THE JOURNAL OF B~LOG~CAL CHEM~STRV Vol. 257. No. 14, Issue of July 25. pp. 8424-8431, 1982 Printed in U.S.A.

A Chinese Hamster Ovary Cell Mutant Deficient in Glucosylation of Lipid-linked Oligosaccharide Synthesizes Lysosomal Enzymes of Altered Structure and Function*

(Received for publication, February 12, 1982)

Sharon S. Krag$ and April R. Robbin@ From the *Department of Biochemistry, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205 and the EGenetics and Biochemistry Branch, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205

B211 is a Chinese hamster ovary cell mutant previ- ously characterized as deficient in the glucosylation of lipid-linked oligosaccharides. We have found that the activities of several acid hydrolases were decreased in extracts of B211; e.g. a-L-iduronidase and j&hexosamin- idase activities were 1 and 208, respectively, of the activities measured in extracts of parental (WTB) cells. A revertant of B211, able to glucosylate lipid-linked oligosaccharides, exhibited hydrolase activity up to 30 times greater than that of B211. The activities of hy- drolases secreted by mutant, revertant, and parent were similar. Sodium dodecyl sulfate gel electrophore- sis of fi-hexosaminidase isolated from secretions of B211 grown with [2-3H]mannose revealed several a and p polypeptides, the 42, values of which differed by + 5000 from those measured for the corresponding poly- peptides from WTB. a-L-Iduronidase secreted by B211 had a molecular weight approximately twice that meas- ured for parental enzyme.

The [3H]mannose-containing oligosaccharides of @- hexosaminidase and a-L-iduronidase were analyzed by enzymatic digestion and gel filtration chromatography. Both enzymes isolated from secretions of WTB cells contained phosphorylated and simple “high mannose” oligosaccharides, as well as oligosaccharides resistant to cleavage by endo-j?-N-acetylglucosaminidase H; the proportion of [3H]mannose in the three classes of oli- gosaccharides differed significantly in the two en- zymes. Neither &hexosaminidase nor a-L-iduronidase from B211 contained phosphorylated or high mannose oligosaccharides; all of the radioactivity was found in oligosaccharides of the complex type. Consistent with the absence of phosphorylated oligosaccharides, P-hex- osaminidase and a-t-iduronidase from B211 were not internalized via the mannose B-phosphate receptor. While B211 failed to phosphorylate endogenous acid hydrolases, membrane preparations from the mutant did phosphorylate exogenously added P-hexosamini- dase.

The oligosaccharide (Glc)s(Man)g(GlcNAc)2 is generally thought to be the principal species transferred from lipid (dolichol pyrophosphate) to protein in the biosynthesis of asparagine-linked oligosaccharides in mammalian cells (for

* This work was supported in part by Public Health Service Grants CA-20421 and CA-00640 awarded by the National Cancer Institute, Department of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

reviews, see Refs. l-3). Comparison in cell-free systems of the rate of transfer of glucosylated versus nonglucosylated lipid- linked oligosaccharides to endogenous proteins demonstrated that the glucosylated species was transferred up to nine times faster (4, 5). A Chinese hamster ovary cell mutant, B211, selected for increased resistance to concanavahn A (6), has been shown to be defective in the glucosylation of lipid-linked oligosaccharides (7). Surprisingly, incorporation of mannose into glycoproteins occurred at near normal rates in B211.

Since examination of total cellular glycopeptides of B211 failed to reveal any significant consequences of the transfer to protein of nonglucosylated oligosaccharides, we have begun to investigate the effects on individual classes of glycoproteins. In this report, we compare the structure and function of acid hydrolases from B211 and parental cells. These enzymes are of interest because they contain a unique moiety, mannose 6- phosphate, on their asparagine-linked oligosaccharides (8-l I). The presence of phosphorylated mannose residues is neces- sary for the uptake of extracellular acid hydrolases into fibro- blasts (12-14) and for the compartmentalization of endoge- nously synthesized hydrolases into lysosomes (11, 15, 16).

We show here that oligosaccharides on acid hydrolases from B211 are not phosphorylated, but are processed to the com- plex type.

EXPERIMENTAL PROCEDURES

Materials

Chemicals and Radiochemicals-4-Methylumbelliferyl a-L-idu- ronide was synthesized (17) and supplied by Dr. Bernard Weissmann, University of Illinois; other 4-methylumbelliferyl substrates were purchased from Research Products International Corp. [2-“HIMan- nose (12-16 Ci/mmol), [““Slmethionine (1000-1500 Ci/mmol), and ?Xabeled methylated proteins (3-30 pCi/mg) were obtained from Amersham Corp. [:‘“P]UDP-GlcNAc (~30 Ci/mmol) was synthesized (18) and provided by Dr. Misao Owada, Genetics and Biochemistry Branch, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. Bovine serum albumin (A grade) was obtained from Calbiochem, sodium dodecyl sulfate was from Sigma, and Immuno- precipitin (formalin-fixed Staphylococcus A cells, 10% (w/v)) was from Bethesda Research Laboratories.

Enzymes and Antisera-Human urinary a-L-iduronidase (13), hu- man placental P-hexosaminidase A and B (20), antisera raised in goats against human kidney a-L-iduronidase (19), and human placen- tal /3-hexosaminidases A, B, and the isolated a subunit (20) were nrovided bv Drs. Leonard Rome, Rachel Myerowitz, Andrej Hasilik, and Elizabeth F. Neufeld, Genetics and Biochemistry Branch, Na- tional Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases.

Endo-P-N-acetylglucosaminidase H was obtained from either Miles Laboratories, Inc., or Health Research Inc., Albany, NY. cu-Mannos- idase (jack bean enzyme), P-N-acetylglucosaminidase (jack bean en- zyme), P-galactosidase (Aspergillus niger), alkaline phosphatase

8424

Glucosylation Mutant Synthesizes Altered Lysosomal Enzymes 8425

(Escherichia coli), and protease, type VI or XIV (pronase), were obtained from Sigma. Neuraminidase (Clostridium perfringens) was obtained from Worthington and Boehringer Mannheim. Endo-P-N- acetylglucosaminidase D (Diplococcus pneumoniae) was obtained from Miles Laboratories, Inc.

