Nonselective Utilization of the Endomannosidase Pathway for

Val. 267, No. 16,Ieaue of June 5,pp. 11573-11578,1992 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemietry and Molecular Biology, Inc.

Nonselective Utilization of the Endomannosidase Pathway for Processing Glycoproteins by Human Hepatoma (HepGZ) Cells* (Received for publication, January 21, 1992)

Catherine RabouilleS and Robert G. SpiroO From the DeDartment of Biological Chemistry and Medicine, Harvard Medical School, and the Elliot P. Joslin Research Laboratory, Boston, MakchGetts 02225

Endo-a-D-mannosidase, a Golgi-situated processing enzyme, provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins (Moore, S. E. H., and Spiro, R. G . (1990) J. Biol. Chern. 265,13104-13112).The present report demonstrates thatat least five distinct glycoproteins secreted by HepG2 cells (al-antitrypsin, transferrin, al-acid glycoprotein, al-antichymotrypsin, and a-fetoprotein) as well as cell surface components can effectively utilize this alternate processing route. During a castanospermine (CST)-imposed glucosidase blockade, these glycoproteins apparently were produced with theirusual complement of complex carbohydrate units, andupon addition of the mannosidase I inhibitor, 1-deoxymannojirimycin (DMJ), to prevent furtherprocessing of deglucosylated N-linked oligosaccharides, Mane-sGlcNAc, but not MansGlcNAc, were identified; the ManeGlcNAc component occurred as the characteristicisomer generated by endomannosidase cleavage. Although the endomannosidase-mediated deglucosylation pathway appeared tobe nonseon the secretion lective, a differential inhibitory effect of the variousglycoproteins was noted in thepresence of CST which was directly related to the number of their N-linked oligosaccharides, ranging fromminimal in a-fetoprotein to substantial (-65%) in al-acid glycoprotein. Addition of DMJ to CST-incubated cells did not further decrease secretion of the glycoproteins, although processing was now arrested at the polymannose stage, and a portion of the oligosaccharides were still in the glucosylated form. These latter findings indicate that complex carbohydrate units are not required for secretion of these glycoproteins and that any effect which glucose residues exert on their intracellular transitwould be related tomovement from the endoplasmic reticulum to theGolgi compartment.

Although the glucose residues of the lipid-linked oligosaccharide (GlcsMan9GlcNAcp)involved in the N-glycosylation reaction play an importantspecifying role, it isequally essential that they must be removed after transfer to protein if processing to complex carbohydrate units is to be accomplished (1, 2). Recent studies from this laboratory have indi-

* This work was supported by Grant DK17477 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “oduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Present address: Dept. of CellBiology, Utrecht University, School of Medicine, Utrecht, Netherlands. $ T Owhom correspondence and reprint requests should be addressed Elliott P. Joslin Research Laboratory, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215.

cated that excision of this sugar, which was previously believed to occur only through the sequential action of the ER1situated glucosidase I and 11, can also be brought about through an alternate deglucosylation mechanism in which an endo-a-mannosidase cleaves the linkage between the glucosesubstituted mannose and the remainder of the polymannose oligosaccharide (3, 4). Indeed, the existence of this enzyme, which is unaffected by glucosidase inhibitors, has provided the basis for understanding the unexpected but frequently observed formation of complex oligosaccharides in the presence of a glucosidase blockade (5-12) or deficiency (13). Studies carried out with HepG2 cells demonstrated that the persistentformation of these complex carbohydrate units (50%of control) in the presence of CST could be accounted for by the appearance of the characteristic endomannosidasegenerated glucosylated mannose saccharides (Glcl-3Man) (14). Since totalglycoproteins, however, wereanalyzed in this investigation, it did not provide insight as to whether the endomannosidase pathway demonstrates a glycoprotein selectivity. In the present study, we have addressed ourselves to this question and for this purpose have continued to focus on HepG2 cells. This cell line is particularly appropriate for such an investigation as it is known to secrete a number of serum proteins with well defined N-linked complex carbohydrate units (15). Indeed, these human hepatoma cells have previously been employed in evaluating the regulation of the intracellular movement and secretion of glycoproteins (6-8, 16). Our studies revealed that all five glycoproteins which we examined circumvent a CST-imposed glucosidase blockade by means of the endomannosidase route and aresecreted with their full complement of complex oligosaccharides although demonstrating in most instances a reduced rate of exit from the cell. Furthermore, we observed that in the presence of a mannosidase I inhibitor, the CST-treated cells secrete these glycoproteins with N-linked carbohydrate units arrested in their processing just beyond the endomannosidase stage. Cell surface glycoproteins were also found to participate in the in vivo action of this enzyme. EXPERIMENTALPROCEDURES

Culture and Radiolabeling of Cells-HepG2 cells (ATCC, Rockville, MD) were grown in RPMI 1640 supplemented with 10% fetal calf serum (Gibco), penicillin (100 units/ml), and streptomycin (100 pg/ ml) at 37 “C in an atmosphere of 95% air, 5% CO,. Radiolabeling was accomplished by incubating cells which had reached 90% confluency on 100-mm dishes (Falcon) with either 0.75 mCi of [35S]methionine ’The abbreviations used are: ER, endoplasmic reticulum; CST, castanospermine; DMJ, 1-deoxymannojirimycin; SDS, sodium dodecy1 sulfate; endo H, endo-P-N-acetylglucosaminidase. All sugars are presumed to be in the D-configuration; reduced sugars are indicated by the designation H, following the symbol of the monosaccharide.

