By
From the * Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139; Department of Medicine, Brigham and Women's Hospital and
Harvard Medical School, Boston, Massachusetts 02115; and the § Abteilung Innere Medizin I,
Albert-Ludwigs-Universität Freiburg, Freiburg, Germany
Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.
MHC class II Cells deficient in the MHC class II-like molecule H-2M
(in humans, HLA-DM) are defective in presentation of exogenous antigens and accumulate Are class II molecules associated with larger Ii remnants
the precursors of peptide-bound complexes in vivo? We
show that this is indeed the case. We treated APC with
protease inhibitors that prevent complete degradation of Ii
and observed the formation of Purified cathepsin D (Cat D) and cathepsin B (Cat B),
the most abundant lysosomal aspartyl and cysteine proteases,
respectively, have been shown to be capable both of destruction of Ii and of generation of antigenic peptides in
vitro (25). The application of class-specific protease inhibitors to APC in culture has also been used to implicate
these enzymes in Ii destruction and in generation of antigenic peptides (32). We have previously identified a
different cysteine protease, cathepsin S (Cat S), as a key enzyme involved in breakdown of Ii in human B lymphoblastoid cells. Cat S, but not Cat B or Cat D, is capable of producing the class II-CLIP complex in vitro from a class II-Ii
substrate (37). Human Cat S is expressed predominantly in
cells that function as professional APC (38).
It is unclear whether the enzymes involved in degradation of Ii in human cells play a similar role in mouse APC.
The effect of the cysteine protease inhibitor leupeptin on
maturation of mouse MHC class II is less pronounced than
in human cells, and the stage at which degradation of Ii is
interrupted by leupeptin differs between the two species
(32, 33, 35, 39, 40). This might reflect species- or cell
type-specific differences in the mechanism involved in destruction of mouse and human Ii. We show here that in
fresh mouse splenocytes, the proteases involved in, and the
pattern of degradation of Ii are similar to human APC. Cat
S acts in concert with other cysteine and noncysteine proteases to degrade Ii in stages, generating discrete intermediates that remain associated with Analysis of maturation of MHC class II molecules in
mice deficient in Cat D reveals that, contrary to suggestions
based on in vitro experiments, this major aspartyl protease
is not required for normal degradation of Ii or for generation of the bulk of peptide-loaded class II complexes in
vivo. Our results highlight the importance of characterizing
the enzymes involved in antigen presentation by MHC
class II molecules. Development of specific inhibitors for
these proteases could enhance the selective manipulation of
immune responses and be used for treatment of autoimmune diseases triggered by specific combinations of MHC
class II-peptide.
Materials.
Leupeptin was from Boehringer Mannheim (Indianapolis, IN). N-morpholinurea-leucine-homophenylalanine-
vinylsulfone-phenyl (LHVS) was provided by Dr. D. Brömme
(Arris Pharmaceuticals, South San Francisco, CA). Purified human Cat S was obtained by expression in Sf9 cells using the baculovirus expression system as described (41). DMEM, RPMI, and
methionine/cysteine-free media were from GIBCO BRL (Gaithersburg, MD).
Antibodies.
N22 is a hamster mAb that recognizes mouse
MHC class II molecules (42), and was a gift of Dr. R.M. Steinman (Rockefeller University, New York). P4H5 is a hamster
mAb raised against a synthetic peptide corresponding to murine Ii
99-116 (43), and was the gift of Dr. J. Miller (University of Chicago, Chicago, IL). Both mAb were used as undiluted culture supernatant for immunoprecipitations. Rabbit sera raised against the
NH2-terminal region of Ii (anti-Nt-Ii) and CLIP were the gift of
Dr. Davoust (Centre d'Immunologie INSERM-CNRS, Marseille, France). An anti-I-A Mice.
Generation of Cat D Metabolic Labeling.
Freshly prepared splenocytes were incubated in 1 ml methionine/cysteine-free medium supplemented
with 10% FCS, 2 mM L-glutamine, penicillin (1:1,000 dilution
U/ml), and 100 µg/ml streptomycin for 45 min. Cells were pulsed
for 30 min with 0.5 mCi/ml [35S]methionine/cysteine (80/20)
(Du Pont New England Nuclear, Boston, MA) and chased in 20 vol of complete RPMI supplemented with FCS, L-glutamine,
penicillin, and streptomycin as above. Protease inhibitors were added
10 min before pulse. [35S]methionine/cysteine incorporation was
similar in control and in leupeptin- or LHVS-treated cells as assayed by TCA precipitation of incorporated radioactivity. At each
time point, cells were spun down, resuspended in 50 µl PBS, frozen in liquid nitrogen, and stored at Immunoprecipitations and Proteolytic Digestion.
