Cathepsin S Regulates the Expression of Cathepsin L and the Turnover of gamma -Interferon-inducible Lysosomal Thiol Reductase in B Lymphocytes*

Karen HoneyDagger §, Meghan DuffDagger , Courtney BeersDagger , William H. Brissette||, Eileen A. Elliott||, Christoph Peters**, Maja MaricDagger Dagger §§, Peter CresswellDagger Dagger , and Alexander RudenskyDagger §Dagger Dagger ¶¶

From the Dagger  Department of Immunology and the § Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98115, the || Department of Immunology, Pfizer Incorporated, Groton, Connecticut 06340, ** Institut fuer Molekular Medizin und Zellforschung, Albert-Ludwigg-Universitaet Freiburg, 76106 Freiburg, Germany, and the Dagger Dagger  Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520-8011

Received for publication, February 28, 2001, and in revised form, April 11, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
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Loading of antigenic peptide fragments on major histocompatibility complex class II molecules is essential for generation of CD4+ T cell responses and occurs after cathepsin-mediated degradation of the invariant chain chaperone molecule. Cathepsins are expressed differentially in antigen presenting cells, and mice deficient in cathepsin S or cathepsin L exhibit severely impaired antigen presentation in peripheral lymphoid organs and the thymus, respectively. To determine whether these defects are due solely to the block in invariant chain cleavage, we used cathepsin-deficient B cells to examine the role of cathepsins S and B in the degradation of other molecules important in the class II presentation pathway. Our data indicate that neither cathepsin S nor B is critical for H-2M degradation or processing of precursor gamma -interferon-inducible lysosomal thiol reductase (GILT) to a mature thiol reductase, but suggest a role for cathepsin S in the turnover of mature GILT and in regulating levels of mature cathepsin L protein in B cells. Despite the presence of mature cathepsin L protein, no enzyme activity could be detected in B cells or dendritic cells. These experiments suggest a novel mechanism by which these functionally important enzymes may be regulated.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Recognition of antigen (Ag) by CD4+ T cells requires presentation of short peptide fragments in the context of major histocompatibility complex (MHC)1 class II molecules (1, 2). MHC class II molecules consist of an alpha /beta heterodimer that is assembled in the endoplasmic reticulum in association with a third glycoprotein, invariant chain (Ii), which promotes the correct folding of the MHC class II molecules (3, 4). A region of Ii known as class II-associated Ii peptide (CLIP) occupies the MHC class II peptide-binding groove to prevent inappropriate peptide loading (5-7), and signals in the cytoplasmic domain of Ii target the MHC class II/Ii chain complex to MHC class II rich endosomal compartments (8-11). Upon entering the endocytic pathway, Ii chain is cleaved in a defined stepwise manner by proteases known as cathepsins (12-14), leaving only CLIP associated with the MHC class II heterodimer. Removal of CLIP and subsequent loading of diverse peptides is mediated by the MHC class II-like molecule HLA-DM (H-2M in mice) prior to MHC class II transport to the cell surface (15, 16).

The cathepsins are a large family of aspartyl (D and E) and cysteinal (B, S, and L) endosomal proteases that have been implicated as critical to MHC class II presentation (12-14, 17). However, recent studies using cathepsin-deficient mice have shown that cathepsin D (catD) and cathepsin B (catB) are unnecessary for MHC class II presentation (18, 19), whereas cathepsin S (catS) and cathepsin L (catL) are important for Ag presentation by discrete populations of cells (20-23). Active catS was detected in B cells, dendritic cells (DCs), and peritoneal macrophages (pMphi s), where it was shown to be involved in the late stages of Ii degradation (21-23). CatL, however, was detected only in Mphi s and cortical thymic epithelial cells, where a defect resulted in severely impaired CD4+ T cell selection. In addition to catS and catL, a third cysteinal cathepsin, cathepsin F, has been recently implicated in Ii degradation and Ag presentation in alveolar Mphi s (24). Thus, it would appear that different APCs utilize distinct cathepsins to mediate late stage Ii degradation and regulate MHC class II presentation.

