ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 283, No. 2, December, pp. 447-457, 1990

Comparison of Cathepsin L Synthesized by Normal and Transformed Cells at the Gene, Message, Protein, and Oligosaccharide Levels’ Nancy A. Stearns, Jianming Dong, Jia-Xiu Pan, David A. Brenner,* and G. Gary Sahagian’ Department of Physiology, Schools of Medicine and Veterinary Medicine, Tufts University, Boston, Massachusetts 02111; and *Department of Medicine, Veterans Administration Medical Center, and University of California, San Diego, California 92161

Received April 20, 1990, and in revised form July 19, 1990

The major excreted protein of transformed mouse fibroblasts (MEP) has recently been identified as the lysosomal cysteine protease, cathepsin L. The synthesis and intracellular trafficking of this protein in mouse fibroblasts are regulated by growth factors and malignant transformation. To further define the basis for this regulation, a cDNA encoding MEP/cathepsin L was isolated from a mouse liver cDNA library and used to compare cathepsin L of normal and Kirsten sarcoma virus-transformed NIH 3T3 fibroblasts. Although cathepsin L message levels were elevated ZO-fold in the transformed fibroblasts, normal and transformed cells displayed similar cathepsin L genomic DNA digest patterns and gene copy numbers, and cathepsin L mRNA sequences appeared identical by RNase protection analysis. These findings indicate that (i) cathepsin L is synthesized from the same gene in normal and transformed cells and (ii) cathepsin L polypeptides made by these cells are translated with the same primary sequence. Cathepsin L polypeptides synthesized by quiescent, growing, and transformed cells displayed similar isoelectric focusing patterns, suggesting similar post-translational modification. Site-directed mutagenesis of the mouse liver cDNA and expression in COS monkey cells was used to examine the glycosylation of mouse cathepsin L. The results indicated that only one of the two potential N-linked glycosylation sites (the one at Asn221) is glycosylated. Analysis by ion exchange chromatography on QAE-Sephadex, and affinity chromatography on mannose (i-phosphate receptor-At&Gel 10, indicated that the cathepsin L oligosaccharide was phosphorylated similarly in normal and transformed i This work was supported by National Institutes of Health Grant DK36632 and the Searle Scholars Program/The Chicago Community Trust. ’ To whom correspondence and reprint requests should be addressed at present address: Department of Physiology, Tufts University, 136 Harrison Ave., Boston, MA 02111. 0003.9861/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

cells. Although several phosphorylated oligosaccharide species were observed, the major species contained two phosphomonoester moieties and bound efficiently to the receptor. These findings suggest that cathepsin L made by normal and transformed mouse fibroblasts are identical and substantiate the hypothesis that trafficking of cathepsin L in these cells is regulated by growth-induced changes in the lysosomal protein transport system. 0 1990

Academic

Press,

Inc.

Malignantly transformed cells are associated with increased release of a number of proteolytic enzymes which are thought to facilitate tumor cell growth and invasiveness [for review see Refs. (l-3)]. These enzymes, in most cases, are thought to exert their effects by modification of collagens or other extracellular matrix components. MEP,3 the major excreted protein of transformed mouse fibroblasts (4), is a lysosomal cysteine protease (5-7) whose synthesis and secretion are regulated by growth factors, phorbol esters, and cellular transformation (812). Recent cloning of cDNAs encoding mouse, rat, and human cysteine proteases has suggested that MEP is the mouse analog of the lysosomal protease, cathepsin L (1317). In quiescent NIH 3T3 fibroblasts, MEP/cathepsin L is synthesized as a 39,000-Da precursor which is processed 3 The abbreviations used are: acetate buffer, 10 mM sodium acetate, pH 5.5; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; IPTG, isopropylthio-fl-galactoside; KNIH cells, NIH 3T3 fibroblasts transformed with Kirsten sarcoma virus; Man-6-P, mannose 6-phosphate; MEM, Eagle’s minimal essential medium; MEP, major excreted protein; NIH cells, NIH 3T3 fibroblasts; PBS, 50 mM sodium phosphate, pH 7.0,0.15 M NaCl; PDGF, platelet-derived growth factor; PMSF, phenylmethylsulfonyl fluoride; TBS, Tris buffered saline: TPA, 12-O-tetradecanoylphorbol-13-acetate; SDS, sodium dodecyl sulfate; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of SDS; SSC, 0.15 M sodium chloride-O.015 M sodium citrate (36). 447

Inc. reserved.

448

STEARNS

to lower molecular weight forms upon delivery to lysosomes (6, 18). Lysosomal delivery of cathepsin L is mediated by mannose 6-phosphate (Man-6-P) receptors which recognize phosphorylated asparagine-linked oligosaccharides of lysosomal proteins during their biosynthesis [see Refs. (19-24) for review]. Treatment of NIH cells with platelet-derived growth factor (PDGF) or the phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA), or transformation with ras-containing Kirsten sarcoma virus, has been shown to produce increased levels of cathepsin L messenger RNA (11, 12) due to increased transcription (25). In addition to regulating cathepsin L synthesis, transformation and PDGF treatment have been shown to produce an alteration in intracellular trafficking of cathepsin L such that the protein is secreted (6, 18, 26). The net effect of these changes is to increase extracellular levels of cathepsin L by as much as 200-fold. Cathepsin L secretion has been shown to correlate with the metastatic potential of ras-transformed cells (27), and it has been suggested that the secreted protein may play a role in modulating interactions of cells with components of the extracellular matrix (7, 13, 17, 28). It has been previously shown that the alteration in intracellular trafficking of cathepsin L induced by viral transformation and PDGF treatment is selective in that other lysosomal proteins are not affected (5,18,26). These results raised the possibility that structural differences in cathepsin L synthesized by quiescent and growing cells might be responsible for the observed differences in trafficking. In the present study, a cDNA encoding cathepsin L was cloned from a mouse liver cDNA library. The cDNA was used to compare cathepsin L in normal NIH cells to that in NIH cells transformed with Kirsten sarcoma virus (KNIH cells). The results indicate that cathepsin L made by quiescent and growing cells are very similar, if not identical, and suggest that the observed alterations in trafficking are not due to structural changes in cathepsin L but instead to alterations in the lysosomal protein transport system.

