THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 20, Issue of July 15, pp. 11864-11871,199O Printed in U.S.A.

Protein Determinants Impair Recognition of Procathepsin L Phosphorylated Oligosaccharides by the Cation-independent Mannose 6-Phosphate Receptor*

(Received for publication, December 28, 1989)

Deborah LazzarinoS and Christopher A. GabelO From the Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

Cathepsin L, a lysosomal cysteine protease, is the major excreted protein of transformed mouse NIH 3T3 cells. Previous studies have shown that asparagine- linked oligosaccharides associated with the secreted hydrolase contain mannose 6-phosphate (Man 6-P), the recognition marker for transport of newly synthesized acid hydrolases to lysosomes. To investigate the mech- anism by which cathepsin L evades targeting to lyso- somes, we determined the structure of the enzyme’s oligosaccharides and analyzed its interaction with the cation-independent mannose 6-phosphate (Man 6-P”) receptor. Oligosaccharides associated with procathep- sin L isolated from the medium of [3H]mannose-labeled 5774 cells were remarkably homogeneous; all of the radiolabeled structures were high mannose-type units that contained two phosphomonoesters and 7 mannose residues. Both the a1,3- and al,6-branches of the oli- gosaccharides were phosphorylated. Oligosaccharides released by endoglycosidase H from [3H]mannose-la- beled procathepsin L bound to a Man 6-P” receptor affinity column. Despite the high affinity binding of these oligosaccharides, the intact glycoprotein was not a good ligand for the Man 6-PC’ receptor. Procathepsin L was internalized poorly by Man 6-P receptor-me- diated endocytosis and the purified acid protease inter- acted weakly with a Man 6-P” affinity column. In contrast, pro-@-glucuronidase (another acid hydrolase produced by 5774 cells) was an excellent ligand for the Man 6-P” receptor as judged by the endocytosis and affinity chromatographic assays. Phosphorylated oli- gosaccharides associated with the J774-secreted pro- /%glucuronidase were heterogeneous and contained both mono- and diphosphorylated species. Tryptic gly- copeptides generated from [3H]mannose-labeled pro- cathepsin L, unlike the intact protein, were excellent ligands for the Man 6-P” receptor. The results indicate that oligosaccharides associated with procathepsin L are processed uniformly to diphosphorylated species that bind with high affinity to the Man 6-P” receptor. Protein determinants inherent within the intact acid hydrolase, however, inhibit the high affinity binding of these oligosaccharides and, as a result, impair the interaction of procathepsin L with the receptor.

* This work was supported in part by Grant GM33342 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 “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported in part by a predoctoral training grant sponsored by the Cancer Institute at Columbia University.

§ To whom correspondence should be addressed: Dept. of Immu- nology and Infectious Diseases, Pfizer Central Research, Eastern Point Rd., Groton, CT 06340.

Soluble acid hydrolases are delivered to lysosomes as a result of the posttranslational formation of a specific mannose 6-phosphate (Man 6-P)’ recognition marker. The marker is synthesized in two steps that involve 1) the transfer of N- acetylglucosamine l-phosphate to the C-6 position of an a1,2- linked mannose residue within a high mannose-type unit and 2) the selective removal of the IV-acetylglucosamine residue to yield a phosphomonoester moiety (l-3). The phosphoryl- ated oligosaccharides allow the newly synthesized lysosomal enzymes to bind to a Golgi-associated Man 6-P receptor and to be delivered to lysosomes (l-4). Phosphorylated oligosac- charides are heterogeneous with respect to the number of phosphates/individual high mannose-type unit and the posi- tion of the phosphorylated mannose residue within the oligo- saccharide (5,6). The mechanism by which this heterogeneity evolves is unknown. Since the various phosphorylated species possess different affinities for the Man 6-P receptors, the oligosaccharide diversity may influence transport of acid hy- drolases to lysosomes (7-10). Previous studies have indicated that acid hydrolases, as a group, contain a common protein- dependent structural element that enables these proteins to be selectively phosphorylated by the lysosomal enzyme:UDP- IV-acetylglucosaminyl phosphotransferase (11-13). Factors that govern the positioning of the phosphate within the oligo- saccharide, however, remain largely unknown. The assembly process occurs in a highly ordered series of reactions that result, in part, from the compartmentalization of the process- ing enzymes to distinct regions of the vacuolar apparatus (14). Moreover, removal of mannose residues from the precursor oligosaccharide by Golgi-associated oc-mannosidases is an im- portant determinant in the maturation process (15).

Cathepsin L, a cysteine protease, is the major excreted protein produced by transformed mouse cell lines (16-19). The synthesis and secretion of the proform of cathepsin L (procathepsin L) are increased by viral transformation and by treatment of cells with phorbol esters and certain growth factors (19-22). The secreted procathepsin L molecules, how- ever, contain the Man 6-P recognition marker (23), and in untransformed cells the hydrolase is delivered efficiently to lysosomes (24, 25). The mechanism by which procathepsin L evades transport to lysosomes within transformed cells is unknown. Binding studies between crude preparations of pro- cathepsin L and the cation-independent Man 6-P (Man 6- PC’) receptor have demonstrated that the acid hydrolase has a lower affinity for the receptor than other acid hydrolases

1 The abbreviations used are: Man 6-P, mannose B-phosphate; Man 6-P”, cation-independent mannose 6-phosphate; Hepes, 4-(2-hydrox- yethyl)-l-piperazineethanesulfonic acid; ConA, concanavalin A, endo H; endo-P-N-acetylglucosaminidase; HPLC, high pressure liquid chromatography; SDS, sodium dodecyl sulfate.

11864

by guest on August 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cathepsin L Phosphorylation

(25). The lower affinity, which may result from modulator proteins or from an inherent property of cathepsin L, is believed to be responsible for hypersecretion of the enzyme (25).

In this paper we analyze the structure of cathepsin L- phosphorylated oligosaccharides to determine if the number and/or position of the Man 6-P residues within high mannose- type units account for the poor interaction of the enzyme with the Man 6-P receptor. Surprisingly, we report that the oligo- saccharides associated with procathepsin L produced by 5774 cells are processed consistently to diphosphorylated structures that bind with high affinity to the Man 6-P” receptor. Despite the presence of the high affinity carbohydrate side chains, intact procathepsin L is a poor ligand. The results indicate that protein determinants associated with procathepsin L direct the processing of its asparagine-linked oligosaccharides and impair recognition of these phosphorylated oligosaccha- rides by the Man 6-P” receptor.