All manipulations
for immunoprecipitations were performed at 4°C. Pulse-chased
samples were thawed in 1 ml lysis buffer (0.5% NP-40, 50 mM
Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM PMSF), incubated for
10 min, and then centrifuged for 10 min at maximum speed in a
microfuge to remove nuclei and cell debris. Lysates were precleared twice for 60 min with 5 µl normal mouse serum, 5 µl
normal rabbit serum (both from ICN Biomedicals, Inc., Aurora,
OH), and 100 µl Staph A (10% wt/vol), followed by another
preclear for 60 min with 100 µl Staph A. Immunoprecipitations
were carried out for 1 h with 100 µl Staph A. Rabbit sera (1-2 µl
per immunoprecipitation) were added directly with the Staph A. Culture supernatants of mAb (0.5 ml) were precoupled for 1 h
before immunoprecipitation. Immunoprecipitates were washed
three times with washing buffer (0.5% NP-40, 50 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 5 mM EDTA), resuspended in sample
buffer containing 5% 2-ME, and analyzed by SDS-PAGE. Radioactively labeled samples were visualized by fluorography using
DMSO-PPO and exposure to Kodak X-OMAT® films (Eastman
Kodak Company, Rochester, NY). For reimmunoprecipitations, Staph A pellets were resuspended in 50 µl PBS containing 1%
SDS and boiled 10 min. 1/10 of the precipitate was retained for
later comparison with reimmunoprecipitates in SDS-PAGE. 1 ml
lysis buffer containing 0.1% BSA was added to the remaining 9/10
of the precipitate. After centrifugation, the supernatant was precleared once and reimmunoprecipitated as above. For proteolytic
digestion with Cat S, precipitates were resuspended in 100 µl of
50 mM Na acetate, pH 5.5, 1% Triton X-100, 3 mM cysteine,
1 mM EDTA, separated into two and incubated 1 h at 37°C
without (control) or with Cat S, respectively. 5 µl of 10× PBS
and 5 µl of 10% SDS was added at the end of digestion and
boiled. 1/5 of each sample was loaded directly on SDS-PAGE
and the remaining portion was reimmunoprecipitated as above.
Gel Densitometry.
Gels were scanned in a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA) and analyzed with the
ImageQuant software. The amount of SDS-stable Labeling of Cysteine Proteases.
Splenocytes were incubated in
complete RPMI with or without protease inhibitors at the concentrations indicated. Detection of active cysteine proteases with Cbz-
[125I]-Tyr-Ala-CN2 was performed as described elsewhere (37).
To assess
the role of cysteine proteases on breakdown of murine Ii,
fresh H-2d splenocytes were pulse-labeled for 30 min and
chased up to 240 min in the absence or presence of leupeptin. At each time point, class II molecules were recovered
by immunoprecipitation with the mAb N22. The samples
were then divided into two and displayed by SDS-PAGE
with or without prior boiling to score for formation of
peptide-bound SDS-stable
Leupeptin is an inhibitor with broad specificity for cysteine proteases (49; and see below) which interferes with
maturation of MHC class II molecules by preventing the
complete destruction of Ii (32, 33, 35, 39, 40). Formation
of mature SDS-stable We have
previously described the involvement of the cysteine protease Cat S in degradation of Ii in human B cells (37). Cat S
can be specifically inactivated in human B cells with low concentrations of the irreversible inhibitor LHVS (37, 52). To assess whether Cat S plays a similar role in mouse APC,
we first determined the content of active cysteine proteases
in splenocytes treated with various concentrations of LHVS
or with leupeptin. After this treatment, splenocytes were
incubated with the iodinated form of Cbz-Tyr-Ala-CN2,
an active site inhibitor that binds irreversibly to cysteine
proteases in proportion to their activity (53). The amount
of active cysteine proteases remaining can thus be visualized
in SDS-PAGE as complexes bound to Cbz-[125I]-Tyr-Ala-
CN2 (Fig. 2 A). Compared with untreated splenocytes, 1-10
nM LHVS completely inactivated Cat S without affecting
Cat B. Thus, comparison of the effect of leupeptin (1 mM)
and LHVS (3 nM) allows an assessment of the relative role
of Cat S in maturation of class II molecules.