Cells and Cell Culture

The isolation of CHO' cell lines WTB, B211, and B211-REV has been previously described (6). All media for cell culture were prepared in the Media Supply Unit of the National Institutes of Health. Growth media (21) and labeling media (22) contained 5% fetal bovine serum or 5% dialyzed fetal bovine serum (both from GIBCO), respectively. Procedures of cell culture have been previously described (23). For maintenance of cells in serum-free medium, we used Waymouth medium MAB 87-3 (24), formulated as described in the GIBCO catalogue to which was added bovine serum albumin (1 mg/ml). Cultures were checked routinely for mycoplasma (25) and were found to be negative.

Labeling of Cells Cells were labeled with [L-'HH]mannose (0.1 mCi/ml) and ["SS]

methionine (250 pCi/ml) for 10 h, and label was chased for 14 h according to the previously described procedures (22). Secretions and cells were harvested and prepared for immunoprecipitation, as pre- viously described (22).

Immunoprecipitation of Radioactive Enzymes, Polyacrylamide Gel Electrophoresis, and Fluorography

Sequential immunoprecipitation of native enzymes was performed as previously described (22). Immunoprecipitates to be used in anal- ysis of oligosaccharides were heated at 100 "C for 5 min in 0.1 ml of 0.1 M Tris-C1, pH 8; immunoprecipitates to be used in electrophoresis were solubilized as previously described (22). For immunoprecipita- tion of the separated a and P chains of P-hexosaminidase, we em- ployed a procedure developed by Dr. Richard L. Proia, Genetics and Biochemistry Branch, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. P-Hexosaminidase in samples of cell extracts or secretions (without added carrier enzyme) was denatured in the presence of 1% SDS by heating at 100 "C for 10 min. The samples were then diluted to 1 ml with 0.15 M NaC1, 0.01 M Tris, pH 7.4, containing 2% Nonidet P-40, 10 mg/ml bovine serum albumin, and 0.02% sodium azide; 50 pl of formalin-fined Immuno-precipitin were added to each sample, and these were incubated for 15 min at 0 "C. The mixtures were clarified by centrifugation at 15,000 X g for 5 min in an Eppendorf Model 5412 bench centrifuge, and then 5 p1 of the appropriate antisera were added. After incubation for 16 h at 4 "C, 50 pl of Immuno-precipitin were added to each sample; after 15 min at 0 "C, the samples were centrifuged as above and the pellets were washed 4 times with 0.01 M Tris-C1, pH 8.6, 0.6 M NaCI, 0.1% SDS, and 0.05% Nonidet P-40, and once with 0.15 M NaCl, 0.01 M Tris-C1, pH 7.4. The immunoprecipitated proteins were separated from the Immuno-precipitin by heating in 60 pl of solubilizer (22) for 5 min at 100 "C, followed by centrifugation.

Electrophoresis on SDS-polyacrylamide gels (12.5%) and fluorog- raphy (26) were carried out according to published procedures (20). Radioactive standards used as markers of molecular weights were myosin (200,000), phosphorylase b (92,500), bovine serum albumin (69,000), ovalbumin (46,000), carbonic anhydrase (30,000), and lyso- zyme (14,300).

Enzyme Assays Mucopolysaccharide a-L-iduronohydrolase (EC 3.2.1.76) was as-

sayed as described (27); 1 unit of enzyme activity represents 1 nmol of substrate hydrolyzed/h at ambient temperature. a-D-Mannosidase (EC 3.3.1.24), P-D-glucuronidase (EC 3.2.1.31), P-N-acetyl-D-glucosa- minidase (EC 3.2.1.30), a-L-fucosidase (EC 3.2.1.51), P-D-galactoside galactohydrolase (EC 3.2.1.23), and acid phosphatase (EC 3.1.3.2) were assayed as previously described (28). For these enzymes, 1 unit of enzyme activity represents 1 nmol of substrate hydrolyzed/h at 37 "C. Protein was measured by the method of Lowry et al. (29).

' The abbreviations used are: CHO, Chinese hamster ovary; SDS, sodium dodecyl sulfate; P-GlcNAc transferase, UDP-N-acetylglu- cosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase; etldo H, endo-P-N-acetylglucosaminidase H endo D, endo-P-N-ace- tylglucosaminidase D; Man, mannose; GlcNAc, N-acetylglucosamine.

The assay of P-GlcNAc transferase (UDP-N-acetylglucosamine: glycoprotein N-acetylglucosamine-1-phosphotransferase) activity was essentially that described by Owada and Neufeld (18). Crude membrane preparations (30) were incubated in reaction mixtures containing 50 mM Tris-C1, pH 7.5, 10 mM MgCln, 10 mM MnC12, 0.25 mM dithiothreitol, 5 mM ATP, 1 mM sodium phosphate, pH 7.5, 1% Triton X-100,5 X lo5 cpm [/3-32P]UDP-GlcNAc, and 5 pg of placental hexosaminidase B in a final volume of 0.1 ml. Incubations were carried out for 2 h at 37 "C, at which time the mixtures were clarified by centrifugation at 120,000 X g for 30 min in a Beckman Airfuge. P-Hexosaminidase was immunoprecipitated and the immu- noprecipitates were electrophoresed on an SDS-polyacrylamide gel (12.5%), as previously described. The gel was stained with Coomassie blue (31) and dried, and radioactivity on the gel was detected by radioautography using an intensifying screen ("Lightning Plus," Du- pont).