MATERIALS AND METHODS

Cells and Metabolic Labeling-The murine macrophage-like cell line 5774 (26, 27) and the Man 6-P” receptor-positive L-cell line (28, 29) were grown in a-minimal essential medium supplemented with 10% new-born calf serum (GIBCO), penicillin (100 units/ml), strep- tomvcin (100 mn/ml). and 10 mM Henes. DH 7.0. 5774 cells were plated at a den&y bf 1 x lo5 cellsjcn?-and allowed to attach overnight. The cells then were incubated for 20-24 h with 0.5-l mCi/ ml of [2-3H]mannose (American Radiolabeled Chemicals Inc., St. Louis, MO; lo-15 Ci/mmol) in a minimal volume of glucose-free (Y- minimal essential medium containing 1 mM glucose and 1% new- born calf serum; the serum was omitted when [3H]procathepsin L was purified from the secretory products. The labeling media were harvested, clarified by centrifugation, and used as a source of secreted procathepsin L and pro-@-glucuronidase.

Inmunopreci~itation-Media samples containing radiolabeled se- creted hydrolases were adjusted (by the addition of concentrated stock solutions) to 20 mM Hepes, pH 7,0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM l,lO-phenanthroline, and 1% Triton X-100. In a typical experiment, 6 ml of culture supernatant were precleared with 0.2 ml of a 10% suspension of Staphylococcus aureus (Pansorbin, Calbiochem). After a 30-min incubation on ice, the bacteria were removed by centrifugation and 0.01 ml of rabbit anti-cathepsin L serum (kindly provided by Michael M. Gottesman of the National Cancer Institute, Bethesda, MD) was added to the supernatant. Immune complexes were allowed to form at 4 “C for 3 h after which they were recovered by adsorption to 0.2 ml of 10% Pansorbin. The bacteria were collected by centrifugation after a 30-min incubation on ice and washed five times with 25 mM Hepes, pH 7, 0.1 M NaCl, 1% Triton X-100.0.2% deoxvcholate. 5 mM Plucose 6-nhosnhate. and once with 50 mM ?‘ris, pH 6.8: The final bacterial pellet’was’suspended in 0.1 ml of disaggregation buffer (23, 30) and boiled for 3 min; the bacteria subsequently were removed by centrifugation and discarded. &Glucuronidase was immunoprecipitated from the cathepsin L-de- pleted supernatants using 0.004 ml of rabbit anti-rat preputial p- glucuronidase serum as described above. The immunoprecipitates were fractionated on 10% polyacrylamide gels, and the 3H-labeled polypeptides were located by fluorography after impregnation of the gel with Amplify (Amersham Corp.).

Isolation and Characterization of Phosphor&ted Oligosaccha- rides-The regions of the dried gels containing radiolabeled proca- thepsin L and pro-@-glucuronidase were excised, and the radioactivity was solubilized by Pronase digestion (29). Glycopeptides were chro- matographed on concanavalin A (ConA)-Sepharose to separate tri- and tetraantennary glycopeptides, biantennary structures, and high mannose-type units as detailed previously (31). VHlOligosaccharides were released from the high m&nose-&e gly&pe&id;?s by endo-p- N-acetylglucosaminidase H (endo H) digestion and the released units were fractionated on QAE-Sephadex columns as described (9). Purity analysis of the suspected ‘H-labeled nhosnhorvlated oligosaccharides was -performed as- detailed previouily (5, 32j. Details of the high performance liquid chromatographic (HPLC) separation of high man- nose oligosaccharides and the hydrolytic cleavage of oll,6-linkages by the acetolysis procedure are provided elsewhere (5, 33, 34).

Receptor-mediated Endocytosis-[[3H]Mannose-labeled 5774 cul-

ture supernatants were adjusted to 40 mM Hepes, pH 7, and to a final concentration of 70% saturation by the addition of solid ammonium sulfate. After stirring for 30 min on ice, precipitated proteins were collected by centrifugation, resuspended in a minimum volume of 10 mM Hepes, pH 7,150 mM NaCl, and dialyzed against the same buffer. Mouse L-cells (nearly confluent on a lo-cm dish) were incubated with 5 x lo5 cpm of the dialyzed polypeptides (containing 11 munits of @- glucuronidase) in the absence or presence of 5 mM Man 6-P; the polypeptides were filter sterilized before addition to the cells. After a 6-h incubation at 37 “C, the post-uptake supernatant was removed and procathepsin L and pro-fi-glucuronidase were recovered by im- munoprecipitation. The immunoprecipitates were analyzed by SDS- gel electrophoresis and autoradiography; the radioactive regions cor- responding to procathepsin L and pro-@glucuronidase were excised from the dried gels, and the radioactivity was solubilized directly into scintillation vials by Pronase digestion.

Purification of 5774 Procathepsin L and Pro-/?-glucuronidase-3 liters of serum-free growth medium from suspension cultures of 5774 cells was prepared as described previously (36). Secretory products were conc&trated in a Minitan Eoncentraior (Millipore Continental Water Svstems, Bedford, MA) after which thev were dialvzed against 20 mM -Tris, PH 8, 5 mM P-glycerol phosphate (buffer A): The dialysate was applied to a DE52 cellulose column (2.5 X 10 cm) equilibrated in buffer A. The column was washed with 100 ml of buffer A, and bound proteins then were eluted with a continuous gradient from 0 to 0.5 M NaCl in buffer A (120 ml of each) at a flow rate of 40 ml/h, 2.5-ml fractions were collected and assayed for cathepsin L and /3-glucuronidase. Cathepsin L was assayed with the svnthetic substrate Z-Phe-Arg-7AMC (Cambridge Research Bio- chemicals Limited, Cambridge, United England) as described previ- ously (37), and p-glucuronidase was assayed with p-NC&-phenyl-P-D- glucuronide (38). The two activities eluted as separate peaks from the ion-exchange column, and peak fractions were pooled separately. fi- Glucuronidase was purified further as described previously (36). The cathepsin L sample was concentrated to a final volume of 2-5 ml in a Centriprep 10 filter (Amicon Division of W. R. Grace & Co., Danvers, MA) and was applied to a Sephacryl S-200 column (1.6 X 46 cm) equilibrated in buffer A; the column was eluted at a flow rate of 8 ml/h, and 30 drop fractions were collected. Cathepsin L activity eluted as a symmetrical peak near the included volume of the column. Peak fractions were pooled and rechromatographed on a DE52 cel- lulose column (0.7 X 17 cm) equilibrated in buffer A; bound proteins were eluted with a linear gradient of NaCl from 0 to 300 mM in buffer A (110 ml of each). The cathepsin L activity bound and eluted as a sharp symmetrical peak; peak fractions were pooled and dialyzed against buffer A containing 50 mM NaCl. The protein concentration of the isolated hydrolases-was determined us&g the BCA reagent (Pierce Chemical Co.). The purification protocol yielded 0.15-0.3 mg of cathepsin L. Coomassie-stained gels of the purified cathepsin L preparation showed a single polypeptide that &grated with an ap- parent molecular mass of 35 kDa. [3H]Mannose-labeled procathepsin L also was isolated by this same purification method. For this purpose, four 15-cm plates of 5774 cells were labeled overnight with [2-3H] mannose as described above. The labeling media was harvested and combined with 3 liters of conditioned growth media isolated from suspension cultures of nonradiolabeled 5774 cells, and the enzyme was concentrated and purified as detailed above.