Mouse splenocytes were pulse chased in the presence of
leupeptin or LHVS and their MHC class II molecules immunoprecipitated with N22 (Fig. 2 B). Treatment with
LHVS resulted in accumulation of LIP10 but not of LIP22,
indicating that LIP22 can be degraded to generate LIP10
by a cysteine protease other than Cat S. The The most direct approach to assess the
involvement of a given protease on antigen presentation is
to study cells from mice deficient in the enzyme. We analyzed the maturation of newly synthesized MHC class II in
mice deficient in Cat D (45), by far the most abundant lysosomal aspartyl protease. Pulse-chase analysis of Cat D-deficient splenocytes showed normal maturation of MHC class II molecules, both in degradation of Ii and in the formation
of SDS-stable dimers (Fig. 3). The mouse cells used in this
experiment only express I-Ab, which explains the simplicity in the pattern of bands in the
The abundance of LIP10 associated with I-Ab
correlated with the presence of an SDS-stable complex
( Unlike H-2d class II molecules, I-Ab immunoprecipitated from leupeptin-treated cells contained very little
LIP22, whereas LIP10 was clearly detectable. Strikingly,
the reduction in the relative amount of SDS-stable The requirement for Ii in the assembly of class II Splenocytes of mice of the d, b, k, s, and u haplotypes
were pulse labeled for 30 min and chased for 4 h in the absence or presence of leupeptin or LHVS. I-A molecules
were then immunoprecipitated with a rabbit serum that reacts specifically with the COOH-terminal segment of the
I-A
In agreement with previous reports, the fraction of mature class II molecules that are resistant to dissociation in
SDS varies among specificities (48, 55). Virtually all I-Ab
and I-As molecules had formed SDS-stable dimers after 4 h
of chase in control cells, whereas the proportion of resistant
I-Ad dimers remained small. This contrasts with the immunoprecipitates obtained from H-2d splenocytes shown in
Figs. 1 and 2, and is due to the presence of I-Ed molecules,
more stable in SDS (data not shown), recovered when the
mAb N22 is used for immunoprecipitation.
To assess the effect of leupeptin and of LHVS on maturation of each allelic product, we determined the amount
of I-A SDS-stable dimers in the precipitates from inhibitor-treated cells relative to the controls (Fig. 4 B). Treatment
with leupeptin resulted in a reduction in the amount of SDS-stable dimers that ranged from 87% (I-Ab) to only 40% (I-Ak
and I-As). These differences were unrelated to the intrinsic
capacity of each allelic product to form SDS-stable dimers
in the absence of drugs, as illustrated by I-Ab and I-Ak. In
all cases, there was an inverse correlation between the fraction of SDS-stable complexes and the amount of LIP10
that remained associated with The effect of LHVS compared with leupeptin was similar for all the allelic products examined: less inhibition of
formation of SDS-stable dimers, decrease in the fraction of
LIP10-containing complexes, and no accumulation of LIP22
(Fig. 4 A). As for leupeptin, the relative effect of LHVS on
maturation of different class II allelic products showed
dramatic variability: formation of SDS-stable I-Ak and I-As
complexes was similar in LHVS and in control cells, whereas
only 35% of the I-Ab complexes generated in control cells
were present in their LHVS-treated counterparts (Fig 4 C).
This suggests that the differences among specificities revealed by leupeptin are the result of inhibiting cleavage of
LIP10 by Cat S, and not of inhibiting the proteases involved in other stages of degradation of Ii or in the generation of antigenic peptides.
The heterogeneity described in the effect of leupeptin and
LHVS among allelic products was consistently observed in
noncongenic animals selected only on the basis of their
MHC, which makes it unlikely that factors other than the
MHC itself are responsible. Nevertheless, to eliminate the
possibility that the cysteine proteases involved in degradation of Ii were affected differentially by leupeptin or LHVS
in cells of different haplotypes, we compared the pattern of
active site-labeled proteases in H-2b, H-2d, and H-2k cells
untreated or treated with the inhibitors using the Cbz- [125I]-Tyr-Ala-CN2 probe (Fig 4 C). The amount of Cat S
relative to Cat B in the control samples varied to some extent among the splenocytes of the three haplotypes. However, this variability cannot account for the different effect
of leupeptin or LHVS on maturation of each MHC class II
allelic product, because both inhibitors, when used under
the conditions that reveal those differences, inactivated their
respective target enzymes to a similar extent in the three haplotypes examined. The residual amount of active Cat B
present in leupeptin-treated H-2k splenocytes is comparable to that in H-2d. Moreover, Cat B is unaffected by
LHVS, eliminating the possibility that the remaining active
Cat B in LHVS-treated cells is responsible for the different
efficiency of maturation of I-Ad, I-Ab, and I-Ak.