MATERIALS

AND

METHODS

Materials Antibodies directed against mouse MEP/cathepsin L (MP-1) were raised in rabbits as previously described (29). The antigen was purified from the culture medium of KNIH cells (30). The specificity of this antibody preparation has been established (18). Cation-independent Man-6-P receptor was isolated from bovine liver by chromatography on a pentamannosyl-phosphate affinity column (31) using previously described methods (29); cation-dependent Man-6-P receptor contained in the preparation was removed by chromatography on a Dictyostelium discoideum lysosomal protein-A&Gel 10 affinity matrix (32) as described (33). D. disciodeum proteins were kindly provided by Dr. Arnold Kaplan at St. Louis University School of Medicine. Recombinant endohexosaminidase H was purchased from Genzyme. pGEM-3 transcription vector and pSVL expression vector were obtained from Promega and Pharmacia/LKB, respectively.

ET AL.

Methods Cells and growth conditions. NIH and KNIH cells were obtained from M. Gottesman at the National Cancer Institute. COS-1 cells were purchased from the American Type Culture Collection. NIH and KNIH cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum, 100 units/ml penicillin, and 100 rg/ml streptomycin, and maintained at 37°C in a humidified atmosphere of 5% COz. COS cells were cultured in a similar manner except that the DMEM used contained antibiotics and 10% fetal calf serum. For isolation of RNA and DNA, isoelectric focusing of secreted proteins and oligosaccharide analysis of cathepsin L, NIH or KNIH cells were seeded at a density of 5 X lo3 cells/cm’ and allowed to grow to confluence without a change of medium. NIH, but not KNIH cells, become growth arrested by this treatment, as determined by [3H]thymidine incorporation into cellular DNA. Biosynthetic labeling of mousefibroblasts. NIH and KNIH cells were grown to confluence under the conditions described above and labeled for 16 h in serum-free Eagle’s minimal essential medium (MEM) containing 50 pCi/ml of [35S]methionine or 200 &i/ml of [3H]mannose and a reduced level of unlabeled precursor in the medium (l/30 of the normal level of methionine for labeling with [?S]methionine and l/50 of the normal level of glucose for labeling with [3H]mannose). For most experiments, 10 mM NH&I was included in the labeling medium to cause quantitative secretion of newly synthesized cathepsin L and other lysosomal proteins. cDNA cloning and sequencing. Cathepsin L cDNA was cloned from a mouse liver Xgtll cDNA library. cDNA was prepared from mouse liver poly(A+)RNA using the RNase H method described by Gubler and Hoffman (34) and modified by EcoRI methylation and the addition of EcoRI linkers. The resulting cDNAs were digested with EcoRI, size selected (>500 bp), and cloned into the EcoRI site of Xgtll DNA, and the recombinant DNA was packaged into phage in vitro (35). Analysis showed that the library contained approximately 6 X lo6 members, 54% of which contained cDNA inserts. Initially, partial cDNAs were obtained by screening the library for @galactosidase-cathepsin L fusion proteins with cathepsin L-specific antiserum using the methods of Young and Davis (36). Two putative cathepsin L cDNA-containing phage were obtained from screening 50,000 phage plaques. Sequencing of the 5’ end of one of these cDNAs (MEP-8.1) and comparison to known sequences revealed that the cloned cDNA was identical to an in frame 789 bp EcoRI restriction fragment contained within the coding sequence of a previously cloned cDNA encoding a mouse macrophage cysteine protease (14). This cysteine protease has since been identified as MEP/cathepsin L (13-17). Screening of the mouse liver library with the antibody-selected cathepsin L cDNA and with an oligonucleotide corresponding to a sequence near the 3’ end of the macrophage cysteine protease cDNA (nucleotides 922 to 945 in Fig. 1) indicated that both probes hybridized to the same plaques, thereby confirming that the two cDNAs represented the same message. To obtain a full length cDNA, the library was screened with the 3’ oligonucleotide probe and a second oligonucleotide probe corresponding to a sequence at the 5’ end of the macrophage cysteine protease cDNA (nucleotides 58 to 78 in Fig. 1). Two cDNAs that hybridized to both probes and to the antibody-selected cDNA were obtained from screening 300,000 plaques. The longest of the two cDNAs (MEP-B.l, approximately 1350 bp) was fully sequenced and used for all of the experiments described below. For screening the library with the antibody-selected cDNA, the cDNA was excised from recombinant phage with EcoRI and labeled with 32P by nick translation (35). Hybridization of the probe to the nitrocellulose filters was carried out using previously described methods (35). Methods described by Wood et al. (37) were used for screening the library with oligonucleotide probes; wash temperatures 4°C below the dissociation temperature for the probe were employed. For sequencing and other analyses, cathepsin L cDNAs were excised from recombinant Xgtll DNAs with either BamHI or EcoRI, and fragments containing cathepsin L sequences were subcloned into pUC18,

CHARACTERIZATION pUC19 or M13mp18. Sequencing of cathepsin L cDNAs was performed by dideoxy-sequencing as described (38) using Ml3 forward and reverse universal primers and oligonucleotides complementary to the published mouse cysteine proteinase sequence (14). Oligonucleotides were made on an Applied Biosystems DNA Synthesizer utilizing fi-cyanoethyl phosphoramidites for synthesis. Site-directed mutagenesis of glycosylation sites. For site-directed mutagenesis, an 1162.bp BamHI restriction fragment of the cDNA, which contains the entire coding sequence, was cloned into the BomHI site of phage M13mp18 DNA. Single base pair mutations in each of the two potential N-linked glycosylation sites were introduced by the method of Zoller and Smith (39) using recombinant single-stranded phage DNA containing the coding strand of the cathepsin L cDNA as a template and mutagenic oligonucleotides, 5’-CCCTGTGTCAGTAGCCACAGC3’ (for substitution of Thr for Asn at position 221) and 5’GGTTCTTGCTGCCACAGTTGG-3’ (for substitution of Gly for Ser at position 270). A pentadecamer phage Ml3 universal sequencing primer (New England Biolabs) was used as the second primer. Phage plaques containing the mutant DNA were identified by screening with 32P-labeled mutagenic oligonucleotide under stringent conditions, and plaque-purified. Mutations were confirmed by dideoxysequencing as described above.

