Alterations in Lipid-linked Oligosaccharide Metabolism in Human Melanoma Cells Concomitant with Induction of Stress Proteins*

Challenge of human A375 melanoma cells with so- dium arsenite induced the synthesis of stress proteins and stimulated r3H]mannose incorporation into a novel component migrating on sodium dodecyl sulfate-poly-acrylamide gel electrophoresis with an apparent mo- lecular mass of 14 kDa (designated M14). Enhanced M14 expression was elicited by heavy metals (zinc, copper, cadmium, and nickel), thiol-reactive agents (iodoacetamide and auranofin), and hyperthermia. The kinetics of M14 induction and recovery from stress were similar to those of the stress proteins, but M14 half-life was only 15 min. Incorporation of ["Hlman-nose into M14 was inhibited by tunicamycin but not by cycloheximide or actinomycin D. M14 was metabolically labeled with [S2P]orthophosphate but not by ["'SI methionine or [" Hlasparagine. Further studies re- vealed that M14 was selectively soluble in chloroform/ methanol/water (10:10:3) and sensitive to both endo-8-N-acetylglucosaminidase H digestion and mild acid hydrolysis. The latter released a water-soluble man-nose-labeled moiety which eluted from Bio-Gel P-6 in a manner similar to GlcaMansGlcNAcZ. Together, these data suggest that M14 is a lipid-oligosaccharide intermediate

resulted in increased incorporation of [3H]mannose into a component with a molecular mass on SDS-PAGE' of 14 kDa (designated M14). p32 and p34 were not detectably mannosylated. We report an initial characterization of the induction of this molecule and its stability and turnover and tentatively identify the molecule as a lipid-linked oligosaccharide whose synthesis is significantly increased in cells exposed to a range of insults. To our knowledge, this is the first report documenting alterations in the metabolism of lipid-linked oligosaccharides under conditions that concomitantly induce stress protein synthesis.
Confluent A375 cultures were heat-shocked by addition of prewarmed (43 "C) DMEM supplemented with 2% fetal bovine serum. Sealed T-25 flasks were submerged in a 43 "C water bath for 30 or 60 min. Following treatment, cultures were replenished for various times with medium prewarmed to 37 "C and radiolabeled during the last hour with 200 &i/rnl [3HJmannose in DMEM.
To study the kinetics of stress-induced alterations in glycosylation, cells were pulse-labeled with [3H]mannose for 1-20 h following the addition of sodium arsenite. The reversibility of stress-induced changes was investigated by challenging cells with arsenite for 8 h, followed by various recovery times in arsenite-free DMEM. During the last hour of each recovery, cultures were radiolabeled with ['HI mannose. To analyze the metabolic turnover of M14, cells were radiolabeled with [3H]mannose in DMEM during the last hour of an 8-h arsenite stress and then incubated with fresh DMEM, with or without arsenite, up to 1 h.
Gel Electrophoresis and Fluorography-Cellular extracts were analyzed as described previously (13). Briefly, cell monolayers were solubilized on ice for 20 min in 10 mM Tris buffer, pH 7.6, containing 1% Nonidet P-40, 0.1% SDS, 0.15 M NaCl, 1% kallikrein inhibitor, and 1 mM phenylmethylsulfonyl fluoride. Following centrifugation in an Eppendorf microcentrifuge, supernatants were mixed with an equal volume of sample buffer and boiled for 3 min. Equal amounts of protein or trichloroacetic acid-insoluble radioactivity were analyzed on one-dimensional SDS-PAGE using the discontinuous buffer system of Laemmli (15) with a 4.5% acrylamide stacking gel and a 12.5% acrylamide resolving gel. Equilibrium two-dimensional SDS-PAGE The abbreviations used are: SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; GlcitolNAc, N-acetylglucosaminitol.