Man S-Pc’ Receptor Affinity Chromatography-The Man 6-P” receptor was isolated from either bovine liver acetone powder (Sigma) or bovine fetal livers (Pelfreeze; Browndeer, WI) as described previ- ously (9). The purified receptor (1 mg) was coupled to 10 ml 0; Affi- Gel 10 or Affi-Prep 10 (Bio-Rad) as described (9). and the coniueate was used as an affinity column. Purified procathepsin L (25 j& and pro-@-glucuronidase (16 pg) were labeled with Na’? (0.1 Ci/mmol; Amersham Corp.) using the chloramine-T method (35). Free lz51 was separated from the radiolabeled proteins using a lo-ml Sephadex G- 25 column equilibrated in 50 mM phosphate, pH 7.5, 1 mg/ml oval- bumin, 2 mg/ml potassium iodide. Excluded iodinated proteins were dialyzed against 20 mM Tris, pH 7, 150 mM NaCl, 5 mM P-glycerol phosphate, and stored on ice. To assess binding to the receptor, the ‘2SI-labeled polypeptides (2 X lo6 cpm) or [3H]oligosaccharides (5000 cpm) were diluted with 0.5 ml of column buffer (50 mM phosphate, pH 7, 150 mM NaCl, 5 mM P-glycerol phosphate, 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 fig/ml ovalbumin, and 0.05% Triton X-100) and loaded onto the affinity column pre-equilibrated in the same buffer at 4 “C. The column was washed with 3-4 column volumes of column buffer and eluted with 2 mM Man 6-P in column buffer; the flow rate was maintained at 8 ml/h and 0.5-l-ml fractions

by guest on August 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

11866 Cathepsin L Phosphmylation

were collected. In the case of the ‘251-laheled hydrolases, peak fractions were pooled, and the radiolaheled polypeptides were precipitated with trichloroacetic acid. The precipitates were collected by centrifugation, washed with cold acetone, then dissolved in sample buffer for analysis on SDS gels (30). “‘I-Labeled proteins were visualized by autoradi- ography using Lightening Plus intensifier screens (Du Pont).

Reduction and Alkylation-Radiolabeled purified procathepsin L was diluted with 0.5 ml of 0.64 M Tris, pH 8.8, 4 mM EDTA, 17 mM Man 6-P, containing 0.14 M P-mercaptoethanol. 0.28 g of urea were added, the sample was flushed with Ne, then incubated at 20 “C for 4 h. At the end of the reduction, the solution was adjusted to 1.4 M iodoacetate, and the reaction mixture was incubated for an additional 15 min. The sample subsequently was dialyzed against either column buffer or 50 mM @-glycerol phosphate, pH 7.2, 2 mM EDTA, 1 mM dithiothreitol (trypsin buffer). The dialyzed samples were clarified by centrifugation; >50% of the starting radioactivity was recovered routinely with the reduced and alkylated polypeptides.

Trypsin Digestion-l.2 X lo6 cpm of intact or reduced and alkylated ‘251-labeled procathepsin L (estimated to be 0.3 pg) in trypsin buffer were incubated with 9 pg of trypsin (Boehringer Mannheim) at 37 ‘C in a total volume of 0.3 ml. At the indicated times, 0.05-ml aliquots of each digest were removed and diluted with an equal volume of cold trypsin buffer containing 5 rg of soybean trypsin inhibitor. After 30 min on ice each sample received 0.01 ml of a mixture of bovine serum albumin (1 mg/ml), hemoglobin (2 mg/ml), and ovalbumin (1 mg/ ml) followed by 1.1 ml of cold acetone. This solution was incubated on ice for 30 min, after which the precipitated proteins were collected by centrifugation and disaggregated for SDS-gel electrophoresis.

FraCIiOn

To isolate tryptic [“Hlglycopeptides, reduced and alkylated [3H] mannose-labeled procathepsin L (28 rg) was incubated in 1 ml of trypsin buffer containing 50 mM Man 6-P and 1 mg of trypsin (pretreated with 5 mM N-tosyl-L-phenylalanine chloromethyl ketone, Boehringer Mannheim) at 37 “C. In pilot experiments the trypsin treatment resulted in a substantial dephosphorylation of the high mannose-type oligosaccharides. Therefore, Man 6-P was included as a competitive substrate for the contaminating phosphatase activity. After 6 h 5 mg of soybean trypsin inhibitor were added to stop the reaction, and the mixture was chromatographed on ConA-Sepharose to isolate high mannose-type [3H]glycopeptides (31). The tryptic glycopeptides were desalted by Sephadex G-25 chromatography then fractionated on the Man 6-P” receptor affinity column.

RESULTS

Procathepsin L Contains a Uniform Diphosphmylated High Mannose-type Oligosaccharide-To determine the structure of the phosphorylated oligosaccharides associated with ca- thepsin L, the acid hydrolase was isolated from the growth medium of mouse 5774 cells. These macrophage-like cells secrete 60-70% of their newly synthesized acid hydrolases (15, 26, 27), and they produce high levels of cathepsin L (16). 5774 cells were cultured in the presence of [2-3H]mannose, after which secreted cathepsin L was recovered from the medium by immunoprecipitation. The major radiolabeled spe- cies detected in the immunoprecipitate by SDS-gel electro- phoresis and radioautography had an apparent molecular mass of 35,000 daltons (not shown); this is the expected mass of the proform of the protease (16,23).

FIG. 1. Characterization of procathepsin L and pro-&glu- curonidase oligosaccharides. 5774 cells were metabolically labeled for 22 h with [2-3H]mannose, after which procathepsin L and pro+ glucuronidase were immunoprecipitated from the medium and puri- fied by SDS-gel electrophoresis. The radioactive regions of the gel were excised and the radioactivity was solubilized by Pronase diges- tion. A, the [3H]glycopeptides derived from procathepsin L were fractionated on ConA-Sepharose; the column was eluted sequentially with 10 mM ol-methylglucoside ((Y-MG) and 100 mM cu-methylman- noside (a-MM) as denoted by the arrowheads. B, the high mannose- type glycopeptides (fractions 11-14 in parwE A) were digested with endo H, and the released oligosaccharides were chromatographed on QAE-Sephadex. The elution positions of the neutral high mannose oligosaccharides (a) and units containing one phosphodiester (b), one phosphomonoester (c), one phosphomonoester and one sialic acid residue (d), and two phosphomonoesters (e) are indicated. C, the cathepsin L oligosaccharides containing two phosphomonoesters were treated with alkaline phosphatase, and the dephosphorylated units were analyzed by HPLC. The arrowhe& denote the elution posi- tions of Man,GlcNAc (5), MansGlcNAc (6), Man,GlcNAc (7), MansGlcNAc (8), and Mam,GlcNAc (9). D, the high mannose-type oligosaccharides recovered from pro-/3-glucuronidase also were frac- tionated on QAE-Sephadex. The elution position of the radiolabeled species can be compared with the phosphorylated standards shown in panel B.