Cathepsins are synthesized in an inactive precursor form, becoming active upon autocatalytic cleavage of the pro-region (25-27), which thus acts as an inhibitor of cathepsin activity (28-30). Alternative mechanisms exist for regulating cathepsin activity, and much effort is now being channelled into defining these. In DCs it has been shown that activity of catS correlates inversely with expression levels of cystatin c (31), a known inhibitor of catS and catL (32, 33). Other studies have suggested intracellular cathepsin expression in Mphi s and Mphi cell lines can be regulated by increasing or decreasing cathepsin mRNA levels (34, 35) or by secretion of the cathepsin from the cell (34). Therefore, it seems that cathepsin activity can be controlled in many ways and determining the role for each regulatory mechanism in vivo would enhance our understanding of Ag presentation.

The importance of cathepsins in regulating the MHC class II presentation pathway is indicated by the phenotype of APCs from catS and catL knockout mice (20, 21). However, their role in mediating late stage Ii degradation is unlikely to explain fully the defects in Ag presentation. The observation that exogenous Ag presentation is defective in catS-deficient B cells and DCs, whereas endogenous Ags are presented normally (21), suggests a role for catS in proteolytic cleavage of some internalized proteins. In addition, recent studies have implicated various cysteinal cathepsins with turnover of the accessory molecule H-2M (36) and cleavage of the precursor form of the thiol reductase GILT (37). This enzyme is expressed constitutively in APCs, where it colocalizes with MHC II molecules (38) and is up-regulated by interferon-gamma (39). Reduction of disulfide bonds is important for efficient MHC II presentation (40, 41), and GILT-mediated reduction is critical for presentation of some protein Ags, as shown by studies of GILT-/- mice.2 No role for GILT in Ii degradation has been observed in these mice, suggesting their cathepsin activity is not impaired in the absence of this thiol reductase.2

In this study, we have explored the mechanism by which catL activity is regulated and the role of cathepsins in regulating the expression of GILT and H-2M in vivo. Here we show that B cells and DCs express a 20-kDa form of catL protein that corresponds to mature catL. However, we were unable to detect activity of this enzyme in these cells. The level of mature catL protein in B cells was dependent upon catS, suggesting that catS plays a role in turnover of mature catL protein. CatS may also play some minor role in the turnover of GILT, while the turnover of the accessory molecule H-2M was independent of both catS and catB, both of which are active in B cells. These results demonstrate catL activity is tightly regulated and suggest B cells and DCs may express a catL-specific inhibitor.

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Mice-- C57BL/6 (BL6) mice were purchased from Taconic Farms Inc. (Germantown, NY) and maintained under specific pathogen-free conditions at the University of Washington. CatS-/-, catL-/-, catB-/-, and catS-/-xcatB-/- animals were bred and maintained under these same conditions. All animals were used at 6-12 weeks of age. All procedures and care of the animals was in accordance with University of Washington guidelines.

Antibodies-- The polyclonal rabbit antisera to mouse catL was a gift of A. Erickson (University of North Carolina, Chapel Hill, NC) and has been described previously (42). The antiserum to mouse GILT was produced by immunizing rabbits with purified His-tagged mouse precursor GILT expressed in Escherichia coli. The rat monoclonal antibody 2C3A was a gift of L. Karlsson (R. W. Johnson Research Institute, San Diego, CA), and rabbit affinity isolated anti-actin antibody was purchased from Sigma-Aldrich.

B Cell, Dendritic Cell, and Macrophage Isolation and Flow Cytometric Analysis-- To isolate highly purified populations of B cells, splenocytes were stained with magnetic MHC class II microbeads (Miltenyi Biotec, Auburn, CA) and positively selected over two columns on an automated magnetic cell separator (AutoMACS, Miltenyi Biotec). Purity of the positive fraction was assessed by flow cytometric analysis. Cells were stained with anti-B220 fluorescein isothiocyanate (Phar- Mingen, San Diego, CA) and analyzed on a FACScan flow cytometer (Becton Dickinson) using Cell Quest (Becton Dickinson) software. All positive fractions were >95% B220hi.

