- or P-pnitrophenyl-D-glucopyranosides, and did not degrade any of the disaccharides. Under these conditions, kojibiose was the only disaccharide inhibitor of Glc:, oligosaccharide glucosidase, whereas both nigerose and maltose inhibited the Glcs/Glcl oligosaccharide glucosidase. None of the,!?-linked glucose disaccharides had an effect. These observations are similar to those previously reported for the rat liver oligosaccharide glucosidases by Ugalde et al. (11) and support the idea that all three glucose residues in the oligosaccharide have the a- configuration (9, ll, 12). The inhibition of the liver enzyme was greater with nigerose than with maltose, and was greater at lower concentrations of both disaccharides than that observed with the yeast enzyme. Deoxynojirimycin was found to be the most potent inhibitor of both yeast and mammalian processing glucosidases. The other glucosidase inhibitors tested, BAY e469 and BAY g5421 (25), had no effect on the yeast enzymes in uitro. Deoxynojirimycin also inhibited the formation of N-linked complex oligosaccharides in intact cells. The results of a- mannosidase treatment of the high mannose oligosaccharides formed in its presence suggest that deoxynojirimycin probably acts by interfering with processing of the oligosaccharides after transfer to protein. It can therefore be used to alter the structure of carbohydrate groups at the cell surface and, like swainsonine which inhibits the a-mannosidases involved in processing (26), it should be useful in modifying the oligosaccharides of various glycoproteins to study the biological function of complex chains. Acknowledgment-We thank Birgitte Bugge for excellent technical assistance. REFERENCES 1. Parodi, A. J., and Leloir, L. F. (1979) Biochim. Biophys. Acta 559, 1-37 2. Hubbard, S. C., and Ivatt, R. J. (1981) Annu. Reu. Biochem. 5, 555-583 3. Parodi, A. J. (1979) J. Biol. Chem. 254, 151-16 4. Lehle, L. (198) Eur. J. Biochem. 19, 589-61 5. Trimble, R. B., Maley, F., and Tarentino, A. L. (198) J. Bid. Chem. 255, 1232-1238 6. Trimble, R. B., Byrd, J. C., and Maley, F. (198) J. Bid. Chem. 255, 11892-11895 7. Sharma, C. B., Lehle, L., and Tanner, W. (1981) Eur. J. Biochmz...~ ~ 116, 11-18 8. Ugalde. R. A,. Staneloni. R. J.. and Leloir, L. F. (1979) BiorhPnl -..-... y, Biophys. Res. Commun. 91, 1174-1181 9. Grinna, L. S., and Robbins, P. W. (1979) J. Bid. Chem. 254, 8814-8818 1. Michael, d. M., and Kornfeld, S. (198) Arch. Biochem. BicJphys. 199, 249-258 11. Ugalde, K. A,, Staneloni, K. J., and Leloir, L. F. (198) Eur. J. Biochem. 113,97-13 12. Spiro, R. G.. Spiro, M. J., and Bhoyroo, V. D. (1979) J. Bid. Chem. 254, 7659-7667 13. Elting, J. J., Chen, W. W., and Lennarz, W..J. (198) J. Bid. Chem. 255, 2325-2331 14. Kilker, K. D., Jr., Saunier, B., Tkacz, J. S., and Herscovics. A. (1981) J. Biol. Chern. 256, 5299-533 15. Inouye, S., Tsuruoka, T., Ito, T., and Niida, T. (1968) Tetrahedron 24, 2125-2144 16. Niwa, T., Inouye, S.. Tsuruoka, T., Koaze, Y., and Niida, T. (197) Agric. Biol. Chem. 34, 966-968 17. Frornrner, W., Junge, B., Muller, L., Schmidt, D., and Truscheit, E. (1979) J. Med. Plant Res. 35, 195-217 18. Schmidt, D. D., Fromrner, W., Muller, L., and Truscheit, E. ( I 979) Naturu,issen.schaften 66, 584 A. Herscovics and B. Saunier, unpublished observations.

Inhibition of N-linked Complex Oligosaccharide Synthesis 14161 19. Sasak, W., Herscovics, A., and Quaroni, A. (1982) Biochem. J. 23. Li, E., Tabas, I., and Kornfeld, S. (1978) J. Bioi. Chem. 253, 7762-21, 359-366 777 2. Herscovics, A., Bugge, B., and,jeanloz, R. W. (1977) J. Bioi. 24. Grinna, L. S., and Robhins, P. W. (198) J. Bioi. Chem. 255, Chem. 252,2271-2277 2255-2258 21. Dunphy, P. J., Kerr, J. D., Pennock, J. F., Whittle, K. J., and 25. Schmidt, D. I)., Frommer, W., Junge, B., Muller, L., Wingender, Feeney, d. (1967) Biochim. Biophys. Acta 136, 136-147 W., and Truscheit, E:. (1977) Natunti.ssenseh~ffen 64, 535-5363 22. Hubbard, S. C., and Hobhins, P. W. (198) J. Bioi. Chem. 255, 26. Elhein, A. I)., Solf, H., Dorling, 1'. H., and Vosheck, K. (1981) 11782-11793 Proc. Nntl. Acad. Sei. U. S. A. 78, 7393-7397