From the 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
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
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ABSTRACT |
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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
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 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 (pM 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 M 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- 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.
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 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 (pM 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 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
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 pM
Protein lysate from BL6 and catS 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
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 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
To assess the kinetics of H-2M degradation, B cells were purified from
BL6, catS
In studies using cathepsin inhibitors, other laboratories have
suggested a role for catB in degradation of the
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
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.
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 M 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
pM Mature catL protein levels in B cells, but not DCs or pM Our finding that, in B cells, catB plays no role in the degradation of
either the 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.
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
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).
s), where it was shown to be involved in the late
stages of Ii degradation (21-23). CatL, however, was detected only in
M
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
M
s (24). Thus, it would appear that different APCs utilize distinct
cathepsins to mediate late stage Ii degradation and regulate MHC class
II presentation.
s and
M
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.
(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
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
, 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.
s) by
intraperitoneal injection of 1 ml of 10% thioglycollate. Cells were
harvested by peritoneal lavage 4 days later.
/
-derived cells
when compared with BL6 cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
, 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.
s from BL6 and catS
/
mice.
The level of catL protein in pM
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.
/
pM
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
/
pM
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 pM
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 M
s
were isolated from the peritoneum of BL6 and
catS
/
mice. Protein lysate from 1 × 106 pM
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.
/
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.
/
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.
/
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.
/
,
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
and
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.
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-2M
and H-2M
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.
/
,
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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s and catL in M
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.
s (100 and 50 × less catL mRNA in B cells and DCs
respectively than in
pM
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 pM
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.
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
M
s (24).
or
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-2M
(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.
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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.
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ABBREVIATIONS |
---|
The abbreviations used are:
MHC, major
histocompatibility complex;
cat, cathepsin;
Ii, invariant chain;
DC, dendritic cell;
M, macrophage;
pM
, peritoneal macrophage;
APC, antigen presenting cell;
GILT,
-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.
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