Glycopeptides generated by Pronase digestion of the radio- active region of the gel bound to ConA-Sepharose and eluted as expected for high mannose-type units (Fig. L4). These high mannose-type glycopeptides were digested with endo H to separate the oligosaccharides from their peptide counterparts, and the released units subsequently were fractionated by QAE-Sephadex chromatography. Phosphorylated high man- nose-type oligosaccharides bind to the anion-exchange resin and elute in characteristic locations (9,32). Greater than 89% of the radiolabeled oligosaccharides isolated from procathep- sin L bound to QAE-Sephadex and eluted in the position expected for an oligosaccharide that contains two phospho- monoesters (Fig. 1B). This uniformity in the structure of the cathepsin L oligosaccharides was surprising in view of the previous demonstration that 5774 cells secrete glycoproteins

that contain several species of phosphorylated high mannose- type units (15). To establish the uniqueness of this phos- phorylation pattern, the oligosaccharides associated with an- other acid hydrolase, @-glucuronidase, were analyzed. p-Glu- curonidase is composed of four identical subunits and each subunit contains 3 or 4 asparagine-linked oligosaccharides (39, 40). The exoglycosidase was immunoprecipitated from the same [3H]mannose-labeled 5774 secretory products used above for the isolation of procathepsin L. The immunoprecip- itate contained a single radiolabeled polypeptide that mi- grated with an apparent molecular mass of the proform of @- glucuronidase (70,000 daltons; not shown). 78% of the radio- labeled glycopeptides derived from the immunoprecipitated exoglycosidase bound to ConA-Sepharose and eluted as high mannose-type glycopeptides. After endo H digestion, the [3H] oligosaccharides eluted from QAE-Sephadex as a heteroge- neous population of structures (Fig. ID); 65% of the radiola- beled oligosaccharides were neutral and failed to bind to the anion-exchange resin and the remainder bound and eluted as species with one phosphomonoester (8%), one phosphomono- ester, and 1 sialic acid residue (4%), and two phosphomono- esters (22%). This diversity in the @-glucuronidase oligosac- charides indicates that the 5774 cells have the potential to produce several types of phosphorylated structures. The pre- cision at which the procathepsin L structures are processed to diphosphorylated species indicates, therefore, that matu- ration of the oligosaccharides attached to the acid protease

by guest on August 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cathepsin L Phosphmylation 11867

occurs in a site-specific process. The structure of the cathepsin L-derived oligosaccharides

was examined in more detail to determine the degree of uniformity. After removal of the phosphates with alkaline phosphatase, all of the oligosaccharides eluted on HPLC as units that contain 7 mannose residues (Fig. 1C). The diphos- phorylated units also were subjected to acetolysis to analyze the position of the phosphorylated mannose residues within the oligosaccharide (5,34). The charged fragments generated by the selective cleavage of &G-linkages migrated on paper chromatography as mannobiose and MansGlcNAcitil (Fig. 2B). The neutral acetolysis fragments, on the other hand, were composed primarily of mannobiose units (Fig. 2A). Thus, the majority of the diphosphorylated cathepsin L oligosaccha- rides contain a single phosphorylated mannose residue on both the (u1,3- and al,6-branch of the core P-linked mannose residue. On the basis of these data, we conclude that the oligosaccharides attached to procathepsin L produced by 5774 cells are processed with a high precision to the same final structure; the proposed structure is depicted in the inset to Fig. 2B.

Diphosphorylated Oligosaccharides Associated with Proca- thepsin L Bind to the Man S-Pc’ Receptor-Diphosphorylated oligosaccharides are known to bind with high affinity to the Man 6-P receptors (7-10). To confirm that the procathepsin L-diphosphorylated oligosaccharides were suitable ligands for the Man 6-P” receptor, bovine liver receptor was conjugated to an insoluble support and the interaction of the QAE- Sephadex purified [3H]mannose-labeled oligosaccharides with the affinity support was determined. 96% of the 3H-labeled two phosphomonoester-containing oligosaccharides isolated

a b c vvv+ s A 3

CM

FIG. 2. Diphosphorylated high mannose-type oligosaccha- rides isolated from procathepsin L contain a phosphate on both their a1,3- and crl,6-branches. The diphosphorylated [3H] oligosaccharides derived from procathepsin L were degraded by ace- tolysis, and the resulting fragments were fractionated by QAE-Seph- adex chromatography to separate neutral and phosphorylated com- ponents. The charged fragments subsequently were dephosphorylated with alkaline phosphatase. The size of the neutral (A) and charged (B, post-dephosphorylation) fragments was determined by descending paper chromatography. The migration positions of Man,GlcNAcitil (a), MansGlcNAci,l (b), mannotriose (c), mannobiose (d), and man- nose (e) are indicated. The inset in panel B depicts the proposed structure for the high mannose-type oligosaccharides of procathepsin L; the cleavage sites for the acetolysis reaction are indicated by the dashed lines. The numbers denote 011,3- (3) and a1,6- (6) linkages, and the @ marks the P-linkage of the core mannose residue. The exact residue phosphorylated on the al,6-branch is not known; potential sites are denoted by an asterisk (5,9). Mannose (IW) and N-acetylglu- cosamine (GZcNAc) residues are indicated as well as the phosphomon- oester groups (P).

from secreted procathepsin L molecules bound to the column and required 2 mM Man 6-P for elution (Fig. 3A). In contrast, oligosaccharides isolated from the total secretory products of 5774 cells that contained only a single phosphomonoester group were retarded by the affinity column but, for the most part, did not require Man 6-P for their elution (Fig. 3B); neutral high mannose oligosaccharides showed no affinity for the receptor and were recovered in the void volume (not shown). Thus, the procathepsin L oligosaccharides and the receptor interact with the high affinity expected for a diphos- phorylated unit (7-10).