Enzymatic Treatments of Immunoprecipitated Enzymes Protease Treatmed-Immunoprecipitates were digested exhaus-

tively with pronase using Procedure A, when retention of phosphate on the glycopeptides was desired, or Procedure B, when dephospho- rylation of glycopeptides was desired. In A, 2 ml of pronase (5 mg/ml) in 0.1 M Tris-C1, pH 8.0, were dialyzed at 4 'C against 200 ml of 0.1 M Tris-C1- pH 8.0, for 2 h, followed by dialysis at 4 "C against 200 ml of 0.1 M Tris-C1,0.02 M sodium phosphate, pH 8.0. This concentration of phosphate inhibited the phosphatase activity present in pronase. The pronase was then incubated at 34 "C for 2 h to destroy contam- inating glycosidase. The immunoprecipit.ates were treated with a total of 0.25 ml of pronase in 3 aliquots during a 72-h incubation at 34 "C under toluene. In B, 2 ml of pronase (10 mg/ml) in 0.1 M Tris-C1, pH 8.0, were dialyzed at 4 "C against 200 ml of 0.1 M Tris-C1, pH 8.0, for 2 h, then incubated at 34 "C for 2 h. An aliquot of 0.20 ml of alkaline phosphatase (4 units), supplied as an ammonium sulfate precipitate, was dialyzed at 4 "C against 200 ml of 0.1 M Tris-CI, 0.004 M MgCL, pH 8.0. The immunoprecipitates were treated with 1 unit of alkaline phosphatase for 30 min at 34 "C. Pronase (0.025 ml) was then added, and the incubation was continued for 24 h at 34 "C under toluene. Subsequently, 2 more units of alkaline phosphatase and 0.1 ml of pronase were added during an additional 48-h incubation. The simul- taneous treatment with phosphatase and pronase was employed, rather than sequential treatment with pronase, then phosphatase, because our attempts to remove phosphate subsequent to pronase digestion (by gel fdtration in water or at pH 4 or by precipitation as a NH4MgP04 complex) all resulted in residual phosphate concentra- tions of at least 40 FM, which was sufficient to inhibit subsequent phosphatase activity on these glycopeptides.

Preparation of Glycopeptides for Subsequent Analyses Samples were either deproteinized by ultrafdtration (Immersible-

CX ultrafilters, Millipore) with greater than 95% recovery of label, or boiled at neutral pH for 5 min. No differences were seen using these two methods for removal or inactivation of pronase. These methods of eliminating pronase activity were chosen over 1) gel fitration at pH 8.0, because the majority of the labeled glycopeptides co-elute with pronase at this pH; 2) gel filtration at pH 4, because under these conditions, only 50% of the radioactivity was recovered separate from the salt peak and 3 ) dialysis, because phosphorylated glycopeptides are retained in the dialysis bag. The glycopeptides were then desalted by passage over a Bio-Gel P-2 or P-6 column eluted with water; this treatment removed the Tris-C1, but not the sodium phosphate.

Endo H Treatment Glycopeptides were tesuspended in 1 ml of sterile buffer (140 mM

NaC1, 7 mM KCI, 1 mM potassium phosphate; the pH of the buffer was adjusted to pH 5.8 with sodium monophosphate). Ten milliunits (in 0.01 ml of water) of endo H were added, and the incubation was begun at 37 "C under toluene. After 2 h, a second aliquot of 10 milliunits was added, and the incubation was continued overnight.

a-Mannosidase Treatment a-Mannosidase (10 units in 0.2 ml), supplied as an ammonium

sulfate precipitate, was dialyzed against 200 ml of 50 mM sodium acetate, pH 6.0, 0.1 mM ZnClz for 2 h, followed by dialysis against 50 mM sodium acetate, pH 4.5, for an additional 2 h. The dialyzed enzyme was then added to the dried sample; treatment was for 16 h at 37 "C under toluene.

8426 Glucosylation Mutant Synthesizes Altered Lysosomal Enzymes

Neuraminidase Treatment Samples were incubated in 0.5 ml of sodium acetate, pH 4.5.

containing 0.3 mg/ml bovine serum albumin and 0.3 unit of neura- minidase (Worthington) for 16 h a t 37 "C under toluene.

Treatment with a Mixture of Glycosidases Immunoprecipitates of a-L-iduronidase and P-hexosaminidase were

treated with pronase in the presence of sodium phosphate, as de- scribed above. The resultant glycopeptides were desalted, boiled to inactivate the pronase, and treated with neuraminidase (12 milliunits, Boehringer Mannheim) in a total of 0.3 ml of 25 mM sodium citrate, 1 mM CaCIZ, pH 5.6, for 16 h a t 37 "C under toluene. The desialylated glycopeptides were desalted, boiled, and incubated with P-galactosid- ase (1 unit) in 0.1 ml of 20 mM sodium citrate, pH 3.5, for 8 h under toluene. The samples were then boiled at pH 3.5 for 15 min in an attempt to inactivate the a-mannosidase contaminating the P-galac- tosidase; 13% of the a-mannosidase activity remained after boiling a t pH 4 for 5 min. The samples were then incubated in a total of 0.2 ml of 40 mM sodium citrate, pH 6, containing a-N-acetylglucosaminidase (2.5 units) and endo D (0.01 unit) for 5 h a t 37 "C under toluene. The products of this treatment were separated by gel filtration chroma- tography.

Gel Filtration Chromatography on Bio-Gel P-6 Samples were applied with standards to a Bio-Gel P-6 column

(200-400 mesh, 1 X 115 cm) and were eluted with 0.1 M Tris-C1.0.16 sodium azide, pH 8.0. Fractions of 1.05 ml were collected; aliquots of 0.9 ml were analyzed for radioactivity using Hydrofluor (National Diagnostics, Inc.) and a Beckman LS7000. Recovery of radioactivity on all columns was greater than 708. The following internal standards were prepared and detected as described (7): dextran (Dl , oligomers of N-acetylglucosamine (Nl-Nd, and mannose ( M ) (see Fig. 3).

RESULTS

Acid Hydrolase Activity-Cell-free extracts of the mutant B211 contained reduced activities of many acid hydrolases (Table I). a-L-Iduronidase and p-glucuronidase activities were only 1 to 3% of those obtained from parental (WTB) cells. p- Hexosaminidase, P-galactosidase, a-mannosidase, and a-fu- cosidase activities ranged from 20 to 50% of parental levels, while acid phosphatase activity was identical in extracts of B211 and WTB. The alteration in lysosomal enzyme activity observed in B211 appears to correlate with the glucosylation defect in that mutant; extracts of a revertant (B211-REV), shown previously to exhibit near normal glucosylation of lipid- linked oligosaccharides (7), contained acid hydrolase activities 233% of those measured with the parent (Table I).