Spleens were enriched for dendritic cells as described previously (22, 43). Briefly, mice were injected subcutaneously with 5 × 106 flt3 ligand secreting B16 melanoma cells (a gift of G. Dranoff, Dana-Farber Cancer Institute, Boston, MA) and spleens were harvested after the tumors reached 2 cm in diameter. Splenocytes were stained with magnetic CD11c microbeads (Milteyi Biotec), positively selected on an AutoMACS and the purity of the positive fraction assessed by flow cytometric analysis as described above. Cells were stained with anti-CD11c fluorescein isothiocyanate and anti-CD11b-phycoerythrin (both PharMingen), and all positive fractions were >96% CD11chi.

The peritoneal cavity was enriched for macrophages (pMphi s) by intraperitoneal injection of 1 ml of 10% thioglycollate. Cells were harvested by peritoneal lavage 4 days later.

Immunoblotting and Cysteine Protease Active Site Labeling-- An equal number of cells per sample (numbers as indicated in the text) were lysed on ice for 40 min in 25 µl of cell lysis buffer (0.5% Nonidet P-40, 0.15 M NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH7.2) supplemented with a cocktail of protease inhibitors. Debris was removed by centrifugation at 8,000 rpm for 10 min, and the lysates boiled for 5 min in SDS-reducing buffer. The total lysate was separated by SDS-PAGE (12% acrylamide, w/v) and the proteins electrophoretically transferred onto nitrocellulose membrane, which was probed for protein using the appropriate primary antibody. Binding was detected using a horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ) diluted 1:1500 and visualized by chemiluminescence (ECL; Amersham Pharmacia Biotech).

An equal number of cells per sample (numbers as indicated in the text) were incubated for 2 h at 37 °C in the presence of 0.25 µM cysteine protease inhibitor Cbz-125I-Tyr-Ala-CN2 (44). This radiolabeled inhibitor binds irreversibly to the active site cysteine via a thioester bond. Cells were lysed as above, separated in 12% SDS-PAGE, and the labeled proteins visualized by exposure to Kodak BioMax MR film.

Metabolic Labeling and Immunoprecipitation-- Purified B cells or splenocytes were preincubated at 37 °C for 1 h and 30 min in methionine/cysteine-free RPMI supplemented with 200 mM L-glutamine, 10 mM HEPES, 50 mg/ml penicillin/streptomycin, and 5% dialyzed FCS. Cells were pulsed for 40 min by addition of 1 mCi/ml [35S]methionine/cysteine (Tran35S-label, ICN) and chased for 0, 1, 3, 6, 15, and 24 h in the presence of 30× unlabeled methionine (3 mM) and cysteine (16 mM). Pulse-labeled cells were washed in PBS and lysed in 1% Nonidet P-40, 0.01 M Tris, pH 7.3, 0.15 M NaCl supplemented with a cocktail of protease inhibitors (Roche Molecular Biochemicals). Prior to precipitation with the antisera or antibody indicated in the text, lysates were precleared with protein A-Sepharose (Amersham Pharmacia Biotech) for rabbit antisera immunoprecipitations or protein G-Sepharose (Amersham Pharmacia Biotech) for H-2M immunoprecipitation and then with 10 µl of normal rabbit serum or 18 µg of normal rat IgG (Caltag Laboratories Inc., Burlingame, CA) as appropriate. Precipitated proteins were boiled in SDS-reducing buffer and separated by 7.5-20% gradient SDS-PAGE. Gels were fixed in 50% methanol, 10% acetic acid, treated with Amplify (Amersham Pharmacia Biotech), dried, and the labeled proteins visualized by exposure to Kodak BioMax MR film.