Procathepsin L Is a Poor Ligand for the Man S-Pc’ Recep- tor-Despite the high affinity interaction between the oligo- saccharide and the receptor, both endocytosis and receptor binding studies have shown that procathepsin L secreted by transformed mouse fibroblasts binds inefficiently to the Man 6-P” receptor (23, 25). To verify that 5774 procathepsin L is also a poor ligand, we assessed the interaction of the intact acid protease with the Man 6-P” receptor. Mouse L-cells that contain the Man 6-P” receptor were incubated with a crude preparation of [3H]mannose-labeled 5774 secretions in the presence and absence of Man 6-P. After a 6-h incubation, procathepsin L and pro-/3-glucuronidase were isolated by im- munoprecipitation from the post-uptake supernatants. The difference in the amount of enzyme recovered in the absence and presence of Man 6-P is assumed to represent enzyme internalized via Man 6-P receptor-mediated endocytosis (28). Only 23% of the [3H]mannose-labeled procathepsin L was internalized by the L-cells during the 6-h incubation (Table I). In contrast, the cells internalized 82% of the [3H]pro-@- glucuronidase during the same time period (Table I). Since a higher percentage of the pro-@-glucuronidase was internalized by the L-cells than was procathepsin L, the exoglycosidase

CPM (X 10 .

Fraction

FIG. 3. High mannose-type oligosaccharides associated with procathepsin L bind to the Man 6-P” receptor. A, [3H] mannose-labeled diphosphorylated high mannose-type oligosaccha- rides recovered from immunoprecipitated procathepsin L were frac- tionated on a Man 6-P” receptor affinity column. B, the elution profile of a mixture of [3H]mannose-labeled oligosaccharides that contain a single phosphomonoester group is shown for comparison. The monophosphorylated species were isolated from total 5774 secre- tory products; the majority of these units are retarded by the column (fractions 16-25) and do not elute in the void volume (fractions 9- 12). Some of the monophosphorylated species bound with a high affinity and required Man 6-P for elution. Previous studies have shown that monophosphorylated units in which the phosphate is attached to the &S-branch of the core b-linked mannose residue have a high affinity for the receptor (9). The radioactivity in each fraction (0.5 ml) was quantitated in a liquid scintillation counter. The arrowheads denote the fraction at which 2 mM Man 6-P was added to elute bound oligosaccharides.

by guest on August 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

11868 Cathepsin L Phosphorylation

TABLE I Procathepsin L is not endocytosed efficiently via the Man 6-P”

receptor 5774 secretions were isolated from cells metabolically labeled with

[2-3H]mannose. L-cells were incubated with the 3H-secretions in the absence (-) and presence (+) of 5 mM Man 6-P. After a 6-h incuba- tion, the post-uptake supernatant (PUS) was recovered, and proca- thepsin L and pro-fl-glucuronidase were immunoprecipitated and analyzed by SDS-gel electrophoresis and fluorography. The radioac- tive regions of the gel were excised, and the radioactivity was solubi- lized by Pronase digestion and quantitated by liquid scintillation counting. Total counts/min (cpm) recovered as each hydrolase is indicated. % Internalized = (cpm recovered + Man 6-P) - (cpm recovered - Man 6-P)/(cpm recovered + Man 6-P).

CPM (X 10 -3 )

Enzyme Man 6-P

Total cpm in PUS

% Internalized

Procathepsin L 1036 23 + 1350

Pro+-glucuronidase - 1560 82 + 8640

appears to be a better ligand for the Man 6-P receptor than does the acid protease.

The poor endocytosis of procathepsin L relative to pro-p- glucuronidase may have resulted from extrinsic factors within the crude 5774 secretory products that selectively impaired the interaction of cathepsin L with the cell surface Man 6-P receptor. To address this possibility, the two individual acid hydrolases were isolated from the medium of unlabeled 5774 cultures and the purified enzymes were labeled with lz51. The radiolabeled polypeptides subsequently were analyzed by Man 6-P” receptor affinity chromatography. The lz51-labeled pro- cathepsin L preparation fractionated into three peaks on the affinity column corresponding to a no affinity (run-through fractions 4-10; 37%), a low affinity (fractions 12-29; 38%), and a high affinity (fractions 34-38; 25%) interaction (Fig. 4A). To correct for radioactivity in the preparation that was not associated with procathepsin L, the column fractions were pooled, and the radiolabeled polypeptides were analyzed by SDS-gel electrophoresis and radioautography (Fig. 5). The regions of the dried gel corresponding to procathepsin L (35 kDa species) were excised and the associated radioactivity was determined in a gamma counter (Table II). The majority (64%) of the ‘251-procathepsin L was recovered in the low affinity fraction whereas the run-through and high affinity fractions contained 11 and 26% of the radiolabeled enzyme, respectively (Table II). In contrast, the majority of 1251 asso- ciated with the pro-/?-glucuronidase preparation bound with a high affinity to the receptor affinity column (Fig. 4B), and all of the radiolabeled polypeptide was recovered in this fraction (Fig. 5; Table II). When an equal mixture of 1251- procathepsin L and ‘251-pro-/3-glucuronidase were chromato- graphed on the receptor affinity column simultaneously, no change in the binding characteristics of either enzyme was observed (Table II), indicating that the lower affinity dis- played by procathepsin L for the receptor did not result from saturation of the available binding sites. These results indi- cate that the purified J774-secreted procathepsin L is an inherently poorer ligand than either pro-p-glucuronidase or the isolated diphosphorylated oliogosaccharides for the Man 6-P” receptor.

Protein Determinants Impair Recognition of the Diphospho- rylated Oligosaccharidees-The differential behavior of the oligosaccharide isolated from procathepsin L and the intact glycoprotein in terms of their ability to bind with a high affinity to the Man 6-P” receptor suggested that protein determinants influence the receptor-ligand interaction. To assess the importance of the enzyme’s three-dimensional con-

Fraction

FIG. 4. Man 6-P” receptor affinity chromatography of pro- cathepsin L and pro-&glucuronidase. ‘Y-Labeled procathepsin L (A), pro-@-glucuronidase (B), and reduced and alkylated procathep- sin L (C) were chromatographed on a bovine liver Man 6-P” receptor affinity column. The chromatograms show the lz51 elution profiles determined by counting equal aliquots of the l-ml fractions. The arrowhead indicates the fraction at which 2 mM Man 6-P was applied to elute bound glycoproteins.