The decreased levels of enzyme activity measured in cell- Cree extracts of the mutant cannot be explained in terms of increased secretion. Activities of acid hydrolases secreted by B211 were similar to those secreted by WTB or B211-REV (Table 11).

Radioactive Polypeptides-The pleiotropic decrease in acid hydrolase activity observed with B211 suggested that the asparagine-linked oligosaccharides present on these enzymes might be aberrant. To examine this possibility, glycopeptides were prepared from P-hexosaminidase and a-L-iduronidase isolated from secretions of B211 and WTB cells grown in [2- "Hlmannose in the presence of 10 mM NH&l. We analyzed the secreted, rather than the cellular, forms of the hydrolases in order to avoid any degradation of oligosaccharides due to lysosomal glycosidases. Ammonia effects the secretion of newly synthesized acid hydrolases (20, 32), increasing the levels of extracellular hydrolases 5- to 10-fold for WTB and 1.5- to 1.8-fold for B211, based on measurements of enzyme activity. Enzymes were isolated by sequential immunoprecip- iAAtion.

To determine the purity of the material, the immunoprecip- itates were electrophoresed on sodium dodecyl sulfate-poly- ecrylamide gels, and the gels were examined following fluo-

TABLE I Acid hydrolase activity in cell-free extracts of B211, B2lI-REV,

and WTB cells B211, B2lI-REV, and WTB were harvested at 5.2 X IO'', 5.3 X lo",

and 4.9 X 10" cells/150-cm2 flask, respectively. Enzvme €321 1 R211-REV WTR

units/rng cell protein

a-L-Iduronidase 0.06 2.2 6.6 P-Glucuronidase 7 123 202 P-Hexosaminidase 32 66 161 a-Mannosidase 41 57 80 B-Galactosidase 48 196 26 1 a-L-Fucosidase 103 641 410 PhosDhatase 1390 1200 1400

TABLE I1 Acid hydrolase activity secreted by B211, B211-REV. and WTB

cells Secretions were collected from cells maintained for 14 h in serum-

free medium. At time of harvest, the densities of B211, B211-REV, and WTB were 3.3 X lo", 3.0 X lo", and 4.3 X 10" cells/150-cm2 flask, respectively. Preparation of secretions for assays of enzyme activity was carried out as previously described (22).

Enz.yne R211 RPll-REV WTB unitdrng cell protein

a-L-Iduronidase 0.45 0.31 0.55 P-Glucuronidase 4.22 11.7 11.7 8-Hexosaminidase 35.7 18.2 27.2 &Galactosidase 5.88 8.18 4.58

/3 Hexosaminidase

MrxlO-3 1 2 3

200 -

93 -

69- u\

P'

P- u-

46-

30 -

FIG. 1. /3"Hexosaminidase from WTB and B211. Cells were incubated for 10 h with [2-"H]mannose, and then label was chased for 14 h; electrophoresis was on SDS-polyacrylamide gels (12.58). 1,

sity at harvest, 5.2 X 10" cells/100-mm dish, 2 dishes) labeled in the precursor enzyme immunoprecipitated from secretions of WTB (den-

presence of 10 mM NH,CI; 2, processed enzyme immunoprecipitated from an extract of WTB cells (5.2 X 10" cells/100mm dish, 1 dish); 3, enzyme immunoprecipitated from secretions of B211 (3.1 X 10" cells/ 100-mm dish, 2 dishes) labeled in the presence of 10 mM NHdCI. a indicates the bands obtained when samples were denatured, then immunoprecipitated with antiserum directed against the a-poly- peptide of human P-hexosaminidase; P indicates the bands obtained when the preparations were denatured, then immunoprecipitated with antiserum to P-hexosaminidase B.

rography. Fig. 1 shows P-hexosaminidase from WTB and B211. Enzyme immunoprecipitated from parental secretions contained two radioactive polypeptides (lane 1). These were identified as the (Y and p polypept.ides of p-hexosaminidase by

Glucosylation Mutant Synthesizes Altered Lysosomal Enzymes 8427

denaturing the enzyme and then immunoprecipitating with antisera directed against the individual polypeptides. P-Hex- osaminidase from secretions of B211 (lune 3) manifested a number of radioactive polypeptides, none of which co-electro- phoresed with the polypeptides from parental secreted en- zyme (lune I ) or parental enzyme that had been proteolyti- cally processed inside the cells (lune 2). The largest poly-

a-L-lduronidase

1 2 3 Mrx10-3

-93

"69

-46

-30

FIG. 2. a-dduronidase from WTB and B211 labeled with [2- 'Hlmannose. 1, precursor enzyme immunoprecipitated from ammo- nia-induced secretions from WTB (density at harvest, 5.2 X lofi cells/ 100-mm dish, 2 dishes); 2, processed enzyme immunoprecipitated from an extract of WTB cells (5.2 X lo6 cells/lOO-mm dish, 1 dish); 3, enzyme immunoprecipitated from ammonia-induced secretions of B211 (3.1 X 10" cells/100-mm dish, 2 dishes). Note that following immunoprecipitation of a-L-iduronidase, these same samples were used for the immunoprecipitation of P-hexosaminidase shown in Fig.

peptide (a doublet) was identified as the a-polypeptide, while the other polypeptides were all identified as P, using antisera direct against the isolated a-chain or against P-hexosamini- dase B (a p-homodimer), respectively. In kinetic experiments, the several a and P polypeptides shown in Fig. 1 were found to be secreted simultaneously. Examination of newly synthe- sized, cell-associated P-hexosaminidase from B211 revealed a pattern similar to that observed with secreted enzyme. We have observed that preparations of P-hexosaminidase from B211 vary in the distribution of radioactivity among the several a and P polypeptides; this variation was found with both [:%]methionine and [2-:'H]mannose-labeled material.

