Effects of the Imino Sugar N-Butyldeoxynojirimycin on the N

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268,No. 1, h u e of January 5,~ p .570-576,1993 , rsnted m U.S.A.

Effects of the Imino Sugar N-Butyldeoxynojirimycin on the N-Glycosylation of Recombinant gp120* (Received for publication, August 24, 1992)

Gunilla B. Karlssont, Terry D. Butters#, Raymond A. Dwek, and Frances M. Platt#f From the Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom and the CSearle Research Group, Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom

The imino sugar N-butyldeoxynojirimycin (NBDNJ) exhibits anti-HIV activity in vitro and inhibits the purified glycoprocessing enzyme al,2-glucosidase I. It has been speculated that the anti-viral activityof this compound may result from inhibition of HIV envelope glycoprotein processing. However, structural evidence that glucosidase inhibition takes place in intact cells at the anti-viral concentration (0.5 mM) is lacking. In this study, N-linked glycosylation of recombinant gp120 expressed in Chinese hamster ovary cells cultured in the presence or absence of NB-DNJ has been characterized. Immunoprecipitation, in conjunction with endoglycosidase H (endo H) digestion and SDSpolyacrylamide gel electrophoresis analysis, revealed that the glycosylation of gp120 wasprofoundly altered in the presence of NB-DNJ. The majority of the gp120 oligosaccharides from untreated cells wereresistant to endo H. However, nearly complete endo H sensitivity was observed following treatment with 0.5 m M NBDNJ indicating that gp120 expressed in treated cells carries immature, high mannose type oligosaccharides. In addition, using metabolic labeling with [3H]mannose, gel filtration chromatography, and digestion with highly purified glucosidases I and 11, we provide the first definitive evidence that glucosidase I inhibition occurs at the anti-viral concentration of NB-DNJ. These data indicate that glucosidase inhibition is a candidate mechanism for the anti-viral activityof this compound.

(1): glucosidase I, whichremoves the terminal al,2-linked glucose residue, and glucosidase I1 hydrolyzing the remaining two a1,3-linked glucoseresidues. Further trimming of the oligosaccharides by ER- andGolgi-located mannosidases permits subsequent processing to complex and hybrid type structures through the action of Golgi resident glycosyl transferases (2, 3). Several compounds that inhibit purified glucosidases have been identified, including deoxynojirimycin,castanospermine, and their derivatives (4-6). It would be predicted that in the presence of these inhibitors complex type oligosaccharide synthesis would be blocked. However, it has been shown in a number of systems that when cells are treated with these compounds some complex type oligosaccharideformation still occurs (7-11). There are several possible reasons why complete inhibition of complex oligosaccharide synthesis is not achieved in cellular systems. First, a high enough inhibitor concentration may not be achieved within the ER. Second, the presence of endomannosidase activity would provide a bypass mechanism to circumvent glucosidase inhibition (1215). Finally, the transfer of non-glucosylated oligosaccharides to proteins hasbeen reported in F9 teratocarcinoma cells (16). It has also been documented that the effects of glucosidase inhibition on cellular glycoproteins are selective (17, 18). Some glycoproteins require correct oligosaccharideprocessing for secretion or cell surface expression (7, 17-20), while for others complete processing of their oligosaccharides is less critical (21). When a range of sugar analogues were screened for antiHIV activity in uitro (22, 23) NB-DNJ was found to be a potent inhibitor of infection and exhibited minimal cytotoxThe biosynthesis of N-linked oligosaccharides involves the icity. This compound inhibited purified a-glucosidase I with cotranslationaltransfer of a GlcsMan9GlcNAc2 precursor a K , of 0.22 &M (24). The ability of anti-viral concentrations from a dolichol carrier ontothe asparagine residue of an Asn- of NB-DNJ to inhibit glucosidase I in cellular systems has to X-Ser/Thr glycosylation sequon of the protein. Terminal date only been inferred from decreased mobility of treated glucose residues are rapidly cleaved by ER-located’ a-gluco- glycoproteins analyzed by SDS-PAGE (9, 25, 26). Definitive sidases of which two distinct activities have been identified structural evidence as towhether treatment with NB-DNJ at this concentration (0.5 mM) results in glucosidase inhibition * The Glycobiology Institute is supported by the Monsanto/Searle of gp120 (or any other glycoprotein) in a cellular system is Co. The costs of publication of this article were defrayed in part by still lacking. the payment of page charges. This article must therefore be hereby The envelope glycoproteins of the HIV virus are heavily N marked “advertisement” in accordance with 18 U.S.C. Section 1734 glycosylated. HIV-1 gp120 has 20-25 potential sites for N solely to indicate this fact. linked glycosylation with the carbohydrate contributing 50% $ Supported by The Swedish National Board for Technical Development (NUTEK) and a GlycobiologyInstitute Research schoIarship. of its apparent molecular weight (27). The positions of the glycosylation sites within the primary amino acid sequence of 11 To whom correspondence should be addressed. ‘The abbreviations used are: ER, endoplasmic reticulum; HIV, gp120 are relatively consistent between different isolates of human immunodeficiency virus; NB-DNJ, N-butyldeoxynojirimycin; HIV-1 (28-30). At least 13 of the glycosylation sitesare PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster conserved, and the remaining sites usually are not located ovary; FCS, fetal calf serum; mAb, monoclonal antibody; TLCK, N”p-tosyl-L-lysine chloromethyl ketone hydrochloride; TPCK, N-tosyl- more than approximately 10 residues from the sites in the L-phenylalanine chloromethyl ketone; PBS, phosphate-buffered sa- reference strain, HIV-lIIIB (30). However, in the absence of crystallographic datathe relative spacial positions of the line; endo, endoglycosidase;g u , glucose unit(s).