11573

11574

Nonselective Use of Endomannosidase Pathway in

(1140 Ci/mmol) or 1 mCi of [U-"C]glucose (315 pCi/pmol), obtained from Du Pont-New England Nuclear, in2 ml of methionine or glucose-free Dulbecco's modified Eagle's medium containing a 2 mM concentration each of glutamine and pyruvate while being subjected to mild agitation on a rocker platform. When the glycosidase inhibitors CST (a gift from Dr. M. Kang, Merrell Dow Research Institute, Cincinnati, OH) and DMJ (purchased from Genzyme) were used, the cells were preincubated with theseagents for 30 min before the addition of the radiolabeled substrate. In pulse-chase studies, a 15min labeling period with [%]methione (200 pCi) was terminated by a wash with medium containing 1 mM methionine, and this was followed by incubations in this unlabeled medium for various time periods; where indicated, the glycosidase inhibitors were present throughout the pulse-chase incubations. Immunoprecipitation-Rabbit antiseraagainst human albumin and al-acid glycoprotein were obtained from Sigma while those against human al-antitrypsin, al-antichymotrypsin, transferrin, and a-fetoprotein were purchased from Accurate. After the addition of specific antisera (25 pl/ml) to thecell media containing phenylmethylsulfonyl fluoride (1 mM) and aprotinin (10 units/ml), an overnight incubation was carried out at 2 "C. Subsequently, Pansorbin cells (Calbiochem) were added as a 10% suspension (170 pl/ml medium) t o bind the antigen-antibody complex during a 2-h incubation at room temperature. The mixture was centrifuged (1000 X g for 15 min) a t 4 "C, and the recovered pellet was washed three times with 0.05 M Tris/HCl, pH 7.4, buffer containing 0.15 M NaC1, 0.1% Triton X100,0.1% SDS, 5 mM EDTA, as well as phenylmethylsulfonyl fluoride and aprotinin before the protein in the immunoprecipitate was solubilized by heating for 5 min at 100 "C in 50 pl of 0.06 M Tris/HCl, pH 6.8, buffer containing 1%SDS and 2% (v/v) 2-mercaptoethanol. In order to enhance the specificity of the immunoprecipitation procedure, all samples were incubated with rabbit nonimmune serum and Pansorbin, in the same sequence as described above, before the addition of the antisera. When labeling was performed with [35S]methionine,separate aliquots of the media were taken for the immunoprecipitation of the various glycoproteins, while after incubation with [14C]glucose,sequential immunoprecipitations were carried out on the entiresample. Endo H Digestion and Polyacrylamide Gel Electrophoresis-Alisolubilized immunoprecipitates quots of the [36S]methionine-labeled, (5-20 pl) were dried by lyophilization and then incubated for 48 h a t 37 "C in 150 pl of 0.2 M sodium citrate buffer, pH 5.2, with or without 4 units of endo H (Genzyme). Subsequently, the samples were examined by electrophoresis which wasperformed on vertical polyacrylamide slab gels (1.5 mm thick) in SDS according to theprocedure of Laemmli (17); the separating gel which consisted of a 7-12% acrylamide gradient was overlaid by a 3% stacking gel. The components were detected by fluorography and quantitated by two-dimensional scanning with a computing laser densitometer (Model 300A, Molecular Dynamics). The molecular weights of the %-labeled proteins were determined from their electrophoretic migration relative to standard proteins which included rabbit muscle myosin (205,000), Escherichia coli 8-galactosidase (116,000),rabbit muscle phosphorylase b (97,000), bovine serum albumin (66,000), hen ovalbumin (45,000), and bovine erythrocyte carbonic anhydrase (29,000). Preparation of Radiolabeled Glycopeptides and OligosaccharidesThe solubilized immunoprecipitates from the incubations with ["C] glucose were dried by lyophilization and then washed with three 1ml portions of 95% (v/v) ethanol to remove the SDS and 2-mercaptoethanol. Subsequently, digestion with pronase (Calbiochem) was carried out under the conditions previously described (la), and the resulting glycopeptides were absorbed on columns of Dowex 1-X2 (acetate form) overlaid with Dowex 50-X2 (H' form), followed by elution with 2 M pyridine acetate, pH 5.0. Cell surface components were released from the HepG2 cells after incubation with [14C]glucosein a mannersimilar to thatdescribed by Sasak et al. (19). In the procedure employed, the monolayers, after several washes with 0.05 M Tris/HCl, pH 7.9, buffer containing 0.2 M NaCl and 0.02 M MgC12,were incubated for 10 min at room temperature with a solution (2 ml) of 0.1% trypsin (Difco) in the same buffer supplemented with 2 mM EDTA. The released material, which contained cell surface glycopeptides, was separated from detached cells by centrifugation at 4 "C (2000 X g for 15 min) and subsequently, after the addition of CaClz to a 10 mM concentration, digested with pronase at 37 'C for 48 h; for comparison, the total proteins from the cell media of the incubations with [14C]glucosewere digested with the pronase after trichloracetic acid precipitation as previously described (14).