RNA Extraction and cDNA Synthesis-- Cells were lysed in RNA STAT-60 (10-20 × 106 cells/ml RNA STAT-60; Tel-Test "B" Inc., Friendswood, TX), the RNA extracted according to the manufacturer's protocol and contaminating DNA removed by treating 2-µg samples of RNA with amplification grade DNase I (Life Technologies, Inc.). First strand cDNA was prepared by reverse transcription using the Life Technologies SuperscriptTM first-strand synthesis system for RT-PCR, as directed by the manufacturer.

Real-time PCR Primers and Probes-- Primer and probe sequences were selected with the assistance of Primer Express software (Applied Biosystems, Foster City, CA) using nucleotide sequences available in the GenBankTM data base (accession numbers NM_013556 and X06086 for HPRT and catL, respectively). Primers (Life Technologies, Inc.) were: HPRT-F, 5'-3' (5'-TGGAAAGAATGTCTTGATTGTTGAA-3'); HPRT-R, 5'-3' (5'-AGCTTGCAACCTTAACCATTTTG-3'); catL-F, 5'-3' (5'-GACCGGGACAACCACTGTG-3'); catL-R, 5'-3' (5'-CTACCCATCAATTCACGAC-3').

Probes were synthesized with a 5' FAM reporter and 3' TAMRA quencher (Biosearch Technologies Inc., Novato, CA): HPRT, 5'-6-FAM-CAAACTTTGCTTTCCCTGGTTAAGCAGTACAGC-TAMRA-3'; catL, 5'-6-FAM-CTCAGGTGTTTGAACCCATGAATCTTTTACTC-TAMRA-3'.

Real-time PCR Amplification and Analysis-- Real-time PCR amplification was performed on an ABI Prism 7700 sequence detector (Applied Biosystems) in optical tubes (Applied Biosystems) and a total volume of 50 µl. All reagents used were obtained from Applied Biosystems unless otherwise specified. PCR amplification of 0.5 µl of cDNA was performed in the presence of 5 µl of 10× Mg2+-free reaction buffer, a final concentration of 3.5 mM Mg2+, the dNTPS (dATP, dCTP, dGTP, and dUTP) each at a final concentration of 0.2 mM, 0.5 units of uracil N-glycosylase, 1.25 units of AmpliTaq Gold, 4 µl of enzyme grade glycerol (Fisher Biotech, Fairlawn, NJ), a final concentration of 0.2 µM fluorescent probe and two primers, each at a final concentration of 0.5 µM for HPRT and 0.2 µM for catL. PCR conditions consisted of 2 min at 55 °C, during which uracil N-glycosylase degrades any contaminating DNA from previous assays, 10 min at 95 °C, followed by 37 cycles of 95 °C for 15 s and 65 °C for 1 min. Triplicates of each cDNA sample were amplified alongside appropriate controls. Each assay was performed on at least three independent occasions.

Relative quantitation of catL expression was determined using the comparative CT method (user bulletin 2, Applied Biosystems). This method was used to calculate the -fold increase in catL cDNA in catS-/--derived cells when compared with BL6 cells.

    RESULTS
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INTRODUCTION
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DISCUSSION
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B Cells and Dendritic Cells Express Cathepsin L Protein-- Previous studies in our laboratory have shown that activity of catS and catL is detected differentially in APCs, e.g. B cells and DCs express only active catS, whereas catB is expressed ubiquitously (21, 45). Here we sought to investigate further the mechanisms regulating discrete expression of catS and catL by analyzing the protein levels of these cathepsins in APCs.

Highly purified B cells were isolated from the spleens of BL6, catS-/-, and catL-/- mice and analyzed by immunoblotting. 2.5 × 106 cell equivalents were loaded per lane and the membranes probed with rabbit antisera specific for catL (Fig. 1A), demonstrating the presence of catL in B cells. The catL detected in the B cells was the same size (20 kDa) as mature catL detected by active site labeling (20).3 In the absence of catS, the level of catL protein was increased when compared with the BL6 B cells. This increase was not due to loading unequal amounts of protein in the different wells as the same membranes probed for actin indicated equal protein loading (Fig. 1B). Similar analysis of protein lysate from 1 × 106 purified DCs isolated from BL6 and catS-/- mice indicated that mature catL was also present in DCs (Fig. 1C). However, the catL protein levels were the same in both wild-type and catS-/- DCs, as were the actin levels, which indicated equal protein loading (Fig. 1C).