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Effects of N-Butyldeoxynojirimycin Recombinant on

gp120

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of NB-DNJ for 1 h before the addition of 100 pCi/ml Tran3%-label for 4 h. The cells were harvested and the supernatantsrecovered and concentrated 10-fold using a 30-kDa cut-off membrane (Amicon, Danvers, MA). Cell pellets were washed twice with PBS, lysed in 100 pl of 2% Triton X-100 in PBS containing 50 pg/ml TLCK, 50 pg/ml TPCK, and 200 pg/ml phenylmethylsulfonyl fluoride (lysing buffer), and incubated on ice for 30 min. For pulse-chase analysis the cells were pulsed for 10 min with 300 pCi/ml Tran3'S-label and chased by the addition of an equal volume of medium supplemented with 10% FCS, 10 mM L-methionine, and 10 mM L-cysteine for defined time periods. NB-DNJ was maintained in the chase medium of treated cultures. The cells were harvested, supernatants retained, concentrated, and thecells lysed as described above. Radiolabeled cell lysates and concentrated supernatants were stored at -20 "C prior to immunoprecipitation. Metabolic Labeling with rH]Mannose and f4C]Mannose-CH0 cells (80% confluent) were harvested mechanically, washed three times with PBS, and resuspended in RPMI 1640 medium (reduced glucose) supplemented with 1%dialyzed FCS. Cultures (10' cells/ml) were preincubated for 1 h in the presence or absence of NB-DNJ before the addition of 1500 pCi/ml [3H]mannoseor 50 pCi/ml ["C] mannose. Cultures were labeled for 4 h, chased for 4 h through the addition of an equal volume of Dulbecco's modified Eagle's culture medium, and supernatants retained and concentrated as described above. Immunoprecipitation Protocol-For analysis by SDS-PAGE, 35SEXPERIMENTALPROCEDURES labeled cell lysates were spun for 15 min (13,000 rpm) andthe Cell Culture-CHO cells, transfected with pEEGHCMVgplZOGS, supernatants retained. Immunoprecipitations were performed as desecreting recombinant HIV-lIIIBgp120, were obtained from Dr. P. scribed (41) except the antibody was not cross-linked to the solid Stevens (MRC AIDS Directed Programme Reagent Project). Cells phase. Briefly, 35S-or 14C-labeledculture supernatants or 35S-labeled were culturedin Glasgow MEM culture medium (Gibco Ltd., lysates were incubated with mAb ABT 1001 at 0.5 pg/lOO p1 of lysate Uxbridge, U. K.) supplemented with 10%fetal calf serum (FCS, or supernatant for 30 min at room temperature followedby the Techgen), 50 units/ml penicillin, and 50 pg/ml streptomycin (Gibco), addition of sheep anti-mouse IgG1-coated magnetic beads (1.2 X lo7 and maintained at 37 "C with 5% COz. High expression of gp120 was beads/sample) for 1 h at 4 "C. The beads were washed three times achieved by maintaining the cultures in 200 p~ methionine sulfoxi- with 2% Triton X-100 in PBS and three times with PBS. gp120 was mine (37). For radiolabeling experiments methionine- and cysteine- eluted from the beads by heating (95 "C, 5 min) in 100 pl of SDS free RPMI 1640 medium andRPMI 1640medium with reduced sample buffer (6% 2-mercaptoethanol) prior to SDS-PAGE analysis. glucose (100 mg/liter) were obtained from ICN Flow Laboratories, Samples for digestion with endo H were eluted from the solid phase High Wycombe, Bucks, U. K. by heating (95 "C, 5 min)with 50 p1 of 100 mM citrate buffer, pH 5.5, Enzymes and Antibodies-Endo H was obtained from Boehringer containing 1%SDS, 5% 2-mercaptoethanol, and 2 mM NaN3. Each Mannheim Ltd. (Lewes, Sussex, U. K.). The glycoprotein processing sample was divided into two equal aliquots, and 25 pl of distilled HzO enzyme al,2-glucosidase I was purified from porcine liver microsomes was added to give a final volume of 50 pl. To one half of each sample by affinity chromatography. Briefly, microsomes were solubilized in 2 p1 of endo H (1 unit/ml) was added while the other was left 0.8% Lubrol PX and subjected to chromatography using N-(5-car- untreated. Digestion was performed at 37 "C for 18 h and terminated boxypentyl) deoxynojirimycin coupled to agarose (38). This