Expression of Cathepsin L cDNAs in COS cells. For expression of cathepsin L cDNAs in COS cells, the 1162-bp BamHI fragments of unmodified MEP or MEP glycosylation mutants were cloned into the BamHI site of the eukaryotic expression vector pSVL. COS cells were transfected with the recombinant DNAs (30 pg/lOO-mm culture dish) using a modification of the DEAE-dextran method (40). Transfected cells were grown for 2 days and then assayed for expression by metabolic labeling with [35S]methionine and immunoprecipitation. Labeling of the cells with [35S]methionine was performed as described above for NIH and KNIH cells except that DMEM containing 10% dialyzed fetal calf serum and 100 rCi/ml of [%S]methionine was used. Immunoprecipitation of cathepsin L from COS cell extracts and media and analysis by SDS-polyacrylamide gel electrophoresis and fluorography were carried out essentially as described (18) except that protease inhibitors and Man-6-P were added to the cell extracts and media and the washing protocols were changed. The inhibitors were 0.05 rig/ml leupeptin, 1mM EDTA, 1 mM PMSF, 5 mM Man-6-P, and 10 mg/ml BSA. Immunoprecipitates from cell extracts were washed four times with RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCI, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 0.02% sodium azide) containing inhibitors, and once with PBS, pH 6.8, containing 1% sodium lauroylsarcosine; media immunoprecipitates were washed three times with RIPA buffer containing 1 mg/ml BSA and once with 0.1 M TrisHCl, pH 6.8. In some experiments cathepsin L was immunoprecipitated from Man-6-P-containing proteins isolated from the culture medium of [35S]methionine-labeled cells using Staphylococcus aureus-bound cationindependent Man-6-P receptor as described previously (18). In other experiments, immunoprecipitated cathepsin L was deglycosylated with endohexosaminidase H as described below under “Oligosaccharide Analysis.” Southern blot analysis of Cathepsin L gerwmic sequences. NIH and KNIH cells, grown to confluence under the conditions described above, were incubated overnight in serum-free DMEM prior to isolation of cellular DNA. DNA from 5 X lo7 cells was isolated and digested to completion with BamHI, EcoRI, HindIII, PstI, PuuII, and BglI using standard procedures (35). The digested DNAs were subjected to electrophoresis on 0.8% agarose gels and transfered to Gene Screen Plus membranes (Dupont/NEN) using an alkaline transfer procedure (41). Membranes were prehybridized for 15 min at 50°C in a solution of 50% formamide, 6X SSC (1X SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 5X Denhardt’s solution, 1 mM EDTA, 200 fig/ml herring sperm DNA, 200 rig/ml wheat germ tRNA, and 1% SDS, and then hybridized for 16 h at 50°C in the same solution containing 6 X lo6 cpm/ ml of cathepsin L cRNA probe. The membranes were washed twice at room temperature for 30 min with 2X SSC, 1% SDS and twice at 65°C

OF CATHEPSIN

L

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for 30 min with 0.1X SSC, 1% SDS. To reduce background radioactivity, membranes were then incubated for 2 h with 1 rig/ml RNase A in 2x SSC, 0.1% SDS and again washed at 65°C for 30 min in 0.1X SSC, 1% SDS. 3ZP-labeled bands were detected by autoradiography. The cathepsin L probe used was a cRNA labeled with [o-32P]UTP by transcription from a recombinant transcription vector (pGEM-3) containing the 1162-bp BamHI fragment of the cathepsin L cDNA (Fig. 1). Conditions for cRNA synthesis were those described below for mRNA analysis. Analysis of MEP messenger RNAs. Total RNA was prepared from cultured cells essentially as described (42). Cells were lysed in 25 mM sodium citrate, pH 7.0, containing 4 M guanidinium isothiocyanate, 0.5% sodium lauroylsarcosine and 0.1 mM /I-mercaptoethanol. RNA was pelleted through a cushion of 5.7 M cesium chloride, extracted twice with phenol:chloroform (1:l) and once with chloroform:isoamyl alcohol (24: l), and then precipitated from 0.3 M sodium acetate with 2 vol of ethanol. For Northern blots, total RNA was subjected to electrophoresis in a 1.2% agarose gel containing formaldehyde and transferred to Gene Screen Plus membrane with 10X SSC (43). The membrane was prehybridized for 30 min at 55’C in 50% formamide containing 1% SDS, 1 M NaCl, 10% dextran sulfate, 50 pg/ml wheat germ tRNA and 50 pg/ ml herring sperm DNA, and hybridized for 16 h in the same solution containing 4 X 10’ cpm/ml cRNA probe. The membrane was washed at room temperature in 2X SSC, treated for 1 h with 1 #g/ml RNase A in 2X SSC containing 0.1% SDS, and washed at 60°C for 30 min in 0.1X SSC containing 1% SDS. Labeled bands were detected by autoradiography. RNase protection assays were conducted as described (44) except that a 16-h incubation period was used for hybridization of cRNA probes with RNAs, and conditions for RNase digestion were varied slightly to ensure degradation of unhybridized probe with minimal degradation of protected sequences. Hybridization mixtures contained 5 X lo6 cpm of probe and cellular RNA plus wheat germ tRNA to give a total of 20 pg per assay. Products of RNase digestions were subjected to electrophoresis in 6% polyacrylamide-8 M urea sequencing gels. Labeled bands were detected by autoradiography. The two cRNA probes used for RNase protection assays were labeled with [~I-~*P]UTP by transcription from pGEM-3 vectors containing cathepsin L cDNA restriction fragments. A 5’ probe was transcribed from a recombinant DNA in which the BamHI-EcoRI fragment of the cDNA (nucleotides I-313) was inserted between the BamHI and EcoRI sites in the polylinker region of the vector. The transcribed probe contained 318 bases corresponding to nucleotides 1 to 318 of the cathepsin L cDNA and 41 bases corresponding to vector sequences. A 3’ probe was synthesized from a recombinant DNA in which the BamHI fragment of the cDNA was cloned into the BamHI site in the polylinker region of the vector. This DNA was linearized within the insert with EcoRV prior to synthesis of the probe. The transcribed 3’ probe contained 431 bases corresponding to nucleotides 737 to 1167 of the cathepsin L cDNA plus 35 bases of vector sequence. High specific activity cRNA probes were generated in 5 ~1 reaction mixtures containing 40 mM Tris-HCl, pH 7.5, 6 mM MgC&, 10 mM dithiothreitol, 2 mM spermidine, 0.5 mM ATP, CTP, and GTP, 12 PM UTP, 0.5 ng linearized DNA template, 6.3 U RNAsin, 125 &i [a32P]UTP (>400 Ci/mmol), and 6 U T7 polymerase. The DNA template was removed by treatment with 0.7 U RNase-free DNase for 20 min at 37°C followed by phenol:chloroform (1:l) extraction and ethanol precipitation. Probes were gel purified prior to use as described previously (45). Isoelectric focusing of cathepsin L. For isoelectric focusing, KNIH and NIH cells were grown as described above to allow NIH cells to become growth arrested. The cells were washed and then incubated for 16 h at 37°C in serum-free DMEM containing 10 mM NH&I to cause secretion of newly synthesized cathepsin L. Washed NIH cells were also incubated for 16 h at 37°C in serum-free DMEM containing NH&I and 10 rig/ml PDGF (Amgen Inc.). Media were concentrated by lyophilization and dialyzed against distilled water. Concentrated media proteins were