Stress-induced Alterations in Glycosylation
was performed according to Bravo (16). Trichloroacetic acid-insoluble radioactivity was estimated as described (13). Protein concentrations were determined using a BCA Protein Assay Kit (Pierce Chemical Co.) with bovine serum albumin as a standard. Molecular weight and PI calibrations were determined using low molecular weight and pH 3.5-10 PI protein standards (Pharmacia P-L Biochemicals), respectively.
Radioactivity associated with electrophoretically separated M14 was estimated by extracting excised gel slices with Protosol (Du Pont-New England Nuclear) for 24 h at 37 'C. Aquasol-I1 (Du Pont-New England Nuclear) was added, and samples were counted 24 h later.
Solubility in Chloroform/Methnnol/ Water-This was performed according to a standard protocol (17) with certain modifications. Cultures were sonicated in 0.02 M Tris buffer, pH 7.4, containing 0.15 M NaCl and centrifuged in a Beckman TL-100 ultracentrifuge at 220,000 X g for 35 min at 4 "C. Supernatants were removed and analyzed by one-dimensional SDS-PAGE. The insoluble pellet was extracted three times with chloroform/methanol/water (3:2:1); the combined lower phases were pooled, washed three times with chloroform/methanol/water (3:48:47), and evaporated to dryness under Nt. The interphase and pellet were washed three times with water and then extracted three times with chloroform/methanol/water (1010:3). Extracts were evaporated and analyzed by one-dimensional Mild Acid Hydrolysis-Conditions for acid hydrolysis were as described (18). Briefly, SDS-solubilized cell extracts were incubated with 0.02 M HCl at 95 'C for 30 min. Acid was removed by repeated dissolution in water and evaporation. The hydrolyzed samples were analyzed by one-dimensional SDS-PAGE.
Characterization of Labeled Oligosaccharides Released from M14 by Mild Acid Hydrolysis-Aliquots of M14 purified from both stressed and unstressed melanoma cells by electroelution from one-dimensional SDS-PAGE were subjected to mild acid hydrolysis in 25% isopropyl alcohol containing 0.02 M HCl at 95 "C for 30 min. After neutralization with 0.02 M NaOH, released oligosaccharides were analyzed by gel filtration on Bio-Gel P-6 (200-400 mesh, 1 X 110 cm), equilibrated, and eluted with 10 mM Tris-HC1, pH 6.8. Fractions were collected and analyzed for radioactivity.

Glycosylation Patterns in Human
Melanoma Cells Challenged with Sodium Arsenite-Treatment of human A375 melanoma cells with sodium arsenite enhanced ['Hlmannose incorporation into a low molecular weight component that migrated on one-dimensional SDS-PAGE with an apparent mass of 14 kDa (Fig. 1). This molecule (referred to as M14) was also present in unstressed cells. Detectable elevation of radioactivity into M14 was elicited by 24 p~ sodium arsenite, whereas maximal stimulation occurred at higher concentrations (72-96 p~) . Similar results were obtained when either equal amounts of radioactivity or total cellular protein were analyzed. Parallel analysis of A375 cultures treated under identical conditions but radiolabeled with either ["S]methionine or 'H-amino acids failed to reveal a concomitant increase of protein synthesis in this molecular weight range, but enhanced synthesis of the major stress proteins (p100, p90, p73/72, and p32 kDa) was readily observed (Fig. 1). ["SI Methionine incorporation into trichloroacetic acid-insoluble cellular material was not significantly inhibited by sodium arsenite (data not shown). Similar results were obtained with human colon carcinoma cells (Colo 201 and HT-29) and fibroblasts (CCD-21Sk and CCD-330) (data not shown).
On two-dimensional SDS-PAGE, radiolabeled M14 migrated as a single acidic spot with a PI of approximately 4.5 (Fig. 2). Parallel analysis of [35 Slmethionine-or [' Hlasparagine-labeled material failed to identify a co-migrating protein (data not shown). M14 extracted from control or stressed cultures displayed similar electrophoretic properties (Fig. 2). In addition, M14 was metabolically labeled with ['*P]orthophosphate as judged by identical migrations on two-dimensional SDS-PAGE (data not shown).

Kinetics of M14 Expression-Enhanced ['Hlmannose
incorporation into M14 was detectable 1 h following challenge of A375 cells with sodium arsenite (48 p~) and peaked after 6-8 h (Fig. 3A). T o investigate whether elevated levels of M14 were sustained in stressed cultures following removal of insult, A375 cells were challenged with arsenite (48 p~) for 8 h and allowed to recover in arsenite-free medium for up to 24 h. During the last hour, cells were radiolabeled with ['Hlmannose. Recovery of M14 to pre-stress levels required approximately 6 h (Fig. 3B).
The stability of M14 expression was examined by challenging cells with arsenite (48 p~) for 8 h and radiolabeling with [3H]mannose for the last hour. The cultures were then replenished with medium and incubated in the presence or absence of arsenite for up to 1 h. Under these conditions, the chase half-life for M14 was approximately 15 min (Fig. 4). A similar value was found for M14 expression in control, unstressed cultures (data not shown).