prepara- by the addition of 50 pl of SDS reducing sample buffer with heating tion hydrolyzed a ['4C]glucose-labeled GlcaMansGlcNAczsubstrate (95 "C, 5 min). For chromatographic analysis, [3H]mannose-labeled with a 5.9%min" release of labeled glucose. No hydrolysis was gp120 was isolated from labeled cell lysates or concentrated culture detected using similarly labeled GlcZor GlclManeGlcNAczsubstrates. medium as described above, but five times more mAb and solid phase cyl,3-GlucosidaseI1 was purified from rat liver microsomes using were used. To release the endo H-sensitive radiolabeled oligosacchaanion exchange and gel filtration chromatography (39). The prepa- rides gp120 was eluted from the magnetic beads in 100 pl of 100 mM ration hydrolyzed ["C]glucose-labeled GlcPMangGlcNAcz with a 5.7% citrate buffer, pH 5.5, containing 1%SDS, 5% 2-mercaptoethanol, min" release of labeled glucose. No a-glucosidase I or a-mannosidase and 2 mM NaN3 by heating (95 "C, 5 min). The samples were diluted activity could be detected in this preparation. Aspergillus phoenicis with 100 pl of distilled HzO, 15 milliunits of endo H was added, and al,2-mannosidase was isolated using similar procedures to those samples were incubated at 37 "C for 18 h. The reaction was stopped previously described (40). The enzyme did not hydrolyze a1,3- or by heating at 95 "C for 5 min. Released oligosaccharideswere desalted al,6-mannose terminating oligosaccharides. Theanti-HIV gp120 by passing the reaction mixture over a column of 100 pl each of monoclonal antibody (mAb) (ABT 1001, murine IgG1) waspurchased Chelex 100, Dowex AG50 X-12 (H+ form), Dowex AG3 X-4A (OHfrom American Biotechnologies, Inc. (Cambridge, MA). form). The eluate was pooled with a wash of five bed volumes of Reagents-The synthesis of NB-DNJ (SC-48334) has been described elsewhere (22). The following chemicals were purchased from distilled HzO, 0.5-pm-filtered, evaporated to dryness, and taken upin Sigma: Triton X-100, Ne-p-tosyl-L-lysinechloromethyl ketone hydro- 200 pl of distilled HzO for analysis by gel filtration chromatography. Gel Filtration Chromatography-Endo H-released oligosaccharides chloride (TLCK), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), phenylmethylsulfonyl fluoride, and 2-mercaptoethanol. from untreated or NB-DNJ-treated gp120were subjected to high [36S]Methionine/cysteine(Tran36S-label, >1100 Ci/mmol) was ob- resolution gel filtration chromatography using two Bio-Gel P-4 (1.5 tained from ICN Flow, and D-[2-3H]mannose(13.7 Ci/mmol) and D- X 100 cm) columns in series. The columns were maintained at 55 "C [U-"Clmannose (270 mCi/mmol) from Amersham International Plc, and eluted with distilled water (1 ml/fraction), and fractions were Amersham, U. K. Magnetic beads (sheep anti-mouse IgG1-coated monitored for radioactivity using liquid scintillation counting. EluDynabeads "450) were purchased from Dynal Ltd., Wirral, Mersey- tion positions in glucose units (gu)were determined by simultaneous side, U. K. Chromatography media were obtained from the following separation of a ladder of partially hydrolyzed dextran and detected sources: Chelex 100, Dowex AG 5OX-12 (H+ form), Dowex AG3 X- on the basis of refractive index. 4A (OH- form), and Bio-Gel P4 (-400 mesh) from Bio-Rad LaboraRESULTS tories Ltd., Watford, Hertfordshire, U. K., and QAE-Sephadex A25 from Pharmacia Ltd., Milton Keynes, Buckinghamshire, U. K. Effects of NB-DNJ on the Electrophoretic Mobility of 35SMetabolicLabeling with p6S]Methionine-CH0 cells were harvested mechanically, washed three times with phosphate-buffered Labeled gpl20"The effect of differing concentrations of NBsaline, 0.1 M, pH 7.2 (PBS), and resuspended in methionine- and DNJ o n the electrophoretic mobility of recombinant a 1 2 0 cysteine-free RPMI 1640 medium supplemented with 1%dialyzed isolated from cell lysates or from culture medium is shown in FCS. Cells (lo7cells/ml) were preincubated in the presence or absence Fig. la. Following a 4-h labeling period (no chase) of untreated