HepG2 Cells

To release polymannose units, the lyophilized glycopeptides (Dowex 50 plus Dowex 1 eluates) from the pronase-digested immunoprecipitates, as well as total cell surface and media proteins, were incubated with 5 milliunits of endo H in 0.2 M sodium citrate buffer, pH 5.5, as previously described (20); after passage of the digests through columns of Dowex 50 and Dowex 1, the neutral oligosaccharides were collected in the effluent and water wash, whereas the endo H-resistant glycopeptides were eluted from the resins with 2 M pyridine acetate, pH 5.0. Before further study, the released oligosaccharides were reduced with NaBH4 (3). Structural and Analytical Procedures-Acetolysis of the reduced oligosaccharides was carried out by the procedure of Varki and Kornfeld (21), and thedesalted products were identified by thin layer chromatography as previously described (3). Quantitation of radiolabeled neutral sugars was carried out after acid hydrolysis (1N HCl, 5 h at 100 "C under nitrogen) followed by passage through coupled columns of Dowex 50 (H+form) and Dowex 1 (acetate form) (22) and separation of the monosaccharides by thin layer chromatography in Solvent System A; radioactivity was determined by scintillation counting after elution of the components from the plate. Chromatographic Procedures-Thin layer chromatographic resolution of monosaccharides through pentasaccharides was carried out on plastic sheets precoated with cellulose (0.1 mm, Merck) for 12 h in pyridine/ethyl acetate/water/acetic acid, 55:3:1 (Solvent System A), while separation of larger oligosaccharideswas achieved on plastic sheets precoated with Silica Gel 60 (0.2 mm thickness, Merck) for 24 hin 1-propanol/acetic acid/water, 3:3:2 (Solvent System B). The chromatography was carried out with a wick of Whatman No. 3MM paper clamped to the top of the thin layer plates. The components were detected by fluorography and quantitatedby scintillation counting after elution with water as previously described (14). For preparative purposes, the eluted oligosaccharides were extracted with peroxide-free ether to remove scintillants and passed through small coupled columns of Dowex 50 (H+) andDowex 1 (acetate). Radiolabeled oligosaccharide standards were prepared in a manner previously reported (4, 14). Radioactivity Measurements-Liquid scintillation counting was carried out with Ultrafluor with a Beckman LS 7500 instrument. Components on electrophoretic gels, and thin layer plates were detected by fluorography a t -70 "C with X-Omatic AR film (Eastman) after treatment with ENHANCE (Du Pont-New England Nuclear) or spraying with a scintillationmixture containing a-methylnaphthalene (23), respectively. RESULTS

Effect of CST and DMJ on the Electrophoretic Migration and Endo H Susceptibility of Several Glycoproteins Secreted by HepG2 Cells-Electrophoretic examination of five distinct glycoproteins secretedby HepG2 cells during incubation with [35S]methioninein the presence of the glucosidase inhibitor, CST (24), revealed that all wereprocessed to an endo Hresistant stage and, moreover, with the exception of al-acid glycoprotein, co-migrated with the proteins produced by cells in the absence of this glucosidase inhibitor(Fig. 1 and Table I); increasing the concentration of CST up to 6 mM did not alter the electrophoretic patterns from that observed at the 1 mM concentration (data not shown). However,when the mannosidase I inhibitor, DMJ (24), was added to the incubations in addition to the CST, the mobility of the glycoproteins was in each case shifted to a lower molecular weight form, and endo H susceptibility became apparent (Fig. 1 and Table I); as anticipated, no change in the migration of albumin, a nonglycosylated protein, occurred under these conditions. These observationsindicated that HepG2 cells can synthesize a number of glycoproteins with apparently a full complement of complex N-linked oligosaccharides during a glucosidase blockade, although in case of the Lul-acid glycoprotein, these carbohydrate units may not have reached completion. In contrast,mannosidase inhibition arrested the processing of each glycoprotein at a stage at which oligosaccharides werestill in the polymannose form; indeed, the N-linked carbohydrate content calculated from the difference in the M,

Nonselective Use of Endomannosidase Pathway in HepG2 Cells of the native and endo H-treated molecule (Table I) corresponded with published analytical values (25-31). Effect of CST and DMJ on the Rate of Secretion of Several Glycoproteins by HepG2 Cells-Quantitation of the glycoproteins in the medium revealed that there was a reduction in CONTROL EndoH

66 -

CST C S T t D M J

T-T "

0

0

~

m

45-

9766-

oTry

.