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Fig. 1.   Cathepsin L protein expression in B cells and DCs. B cells were purified from BL6, catS-/-, and catL-/- spleens by positive selection on an automated MACS following staining with MHC class II specific magnetic beads. All populations were >95% B220hi. 2.5 × 106 cells of each type were lysed in the presence of protease inhibitors, reduced, separated by SDS-PAGE on a 12% gel, and electrophoretically transferred onto nitrocellulose membrane. Membranes were probed with either catL-specific rabbit antiserum (A) or actin-specific affinity isolated rabbit antibody (B). These results are representative of five independent B cell preparations. C, DCs were isolated from BL6 and catS-/- spleens by positive selection on an automated MACS following staining with CD11c-specific magnetic beads. Purity of the positive fraction was >95% CD11chi. Protein lysate from 1 × 106 cell equivalents was analyzed for catL and actin protein as above. These results are representative of three independent DC isolations. D, 2.5 × 106 B cells and 1 × 106 DCs were incubated in the presence of the irreversible cysteine protease inhibitor Cbz-125I-Tyr-Ala-CN2, lysed in the presence of protease inhibitors, reduced, and separated by SDS-PAGE on a 12% gel.

To determine whether the catL protein observed in B cells and DCs was active, the same number of purified cells as used for immunoblotting were incubated in the presence of the irreversible cysteine protease inhibitor Cbz-125I-Tyr-Ala-CN2 (Fig. 1D). No catL activity could be detected in either B cells or DCs, although both active catS and catB were present, indicating that the inhibitor had been internalized by the cells and that culture conditions were permissive for its binding to active site cysteines.

Macrophage Cathepsin L Protein Levels Are Independent of Cathepsin S-- Having observed that the level of catL protein in B cells is increased in the absence of catS, whereas in DCs the level is constant in the presence or absence of catS, we wanted to analyze catL protein levels in pMphi s from BL6 and catS-/- mice. The level of catL protein in pMphi s is much greater than in either B cells or DCs3; therefore, a serial dilution of protein was performed to enable us to detect any potential differences between samples.

Protein lysate from BL6 and catS-/- pMphi s was serially diluted 2-fold, from 40 µg of protein to 312.5 ng of protein, the proteins separated by SDS-PAGE, and the membranes probed with anti-catL rabbit sera (Fig. 2). For both BL6 and catS-/- pMphi s, the mature, 20-kDa form of catL could be detected at the first three protein concentrations and the 36-kDa pro-form of catL at the first five. This demonstrated that in pMphi s, as in DCs, catL protein levels are independent of catS.


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Fig. 2.   Cathepsin L protein in BL6 and catS-/- macrophages. Thioglycollate-elicited Mphi s were isolated from the peritoneum of BL6 and catS-/- mice. Protein lysate from 1 × 106 pMphi s from each animal was assayed for protein content (52). Protein from 2-fold serial dilutions of lysates, from 40 µg of protein to 312.5 ng of protein, was separated by 12% SDS-PAGE and the membranes probed with catL-specific rabbit antiserum as described in Fig. 1. The data are representative of three independent experiments.

Increased Cathepsin L Protein in B Cells in the Absence of Cathepsin S Is Not a Result of an Increase in Cathepsin L mRNA-- The finding that, in B cells, the level of catL protein is enhanced in the absence of catS led us to consider two possibilities for this: 1) transcription of catL mRNA is increased, or 2) degradation of catL protein is mediated either directly or indirectly by catS. We aimed to investigate the first possibility using a quantitative PCR technique.