M14 Expression in Response to Different Insults-Challenge
of A375 cells with heavy metals (zinc, copper, cadmium, and nickel), sulfhydryl-reactive reagents (iodoacetamide and auranofin), amino acid analogs (~-azetidine-2-carboxylic acid and L-canavanine), disulfiram (a copper-chelating agent), or the calcium ionophore A23187 induced significant incorporation of ['Hlmannose into M14 (Table I). Treatment with hyperthermia gave equivocal results. Heat shock (43 "C for 30 or 60 min) significantly inhibited cellular uptake of [3H] mannose, but incorporation of radiolabel into M14 was less severely affected (Fig. 5). At 1 h post-recovery, M14 expression, as determined by densitometry, was decreased 62% compared to control; but by 4 h, it had increased 27% over control values. Recovery to pre-stress levels required approx-

13~1
? 0 A375 melanoma cells were radiolabeled with [3 Hlmannose in DMEM. Cultures were then incubated in the presence of arsenite for different times (0-60 min). Cell extracts containing equivalent amounts of trichloroacetic acidinsoluble radioactivity were analyzed by one-dimensional SDS-PAGE and fluorography. Quantitation of radioactivity contained within the M14 bands was determined as described under "Experimental Procedures." imately 8 h. Under the same conditions, increased synthesis of the major stress proteins (p100, p90, and p73/72) was readily detected (data not shown).
Metabolic Inhibitors and M14 Expression-Treatment of control or arsenite-stressed A375 cells with either actinomy- Calcium ionophore

Heavy metals and sulfhydryl reagents
Copper-chelating agent cin D (1 pg/ml) or cycloheximide (5 pg/ml) had minimal effect on incorporation of [3H]mannose into M14 (Fig. 6). Both drugs effectively blocked protein synthesis. Tunicamycin Al (250 ng/ml), which does not inhibit protein synthesis (23), caused nearly complete inhibition of [3H]mannose incorporation into M14 and other mannosylated material (Fig. 6). Enzymatic Digestion ofM14"SDS-PAGE analysis revealed . A375 cultures were stressed for 8 h with sodium arsenite (48 p~) and radiolabeled with [3H]mannose for the last 4 h. Cells were sonicated in Tris buffer and centrifuged. The insoluble pellet was sequentially extracted with a 3:2:1 mixture of chloroform/methanol/water ( l a n e 3,40,000 cpm), followed by extraction with a 10:10:3 solution of chloroform/methanol/water ( l a n e 4, 100,000 cpm). The insoluble pellet remaining after organic extractions ( l a n e 5, 40,000 cpm) and total cellular one-dimensional SDS lysates from control ( l a n e 1,50,000 cpm) and arsenite-stressed ( l a n e 2,50,000 cpm) cultures are all included for comparison. All samples were dissolved in one-dimensional SDS-PAGE sample buffer and analyzed as described under "Experimental Procedures." that M14 isolated by electroelution from stressed A375 cells was partially degraded within 2 h by endoglycosidase H, an enzyme that hydrolyzes the di-N-acetylchitobiose linkage of high mannose oligosaccharides which are N-linked to proteins or joined through a pyrophosphate bridge to dolichol carrier (19). Complete digestion required 24 h. In contrast, M14 was stable to prolonged (5 days) Pronase digestion and was not a substrate for peptide:N-glycosidase F. Control experiments indicated that the three enzymes were active against appropriate substrates (data not shown).
Acid Hydrolysis of M14"Extracts of arsenite-challenged melanoma cells were treated with acid and analyzed on onedimensional SDS-PAGE. Mild acid treatment caused the total loss of M14-associated radioactivity, whereas other mannosylated bands were unaffected (Fig. 8). were heated for 30 min at 95 "C in the presence (+) or absence (-) of 0.02 N HCI. Following hydrolysis, samples were analyzed as described under "Experimental Procedures."

MI4 Chromatography on Bw-Gel P-6-Gel filtration on
Bio-Gel P-6 of radioactivity released from acid-hydrolyzed M14 showed one major component slightly higher in molecular weight than the standard oligosaccharide Man,GlcitolNAc (Fig. 9). The elution profile was similar to that observed for GlcnMangGlcNAc2 by others using identical conditions (21). Extracts from both control and stressed cultures displayed similar patterns of elution (Fig. 9).