-

-

Fe' "

.~

Alb

45-

FIG. 1. Effect of glycosidase inhibitors on the electrophoretic migration in polyacrylamide gels of various protein secreted by HepG2 cells. After incubation of HepG2 cells for 4 h with [:%]methionine (750 pCi) in the ahsence (CONTROL)or presence of the indicated glycosidase inhibitors (CST, 1 mM, f DMJ, 2 mM), aliquots of the medium were immunoprecipitatedwith antibodies against specific proteins and submitted with (+) or without (-) prior endo H digestion to polyacrylamide gel electrophoresis in SDS as described under "Experimental Procedures."The components were visualized by fluorography; the designated molecular weight markers were rahhit muscle phosphorylase b (97,000), hovine serum albumin (66,000),and hen ovalhumin (45,000). The proteins shown are n l antitrypsin (aTry), n,-acid glycoprotein (AC), n-fetoprotein (Fet), and albumin ( A h ) . Since the aliquots of the samples ( 2 4 % of the total medium) were notuniform, this variation was taken into account in quantitating the hands by densitometry (seeFig. 2).

11575

secretionduringincubation of the HepC2 cellswith CST which varied, from negligible in the case of n-fetoprotein to substantial (>65%) in nl-antitrypsin and nl-acidglycoprotein be (Fig.2). The selectivity of thisinhibitionappearedto related to the N-linkedoligosaccharide content of the glycoprotein with a greater decrease in secretion being apparent in those with a high carbohydrate content (cf. Fig. 2 and Table I). It can be noted that al-acid glycoprotein ( 2 8 ) and a,antichymotrypsin (30) with 6 and 4 oligosaccharides, respectively, demonstrated the most reduction, while n-fetoprotein (32) with a single carbohydrate unit the least; transferrin (27) and al-antitrypsin (26) which have 2 and 3 oligosaccharides, respectively, occupied an intermediate place. When the incubations with CST were carried out in conjunction with DMJ, no further inhibition in glycoprotein secretion was observed (Fig. 2) although, as already noted (Fig. l ) , the glycoproteins remained sensitive to endo H digestion (Fig. 1). Pulse-chase studies carried outwith nl-antitrypsin (Figs. 3 and 4) andtransferrin(datanotshown) provided similar results as obtained under the steadystate conditions (Figs. 1 and 2). The rate of secretion was reduced in the presence of the glycosidase inhibitors, and the glycoproteins which appeared in the medium during the glucosidase or glucosidasemannosidase blockades were endo H-resistant and sensitive, respectively. Evidence That Variouc Secreted Glycoproteins Are Processed by the Endomannosidace Pathway during Glucosidase Blockade-In order to determineif deglucosylation of individual secreted glycoproteins was brought about by the endomannosidase route in the glucosidase-inhibited cells, an examination of theN-linkedpolymannose oligosaccharides present in several of these proteins, including nl-antitrypsin, transferrin, and a,-acid glycoproteins, was undertaken after incubations of the HepC2 cells with ["C]glucose. As anticipated from the electrophoretic studies (Fig. 1 and Table I), endo H treatment of the glycoproteins released no oligosaccharides from control incubations, while only a single com5% of thetotalsaccharide ponent,representinglessthan radioactivity, which migrated on thin layer chromatography was to the position of Glc3Man7GlcNAc/GlclMan9GlcNAc excised from the proteinsproduced by CST-treated cells (Fig. 5). In contrast, endo H effected a release of the carbohydrate units in the glycoproteins secreted by cells incubated with CST in the presence of DMJ as the latter inhibitor prevented the further processing of the N-linked oligosaccharides after deglucosylation (Fig. 5 and Table 11). The chromatographic

TABLEI Effect of glycosidase inhibitors and endo H treatment on the molpcular weight of several proteins secreted hv H e f l 2 cells Inhibitor ST

Control

+ DM.)

Protein"

n.