B cells were purified from the spleens of BL6 and catS-/- mice, RNA was extracted and cDNA synthesized. Real-time PCR amplification and subsequent quantitation indicated the level of catL mRNA was not significantly different in BL6 and catS-/- mice (Fig. 3).


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Fig. 3.   Cathepsin L mRNA levels in BL6 and catS-/- B cells. cDNA was synthesized from RNA extracted from highly purified populations of B cells (>95% B220hi) obtained from BL6 and catS-/- mice. Real-time PCR was used to determine relative catL mRNA levels in BL6 and catS-/- mice. The data are plotted as the amount of catL mRNA detected in catS-/- B cells relative to BL6 B cells, with a value of 1 being equal levels. cDNA derived from three independent DC preparations was assayed on a minimum of three independent occasions, and the data are shown as the mean and standard deviation of triplicates in each individual assay.

Having excluded the possibility that catL transcription was increased in the absence of catS, we attempted to determine whether the kinetics of catL degradation were altered in catS-/- mice. However, despite numerous attempts, we were unable to specifically detect catL protein by metabolic labeling and immunoprecipitation. Thus, we have shown that an increase in transcription of catL mRNA is not the mechanism by which the mature form of catL protein is accumulated in catS-deficient B cells, and we suggest that this may be due to slowed protein degradation.

Neither Cathepsin S nor Cathepsin B Play a Role in H-2M Degradation-- We have shown previously that, in catS-deficient B cells, exogenous antigen presentation is severely impaired and that the late stages of Ii chain cleavage are blocked (21). These observations suggested the block in Ii chain degradation may be a major factor resulting in deficient antigen presentation in catS-/- B cells. Our data presented here, indicating a role for catS in regulating protein levels of catL, led us to study the role of catS in the degradation of H-2M and GILT, two other important molecules in the antigen presentation pathway.

To assess the kinetics of H-2M degradation, B cells were purified from BL6, catS-/-, catB-/-, and catS-/-xcatB-/- spleens; pulse-labeled with [35S]methionine/cysteine; and chased for 0, 1, 5, 15, and 24 h before immunoprecipitation with the H-2M-specific rat monoclonal antibody, 2C3A. Fig. 4 shows that degradation of both the alpha  and beta  chains of H-2M have the same kinetics in BL6 control B cells as in cells derived from catS-deficient mice, indicating no major role for catS in the turnover of this important accessory molecule.


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Fig. 4.   H-2M degradation in cathepsin-deficient B cells. Highly purified B cell populations (>95% B220hi) were isolated from BL6, catS-/-, catB-/-, and catS-/-xcatB-/- spleens using the AutoMACS. The cells were pulse-labeled with [35S]methionine/cysteine and chased for 0, 1, 3, 15, and 24 h before immunoprecipitation with the H-2M-specific rat monoclonal antibody, 2C3A. Immunoprecipitates were separated by SDS-PAGE in 7.5-20% gradient gels. The data shown are representative of two independent experiments.

In studies using cathepsin inhibitors, other laboratories have suggested a role for catB in degradation of the beta  chain of H-2M in mature bone marrow-derived DCs (36). By using catB-deficient mice, we show here that in B cells the kinetics of both H-2Malpha and H-2Mbeta turnover are unaffected by the presence or absence of catB (Fig. 4). B cells from mice deficient in both catS and catB also degrade H-2M with the same kinetics as the control (Fig. 4), indicating no redundancy between these two cathepsins in H-2M turnover.

As previously observed in B cells by others (46), following precipitation of H-2M we were able to detect Ii chain fragments (Fig. 4), highlighting the interaction between H-2M and MHC class II molecules bound to Ii degradation intermediates. In the absence of catS, we also observed characteristic accumulation of the 14-kDa small leupeptin-induced protein and 10-kDa leupeptin-induced protein fragments of Ii.