glycosylation sites cannotbe accurately defined. Studies have shown that a diverse range of high mannose, hybrid, bi-, tri-, and tetra-antennary structures are present bothon recombinant CHO expressed and virally derived gp120 and that the proportions of the different structures are similar between the two systems (31-34). Analysis of gp120 mutants suggests that N-glycosylation of either gp120 or gp41 is necessary for a post-CD4 binding event, such as the fusion of the viral and cellular membranes (35, 36). We wished to determine whether NB-DNJ could inhibit glycoprotein processing in intact cells at concentrations equivalent to those which inhibit HIV replication in uitro. Using metabolic labeling with [3H]mannose, endo H release of labeled oligosaccharides, and gel filtration chromatography we have demonstrated that treatment with anti-viral concentrations of NB-DNJ profoundly alters theprofile of oligosaccharide structures present onsecreted recombinant gp120. Using both highly purified al,2-glucosidase I and d,3-glucosidase I1 we provide definitive structural evidence that theterminal sequence Glca1,2Glcal,3Glca!l,3Manis present on gp120 oligosaccharides following treatment with 0.5 mM NB-DNJ.

572

gp120

Effects of N-Butyldeoxynojirimycin Recombinant on a) cells kD

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FIG.1. Effects of NB-DNJtreatment on the electrophoretic mobility of recombinant -120. Cultures were labeled with Tran35S-labeland immunoprecipitated either from cell lysates ( a )or from culture supernatants ( b ) . Cultures were left untreated (lane 1 ), or differing concentrations of NB-DNJ (lanes 2-12) were added to cultures 1 h prior to labeling and were maintained throughout the 4h pulse. Samples were immunoprecipitated, separated on 6.5% SDSPAGE, and visualized by autoradiography.