Fig. 2 shows a-L-iduronidase from WTB and B211. Enzyme secreted by the parent (lune 1 ) had an M, = 82,000; lune 2 shows the intracellular proteolytically processed enzyme from WTB cells. The only band obtained from secretions of B211 (lune 3) had an M, = 180,000. This same band was obtained on immunoprecipitation of cell-associated enzyme from B211 following a short pulse (30-60 min). On pulse-chase experi- ments, no cell-associated a-L-iduronidase was detectable in B211. To determine whether this material bore any relation- ship to a-L-iduronidase, secretions from cells grown with ["S] methionine were chromatographed on Sephadex G-100 (33), and the fractions obtained were tested for enzymatic activity. a-L-Iduronidase activity from preparations of the mutant eluted in the void volume; activity from the parent eluted close to the position of bovine serum albumin. Immunoprecip- itation of the fractions exhibiting enzyme activity followed by electrophoresis and fluorography yielded radioactive polypep- tides identical with those shown in Fig. 2. Thus, the a+ iduronidase activity present in B211 secretions appears to correlate with the mannose-labeled polypeptide of Mr = 180,000. Dimerization of human urinary a-L-iduronidase under conditions of low salt (10 mM) has been reported (34). In our preparations of enzyme from secretions of B211, the salt

Pronase PhosDhatase

D N, N, N, N, N, M Endoglycosidase H

200- 1 ,~~ ! ~

2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

FIG. 3. Glycopeptides of 8-hexos- aminidase and a-L-iduronidase from WTB. Immunoprecipitates were di- gested with pronase in the presence of 20 mM sodium phosphate, then chromato- graphed on Bio-Gel P-6 columns before (A and D) or after ( B and E ) treatment with endo H. C and F, the profiles ob- tained after treatment of the immuno- precipitates with pronase (in the absence of added phosphate) plus alkaline phos- phatase followed by endo H. D, dextran; M, mannose; N, oligomers of N-acetyl- glucosamine.

Fraction Number Fraction Number

8428 Glucosylation Mutant Synthesizes Altered Lysosomal Enzymes

concentrations were 150 (dialysis) or 500 mM (gel filtration, immunoprecipitation).

Several independent preparations of ["H]mannose-labeled enzymes were employed in the following analyses of oligosac- charides. These were all monitored for homogeneity by elec- trophoresis followed by fluorography. The polypeptides ob- tained were identical with those shown in Figs. 1 and 2.

Oligosaccharide Analysis-A number of oligosaccharides were obtained from both /?-hexosaminidase and a-L-iduroni- dase secreted by WTB cells. Treatment of glycopeptides from p-hexosaminidase (Fig. 3A) with endo H (Fig. 3B) yielded a t least five oligosaccharides: HI (15% of total radioactivity) appeared resistant to endo H. HS1 and HS2 (47 and 14% of total radioactivity, respectively) eluted from Bio-Gel P-6 be- tween the standards dextran and (G1cNAc)s. HS3 (13%) and HS4 (6%) co-eluted with the (GlcNAc)4 and (GlcNAc)s stand- ards, respectively. Treatment of glycopeptides from a-L-idu- ronidase (Fig. 3 0 ) with endo H (Fig. 3E) resulted in a similar profile; however, at least 40% of the radioactivity in U-L- iduronidase glycopeptides was resistant to endo H.

Terminal mannose residues on oligosaccharides HS1 and HS2 appear to be substituted. As shown in Fig. 4A, HS1 was completely resistant to digestion with a-mannosidase. HS2 exhibited some sensitivity to treatment in that 24% of the radioactivity was released as mannose; however, the elution position of the treated oligosaccharide did not change (Fig. 4B). HS3 and HS4 (Fig. 4C) were digested to mannose and a compound that eluted at the position predicted for the disac- charide (GlcNAc)(Man).

Phosphate is present on HS1 and HS2. When immunopre- cipitated /?-hexosaminidase was treated with pronase and alkaline phosphatase (in the absence of inorganic phosphate) and the resultant glycopeptides were treated with endo H, little radioactivity was observed in the position of HS1 and HS2 (Fig. 313. Instead, about 60% of the total radioactivity eluted between the standards (G1cNAc)Z and (GlcNAch. This sequential enzymatic treatment yielded similar results with a-L-iduronidase (Fig. 3 F ) . Digestion of the dephosphory- lated oligosaccharide with a-mannosidase yielded mannose and radioactivity eluting in the expected position of the (GlcNAc)(Man) disaccharide.

We estimate the composition of most of the dephosphory- lated HS1 to be (GlcNAc)(Man)5, based on its elution position with respect to the GlcNAc homopolymers. In the gel filtra- tion system employed here, displacement of one GlcNAc

1 1 0 0

Fraction Number

FIG. 4. a-Mannosidase digestion of oligosaccharides of P- hexosaminidase from WTB. Immunoprecipitates were treated with pronase (in the presence of sodium phosphate), desalted, treated with endo H, and chromatographed on Bio-Gel P-6 (as shown in Fig. 3 8 ) . Small aliquots of the fractions were counted, and then fractions were pooled as indicated by the solid bars. The pooled fractions were desalted, treated with a-mannosidase, and then chromatographed on Bio-Gel P-6. A, oligosaccharide HS1; B , HS2; C, HS3 and HS4. Abbreviations are as in Fig. 3.

0-Hexosaminidase o-r-lduronidase

A Pronase 30 I t

FIG. 5. Glycopeptides of P-hexos- aminidase and a-L-iduronidase from B211. Immunoprecipitates were di- gested with pronase in the presence of 20 mM sodium phosphate ( A and C ) or pro- nase (in the absence of added phosphate) plus alkaline phosphatase, followed by endo H ( B and D). Glycopeptides were chromatographed on Bio-Gel P-6. Ab- breviations are as in Fig. 3.

Fractlon Number Fraction Numbel

Glucosylation Mutant Synthesizes Altered Lysosomal Enzymes 8429

residue equals two hexoses. The breadth of the peak obtained following treatment with phosphatase and endo H suggests that phosphorylated oligosaccharides may range in composi- tion from (GlcNAc) (Man)4 to (GlcNAc) (Man)s. The dramatic shift in elution position following dephosphorylation is con- sistent with the sensitivity of this chromatographic system to charge. For example, mannose 6-phosphate elutes in the po- sition of (G1cNAc)s.