450

STEARNS

subjected to isoelectric focusing for 90 min at 500 V in ultrathin agarose slab gels, pH 3-10 (IsoGel, FMC) using methods specified by the vendor. The focused proteins were transferred to nitrocellulose by press blot transfer (as described by FMC) and cathepsin L bands were detected by immunoblotting (46) with MP-1 serum (diluted 1:lOOO) and phosphatase-labeled goat anti-rabbit IgG (diluted 1:2000). Isoelectric points were determined by comparison to protein isoelectric point standards (FMC). In some experiments, the focused proteins were subjected to polyacrylamide gel electrophoresis in the presence of SDS by cutting out a strip of the focused agarose gel, fixing the strip in place above the stacking gel of a Laemmli gel system with agarose, and electrophoresing in the usual manner (18). Oligosacchuride analysis. For preparation of radiolabeled cathepsin L oligosaccharides, NIH or KNIH cells were labeled with [3H]mannose in the presence of NH&l and cathepsin L was purified from the culture medium by immunoprecipitation (18). Labeled cathepsin L was dissociated from immunoprecipitates by incubation at 95°C for 10 min in 50 pl of acetate buffer (10 mM sodium acetate, pH 5.5) containing 0.3% SDS. After clarification, the samples were diluted threefold with acetate buffer, endohexosaminidase H (5 mu) was added, and the samples were incubated at 37°C for 16 h. The phosphorylation state of cathepsin L oligosaccharides was determined by chromatography on QAE Sephadex using methods modified from those described by Varki and Kornfeld (47). Endohexosaminidase H incubation mixtures containing the [3H]mannose-labeled oligosaccharides were diluted to 0.75 ml with 2 mM Tris-HCl, pH 8.0, and applied to l-ml columns of QAE Sephadex (Sigma) equilibrated with the same buffer. After washing the columns with five 0.75-ml aliquots of the Tris buffer, bound oligosaccharides were eluted from the columns with sequential washes (6 X 0.75 ml) of Tris buffer containing NaCl at concentrations of 20, 40,60,80, 100, 120, 140, 160, 180, 200, and 400 mM. Five peaks of labeled oligosaccharides were eluted from the column: peak 1 was composed of uncharged oligosaccharides, peak 2 of oligosaccharides containing 1 phosphodiester, peak 3 of oligosaccharides containing 1 phosphomonoester or 2 phosphodiesters, peak 4 of oligosaccharides containing 1 phosphodiester and 1 phosphomonoester, and peak 5 of oligosaccharides containing 2 phosphomonoesters. The composition of each peak was determined by treating individual peaks with combinations of mild acid, alkaline phosphatase, and neuraminidase as previously described (48). Analysis of the binding of [3H]mannose-labeled cathepsin L and cathepsin L oligosaccharides to immobilized Man-6-P receptor was carried out as previously described (49). The immobilized receptor was prepared by covalent attachment of bovine cation-independent Man-6-P receptor to Affi-Gel 10 using the method described by the vendor (Bio-Rad).

RESULTS Characteristics

of Cathepsin L cDNA

A cDNA containing the cathepsin L coding sequence and untranslated 5’ and 3’ sequences was isolated from a Xgtll library constructed from mouse liver mRNA as described under Methods. The complete nucleotide sequence of the cDNA and the deduced amino acid sequence of the protein is shown in Fig. 1. The protein sequence is the same as those encoded by previously cloned mouse MEP/ cathepsin L cDNAs (13,14,16,17). However, differences in nucleotide sequence were observed in untranslated regions. The mouse liver cDNA contains an 89-bp insert at the 3’ end of the cDNA immediately preceding the poly(A) tail that is absent from the cDNA cloned from a mouse macrophage cell line (14) but present in the cDNAs cloned from transformed NIH 3T3 fibroblasts (16, 17). In addition, the mouse liver cDNA is missing a G at position

ET AL.