samples, a 100-kDa species was immunoprecipitated from the cellular fraction (Fig. la, lane 1 ) while a species migrating with the expected apparent molecular mass, 120 kDa, was immunoprecipitated from the culture medium (Fig. lb, lane 1 ). It was apparent that the the mobility of cellular gp120 was profoundly affected by treatment with NB-DNJ andwas dosedependent (Fig. la, lanes 1-12). The presence of glucosylated, untrimmed high mannose oligosaccharides on gp120 would be expected to increase the apparentmolecular weight of gp120. The decreased mobility on SDS-PAGE was therefore consistent with the maintenance of immature structures due to treatment with the a-glucosidase inhibitor NB-DNJ. It was also apparent that the effect was near completion at the 0.5 mM dose, a t which NB-DNJ exhibits anti-HIV activity in uitro (22, 23). The maximal effect was observed at 1.0 mM NB-DNJ resulting in a homogeneous gp120 species migrating with an apparent molecular mass of 125 kDa. Increasing concentrations of the imino sugar (2-5 mM) did not result in any further increase in apparent molecular weight. Concentrations of NB-DNJ below 0.02 mM did not significantly affect the mobility of gp120. The change in electrophoretic mobility obtained with NBDNJ-treated gp120 isolated from the culture medium was less profound. The electrophoretic mobility of gp120 a t doses of NB-DNJ less than 0.1 mM was similar to untreated gp120 (Fig. lb, lanes 2-7). However, at NB-DNJ concentrations of approximately 0.1 mM and above (Fig. lb, lanes 7-12) there was a small increase in the apparent molecular mass of the secreted form to 125 kDa. Pulse-chaseexperiments were performed to determine whether the 100-kDa species observed in the cellular fraction

(Fig. la, lane a) represented an immature form of gp120 which would, with time, acquire a molecular mass of 120 kDa and be secreted, or whether it represented a resident intracellular pool of gp120 molecules, possibly differentially glycosylated relative to the secreted form. Fig. 2 shows gp120 immunoprecipitated from the cells and medium in the presence or absence of endo H. A heterogeneous 100-110-kDa form ofgp120was initially detected in untreated cells (Fig.2a, lane 1). This species was totally sensitive to endo H (Fig. 2a, lane 9 ) , resulting in a 60-kDa species that is consistent with the predicted molecular weight of the gp120 polypeptide alone. After a 1-h chase the heterogeneous form of gp120 resolvedinto two species, a major form migrating with increased mobility and a less abundant form of decreased mobility (Fig. 2a, lane 3). Digestion with endo H revealed that the majority of gp120 at this chase point was totally sensitive to hydrolysis with only a minor population of molecules being partially resistant to the action of endo H resulting in a 100-kDa species (Fig. 2a, lane 11 ). After 4 h of chase no gp120 molecules carrying endo H-resistant structures were detected in the cells. Following treatment with 0.5 mM NB-DNJ an approximately 130-kDa species of gp120 was precipitated from the cells (Fig. 2a, lane 5), which was totally sensitive to endo H (lane 13). When the label was chased there appeared to be a minor reduction in molecular mass (Fig.2a, lanes 6-8) to yield a 125-kDa species, but there was no acquisition of endo H resistance (lanes14-16). As observed with untreated gp120 most of the label was chased out of the cell after 4 h. When gp120 was immunoprecipitated from the medium it a) cells kD

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FIG.2. Pulse-chase analysis and endo H sensitivity of gp120 generated in the presence or absence of 0.5 mM NBDNJ. Cultures, untreated (lanes 1-4 and 9-12) or treated with 0.5 mM NB-DNJ (lanes 5-8 and 13-16), were labeled for 10 min with Tran"S-labe1 and chased for the time periods indicated. Immunoprecipitates from the cells ( a ) or from the medium ( b ) were divided in two halves and incubated for 18 h at 37 "C in the presence (lanes 916) or absence (lanes 1-8) of 2 milliunits of endo H. Samples were analyzed by 6.5%SDS-PAGE and visualized by autoradiography.