Oligosaccharides from enzymes secreted by the mutant €3211 were not affected by either phosphatase or endo H. Fig. 5 shows the glycopeptides generated from P-hexosaminidase ( A ) and a-L-iduronidase (C) after digestion with pronase in the presence of inorganic phosphate. Essentially identical profiies were obtained after treatment of these enzymes with pronase plus alkaline phosphatase followed by endo H (Fig. 5, B and D ) . Less than 5% of the radioactivity was found to elute in the position of dephosphorylated HSl.

The oligosaccharides on &hexosaminidase and a-L-iduron- idase from B211 appear to be of the complex type. Treatment of glycopeptides, generated in the presence of inorganic phos- phate, with neuraminidase resulted in a shift of the elution profiies (Fig. 6). Further sequential treatments of these gly- copeptides with &galactosidase, P-N-acetylglucosaminidase, endoglycosidase D, and a-mannosidase (present at low levels in the P-galactosidase preparation) resulted in the release of 60 to 80% of the radioactivity as mannose plus a compound that eluted in the position predicted for the trisaccharide (GlcNAc) (Man)z. Treatment with neuraminidase, P-galacto- sidase, and a-mannosidase without treatment with /?-N-ace- tylglucosaminidase resulted in no release of mannose and had little effect on the elution profiles of p-hexosaminidase and

a-L-iduronidase beyond that obtained with neuraminidase alone (Fig. 6).

A summary of our analyses of the oligosaccharides of p- hexosaminidase and a-L-iduronidase from WTB and B211 is presented in Table 111.

P-GlcNAc Transferase Activity-Secretion of acid hydro- lases with oligosaccharides lacking or deficient in phosphate has previously been reported for fibroblasts from patients with mucolipidosis I1 (11, 16). These patients have an inher- ited deficiency in UDP-N-acetylglucosamine: glycoprotein N - acetylglucosamine-I-phosphotransferase, the enzyme that catalyzes the fist step in the formation of the mannose 6- phosphate recognition marker (35, 36). However, B211 ap- pears to contain an active P-GlcNAc transferase. As shown in Fig. 7, particulate fractions prepared from B211 and WTB cells transferred radioactivity from [P-3ZP]UDP-GlcNAc to exogenously added /3-hexosaminidase B.

Internalization and Compartmentalization of Enzymes- Since uptake of acid hydrolases into human fibroblasts and

TABLE I11 Summary of oligosaccharide structure

Distribution of [ 'Hlmannosyl peptides

Cell Enzyme Endo H-re- Endo H-sensitive

sistant Phospho- Nonphos- rylated phorylated

% % WTB /-Hexosaminidase 14 65 21 WTB a-L-Iduronidase 50 37 13 B211 /-Hexosaminidase >95 <5 <I B211 a-t-Iduronidase >99 <I <1

P-Hexosaminidase

A a-1-lduronidase

Pronase C 140 - - 70

N4N3 Nz NI M t t t t t

-60

-50

-40

N6N5N4N3 Nz NI M t o t t t 4 -30

- 20

- 10 8 a

2

Neuraminidase. F O f

h n. 9

E 0

s 140- I

1 -70 ' Pronase

- -60

I -50

- -40 N4N3 N2 NI M i t t t t N6N5N4N3 N2 N1 M

t t t t i t t -30

40- -m

- 10

Fraction Number Fraction Number

FIG. 6. Neuraminidase digestion of glycopeptides of P-hexosamini- dase and a-L-iduronidase from B211. Immunoprecipitates were treated with pronase (in the presence of 20 mM so- dium phosphate). Glycopeptides were desalted and chromatographed on Bio- Gel P-6 before (A and C ) or after ( B and D) treatment with neuraminidase, Ab- breviations are as in Fig. 3.

8430 Glucosylation Mutant Synthesizes Altered Lysosomal Enzymes

CHO cells is dependent on the presence of mannose 6-phos- phate on those enzymes (12-14, 23), /?-hexosaminidase and a-L-iduronidase secreted by B211 should be of "low uptake" form. Indeed, no uptake of a-L-iduronidase was observed when growth medium conditioned by R211 was added to human fibroblasts lacking that enzyme (Table IV). Uptake was ob- served when recipient cells were incubated with media con- ditioned by WTB or B211-REV (Table IV). Results identical with those in Table IV were obtained when secretions were prepared under the conditions employed in the preceding analysis of oligosaccharides, ( i e . growth of the cells in medium containing 10 mM NH4Cl, 15 mM N-[2-hydroxy-l,l-bis(hy- droxymethyl)ethyl]glycine, and 0.14 mM glucose). To ascer- tain that enzyme secreted by B211 was not taken up, then rapidly degraded, secretions from B211 grown with ["S]me- thionine were added to recipient cells. All radioactive /3-hex-

B211 WT - Hex B + - - +

Mr x 10-3

93 - 69-

46-

30-

14 -

FIG. 7. P-GlcNAc transferase activity in membranes from B211 and WTB. Membranes of B211 (561 pg of protein/assay) and WTB (879 pg of protein/assay) were incubated with [8-'*P]UDP- GlcNAc in the presence (Hex B+) or absence (Hex B- ) of human placental P-hexosaminidase B (5 pg/assay). Following clarification by centrifugation, P-hexosaminidase B (5 pg) was added to the Hex B- samples, enzyme was immunoprecipitated, and the immunoprecipi- tates were electrophoresed on an SDS-polyacrylamide gel. After autoradiography, the labeled bands were excised from the gel and counted B211,102 cpm; WTB, 68 cpm.

TABLE IV Uptake of a-L-iduronidase from B211, B211-REV, and WTB into

human fibroblasts Media were conditioned for 48 h by B211, B211-REV, and WTB

(density at harvest, 6.1 X lo", 6.8 X IO6, and 8.2 X 10" cells/150-cm2 flask, respectively). Conditioned media from 3 flasks of each cell type were concentrated, dialyzed, and assayed for enzymatic activity as previously described (22). These secretions were each added to two 100-mm dishes of a-L-iduronidase-deficient human fibroblasts; after 24 h, the recipient cells were harvested (0.48-0.56 mg of cell protein/ 100-mm dish) and enzyme activity was determined. Recipient cells contained 0.055 unit of a-L-iduronidase/mg of protein, and the values reported have been corrected for this residual activity.