32 which is present in the sequences from the transformed fibroblasts but absent in the macrophage sequence and has a G at position 1190 which is present in one of the other sequences (17). Whether or not these differences have functional significance is not known. Comparison of ,Cathepsin L Synthesized Transformed Cells

by Normal and

A comparison of cathepsin L made by normal and transformed cells was undertaken in order to determine the basis for growth-related changes in synthesis and trafficking of cathepsin L. It is known that cathepsin L synthesized by mouse fibroblasts displays considerable charge heterogeneity and it has been suggested that multiple cathepsin L mRNAs may exist or that a single mRNA may be translated in a number of ways (30). Therefore, a plausible explanation for the observed difference in trafficking of cathepsin L in normal and transformed cells is that synthesis of secretory forms of cathepsin L are induced by transformation. Alterations in expression of cathepsin L isoforms could potentially be caused by transformationinduced changes in the structure of cathepsin L genomic sequences, changes in processing of cathepsin L transcripts, or alterations in post-translational modification of cathepsin L polypeptides. Genomic sequences. Comparison of cathepsin L genomic sequences in normal and transformed cells is shown in Fig. 2. In this experiment, genomic DNAs from NIH and KNIH cells were digested to completion with each of the six indicated restriction enzvmes and nrobed with radiolabeled antisense cRNA transcribed from the coding strand of the cloned cathepsin L cDNA. The observed restriction patterns and band intensities for NIH and KNIH cells were identical for all digests and the sizes of the restriction fragments were consistent with those reported previously for NIH cells (50). These results suggest that cathepsin L aenomic sequences in the virally transformed cells are the same as those in the untransformed cells. Since NIH cells have been shown to contain only a single cathepsin L gene (50), it appears that increased synthesis of cathepsin L mRNA in transformed cells results from increased transcription of the same gene. Messenger RNAs. A Northern blot of cathepsin L mRNAs present in NIH and KNIH cells is shown in Fig. 3. The antisense cRNA probe hybridized to a single mRNA species of approximately 1.4 kb for both normal and transformed cells. In agreement with a previous study (ll), the level of cathepsin L mRNA was found to be approximately 20-fold greater in KNIH cells than in NIH cells. An RNase protection experiment using the same RNA preparations is shown in Fig. 4. Bases in the antisense RNA probes which corresponded to either the 3’ or the 5’ end of the cloned cDNA were completely protected from RNase digestion by hybridization to either NIH or KNIH mRNA. Since this assay is capable of detecting

CHARACTERIZATION

OF CATHEPSIN

L

451

GGATCCGAGTTTGCAGACTTCTTGTGCGCACTAGCCGCCTCAGGTGTTTGAACC ATG AAT CTT TTA CTC Met As-n Leu LeU Leu 1 TTT GAT CAA ACC TTT Phe Asp Gin Thr Phe GGC ACG Gly Thr

CTT TTG GCT GTC CTC TGC TTG GGA ACA GCC TTA GCT ACT LeU Leu Ala Val Leu Cys Leu Gly Thr Ala Leu Ala Thr 18 ACT GCA GAG TGG CAC CAG TGG AAG TCC ACG CAC AGA AGA Ser Ala Glu Trp His Gin Trp Lys Ser Thr His Arg Arg

AAT GAG GAA GAG TGG AGG AGA GCG ATA TGG GAG AAG AAC ATG AGA ATG Asn Glu Glu Glu Trp Arg Arg Ala Ile Trp Glu Lys Asn Met Arg Met

CTA CAC AAC GGG GAA TAC AGC Leu His Asn Gly Glu Tyr Ser

AAC GGG CAG CAC GGC TTT TCC ATG GAG ATG AAC Asn Gly Gin His Gly Phe Ser Met Glu Met Asn

GGT GAC ATG ACC AAT GAG GAA 'IiC AGG CAG GTG GTG AAT GGC TAT CGC CAC CAG Gly Asp Met Thr Asn Glu Glu Phe Arg Gln Val Val Asn Gly Tyr Arg His Gln AAG AAG GGG AGG CTT TTT CAG GAA CCG CTG.ATG Leu Phe Gln Glu Pro Leu Met Lys Lys Gly Arg

CTT AAG ATC Leu Lys Ile 114 AGA GAA AAG GGT TGT GTG ACT CCT GTG AAG AAC CAG GGC CAG Arg Glu Lys Gly Cys Val Thr Pro Val Lys Asn Gln Gly Gin

TTT AGC GCA Phe Ser Ala

CCC AAG Pro Lys

TCT GTG Ser Val

TGC GGG TCT TGT Cys Gly Ser Cys

TCG GGT TGC CTA GAA CGA CAG ATG TTC CTT AAG ACC Ser Gly Cys Leu Glu Gly Gln Met Phe Leu Lys Thr

GGC AAA Gly Lys

CTG Leu

CTG AGT GAA CAG AAC CTT GTG GAC TGT TCT CAC GCT CAA GGC AAT CAG GGC TGT Leu Ser Glu Gln Asn Leu Val Asp Cys Ser His Ala Gln Gly Asn Gln Gly Cys GGC CTG ATG GAT TTT GCT TTC CAG TAC ATT AAG GAA AAT GGA GGT CTG GAC TCG Gly Leu Met Asp Phe Ala Phe Gln Tyr Ile Lys Glu Asn Gly Gly Leu Asp Ser TCT TAC CCC TAT GAA GCA AAG GAC GGA TCT TGT AAA TAC AGA GCC GAG TTC GCT Ser Tyr Pro Tyr Glu Ala Lys Asp Gly Ser Cys Lys Tyr Arg Ala Glu Phe Ala AAT Asn 0 221 ACT Thr

GAC ACA GGG TTC GTG GAT ATC CCT CAG CAA GAG AAA GCC CTC ATG AAG GCT Asp Thr Gly Phe Val Asp Ile Pro Gln Gln Glu Lys Ala Leu Met Lys Ala GTG GGG CCT ATT TCT GTT GCT ATG GAC GCA AGC CAT CCG TCT CTC CAG TTC Val Gly Pro Ile Ser Val Ala Met Asp Ala Ser .His Pro Ser Leu Gln Phe

TCA GGC ATC TAC TAT GAA CCC AAC TGT Ser Gly- Ile Tyr Tyr Glu Pro Asn Cys 0 268 GTG GGC TAT CCC TAT GAA GGA ACA GAT Val Gly Tyr Gly Tyr Glu Gly Thr Asp

AGC AGC AAG MC Ser Ser Lys Asn

CTC GAC CAT GGG GTT Leu Asp His Gly Val

4 TCA AAT AAG AAT AAA TAT TGG CTT GTC Ser Asn Lys Asn Lys Tyr Trp Leu Val

CCA AAA Pro Lys 20 CTG TAT Leu Tyr 40 ATC CAG Ile Gln 60 GCC TTT Ala Phe 80 AAG CAC Lys His 100 GAC TGG Asp Trp 120 TGG GCG Trp Ala 140 ATC TCA Ile Ser 160 AAC GGA Asn Gly 180 GAG GAG Glu Glu 200 GTG GCT Val Ala 220 GTG GCG Val Ala 240 TAT AGT Tyr Ser 260 CTG TTG Leu Leu 280 AAG AAC Lys Asn