M,x IO-"' al-Antitrypsin

53 82 52 72 73 66

53 82 49 52 72 73 66

53 82

N-Linkd cRrhnhvdrRteh

53 82

51

47 70

11 15 44 33

75 Transferrin 49 29 41 nl-Acid glycoprotein 57 48 72 72 a,-Antichymotrypsin 73 73 72 70 4 a-Fetoprotein 66 66 0 66 66 Albumin " After incubation of HepC2 cellswith ["Sjmethionine underthe conditions describedin Fig. 1, the listed proteins were immunoprecipitclted from the medium and submitted to polyacrylamide gel electrophoresis in SDS with (+) or without (-) prior endo H treatment. The N-linked Carbohydrate content of the native molecule (percent of weight) has heen calculated as the difference in t h e M,values of the protein produced under control conditionsand that from CST + DMJ incuhations after endo H digestion. 'The molecular weights were estimated from the electrophoretic migration of the predominant (>90% of total) protein hand relative to standard proteins.

Nonselective Use of Endomannosidase Pathway in

11576

try

AG

achy

Trf

Alb

Fct

FIG. 2. Effect of glycosidase inhibitors on the amount of various proteins secreted by HepG2 cells. After incubation of HepGP cells with [%]methionine under the conditions described in Fig. 1, the indicated proteins from the medium were immunoprecipitated and submitted to polyacrylamide gel electrophoresis in SDS. After fluorography, the predominant bandfrom each gel was quantitated by scanning with a laser densitometer and calculated as total densitometric units for each incubation. The effect of the inhibitors + DMJ onsecretion is presented relativeto thecontrol CST and CST incubations. The abbreviations of the proteins are al-antitrypsin, aTry; nl-acid glycoprotein, A G transferrin, Trf;al-antichymotrypsin, achy; a-fetoprotein, Fet; and albumin, Alb. Chosefmin) E

0 n

30

60 d

120 o

0

240 H

~

420

480

240

~

FIG.4. Quantitation of the effect of glycosidase inhibitors on the time course of al-antitrypsin secretion by HepG2 cells. After pulse-labelingwith["S]methionine, the HepGP cells were chased for various times as described in Fig. 3. The al-antitrypsin immunoprecipitated from an aliquot of the medium was submitted to polyacrylamide gel electrophoresis in SDS and, after fluorography, was quantitated by scanning with a laser densitometer. The course of secretion determined from the densitometric units of each time is plotted for the control (0)incubations, as well as for those carried out in the presence of CST (0)or CST + DMJ (x), asa percentage ~ of the ~value obtained at~ the240-min control chase. TRANSFERRIN CST Contr

CST+DMJ

60

CHASE f m i n )

ANTITRYPSIN

CST

HepG2 Cells

CSTfDMJ

Contr

CST CST+DMJ

-0""

"-"a0*

FIG. 3. Electrophoretic examination of the effect of glycosidase inhibitors on the time course of a,-antitrypsin secretion by HepC2 cells. The cells were incubated for 15 minwith [''SI methionine (200 pCi) and then chased for various times as described under "Experimental Procedures"; glycosidase inhibitors were either absent (CONTROL)or present (CST,1mM, & DMJ, 2 mM) throughout both thepulse and chase incubations. a,-Antitrypsin was immunoprecipitated from an aliquot of the medium and submitted with (+) or without (-) prior endo H digested to polyacrylamide gel electrophoresis in SDS. The components were visualized by fluorography.

pattern (Fig. 5) and the molar distribution (Table 11) of the endo H-susceptible oligosaccharidesfrom al-antitrypsin, transferrin, and Lul-acid glycoprotein were very similar and characteristic of processing by endomannosidase (14). The presence of substantial amounts of ManEGlcNAc, Man7GlcNAc, and Man6GlcNAc,while MangGlcNAcwas essentially absent, was consistent withthe deglucosylation of the triglucosylated oligosaccharides (Glc3Man7-9GlcNAc)by endomannosidase action through the excision of Glc3Man (Table 11). Since adistinctive MansGlcNAc variant (isomer A), in which the terminal mannose of the al,3-linked branch is missing, is formed by the action of endomannosidaseon glucosylated MangGlcNAc (3), characterization of this oligosaccharide isolated from the glycoproteins was undertaken. Chromatographic examination of the fragments provided by acetolysis indicated that the ManEGlcNAc from al-antitrypsin, transferrin, and al-acid glycoprotein was indeed exclusively or predominantly present as the A isomer (Fig. 6); in particular, the formation of ManaGlcNAc distinguished this MansGlcNAc oligosaccharide from the isomer formed in uninhibited cells (isomer B), in which the terminal mannose from the middle branch is missing (14), as well as from the third possible variant (isomer C ) .

c o FIG.5. Thin layer chromatographic examination of the endo H-susceptible oligosaccharides occurring in transferrin and a]-antitrypsin secreted by HepG2 cells in the absence and presence of glycosidase inhibitors. After incubation of the cells with [14C]glucose(1 mCi) for 4 h in the absence (CONTROL) or presence of glycosidase inhibitors (CST, 1 mM, & D M J , 2 mM), a1antitrypsinandtransferrin were sequentiallyimmunoprecipitated from the medium as described under "Experimental Procedures"; the reduced endo H-released oligosaccharides from these proteins were chromatographed on a silicagel-coated plate in Solvent System B for 24 h. Equal aliquots from each protein were applied to the plate, and the components were visualized by fluorography. The migrations of radiolabeled standard oligosaccharides are indicated by the following G,NR, GlcnMansGlcNAcHp; abbreviations: G a s , GlcnManQGlcNAcHz; G3M7,Glc3Man7GlcNAcHz;Mg,M9GlcNAcHz; MR, MansGlcNAcHZ; .M7,Man7GlcNAcH2;Me,ManeGlcNAcH,; Ms, ManSGlcNAcHz.