Source of secretions a-L-Iduronidase activity

Added Received units units/mgprotein

B211 1.52 0.001

WTB 2.18 0.393 B211-REV 1.52 0.235

osaminidase and a-L-iduronidase was recovered from the me- dium.

Uptake of normal acid hydrolases into B211 is not impaired. Following incubation of the mutant with human urinary a-L- iduronidase or bovine testicular /3-galactosidase, the enzyme activity in the recipient B211 cells was increased to the same degree as in control cells. In addition, uptake of labeled p- hexosaminidase and a-L-iduronidase from ammonia-induced secretions of WTB cells grown with ["'SS]methionine was the same in B211 and WTB cells. In all of these experiments, uptake of enzyme into B211 was inhibited by 290% in the presence of 2 mM mannose 6-phosphate.

Interaction between the mannose 6-phosphate receptor and the recognition marker appears to be necessary for proper compartmentalization of endogenous hydrolases (15, 22, 37). Fractionation of cell-free extracts of B211 on gradients of colloidal silica (28, 38) resulted in sedimentation of acid hy- drolase activity (P-hexosaminidase, /3-galactosidase, and acid phosphatase) in a single light peak that co-sedimented with the Golgi and GERL fractions. Fractionation of extracts of WTB and B211-REV resulted in the usual bimodal distribu- tion of acid hydrolase activity (28,38), with a peak of activity corresponding to residual bodies as well as the lighter peak observed with B211.

DISCUSSION

We have shown that B211, a mutant deficient in glucosyl- ation of lipid-linked oligosaccharide (7), synthesizes acid hy- drolases of altered structure and function. While P-hexosa- minidase and a-L-iduronidase from parental cells contain phosphorylated, high mannose oligosaccharides, enzymes from B211 contain only oligosaccharides of the complex type. Hydrolases from B211 were not internalized by other fibro- blasts and appear to be compartmentalized aberrantly within the mutant. Both of these phenomena probably reflect ab- sence of the mannose 6-phosphate recognition marker from the acid hydrolases, and the consequent inability of these hydrolases to interact with the mannose 6-phosphate receptor.

All of the phosphate residues on oligosaccharides of p- hexosaminidase and a-L-iduronidase secreted by wild type CHO cells appeared to be in monoester linkage. This is in contrast to results obtained with other cell types in which some phosphates were found to persist in the blocked, diester form (39, 40). While loss of the blocking group could have occurred during preparation of the CHO acid hydrolases, this seems unlikely; hydrolases maintained in the presence of 5 mM GlcNAc, a potent inhibitor of the diesterase (41), during labeling of the cells, preparation of the secretions, immuno- precipitation of the enzymes, and preparation of the glycopep- tides contained oligosaccharides with phosphates exclusively in monoester linkage, identical with those shown above. Thus, it seems more likely that the absence of phosphodiesters from the secreted CHO cell hydrolases reflects the efficiency of the phosphodiesterase in those cells.

The a and B polypeptides of 8-hexosaminidase from B211 were found to be heterogeneous after electrophoresis on SDS- polyacrylamide gels. The differences observed between the several a and /3 chains of B211 and the corresponding poly- peptides from the parent are such that they could reflect different degrees of glycosylation (42) or sialylation (43). The difference between a-L-iduronidase from mutant and parent is much more extreme; the M, of enzyme isolated from B211 is approximately twice that of a-L-iduronidase from wild type cells. No such polypeptide has been obtained from parental cells even after very short labeling periods;' thus, it is unlikely

A. R. Robbins, unpublished data.

Glucosylation Mutant Synthesizes Altered Lysosomal Enzymes 843 1

that a-L-iduronidase as isolated from B21l represents a nor- mal precursor prior to proteolytic cleavage. The absence of glucose from the oligosaccharides of newly synthesized a - ~ - iduronidase in B211 may lead to dimerization of the enzyme or to formation of a complex with heterologous protein; the homo- or heteropolymer formed must be stable under the reducing and denaturing conditions of electrophoresis.

Of the mutants described to date, B211 is unique in that it fails to phosphorylate at least two of its acid hydrolases, yet it contains an enzymatic activity capable of phosphorylating exogenously added /?-hexosaminidase. Since B211 transfers oligosaccharides to its acid hydrolases, why is it unable to phosphorylate them? 1) The transfer of GlcNAc-1-P in vivo is to oligosaccharides that retain at least one glucose residue. The earliest detectable phosphorylated oligosaccharide on p- glucuronidase from a macrophage cell line contained no glu- cose (40); indeed, it appeared that glucose was removed more rapidly from p-glucuronidase than from other glycoproteins. 2) Glucose protects some structural feature of the oligosac- charide that is required for phosphorylation en route to the P-GlcNAc transferase, e.g. a-mannosidase action on the a 1-3 branch is prevented by glucose. Interestingly, secretion of phosphorylated acid hydrolases was demonstrated in a gly- cosylation mutant that transfers glucosylated pentamannosyl oligosaccharides in which only the a 1-3 branch is intact (44). 3) Phosphorylation occurs in a subcellular compartment from which the acid hydrolases of B211 are excluded. This could result either from the absence of glucose from the hydrolases themselves or, since glycosylation deficiencies are perforce pleiotropic, from alteration in the function of some unrelated protein.

Like acid hydrolases secreted by human fibroblasts deficient in the P-GlcNAc transferase (I cells) (45,46), acid hydrolases from B211 contain complex oligosaccharides rather than sim- ple, high mannose structures. It appears that, in the absence of phosphorylation, oligosaccharides on acid hydrolases are subject to further modification. This suggests that in normal cells phosphorylation occurs prior to exposure of the hydro- lases to the Golgi-associated mannosidase and glycosyl trans- ferases. B211 differs from human I cells not only in that it manifests P-GlcNAc transferase activity, but also in that total (secreted plus cellular) acid hydrolase activity is diminished in B211. Our results suggest that this may reflect a decrease in both the stability and specific activity of hydrolases from B211.