291

54 114

174

234

294

354

414

414

534

594

654

714

774

834

894

954

300

AGC TGG GGA ACT GAA TGG GGT ATG GAA GGC TAC ATC AAA ATA CCC AAA GAC Ser Trp Gly Ser Glu Trp Gly Met Glu Gly Tyr Ile Lys Ile Ala Lys Asp

1014

CGG GAC AAC Arg Asp Asn 320 CAC TGT GGA CTT GCC ACC GCG GCC AGC TAT CCT GTC GTG AAT TGA TGGGTAGCGGTAATGAGGA His Cys Gly Leu Ala Thr Ala Ala Ser Tyr Pro Val Val Asn End 334 CTTATGGACACTATGTCCAAAGGAATTCAGCTTAAAACTGACCAAACCCTTATTGAGTCAAACCATGGTACTTGAATCA

11.57

TTGAGGATCCAAGTCATGATTTGAATTCTGTTGCCATTTTTACATGGGTTAAATGTTACCACTACTTAAAACTCCTGTTA

1237

TAAACAGCTTTATAATATTGAAAACTTAGTGCTTAATTCTGAGTCTGGAATATTTGTTTTATATAAAGGTTGTATAAAA

1316

CTTTCTTTACCTCTTAAAAATAAATTTTAGCTCAGTGTGTGTGT(Poly I 1

1360

A)

1078

L cDNA. Numbering of the nucleotide sequence begins at the first base of FIG. 1. Nucleotide and deduced amino acid sequence ofcathepsin the cDNA insert and numbering of the amino acid sequence begins at the presumed initiation codon. Nucleotide sequences corresponding to the 5’ and 3’ oligonucleotides used for screening of the library are underlined. The 3’ insert described in the text is overlined, the polyadenylation signal is bracketed and the two potential N-linked glycosylation sites are boxed. Predicted sites of proteolytic processing are shown by arrows.

STEARNS

452 I Pvu II I Pst l

IK

NIK

EcoRI 1 BarnHI IHind III

I Bgl l

NIK

N(IK

NIK

NIK

NI

FIG. 2. Southern blot of cathepsin L genomic sequences in NIH and KNIH cells. DNA (20 pg) from NIH (N) and KNIH (K) cells was digested with the indicated restriction enzyme. After electrophoresis the DNA fragments were transferred to Gene Screen Plus and probed with 32Plabeled cathepsin L cRNA. An autoradiogram of the blot after a l-week exposure is shown. Migration of DNA standards is indicated for each blot; numbers refer to sizes in kilobases.

most single base mismatches (51), these results suggest that cathepsin L mRNAs present in NIH and KNIH cells have the same sequence. Isoelectric focusing patterns. Cathepsin L proteins made by NIH and KNIH cells were also found to be very similar by isoelectric focusing (Fig. 5). Precursor cathepsin L contained in the culture media of quiescent and PDGFtreated NIH cells and KNIH cells grown in the presence of NH&l to cause quantitative secretion of the protein (52, 53), were found to be comprised of several species with isoelectric points between 6.7 and 8.6. With the exception of minor species (>pI 7.7) present at higher levels in KNIH medium than in NIH media, little difference was observed in the pattern of bands for the two cell types. These minor species were not detected on two-dimensional gels, suggesting that they probably represent low molecular weight degradation products of cathepsin L. Cathepsin L species from both cell types were converted to a major species of pI 7.7 by treatment with endohexosaminidase H, indicating that the various forms of the protein were to a large extent due to heterogeneity of the phosphorylated oligosaccharide (data not shown). These results provide further evidence that cathepsin L proteins synthesized by normal and transformed cells have the same primary structure and indicate that the proteins undergo similar post-translational modifications. Glycosylation. The glycosylation of cathepsin L was examined by transient expression of the mouse liver cDNA in COS cells. The utility of the COS cell system for studying the glycosylation of cathepsin L is illustrated by the results shown in Fig. 6. The level of expression of the mouse protein 48 h after transfection was approxi-

ET AL.

mately the same as the level of expression of endogenous cathepsin L in NIH 3T3 cells (Fig. 6A). The size of the expressed protein both before and after treatment with endohexosaminidase H was similar to that of the NIH cell protein, indicating that COS cells glycosylate mouse cathepsin L normally. Mouse cathepsin L expressed by COS cells was also phosphorylated on mannose as demonstrated by its ability to bind specifically to immobilized Man-6-P receptor (Fig. 6B). Mouse cathepsin L contains two potential sites for asparagine-linked glycosylation, one at Asn221 and the other at Asn268. Utilized sites were identified by examination of mutant proteins in which the glycosylation sites were abolished by site-directed mutagenesis of the cDNA (Fig. 7). Mutation of glycosylation site 1 at Asn221 (MEPGSl) resulted in expression of a mutant protein (Mr 36,000) with increased electrophoretic mobility relative to wild type cathepsin L (Mr 39,000). The mutant protein migrated slightly faster than the deglycosylated wild type protein (Mr 37,500) and its migration was not altered by treatment with endohexosaminidase H, indicating that the mutation prevented glycosylation of the protein. The slightly smaller size of the mutant protein is likely due to partial degradation during the course of the experiment.

FIG. 3. Northern blot of cathepsin L mRNA in NIH and KNIH cells. Total RNA from NIH (0.4 pg) and KNIH (0.02 pg) cells were electrophoresed, transferred to Gene Screen Plus, and probed with “P-labeled cathepsin L cRNA. An autoradiogram of the blot after a 24-h exposure is shown. Migration of the 28 S and 18 S ribosomal RNAs contained in the total RNA preparations is indicated.