Demonstration That Cell Surface GlycoproteinsCan Also Be Processed by Endomannosiduse-Examination of the endo Hsusceptible oligosaccharides present on the cell surface glycoproteins after incubation of HepGP cells in the presence of CST and DMJrevealed a chromatographicpattern similar to that observed in individual secreted as well as in total medium glycoproteins, Man7GlcNAcand Man8GlcNAcwere again the

Nonselective Use of Endomannosidme Pathway in HepG2 Cells TABLE I1 Molar dbtributwn of endo H-susceptible N-linked oligosaccharides occurring in several glycoproteins secreted by HepG2 cellsin the presence of CST and D M J Oligosaccharide”

Antitrypsin

Transferrin

Acid elvcoDrotein

molar Sb Glc3MangGlcNAc 12 15 16 11 9 GlcaManRGlcNAc 11 5 Glc3Man,GlcNAc‘ 10 4 2 1 MangGlcNAc 1 32 25 ManRGlcNAc 30 24 Man?GlcNAc 27 24 Man6GlcNAc 13 14 14 Total (dpm X 10-3)d (61) (56) (46) After incubation of HepG2 cells with [14C]glucoseunder the conditions described in Fig. 5,al-antitrypsin, transferrin, and al-acid glycoprotein were sequentially immunoprecipitated from the medium, and their reduced endo H-released oligosaccharides were resolved by preparative thin layer chromatography as described under “Experimental Procedures.” *The molar distribution of the oligosaccharides was established by scintillation counting of the eluted components; the values for the glucosylated oligosaccharides were corrected for the experimentally determined difference in specific activity of the glucose and mannose residues (Glc/Man = 1.2). Small amounts of GlclMangGlcNAc were intermixed with this component. Values in parentheses indicate the totalradioactivity of the endo H-released oligosaccharides.

M

M2

M9 ME8MEA

oTry Trf AG

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this enzyme; however, the formation of complex oligosaccharides was reduced (44% of control) in the presence of the glucosidase inhibitor. Analysis of Complex Oligosaccharides Formed during Glucosidase Blockade-Although the endo H resistance acquired by glycoproteins produced by the HepGP cells in the presence of CST indicated the presence of complex carbohydrate units, this finding did not provide information as to whether these oligosaccharides were comparable to those formed under control conditions. We therefore determined the extentof galactosylation of the endo H-resistant oligosaccharides as an index of complex carbohydrate unit processing (Table 111). These analyses indicated that the incorporation of galactose into the oligosaccharides of cq-antitrypsin and transferrin during glucosidase blockade was indistinguishable from that occurring in cells incubated without inhibitor; the small decrease in thegalactose of al-acid glycoprotein indicates a lack of completion of some saccharide chains whichwas also suggested by the slightly lower hi, which this glycoprotein from CST-treated cells exhibited (Fig. 1 and Table I). DISCUSSION

The studies reported in this paper suggest that endomannosidase functions without selectivity in processing the glycoproteins of HepG2 cells. During a CST-imposed glucosidase blockade, all five of the distinct glycoproteins which we examined were secreted by these cells with apparently a full complement of N-linked complex carbohydrate units, and such extensively processed oligosaccharides were also found to be present on the surface proteins of these cells. Characterization of the N-linked polymannose oligosaccharides revealed that in each case circumvention of the glucosidase blockade, to make complex oligosaccharide formation possible, was achieved by the endomannosidase route which is known to function effectively in HepGP cells (14). In the presence of DMJ to prevent processing by mannosidase I, MaGGlcNAc (isomer A), Man7GlcNAc, and Man6GlcNAc, rather than MangGlcNAc,were the predominant deglucosylated oligosaccharide species and are presumed to have originated by cleavage of Glc3Man from Glc3MangGlcNAc, Glc3Man8GlcNAc,and Glc3Man7GlcNAc, respectively. While the existence of a nonselective endomannosidasemediated deglucosylation pathway resolves the previously