A 6 C

FIG. 5. Characterization of polypeptides isolated by recep- tor affinity chromatography. Fractions recovered after affinity chromatography (Fig. 4) corresponding to the column run-through (RT) and the low (L) and high (H) affinity interactions were pooled and the radiolabeled polypeptides were precipitated with trichloroa- cetic acid. The precipitates were analyzed by SDS-gel electrophoresis; an equal quantity of radioactivity was loaded into each lane. The autoradiogram shows the distribution of A, ‘2”I-pro-/3-glucuronidase; B, ‘*‘I-procathepsin L; and C, reduced and alkylated “‘1-procathepsin L.

formation in this interaction, 1251-labeled procathepsin L was denatured through chemical reduction and alkylation. The effectiveness of the chemical modification was apparent from the decreased electrophoretic mobility and the increased tryp- sin sensitivity of the reduced and alkylated enzyme (Fig. 6). Native procathepsin L was resistant to trypsin digestion throughout a 30-min incubation at 37 “C (Fig. 6, lanes l-5). The reduced and alkylated enzyme, on the other hand, was proteolyzed rapidly under these same experimental condi- tions. After just 1 min of incubation the majority of the radiolabeled polypeptide was lost, and by 5 min only low molecular weight fragments were detected (Fig. 6, lanes 6- 10). This enhanced trypsin sensitivity indicates that the chemically modified enzyme contains a dramatically altered conformation.

When the reduced and alkylated ‘251-procathepsin L was chromatographed on the Man 6-P” receptor affinity column

by guest on August 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cathepsin L Phosphorylation 11869

TABLE II Quantitation of the binding of ‘L51-labeled procathepsin L and pro-&

glucuronidase to the Man 6-P” receptor column The radioactive regions of the gel shown in Fig. 5 were excised and

the associated i*‘I was quantitated in a y-counter. Percentages of the “‘I-labeled procathepsin L (L), pro-fl-glucuronidase (P-Glut), and reduced and alkylated procathepsin L recovered in the indicated peak fractions are shown; these values were adjusted to reflect the total radioactivity recovered from the receptor affinity column. The mix- ture sample refers to an experiment where equal amounts of proca- thepsin L and pro-@-glucuronidase were applied to the receptor aftin- ity column.

% of protein Sample Peak fraction species

L P-Glut Procathepsin L Run-through 11

Low affinity 64 High affinity 26

Pro-fi-glucuronidase

Mixture

Run-through 0.2 High affinity 99.8

Run-through 5 0.5 Low affinity 51 0.2 High affinity 43 99

Reduced/alkylated procathepsin L Run-through 11 Low affinity 64 High affinity 25

O..aC l -

m5m

FIG. 6. Trypsin sensitivity of native and reduced and al- kylated ‘*‘I-procathepsin L. 1.2 x lo6 cpm of ‘*“I-procathepsin L (lanes I-5) and its reduced and alkylated counterpart (lanes 6-10) were digested with trypsin for 0 (lanes 5 and 6), 5 (lanes 1 and 7), 10 (lanes 2 and 8), 15 (lanes 3 and 9), and 30 (lanes 4 and 10) min at 37 “C. At the end of the incubation, trypsin was inhibited by the addition of soybean trypsin inhibitor, and the digestion products were precipitated with trichloroacetic acid and separated by SDS-gel elec- trophoresis. An autoradiogram of the gel is shown. Note the slower mobility of reduced and alkylated procathepsin L (lone 6) relative to the native enzyme (lane 5).

(Fig. 4C) the denatured protease bound to the receptor with the same apparent affinity as the native enzyme (Fig. 5 and Table II). The majority of the reduced and alkylated proca- thepsin L was recovered in the low affinity fraction (Table II). Thus, a native three-dimensional conformation is not necessary to mask the high affinity interaction between the receptor and the diphosphorylated oligosaccharide.

To eliminate additional protein determinants that may influence receptor recognition, glycopeptides were isolated from procathepsin L after trypsin digestion. For this analysis [3H]procathepsin L was purified from the growth medium of 5774 cells metabolically labeled with [2-3H]mannose. The 3H- labeled acid hydrolase was reduced and alkylated then di- gested with an excess of trypsin. The resulting 3H-labeled glycopeptides were fractionated on ConA-Sepharose. 85% of the radiolabeled units bound and were eluted as high man- nose-type oligosaccharides as was observed earlier for the Pronase-derived glycopeptides (Fig. 1A). The ConA-isolated glycopeptides were chromatographed on the Man 6-P” recep- tor affinity column, and 84% of the [3H]glycopeptides bound to the receptor column with a high affinity that required the addition of Man 6-P for elution (Fig. 7A). Endo H digestion

CPM (X 10.

L-AJ 20 40 50 80 Fraction

FIG. 7. Tryptic [3H]glycopeptides derived from procathep- sin L hind with high affinity to the Man 6-P” receptor column. [3H]Mannose-labeled procathepsin L was purified from 5774 secre- tions and was reduced and alkylated. The denatured enzyme was digested with an excess of trypsin and the resulting 3H-labeled high mannose-type glycopeptides were isolated by ConA-Sepharose chro- matography. The 3H-labeled high mannose-type units subsequently were chromatographed on the Man 6-P” receptor affinity column before (A) and after (B) digestion with endo H. 0.5-ml fractions were collected and counted directly in a liquid scintillation counter. The arrowheads indicate the fractions at which 2 mM Man 6-P was added to elute the bound units.

of the 3H-labeled glycopeptides prior to their chromatography did not affect their apparent binding affinity (Fig. 7B). The endo H-released oligosaccharides bound to QAE-Sephadex and eluted as diphosphorylated species (not shown). The tryptic glycopeptides, therefore, bind to the Man 6-P” recep- tor with the same apparent affinity as the free oligosaccha- rides. Importantly, the [3H]mannose-labeled enzyme purified from 5774 secretions contained the same type of oligosaccha- rides as found associated with enzyme directly immunoprecip- itated from 5774 conditioned media; that is, all of the radio- labeled oligosaccharides contained two phosphomonoesters. The inability of the ‘251-labeled procathepsin L to bind with a high affinity to the Cl Man 6-P receptor, therefore, did not result from a dephosphorylation of the enzyme’s oligosaccha- rides during the purification procedure.

DISCUSSION

Hypersecretion of an individual acid hydrolase by trans- formed cells is not restricted to cathepsin L. A cathepsin B- like protease, for example, is found in the ascitic fluid of patients with ovarian carcinoma (41) and this enzyme also is secreted by cultures of malignant human breast tumors (42). Cathepsins B and L are similar in that both are thiol pro- teases, and they share significant sequence homology (43). In addition, estrogen is known to induce the synthesis and se- cretion of procathepsin D from MCF7 breast cancer cells (44), and the secreted enzyme contains the Man 6-P recognition marker (45). The mechanisms by which transformed cells divert an enzyme normally destined for lysosomes to the secretory pathway remain unknown. Evidence is accumulat- ing, however, that the extracellular hydrolases are active and may facilitate metastasis (19, 42, 44, 46).