Cathepsin S but Not Cathepsin B Plays a Minor Role in the Turnover of GILT-- Cleavage of precursor GILT at both the N terminus and C terminus is required for generation of the mature thiol reductase (39). It has been shown previously in vitro that catS is able to cleave the N-terminal propeptide completely and the C-terminal propeptide partially, whereas catB cleaved the C-terminal propeptide only (37). We therefore, sought to establish whether in vitro activity of these cathepsins correlated with a critical function in GILT maturation in vivo.

To determine the kinetics of GILT degradation, purified B cells from BL6, catS-/-, catB-/-, and catS-/-xcatB-/- spleens were metabolically labeled and chased for 0, 1, 3, 6, 15, and 24 h. Protein lysates were immunoprecipitated with GILT-specific rabbit antiserum and analyzed by SDS-PAGE (Fig. 5). In the absence of catB, i.e. catB-/- and catS-/-xcatB-/- B cells, we observed that, although the initial amount of precursor GILT was greater than in wild-type control and in catS-/- B cells, the kinetics of precursor cleavage to form mature GILT were not significantly delayed, occurring within 1 h of chase.


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Fig. 5.   GILT degradation in cathepsin-deficient B cells. Highly purified B cell populations (>95% B220hi) were isolated from BL6, catS-/-, catB-/-, and catS-/-xcatB-/- spleens using the AutoMACS. The cells were pulse-labeled with [35S]methionine/cysteine and chased for 0, 1, 3, 6, 15, and 24 h before immunoprecipitation with GILT-specific rabbit antiserum. Immunoprecipitates were separated as described previously. The data shown are representative of three independent experiments.

In the absence of catS, the kinetics of precursor cleavage to form mature GILT were not significantly delayed when compared with BL6 B cells. However, mature form protein was still detected throughout 15 and 24 h of chase (Fig. 5), whereas in control cells it was detectable for only 5 h. This observation indicates that, although catS plays no role in the in vivo generation of mature GILT from its precursor, it plays a role in turnover of the mature protein.

In summary, we observed no major role in vivo for either catS or catB in the generation of mature form GILT, as the kinetics of degradation were not significantly affected by a deficiency in either cathepsin. Therefore, we assume that in vivo regulation of GILT by proteolytic processing of its precursor can be mediated by proteases other than catS or catB. CatS was, however, shown to play a role in the degradation of the mature form of GILT.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies have shown that activity of the cysteine proteases catS and catL is detected differentially in APCs, with catS present in B cells, DCs, and Mphi s and catL in Mphi s and thymic epithelial cells (20, 21, 23). The importance of these enzymes is highlighted by the profound defect in MHC class II Ag presentation in their absence (20-22). Such defects are thought to be largely a result of the critical role played by catS and catL in the late stages of Ii degradation (20-22); however, evidence is accumulating to suggest a role for cathepsins, including catS, catL, and the aspartyl protease asparaginyl endopeptidase (47), in the generation of peptide fragments for MHC class II loading (21, 48). Here we studied the role of catS and catB in the degradation and processing of two other molecules important in the MHC class II presentation pathway, H-2M and GILT. Using cathepsin-deficient B cells, we show that neither catS nor catB play a role in degradation of H-2M or proteolytic cleavage of precursor GILT; however, a role for catS in the turnover of mature GILT was observed. Previous in vitro studies have shown that processing of precursor GILT to a mature thiol reductase can be mediated by both catS and catB (37); however, our data indicate that neither cathepsin is essential for this maturation in vivo. Therefore, it is possible that neither catS nor catB are involved in GILT precursor cleavage in vivo, or that there is redundancy among enzymes capable of mediating this step. Our data indicate that, although the major function of catS in the MHC class II presentation pathway remains degradation of Ii, it also regulates turnover of another important accessory molecule, GILT. In addition, we report here a dependence on catS for regulation of mature catL protein levels in B cells.