Acknowledgments-We thank David Donovan and Helen Lei for their skillful technical assistance, and Dr. Elizabeth F. Neufeld for her careful consideration of this manuscript.

REFERENCES 1. Hubbard, S. C., and Ivatt, R. J. (1981) Annu. Reu. Biochem. 50,

2. Phelps, C. F. (1980) in The Enzymology of Post-Translational Modification of Proteins, (Freedman, R. B., and Hawkins, H. C., eds) Vol. 1, pp. 105-155, Academic Press, New York

3. Struck, D. K., and Lennarz, W. J. (1980) in The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, W. J., ed) pp. 35-83, Plenum Press, New York

4. Turco, S. J., Stetson, B., and Robbins, P. W. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,4411-4414

5. Spiro, M. J., Spiro, R. G., and Bhoyroo, V. D. (1979) J. Biol. Chem. 254, 7668-7674

6. Cifone, M. A., Hynes, R. O., and Baker, R. M. (1979) J. Cell. Physiol. 100, 39-54

7. Krag, S. S. (1979) J. Biol. Chem. 254, 9167-9177 8. Von Figura, K., and Klein, U. (1979) Eur. J. Biochem. 94,347-354

555-583

9.

10.

11.

12.

13. 14.

15.

16.

17. 18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41. 42.

Distler, J., Hieber, V., Sahagian, G. G., Schmickel, R., and Jour- dian, G. W. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,4235-4239

Natowicz, M. R., Chi, M. M.-Y., Lowry, O.H., and Sly, W. S. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,4322-4326

Hasilik, A,, and Neufeld, E. F. (1980) J. Biol. Chem. 255, 4946-4950

Kaplan, A,, Achord, D. T., and Sly, W. S. (1977) Proc. Natl. Acad. Sci. U. S. A . 74,2026-2030

Sando, G. N., and Neufeld, E. F. (1977) Cell 12, 619-627 Ullrich, K., Mersmann, G., Weber, E., and von Figura, K. (1978)

Hickman, S., and Neufeld, E. F. (1972) Biochem. Biophys. Res.

Bach, G., Bargal, R., and Cantz, M. (1979) Biochem. Biophys.

Weissrnann, B. (1978) Methods Enzymol. 50, 141-150 Owada, M., and Neufeld, E. F. (1982) Biochem. Biophys. Res.

Rome, L. H., Garvin, A. H., and Neufeld, E. F. (1978) Arch.

Hasilik, A., and Neufeld, E. F. (1980) J. Biol. Chem. 255,

Cantz, M., Kresse, H., Barton, R., and Neufeld, E. F. (1972)

Robbins, A. R., and Myerowitz, R. (1981) J. Biol. Chem. 256,

Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622

Gorham, L. W., and Wayrnouth, C. (1975) Proc. SOC. Exp. Biol. Med. 119,287-290

Becker, B. G., and Levine, E. M. (1976) in Tissue Culture Asso- ciation Manual (Evans, V. J., Perry, V. P., and Vincent, M. M.,

MD eds) Vol. 2, pp. 305-308, Tissue Culture Association, Rockville,

Bonner, W. M., and Laskey, R. A. (1974) Eur. J. Biochem. 46,

Rome, L. H., Weissmann, B., and Neufeld, E. F. (1979) Proc.

Robbins, A. R. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,

Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

Krag, S. S., and Robbins, P. W. (1977) J . Biol. Chem. 252,

Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli,

Gonzalez-Noriega, A., Grubb, J . H., Talkad, V., and Sly, W. S.

Shapiro, L. J., Hall, C. W., Leder, I. G., and Neufeld, E. F. (1976)

Barton, R. W., and Neufeld, E. F. (1971) J. Biol. Chem. 246,

Hasilik, A., Waheed, A., and von Figura, K. (1981) Biochem.

Reitman, A. L., Varki, A., and Kornfeld, S. (1981) J. Clin. Inuest.

Fischer, H. D., Gonzalez-Noriega, A,, and Sly, W. S. (1980) J.

Rome, L. H., Garvin, A. J., Allietta, M. M., and Neufeld, E. F.

Hasilik, A., Klein, U., Waheed, A., Strecker, G., and von Figura,

Goldberg, D. E., and Kornfeld, S. (1981) J. Biol. Chem. 256,

Varki, A., and Kornfeld, S. (1980) J. Biol. Chem. 255, 8398-8401 Robbins, P. W., Hubbard, S. C., Turco, S. J., and Wirth, D. F.

Biochem. J. 170,643-650

Commun. 91,976-981

Res. Commun. 91,976-981

Commun. 105,814-820

Biochem. Biophys. 189,344-353

4937-4945

Methods Enzymol. 28,884-897

10623-10627

83-88

Natl. Acad. Sci. U. S. A. 74,2026-2030

1911-1915

(1951) J. Biol. Chem. 193,265-275

2621-2629

U. K. (1977) J. Biol. Chem. 252, 1102-1106

(1980) J. Cell Biol. 85, 839-852

Arch. Biochem. Biophys. 172, 156-161

7773-7779

Biophys. Res. Commun. 98,761-767

67, 1574-1579

Biol. Chem. 255,5069-5074

(1979) Cell 17, 143-153

K. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7074-7078

13060-13067

(1977) Cell 12.893-900 43. Segrest, J. P., Jackson, R. L., Andrews, E. P., and Marchesi, V. T.

(1971) Biochem. Biophys. Res. Commun. 44, 390-395 44. Stoll, J., Robbins, A. R., and Krag, S. S . (1982) Proc. Natl. Acad.

Sci. U. S. A. 79,2296-2300 45. Vladutiu, G. D., and Ratazzi, M. (1975) Biochem. Biophys. Res.

Commun. 67,956-964 46. Miller, A. L., Kress, B. C., Stein, R., Kinnon, C., Kern, H.,

Schneider, J. A., and Harms, E. (1981) J. Biol. Chem. 256, 9352-9362