CHARACTERIZATION 3’ PROBE

5’ PROBE

800 603 -

1s 3871

209-

171-

OF CATHEPSIN

453

L

phosphorylation state of oligosaccharides of NIH and KNIH cathepsin L was examined. Proteins synthesized from both cell types possess a single asparagine-linked oligosaccharide at position 221 [(49), experiments described above]. Analysis by ion exchange chromatography on QAE-Sephadex indicated that the predominant oligosaccharide species of both proteins were those with 2 phosphomonoester moieties; minor portions of species with 1 phosphomonoester, 1 phosphomonoester and 1 phosphodiester, 1 phosphodiester or 2 phosphodiesters were also observed (Fig. 8, top). A large portion of the oligosaccharide species bound to immobilized Man-6-P receptor and were eluted with the competitive inhibitor, Man-6-P (Fig. 8, bottom); the bound species were those containing 2 phosphomonoester moieties (data not shown). The relative amounts of the various species were similar in normal and transformed cells (Table I). In a separate experiment the binding of intact cathepsin L to immobilized receptor was examined. Only a portion of the total cathepsin L present in the medium of NH<reated [3H]mannose-labeled cells was capable of binding to the column. The portion was 51% for NIH cells and 49% for KNIH cells. In both cases, eathepsin L which

z 3

FIG. 4. Comparison of NIH and KNIH cathepsin L mRNA by RNase protection. 32P-labeled cRNA probes corresponding to nucleotides 1 to 318 (5’ probe) or 736 to 1167 (3’ probe) were hybridized to NIH (20 pg) or KNIH (1 rg) RNA, digested with RNase, and electrophoresed as described under Methods. Controls containing probe and tRNA were subjected to the same treatments. RNase-treated samples were electrophoresed alongside of undigested probe. An autoradiogram of the gel after a 2-h exposure is shown. Migration of each probe (P) and of the protected fragment of the 3’probe (A) and the 5’ probe (B) are indicated by arrowheads. Migration of size markers is also indicated. The untreated probes were slightly larger that the protected RNA fragments due to the vector sequence contained in each probe.

zz Stds.

-4.2 - 5.5 -6.1

Mutation of glycosylation site 2 at Asn268 (MEP-GS2) resulted in expression of a protein that had the same electrophoretic mobility as wild type cathepsin L (MEP) both before and after treatment with endohexosaminidase H. Mutation of both glycosylation sites (MEP-GS12) produced a mutant protein which behaved like the MEPGSl mutant. These results demonstrate that the glycosylation site at Asn221 (site 1) is utilized whereas the site at Asn268 (site 2) is not. When the labeling period was increased from 2 to 16 h, wild type cathepsin L (MEP) and the MEP-GS2 mutant accumulated in the medium whereas the MEP-GSl mutant and double mutant were not recovered from cells or medium, and were apparently degraded. Finally, because lysosomal targeting and delivery of cathepsin L depend on oligosaccharide phosphorylation, the

6.71 -7.0 7.76.2.

- 6.2

6.6-

5. Isoelectric focusing of NIH and KNIH cathepsin L. Media from KNIH cells (l), NIH cells (2), and PDGF-treated NIH cells (3) were subjected to isoelectric focusing and bands reacting with cathepsin L-specific antiserum were visualized as described under Methods. Isoelectric points of cathepsin L species (left) and protein standards (right) are indicated. FIG.

454

STEARNS

1-6-P

-MEP

A

ET AL.

factors that have yet to be identified. These same factors are presumably responsible for regulation of cathepsin L synthesis in untransformed cells by growth factors and tumor promoters. Since cathepsin L mRNAs synthesized by the normal and transformed cells are transcribed from the same gene and were indistinguishable by Northern blotting and RNase protection analysis, it would appear that cathepsin L proteins made by these cells are translated with the same primary sequence. These findings, taken together with the observed similarities in isoelectric focusing patterns, oligosaccharide number and phosphorylation, and binding to the cation-independent Man-6-P receptor, provide strong evidence that the cathepsin L proteins synthesized by normal and transformed cells are identical. In addition, cathepsin L synthesized by PDGF-treated NIH cells displayed the same isoelectric focusing pattern as cathepsin L synthesized by quiescent NIH cells, suggesting that the primary sequence and post-translational

B

FIG. 6. Expression of cathepsin L cDNA in COS-1 cells. NIH cells and COS-1 cells transfected with pSVL vector containing cathepsin L cDNA (pSVL-MEP) or pSVL vector alone, were labeled with [%]methionine for 16 h in the presence of NH&l. (A) Fluorogram of SDS-PAGE gel comparing cathepsin L secreted by NIH cells and transfected COS-1 cells before and after treatment with endohexosaminidase H. (B) Fluorogram of SDS-PAGE gel showing specific binding to immobilized Man-6-P receptor of total Man-6-P-containing proteins and cathepsin L secreted by NIH cells and by COS-1 cells transfected with pSVL-MEP. 69-

bound to the column contained the oligosaccharide species with two phosphomonoester moieties whereas cathepsin L which did not bind contained the incompletely processed oligosaccharide species (data not shown).

46-

DISCUSSION

In this study, a MEP/cathepsin L cDNA was cloned from a mouse liver cDNA library and used to characterize cathepsin L of normal and transformed cells. Several conclusions concerning the regulation of this protein can be made from the results. It has been previously shown that viral transformation of mouse fibroblasts is associated with a 15. to 20.fold increase in cathepsin L mRNA (11,12) and that this increase is due to increased message synthesis (25). In this study, no differences were detected in the number or restriction digest pattern of cathepsin L genomic sequences between normal and transformed cells. Since NIH cells are known to have one cathepsin L gene, these results suggest that cathepsin L expressed by normal and transformed cells is encoded by the same gene. Thus, it appears likely that synthesis of cathepsin L messenger RNA is regulated by transformation-dependent transcription

MSFIG. 7. Expression of cathepsin L glycosylation mutants in COS-1 cells. COS-1 cells transfected with pSVL, pSVL-MEP, pSVL-MEPGSl (site 1 mutant), pSVL-MEP-GS2 (site 2 mutant), or pSVL-MEPGS12 (site 1 and site 2 mutant) were labeled with [%]methionine for 2 h in the presence of NH&l. Cathepsin L was immunoprecipitated from detergent extracts of the cells, treated with endohexosaminidase H, and subjected to SDS-PAGE. Radioactive bands migrating at 51 and 49 kDa presumably represent glycosylated and deglycosylated forms of endogenous monkey cathepsin L, respectively. The origin of bands migrating in the 32 to 34-kDa region in cells expressing mutant and wild type cathepsin L is unknown.