FIG.6. Characterization of the fragments obtained by aceTABLE I11 tolysis of ManaGlcNAc from several glycoproteins secreted Evaluation of the effect of CST on thegalactosylatwn of complex by HepG2 cells in the presence of CST and DMJ. Acetolysis oligosaccharidesfrom several glycoproteins secreted by HepG2 cells was carried out on the reduced ‘.C-labeled MansGlcNAc released by endo H from immunoprecipitated at-antitrypsin (aTry), transferrin Acid Antitrypsin ( T r f ) ,and al-acid glycoprotein (AG), after its purification by thin glycoprotein Sugar” layer chromatography and NaBH. reduction as described under “ExControl CST Control CST Control CST perimental Procedures.” The acetolysis products (-2000 dpm) were dpm X IO-=‘ resolved by chromatography on a cellulose-coated plate for 12 h in Solvent System A, and the components were visualized by fluorog9.4 9.9 11 Mannose 25 20 18 raphy for comparison; acetolysis was carried out on “C-labeled, (1.0) (1.0) (1.0) (1.0)C (1.0) (1.0) reduced standard MangGlcNAc (Mg), MaaGlcNAcisomer A (MsA), Galactose 15 18 25 13 34 30 and MamGlcNAc isomer B (M8& the latter standard contained about (1.2) (1.2) (1.4) (1.5) (1.9) (1.6) 10% of the isomer A. The migration of acetolysis products are a After incubation of HepG2 cells with [14C]glucose with or without indicated by the following abbreviations: M , mannose; M2, mannoCST (1mM) under the conditions described in Fig. 5,a]-antitrypsin, biose; M3, mannotriose; M3GN, Man3GlcNAcH2; and M.GN, transferrin, and al-acid glycoprotein were sequentially immunopreMan,GlcNAcH2. cipitated from the medium, and their endo H-resistant glycopeptides were submitted to acid hydrolysis as described under “Experimental predominant nonglucosylated oligosaccharide species, and ac- Procedures” in order to determine the radioactivity associated with etolysis of the latter revealed that it was exclusively present the mannose and galactose components. * Determined by scintillation counting afterelution from the chroas the A isomer (data not shown). As in the secreted glycoproteins, the cell surface carbohydrate unitsfrom CST-tested matograph. Figures in parentheses represent the ratio of Gal to Man radiocells were, like those from control incubations, predominantly activity; these are relative values as after [14C]glucose-labelingmanresistant to endo H, and only a small proportion (less than nose and galactose have different specific activities with the latter 15% of the total radioactivity in mannose) was released by being higher (33).