The mechanism by which catS and catL activity is differentially regulated has hitherto been undetermined, and here we report for the first time the presence of mature catL protein in B cells and DCs in the absence of detectable enzyme activity (Fig. 1). The detection of high levels of the cysteinal proteases catS and catB in these same cells indicates that the absence of catL activity cannot be attributed to inappropriate conditions for inhibitor binding, e.g. an oxidative environment. Previous investigations have shown that binding of the irreversible inhibitor used in these studies can be blocked by the presence of a reversible inhibitor of cysteinal cathepsins (18). Therefore, our observations suggest it is possible that catL activity in B cells and DCs may be modulated by a specific inhibitor. However, as the level of catL in these cells is much lower than that detected in pMphi s (100 and 50 × less catL mRNA in B cells and DCs respectively than in pMphi s),4 we cannot at present rule out the possibility that the catL activity is below the detection threshold of the assay. Many molecules have been implicated as inhibitors of cathepsins, including cystatin c (33), the propeptide regions of the cathepsins (28-30), and the p41 isoform of Ii (49, 50). Using Ii knockout mice, we have shown that p41 does not inhibit catL activity in pMphi s3 or in B cells.4 Therefore, as other described inhibitors of cathepsin activity are highly nonspecific, and B cells and DCs express high levels of active catS and catB, we suggest a novel mechanism of catL specific inhibition may exist in B cells and DCs.

Mature catL protein levels in B cells, but not DCs or pMphi s, were shown to increase in the absence of catS (Fig. 1), suggesting that in B cells catL protein levels are regulated by catS. This regulation is not mediated through transcription, and we propose that catS plays a role in the degradation of mature catL protein. Furthermore, we suggest the difference between APCs in dependence on catS for regulating mature catL protein levels may indicate cell type-specific proteolytic environments, e.g. cathepsin F has been detected only in Mphi s (24).

Our finding that, in B cells, catB plays no role in the degradation of either the alpha  or beta  chains of H-2M (Fig. 4) contrasts with a previous study using bone marrow-derived DCs from Ii-deficient mice, which suggested catB plays a major role in turnover of H-2Mbeta (36). One potential explanation for the discrepancy in these data is that maturation of B cells requires Ii (51) and it is possible that in the absence of Ii, endosomal maturation and therefore protease and H-2M trafficking and function may be perturbed not only in B cells4 but in all APCs derived from these mice.

In conclusion, we have shown here that in B cells, degradation of the accessory molecule H-2M and proteolytic maturation of GILT is not dependent on either catS or catB, implicating the profound block in Ii cleavage as the critical event in impaired Ag presentation by catS-deficient B cells. CatS was, however, observed to play a role in degradation of mature GILT protein and in regulating levels of catL protein in B cells, and we suggest that this is through turnover of the mature protein. However, activity of the mature catL protein detected in B cells and DCs was not observed, and it is possible that this may be due to the presence of a catL-specific inhibitor.

    FOOTNOTES

* This work was supported in part by the Howard Hughes Medical Institute (to K. H., A. R., and P. C.) and grants from the National Institutes of Health (to C. B. and A. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Affiliated with the Molecular and Cellular Biology graduate program at the University of Washington.

§§ Recipient of a fellowship from the Cancer Research Institute.

¶¶ To whom correspondence should be addressed. Tel.: 206-685-9310; Fax: 206-685-3612; E-mail: aruden@u.washington.edu.

Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M101851200

2 M. Maric and P. Cresswell, manuscript in preparation.

3 C. Beers, K. Honey, and A. Rudensky, manuscript in preparation.

4 K. Honey and A. Rudensky, unpublished data.

    ABBREVIATIONS

The abbreviations used are: MHC, major histocompatibility complex; cat, cathepsin; Ii, invariant chain; DC, dendritic cell; Mphi , macrophage; pMphi , peritoneal macrophage; APC, antigen presenting cell; GILT, gamma -interferon-inducible lysosomal thiol reductase; CLIP, class II-associated Ii peptide; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; HPRT, hypoxanthine-guanine phosphoribosyl transferase; Cbz, benzyloxycarbonyl.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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