CHARACTERIZATION

OF CATHEPSIN

455

L

NIH

KNIH

Receptor column

Receptor column

0

10

20

30

40

50

Fraction

Fraction

FIG. 8. Oligosaccharide phosphorylation of NIH and KNIH cathepsin L. [3H]Mannose-labeled oligosaccharides of cathepsin L synthesized by NIH and KNIH cells were obtained by immunoprecipitation and endohexosaminidase H treatment and analyzed by chromatography on QAESephadex and Man-6-P receptor affinity matrix as described in the Methods section. The arrows shown on the QAE-Sephadex elution profiles indicate the positions of elution of oligosaccharide species with zero, one, two, three or four charges. The phosphorylation state of oligosaccharide species eluting at these positions is given in Table I. The arrow on receptor column profiles indicates the point at which elution with Man-6-P was started.

modification of the protein is not affected by growth factor treatment. Consequently, altered trafficking of cathepsin L that occurs in transformed (6, 18) and growth factortreated cells (26) cannot be explained by growth-related changes in the structure of the protein. Cathepsin L from normal and transformed cells displayed considerable charge heterogeneity by isoelectric focusing. This heterogeneity has been observed previously (6, 10, 30) and has raised the possibility that multiple cathepsin L mRNAs exist or that a single mRNA may be translated in a number of ways (30). The RNase protection

experiments

described

here

suggest

that

only

a single

species of cathepsin L mRNA exists in both normal and transformed cells, making the former possibility unlikely. We attribute most of the charge heterogeneity of mouse cathepsin L to heterogeneity of the phosphorylated oli-

gosaccharide although evidence to the contrary has been reported (30). In any case, the spectrum of isoforms of the protein is similar in normal and transformed cells and we could find no evidence that expression of the various isoforms are effected differentially by transformation or growth factor treatment. All available data indicate that in quiescent NIH cells, cathepsin L is targeted to lysosomes via the Man-6-P recognition system. Lysosomal delivery can be inhibited by antibodies directed against cation-independent Man-6-P receptor (Prence and Sahagian, unpublished data) but is minimally affected by Man-6-P (26), indicating that the transport process is mediated by this receptor and occurs via the typical intracellular delivery pathway. Although mouse cathepsin L has an affinity for the receptor that is at least lo-fold lower than that for other lysosomal

456

STEARNS ET AL. TABLE1

Composition

of Oligosaccharides

of NIH

and KNIH

MEP”

% of total Oligosaccharide fraction 0 (Neutral) 1 (1PD) 2 (1PM or 2PD) 3 (1PM and 1PD) 4 (2PM) Receptor-bound

NIH 8.9 2.8 10.1 11.9 66.2 57.0

KNIH 3.6 4.3 13.9 14.4 63.8 56.0

a Calculated from Fig. 8. PD, Man-6-P moiety in phosphodiester age with GlcNAc; PM, Man-6-P phosphomonoester moiety.

link-

proteins (18), its affinity is apparently high enough to allow efficient sorting and lysosomal delivery in quiescent cells. In transformed and growth factor-treated NIH cells, cathepsin L is secreted. One explanation for the high level of secretion by transformed cells is that intracellular Man6-P receptors become saturated by the high level of cathepsin L produced (18); a lack of interaction with the receptor would be expected to result in secretion of the protein by default. This explanation can also account for oversecretion of cathepsin L by transfected cells expressing similarly high levels of the protein (54). However, studies with PDGF have shown that growth factor-induced changes in cathepsin L trafficking are not obligatorily linked to changes in cathepsin L synthesis (26). Thus, while overexpression may contribute to increased secretion of cathepsin L under some conditions, cells are capable of regulating cathepsin L trafficking directly. This regulation does not appear to involve alterations in cathepsin L, so it must alternatively be effected by growthinduced changes in the cation-independent Man-6-P receptor or other components of the cellular lysosomal protein transport machinery. Treatment of NIH cells with PDGF also causes redistribution of cation-independent Man-6-P receptors (26). Further studies will be required to determine if this effect is responsible for regulating cathepsin L secretion. Site-directed mutagenesis of the two potential asparagine-linked glycosylation sites of mouse cathepsin L indicates that only the site at Asn221 is utilized. The oligosaccharide at this site is composed primarily of species with two phosphomonoester Man-6-P residues that bind to immobilized cation-independent Man-6-P receptor, indicating efficient recognition by the lysosomal protein targeting enzymes, N-acetylglucosaminylphosphotransferase and N-acetylglucosamine-I-phosphodiesterase. The existence of only a single phosphorylated oligosaccharide on mouse cathepsin L is responsible for its low affinity for the cation-independent Man-6-P receptor (49). This property could provide cathepsin L with an increased

sensitivity to reductions in receptor levels or affinity and may thereby be responsible for the ability of cells to regulate trafficking of this protein specifically. Mutant unglycosylated cathepsin L proteins were rapidly degraded by COS cells. These results suggest that glycosylation is important for proper folding of cathepsin L in Go. However, glycosylation does not appear to be an absolute requirement, at least for the human protein, since human cathepsin L expressed in Escherichia coli can be refolded after denaturation with urea to yield small quantities of active enzyme (55). Native cathepsin L is still active after deglycosylation, indicating that glycosylation is apparently not required to maintain the folded conformation once it has formed (56). An alternative explanation is that the unglycosylated mutant cathepsin L proteins are folded normally but are recognized as abnormal proteins (perhaps in the endoplasmic reticulum by BiP or other polypeptide chain binding proteins) and targeted for degradation. ACKNOWLEDGMENTS The authors thank Dr. J. Fred Dice for critical review of the manuscript, Dr. Stephen A. Goff for helpful discussions, and Teresa L. Paluszcyk and Monica L. Mina for excellent technical assistance.

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Comparison of cathepsin L synthesized by normal and transformed cells at the gene, message, protein, and oligosaccharide levels.

The major excreted protein of transformed mouse fibroblasts (MEP) has recently been identified as the lysosomal cysteine protease, cathepsin L. The sy...
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