11578

Nonselective Use of Erzdomannosidase Pathway inHepG2 Cells

2. Kornfeld, R., and Kornfeld, S. (1985) Annu. Rev. Biochem. 5 4 , puzzling observation, made by a number of investigators, that 631-664 various glycoproteins produced by HepG2 cells (6-8) or pri3. Lubas, W. A., and Spiro, R. G. (1987) J. Bwl. Chem. 262,3775mary cultures of rat hepatocytes (9) can in their processing 3781 circumvent a glucosidase blockade, it does not clarify our 4. Lubas, W. A., and Spiro, R. G. (1988) J.Bwl. Chem. 2 6 3 , 3990finding and that of others (6-8,34) that in thepresence of a 3998 glucosidase inhibitor there is a differential effect on the se5. Pan, Y. T., Hori, H., Saul, R., Sanford, B.A., Molyneux, R. J., cretion of the various glycoproteins. Clearly, our studies inand Elbein, A. D. (1983) Biochemistry 22,3975-3984 6. Lodish, H. F., and Kong, N. (1984) J. Cell Biol. 98,1720-1729 dicate that glucose removal from N-linked oligosaccharides is not a requirement for exit of glycoproteins from the ER as in 7. Sasak, V. W., Ordovas, J. M., Elbein, A. D., and Berninger, R. W. (1985) Biochem. J. 2 3 2 , 759-766 order to achieve deglucosylation by endomannosidase they 8. Parent, J. B., Yeo, T.-K, Yeo, K.-T., and Olden, K. (1986) Mol. have to enter the Golgi compartment in which this enzyme is Cell. Bwchem. 72,21-33 located (3). The presence of multiple glucose residues may 9. Gross, V., Tran-Thi, T. A., Schwarz, R. T., Elbein, A. D., Decker, however retard transit of glycoproteins from ER to Golgi or K., and Heinrich, P. C. (1986) Bwchem. J. 236,853-860 possibly even promote degradation in the former organelle 10. Foddy, L., and Hughes, R. C. (1988) Eur. J. Biochem. 1 7 5 , 291299 (35). Indeed, we noted that the reduction during glucosidase inhibition was directly related to the number of N-linked 11. Duronio, V., Jacobs, S., Romero, P. A., and Herscovics, A. (1988) J.Biol. Chem. 263,5436-5445 oligosaccharides present in the glycoprotein, ranging from K.,McDowell, W., Schwarz, R.T., and Hauri, H.-P. minimal in a-fetoproteinto extensive in theal-acid glycopro- 12. Matter, (1989) J. Bwl. Chem. 264,13131-13139 tein. In addition to these observations on secreted glycopro- 13. Reitman, M.L., Trowbridge, I. S., and Kornfeld, S. (1982) J. teins, it has been reported that the translocation to the cell Bwl. Chem. 257,10357-10363 surface of insulin and insulin-like growth factor I receptors 14. Moore, S. E.H., and Spiro, R.G. (1990) J. Bwl. Chem. 2 6 5 , 13104-13112 in HepG2 cells is retarded in the presence of a glucosidase inhibitor although complex oligosaccharide formation does 15. Knowles, B.B.,Howe, C.C., and Aden, D. P. (1980) Science 209,497-499 take place (11);since these trypsin-sensitive receptors would H.F., Kong, N., Hirani, S., and Rasmussen, J. (1987) J. be included in the cell surface glycopeptides which we ana- 16. Lodish, Cell Biol. 104,221-230 lyzed, it is likely that they are processed by the endomanno- 17. Laemmli, U. K. (1970) Nature 227,680-685 sidase pathway. 18. Spiro, M. J., Spiro, R. G., and Bhoyroo, V. D. (1979) J. Bwl. Our studies in which cells were treated with CST in conChem. 254,7668-7674 junction with DMJ makes it unlikely that the rate-limiting 19. Sasak, W., Herscovics, A., and Quaroni, A. (1982) Biochem. J. 201,359-366 step in the translocation of glycoproteins during aglucosidase . R. G. (1983) J. Biol. Chem. 2 5 8 , blockade can be attributed to a restricted capacity of the 20. Anumula. K.R., and SDiro, 15274-i5282 Golgi-situated endomannosidase to act on the glycoproteins Varki, A., and Kornfeld, S. (1983) J.Bwl. Chem. 258,2808-2818 which exit from the ER so that the formation of complex 21. 22. SDiro. R. G. 11966) Methods Enzvml. 8. 26-52 oligosaccharides can take place. We noted that the addition 23. Spiro; M. J.,'and Spiro, R. G. (1985) J.Bwl. Chem. 2 6 0 , 5808of the mannosidase inhibitor to cells incubated with CST did 5815 not further decrease the secretion of the various glycoproteins 24. Elbein, A. D. (1987) Annu. Rev. Biochem. 5 6 , 497-534 despite that fact that a portion of their carbohydrate units 25. Hodges, L. C., and Chan, S.-K. (1982) Biochemistry 2 1 , 28052810 was still in the glucosylated form; indeed this retarded deglucosylation in the presence of DMJ is presumed to be due to 26. Mega, T., Lujan, E., and Yoshida, A. (1980) J. Biol. Chern. 255, 4053-4056 the decreased formation of oligosaccharides with truncated B.,LBger,D., Wieruszeski, J.-M., Montreuil, J., and mannose branches which are the optimal substrates for en- 27. Campion, Spik, G. (1989) Eur. J. Biochem. 184,405-413 domannosidase (4). It would therefore appear that although 28. Yoshima, H., Matsumoto, A., Mizuochi, T., Kawasaki, T., and the ERglucosidases mayexert aregulatory action on the rate Kobata, A. (1981) J. Biol. Chem. 256,8476-8484 of egress of glycoproteins from this compartment, further 29. Travis, J., Garner, D., and Bowen, J. (1978) Biochemistry 1 7 , processing of N-linked oligosaccharides in the Golgi complex 5647-5651 30. Laine, A., Hachulla, E., Strecker, G., Michalski, J.-C., and Widoes not have a major influence on theirtransittothe eruszeski, J.-M. (1991) Eur. J. Bwchem. 1 9 7 , 209-215 periphery of the cell since it occurs to thesame extent whether complex oligosaccharides are formed or polymannose units 31. Alpert, E., Drysdale, J. W., Isselbacher, K. J., and Schur, P.H. (1972) J. Biol. Chem. 247,3792-3798 are retained. Nevertheless, the occurrence of a Golgi-situated 32. Yoshima, H., Mizuochi, T., Ishii, M., and Kobata, A. (1980) endomannosidase assures that even duringa glucosidase Cancer Res. 40,4276-4281 blockade all proteins which do emerge from the ER can take 33. Spiro, R. G., and Spiro, M. J. (1966) J. Bwl. Chem. 2 4 1 , 1271part in the processing reactions ultimately leading to the 1282 biologically important complex carbohydrate units. 34. Gross, V., Andus, T., Tran-Thi, T.-A., Schwarz, R. T., Decker, K., and Heinrich, P. C. (1983) J. Bwl. Chem. 2 5 8 , 12203-

REFERENCES 1. Spiro, R. G., and Spiro, M. J. (1982) Philos. Trans. R. SOC. hnd. B. Biol. Sci. 3 0 0 , 117-127

12209 35. Klausner, R. D., Lippincott-Schwartz, J., and Bonifacino, J. S. (1990) Annu. Rev. Cell Biol. 6,403-431