Effects of N-Butyldeoxynojirimycin on

Recombinant gp120

573

was apparent that a chase time of at least 1 h was required oligosaccharide formation in the presence of 0.5 mM NB-DNJ for efficient secretion of gp120, independent of treatment with did not result from the transfer of non-glucosylated oligosacNB-DNJ. In untreated cultures a species of 120 kDa was charides onto theprotein or of endomannosidase activity but secreted (Fig. Zb, lanes 3 and 4 ) . This form exhibited partial was a consequence of the dose of NB-DNJ used, leading to resistance to endo H (Fig. 2b, lanes 11 and 12),resulting in a partial glucosidase inhibition at concentrations below 5 mM. r4CJMannose Labeling of Untreated and NB-DNJ-treated 100-kDa form similar to theminor population of gp120 exhibiting partialendo H resistance detected in the cellular fraction gpl20-To demonstrate that the oligosaccharides from NB(Fig. 2a, lanes 10-11). The reduction in apparent molecular DNJ-treated secreted gp120couldbe nearly quantitatively mass of approximately 20 kDa suggested that approximately removed by treatment with endo H, cultures were labeledwith 10 out of 24 N-glycosylation sites carried high mannose type ['4C]mannose, and immunoprecipitated gp120 was subjected structures, which is in agreement with previous reports (31). to SDS-PAGE (Fig. 4). A small increase in the molecular Following treatment with 0.5 mM NB-DNJ a125-kDa species mass of gp120 was observed following NB-DNJ treatment was precipitated from the medium (Fig. 26, lanes 7 and 8). (Fig. 4, lanes 2 and 3 ) , which is in agreement with with the Digestion with endo H demonstrated that the glycosylation [35S]methionine-labeledgp120 (Fig. 3, lanes 4 and 6). Furof gp120 was profoundly altered by NB-DNJ treatment and thermore, endo H digestion of gp120 precipitated from unresulted in a mixed population of molecules with molecular treated cultures resulted in only partial removal of the manmasses between 60 and 70 kDa (Fig. 2b, lanes 15 and 16). To nose label, indicating that these molecules carry a mixture of confirm that the absence of a single 60-kDa deglycosylated high mannose and complex type oligosaccharides (Fig. 4, lane form of gp120 wasnot a result of incomplete endo H digestion 4 ) . When gp120 was generated in the presence of 0.5 or 5.0 a time course experiment was performed. It was confirmed mM NB-DNJ the label was almost entirely removed with endo that the conditions used for endo H digestion were optimal H (Fig. 4, lanes 5 and 6). This suggested that theoligosacchawith complete hydrolysis occurring after as littleas 15 min of rides from treated gp120 had been retained in an immature, incubation with the enzyme (data notshown). It was therefore high mannose form. In addition, these data show that the concluded that some NB-DNJ-treated molecules carried com- preparation of gp120 was free from contaminating glycoproplex type oligosaccharides resulting in the observed hetero- teins, confirming that this method of metabolic labeling and oligosaccharide release was suitable for performing detailed geneity. To investigate the glycosylation status of secreted gp120 chromatographic analysis of gp120 N-linked oligosaccharides. Analysis of Oligosaccharides by Gel Filtration Chromutogragenerated at varying concentrations of NB-DNJ, cultures were35S-labeled for 4 h followedby a 4-h chase. A dose- phy-NB-DNJ-treated or untreated recombinant CHO cells dependent increase in endo H sensitivity was observed (Fig. were labeled with [3H]mannose, chased for 4 h, and gp120 3). Treatment with 0.5 mM NB-DNJ resulted in a heteroge- isolated from the medium. Oligosaccharides were released neous population of gp120 molecules,comprising four distinct with endo H and analyzed by gel filtration chromatography. species, following digestion with endo H (Fig. 3, lane 10) as The profile obtained from untreated gp120 contained a mixwas also observed in the pulse-chase analysis (Fig. 2b, lanes ture of at least five different oligosaccharide structures, prob15 and 16). Thelowest molecular weight species migrated as ably representing the oligomannose series, eluting between a 60-kDa protein. The difference in molecular mass between 6.1 and 9.6 gu (Fig. 5a,peaks A-E). When the oligosaccharides each of the four detected species was 2-3 kDa, indicative of from gp120 generated in the presence of either 0.5 or 5.0 mM four populations of gp120 moleculeshaving zero, one, two, or NB-DNJ were analyzed (Fig. 5, b and c, peaks F, G, and H ) three sitescarrying complex (endo H resistant)type oligosac- three peaks of 11.0, 12.0, and 12.6 gu were identified. Followcharides. The relative proportions of these populations shifted ing treatment with 0.5 mM NB-DNJ a minor (