We have shown previously that the formation of phos- phorylated high mannose-type oligosaccharides occurs in a multistep process that involves a potential competition be- tween the lysosomal enzyme phosphotransferase and Golgi- associated a-mannosidase I (14,15). A single mannose residue within the al,6-branch of the oligosaccharide is phosphoryl-

by guest on August 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

11870 Cathepsin L Phosphorylation

ated at a pre-Golgi location, but a second phosphate is at- tached only after 1 or 2 mannose residues are removed from the precursor oligosaccharide within the Golgi apparatus. Removal of additional mannose residues, however, may pre- vent the second site phosphorylation and lead to the forma- tion of monophosphorylated and sialylated-phosphorylated hybrid species (15). Transformation often leads to alterations in the structures of many cellular glycoprotein oligosaccha- rides as a result of changes in the activity of various oligosac- charide processing enzymes (47, 48). If the relative activities of the Golgi-associated lysosomal enzyme phosphotransferase and a-mannosidase were altered upon transformation, then a change in the processing of the oligosaccharides attached to newly synthesized acid hydrolases may occur as a result of a shift in the competitive balance. Changes in the structure of the phosphorylated oligosaccharides could, in turn, lead to altered recognition by the Golgi-associated Man 6-P receptors and, thus, altered protein trafficking patterns. Interestingly, the specific activity of the phosphotransferase is known to increase in ovarian tumors and in some transformed cell lines (49). We investigated the structure of the procathepsin L oligosaccharides, therefore, in an attempt to explain the basis for the enzyme’s hypersecretion.

Oligosaccharides associated with 5774 cell secreted proca- thepsin L were uniformly processed to high mannose-type units that contained two phosphomonoesters and 7 mannose residues. The phosphorylated mannose residues were posi- tioned to both the a1,3- and al,6-branches of the oligosac- charides. In comparison, pro-p-glucuronidase contained a di- verse population of oligosaccharides, including one and two phosphomonoester species and hybrid-type molecules. The monomeric subunits of mouse ,&glucuronidase contain at least 3 asparagine-linked glycosylation sites (39, 40), and each site of the mature glycoprotein contains a mixture of oligosaccha- ride structures (39). Processing of the /3-glucuronidase oligo- saccharides, therefore, does not appear to occur in a site- specific manner. Procathepsin L, on the other hand, is mon- omeric and the mouse enzyme is thought to contain a single asparagine-linked oligosaccharide (46); the cDNA sequence predicts two potential glycosylation sites but only one of these apparently is utilized (16, 46). The structural uniformity of the procathepsin L oligosaccharides sharply contrasted the diverse phosphorylated products associated with pro+glu- curonidase. Since both acid hydrolases were produced simul- taneously by the same cells, protein determinants associated with procathepsin L apparently directed the processing of its oligosaccharide to yield a homogeneous final product. Site- specific processing of complex-type asparagine-linked oligo- saccharides has been reported previously (50, 51), but this is the first example that maturation of phosphorylated oligosac- charides also may occur in a directed process. Perhaps the apparent heterogeneity of the phosphorylated oligosaccha- rides attached to /3-glucuronidase does not result from random processing but, rather, reflects that an oligosaccharide at a specific site within each of the 4 subunits may not be exposed to the same microenvironment due to conformational con- straints imposed by the quaternary structure of the holoen- zyme. As a result of these different environments, the same glycosylation site on the four monomeric subunits may mature in a non-random process, but not to the same final structure (51, 52).

In contrast to our initial expectations, the phosphorylated oligosaccharides associated with procathepsin L were high affinity ligands for the Man 6-P” receptor. Previous studies have indicated that the structure of a phosphorylated oligo- saccharide affects its interaction with the Man 6-P” receptor.

Oligosaccharides that contain phosphomonoester units, for example, bind more strongly to the Man 6-P” receptor than phosphodiester-containing units (7-10). Moreover, oligosac- charides with two phosphomonoesters are better ligands (loo- fold greater) than units with a single phosphomonoester group, and diphosphorylated oligosaccharides that possess phosphates on terminal mannose residues are the best ligands (9). The structure that we derived for the procathepsin L- diphosphorylated oligosaccharide contains many of the fea- tures expected for a high affinity ligand. Thus, structural characteristics of the oligosaccharide do not account for the poor binding of procathepsin L to the Man 6-P receptor.

Relative to pro+-glucuronidase, procathepsin L was a poor substrate for the Man 6-P” receptor. In both an endocytic and an affinity chromatographic assay pro+glucuronidase displayed a higher affinity for the receptor. In part, this differential affinity may occur because the intact P-glucuron- idase tetramer contains multiple phosphorylated oligosaccha- rides that make the hydrolase a multivalent ligand. Since the Man 6-P” receptor binds only 1 diphosphorylated oligosac- charide/mol of receptor, any multivalent effect must occur as a result of the binding to more than one receptor molecule (53, 54). Monomeric procathepsin L, on the other hand, contains only 1 phosphorylated oligosaccharide. This oligo- saccharide, however, is sufficient on its own to mediate a high affinity interaction with the Man 6-P” receptor. The poor affinity of intact procathepsin L for the receptor, in either a native or a reduced and alkylated state, indicates that the polypeptide contains determinants that impair binding of the diphosphorylated oligosaccharides. Glycopeptides generated by trypsin digestion of procathepsin L, in contrast, bind with a high affinity to the receptor. Based on the protein sequence predicted from the corresponding cDNA, the tryptic glycopep- tides are expected to contain 19 or 35 amino acids, depending on which of the glycosylation sites is utilized (16). The pres- ence of a number of attached amino acids, therefore, is not detrimental to a high affinity interaction between the phos- phorylated oligosaccharide and the receptor. The negatively charged phosphomonoester groups may interact ionically with positively charged lysine or arginine side chains located at a distance from the glycosylated asparagine residue and impair Man 6-P” receptor recognition. Alternatively, a protein de- terminant of procathepsin L may interact with the Man 6- PC1 receptor and reduce the affinity of the oligosaccharide- receptor interaction. The internalization of a-L-iduronidase, for example, is inhibited when the hydrolase is pretreated with reagents that alter arginine residues (55), suggesting a role for protein determinants in receptor-ligand binding. Fur- ther studies will be required to delineate the exact structural elements that are involved in the modulation of Man 6-P” receptor recognition,

Acknowledgments-We wish to thank Dr. Kurt Drickamer for his suggestions and efforts.

REFERENCES

1. van Figura, K., and Hasilik, A. (1986) Annu. Reu. Biochem. 66, 167-193

2. Kornfeld, S. (1987) FASEB J. 1, 462-468 3. Kornfeld, S., and Mellman, I. (1989) Annu. Reu. Cell Bid. 5,483-

525 4. Brown, W. J., and Farquhar, M. G. (1984) Cell 36, 295-307 5. Varki, A., and Kornfeld, S. (1980) J. Biol. Chem. 255, 10847-

10858 6. Natowicz, M., Baenziger, J. U., and Sly, W. S. (1982) J. Biol.