DISCUSSION
In a previous investigation (13), we described the induction of a 32-kDa protein (p32) in human normal and neoplastic cells stressed with sodium arsenite. The original objective of this study was to examine whether p32 was glycosylated and whether the extent of glycosylation was influenced by stress. Preliminary experiments on cultures challenged with sodium arsenite (and other insults) and radiolabeled with [3H]mannose revealed that p32 was not detectably mannosylated in either control or stressed cells. However, under identical conditions, we observed in arsenite-treated cultures significantly increased incorporation of [3H]mannose into a component with an apparent molecular mass on one-dimensional SDS-PAGE of 14 kDa. We refer to this molecule as M14; and in this report, we provide an initial description of its induction, turnover, and stability in stressed cells and tentatively identify it as a lipid-linked oligosaccharide whose steady-state level is substantially enhanced in cultures exposed to noxious insult.
Expression of M14 in arsenite-challenged cells has not been reported previously. Several lines of evidence indicate that its induction is a stress-related event. Although sodium arsenite was the most potent inducer of M14, a broad range of other chemical insults, including a thiol-reactive reagent (iodoacetamide), heavy metals (zinc, copper, cadmium, and nickel), amino acid analogs (~-azetidine-2-carboxylic acid and L-canavanine), the calcium ionophore A23187, and disulfiram, increased significantly ['Hlmannose incorporation into M14. These reagents also induced synthesis of the major stress proteins (13). Exposure of cells to auranofin (2,3,4,6-tetra-Oacetyl-l-thio-B-D-glucopyranosato-S-triethylphosphine gold (I) (RidauraTM)), an antiarthritic compound recently reported to induce the synthesis of stress proteins (22). also enhanced M14 expression in a manner similar to other tested agents. Hyperthermia, the insult originally used to identify heatshock (stress) proteins (1). also elevated M14 levels in A375 cultures allowed to recover from thermal insult. In addition, the kinetics of M14 induction and recovery paralleled those described previously for the major stress proteins (13). Peak expression of M14 occurred within 6-8 h of arsenite treatment, and recovery to pre-stress levels required approximately 8 h in arsenite-free medium.
Whereas these initial data suggested that M14 was a novel, glycosylated stress protein, studies with metabolic inhibitors demonstrated that this was not the case. Actinomycin D and cycloheximide failed to block M14 induction in arsenitechallenged cells, indicating that expression of this molecule was independent of both RNA and protein synthesis. Under identical conditions, induction of stress proteins was completely inhibited (13). In contrast, tunicamycin, which prevents N-linked protein glycosylation by inhibiting formation of lipid-linked oligosaccharides (23) (24). Resistance to Pronase provides additional support, although it should be noted that certain glycoproteins, particularly those which are heavily glycosylated or associated with fatty acids, are insensitive to Pronase treatment (25,26). Second, M14 sensitivity to digestion with endoglycosidase H indicates that the carbohydrate component of M14 is "high mannose" and suggests that it contains the minimal structure ManGGlcNAcz since smaller lipid-linked species (Man,GlcNAc2) are endoglycosidase H-resistant (27,28).
Third, the chase half-life of M14 from either control or stressed cultures was only 15 min, which compares to the reported half-life of other oligosaccharide lipid-linked intermediates (8-15 min) (29,30).
Finally, other investigators (33-35), studying hepatocytes and oviduct membranes, have observed alterations in dolichol-linked oligosaccharides induced by steroids. Since steroids have also been shown to induce stress protein synthesis and thermal tolerance (6, 36, 37), these observations are consistent with our own and identify the lipid-linked oligosaccharides as potentially important mediators of the cellular response to stress and define a new component of cellular response to injury.
One possible explanation for enhanced M14 levels in stressed cells is its decreased utilization due to inhibition of protein synthesis and thus lack of available "acceptor substrates." We consider this highly unlikely because under conditions in which sodium arsenite induced significantly increased levels of M14, protein synthesis was essentially unaffected. Similarly, cycloheximide blocked protein synthesis by more than 90% but failed to enhance M14 expression. Both pieces of evidence support the view that stress agents like sodium arsenite and amino acid analogs increase M14 levels by promoting its synthesis rather than blocking utilization.
Together, these data provide strong, although indirect, evidence that stressing of A375 melanoma cells can induce enhanced synthesis of large (six or more mannose moieties) lipid-linked oligosaccharides in addition to other well-documented changes in protein synthesis. Confirmation of these conclusions will require isolation of M14 in sufficient quantities for unequivocal structural determination.