Chem.257,4412-4420 7. Creek, K. E., and Sly, W. S. (1982) J. Biol. Chem. 257, 9931-

9937

by guest on August 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cat&p&n L Pho~ph~~latio~ 11871

8. Fischer, D. H., Creek, K. E., and Sly, W. S. (1982) J. Biol. Chem. 32. Gabel, C. A,, Goldberg, D. E., and Kornfeld, S. (1982) J. Cell Biol. 257,9938-9943 95,536-542

9. Varki, A., and Kornfeld, S. (1983) J. BioE. Ck.em. 258,2808-2818 33. Mehis, S. J., and Baenziger, J. II. (1981) Anal. Biochem. 114, 10. Hoflack, B., Fujimoto, K., and Kornfeld, S. (1987) J. Biol. Chem 276-280

262,123-129 34. Karston, E. M., and Ballou, C, E, (1978) J. BioL Chem. 253, Il. Reitman, M. L., and Kornfeld, S. (1981) J. Biol. Chem. 256, 680-685

11977-11980 35. Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963)

12. Waheed, A., Hasilik, A., and von Figura, K. (1982) J. Biai. Chem. Biochem. J. 89,114-123 267,12322-12331 36. Gabei, C, A., and Foster, S. A. (1987) J. Cell Biol. 105, 1561-

13. Lang, L., Reitman, M., Tang, J., Roberts, R. M., and Kornfeld, 1570

S. (1984) J. Biol. Chem. ZEiS, 14663-14671 37. Troen, B. R., Ascherman, IX, Atlas, D., and Gottesman, M. M.

14. Lazzarino, D. A., and Gabel, C. A. (1988) J. Biat. Chem. 263, (1988) J. Biol. Chem. 263,254~261

10118-10126 38. Hall, C. W., Liebaers, I., Natale, P. D., and Neufeld, E. F. (1978)

15. Lazzarino, D. A., and Gabel, C. A. (1989) J. Biol. Chem. 264, ~e~hads ~~~~~. E&,439-456

39. Goldberg, D. E., and Kornfeld, S. (1981) J. Bial. &em. 256, 5015-5023 1306~13~7

16. Portnoy, D. A., Erickson, A. H., Kochan, J., Ravetch, J. V., and 40. Nishimura, Y., Rosenfeld, M. G., Kreibich, G., Gubler, U., Sa- Unkeiess, J. 6. (1986) J. Biol Gem. 261,1469?-14703 batini, D. D., Adesnik, M., and Andy, R. (1986) Proc. Nati.

17. Troen, B. R., Gal, S., and Gottesman, M. M. (1987) Biochem. J. Acad. S-i. U. S. A. 83,7X%7296 246,731-735 41. Mort, J. S., Leduc, M., and Recklies, A. D. (1981) Biochim.

18. Mason, R. W., Gal, S., and Gottesman, M. M. (1987) Bfochem. Biophys. Acta 662,173-180 J. 248,449-454 42. Mart, J. S., Recklies, A. D., and Poole, A. R. (1980) Biachim.

19. Denhardt, D. T., Hamilton, R. T., Parfett, C. L. J., Edwards, D. Biophys. Acta 614,134-143 R., St. Pierre, R., Waterbouse, P., and Nileen-Hamilton, M. 43. Dufour, E. (1988) Biochimie 70,1335-1342 (1986) Cancer Res. 46,45QO-4593 44. Capony, F., Rougeot, C., Montcourrier, P., Cavailles, V., Salazar,

20. Gattesman, M. M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, G., and Rochefort, H. (1989) Cancer Iles. 49,3904-3909

2767-2771 45. Capony, F., Morisset, M., Barrett, A. J., Capony, J. P., Broquet,

21, Gottesman, M. h4. (1980) Cell 19,449-455 P., Vignon, F., Chambon, M., Louisot, P., and Rochefort, H

22. Nilsen-Hamilton, M., Hamilton, R. T., Allen, W. R., and Mas- (1987) J. Cell Biol. 104,253~262

soglia, S. L. (1981) bairn. ~io~hys. Res. Camm~n. 101,411- 46. Smith, S. M., Kane, S. E., Gal, S., Mason, R. W., and Got~sman,

M. M. (19891 ~~ochem. J. 262.931-938 417

_

23. Sahagian, G. G., and Gottesman, M. M. (1982) J. Biol. Chem. 257,11145-11150

24. Gal, S., Willin%ham, M. C., and Gottesman, M. M. (1985) J. Cell Biol. 100,535-544

25. Dong, J., Prence, E. M., and Sahagian, G. G. (1989) J. Bial. Chem, 264,7377-7383

26. Jessup, UT., and Dean, R. T. (1980) Biochem. J. 190,847~850 27. Gabel, C. A., Goldberg, D. E., and KornfeM, S. (1983) Proc. N&l.

Acad. Sci. U. S. A. 80,775-779 28. Gabel, C. A., and Foster, S. A. (1986) J. Cell Biol. 102,943-950 23. Gabel, C. A., and Foster, S. A. (1986) J. Cell Aiol. 103, 1817-

1827 30. Laemmli, U. K. (1970) Nature 227,680-685 31. Cummings, R. D., and Kornfeld, S. (1982) J. Biol. Chem. 287,

11235-11240

47. Holmes, E. H.; and Hakomori, g-1. (1983) J. Biol. Chem. 258, 3706-3713

48. Dennis, J, W., Laferte, S., Waghorne, C., Breitman, M. L., and Kerbel, R. S. (1987) Science 235,582-585

49. Madiyalakan, R., Mueller, 0. T., Shows, T. B., and Matta, K. L. (1987) Cancer Pnuest. 5,553-558

50. Swiedler, S, J., Hart, G. W., Tarentino, A. L., Plummer, T. H., Jr., and Freed, J. H. (1983) J. Biol. Chem 2Fi8,11515-11523

51. Hubbard, S. C. (1986) J. Bid. Chem. 265,19303-19317 52. Dahms, N. M., and Hart, G. W. (1986) J. Biol. Chem. 261,

13186-13196 53, Tong, P. Y., Gregory, W., and Kornfeld, S. (1989) J. Biol. Chem.

264,7962-7969 54. Fischer, 11. D., Natowicz, M., Sly, W. S., and Bretthauer, R. K.

(1980) J. Cell Biol. 84, 77-86 55. Rome, L. H., and Miller, J. (1980) g~chern. Biophys. Res. Cam-

mun. S&2,986-993

by guest on August 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

D Lazzarino and C A Gabeloligosaccharides by the cation-independent mannose 6-phosphate receptor.Protein determinants impair recognition of procathepsin L phosphorylated

1990, 265:11864-11871.J. Biol. Chem. 

  http://www.jbc.org/content/265/20/11864Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/265/20/11864.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on August 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from