Another explanation for the allele-dependent heterogeneity could be that the These results are consistent with I-As, and particularly
I-Ak, being capable of converting the LIP10-associated MHC class II molecules in many respects behave as do
other residents of the endosomal-lysosomal system (57,
58). Lysosomal hydrolases are synthesized as inactive precursors, equipped with a prosequence that must be removed by proteolysis to generate the active enzyme, a conversion that takes place in the endosomal system itself. Class
II molecules are synthesized with the structural equivalent
of such a propiece, namely the invariant chain. The noncovalently associated Ii directs the class II molecules into the
endocytic route, and preserves their peptide-binding capacity until they reach the compartments where antigenic peptides reside. Once there, Ii is eliminated to restore the peptide-binding capability of the class II heterodimers. This is
accomplished by the interplay of two different activities
that colocalize with the class II molecules. The first is the
proteolysis by endosomal enzymes, and the second is the
action of H-2M/HLA-DM, which facilitates the release of
CLIP and its substitution with antigenic peptides. Because
of the heterogeneity of the endocytic pathway in terms of
pH , redox potential, and protease content, the exact timing and location at which class II molecules acquire their
peptide binding capacity will determine the sets of antigenic determinants that the class II molecule will finally
present at the cell surface. Here, we describe the ordered
progression of Ii removal and formation of class II complexes competent for antigen binding in mouse APC and
address the role of different proteases of the endocytic route in this process. Our results also show that the extent to
which Ii must be degraded to obtain Using protease inhibitors, breakdown of Ii can be blocked
at different stages in both mouse and human cells (32, 33,
35, 39, 40). Degradation intermediates are observed that
also occur in cells not exposed to these protease inhibitors.
Our results indicate that the majority of mouse Ii is converted to the LIP22 fragment by a noncysteine protease,
which is therefore not inhibited by leupeptin. LIP22 would
be cleaved COOH terminally of CLIP by a cys protease(s)
distinct from Cat S, generating LIP10. Finally, Cat S cleaves
LIP10 NH2 terminally of CLIP, yielding The process of degradation of Ii that we have described occurs in stages,
through sequential cleavages at specific sites. This model
implies that either the structure of each substrate prevents
the use of cleavage sites downstream in the degradation pathway, as supported by experiments in vitro (59), or that the different protease activities are compartmentalized. This could be achieved physically (i.e., in the extreme version,
each protease would be present and active at a different compartment of the endocytic route) or functionally, so that
different proteases accompanying the newly synthesized
The effect of leupeptin on maturation of mouse class II
molecules expressed in the B lymphoma cell line A20 (40,
61) appears less pronounced than in the unstimulated splenocytes employed in this study. Furthermore, LIP22 is not
observed in association with I-Ad in leupeptin-treated A20
cells. This suggests that the proteolytic events involved in
maturation of MHC class II may vary among cell types or at
different stages of differentiation. Indeed, differences in antigen processing between resting and activated B cells have been observed (68, 69). This underlines the importance of analyzing the process of degradation of Ii as a possible
mechanism employed by professional APC to modulate antigen presentation according to their state of differentiation
(40, 54).
The role of aspartyl proteases on maturation of MHC
class II molecules has aroused considerable interest. Immunoelectron microscopy and cell fractionation studies carried
out on human B cells showed the presence of Cat D in
MIIC compartments. This finding suggested a possible role
for this enzyme in the degradation of Ii, generation of antigenic determinants, or both (62, 70, 71). Indeed, Cat D
can completely degrade full-length Ii in vitro (27, 31), and
class-specific inhibitors of aspartyl proteases block degradation of Ii in human B cells (34). In vitro degradation of
ovalbumin and hen egg lysozyme by Cat D yields peptides
that can be presented to T lymphocytes by MHC class II
molecules on the surface of fixed APC (28, Rodriguez,
1992 #1123) (30). The pathway for degradation of Ii that
we have described involves noncysteine protease(s), yet
degradation of Ii and generation of mature MHC class II
molecules in Cat D-deficient splenocytes proceed normally.
Moreover, inactivation of cysteine proteases with leupeptin
had a similar effect on both Cat D+/ The capacity of different MHC class II allelic products to bind antigenic peptides in a DM-deficient background is variable (27, 56). Likewise, we show here that
the effect of protease inhibition on maturation of class II
molecules varies widely among class II specificities. The
conversion of LIP22 into LIP10 in the presence of leupeptin is less efficient for I-Ad than for the other specificities
analyzed. Also, the extent to which Two mechanisms could account for the differences observed. First, conversion of LIP22 into LIP10, and of LIP10
into CLIP require proteolysis at a site immediately COOH
and NH2 terminally of CLIP, respectively (Fig 1 C). Because
CLIP is inserted into the peptide-binding groove of the Regardless of the actual mechanism involved in these allele-specific differences, these findings have implications for
addressing the requirement for newly synthesized MHC
class II molecules in antigen presentation. Efficient presentation by APC treated with protease inhibitors has been
used as a criterion to establish whether or not presentation
of a given determinant requires newly synthesized Studies of antigen presentation in the presence
of protease inhibitors have attempted to define which proteases are required to generate specific antigenic determinants (28, 36, 74, 77). However, the complexity of the
proteolytic events involved in antigen presentation by
MHC class II molecules confound the interpretation of
those studies. First, most inhibitors have broad specificity
and do not inhibit a single protease (49; Fig. 2 A). Second,
the same proteases involved in degradation of antigens may
be necessary for destroying Ii, making it difficult to determine whether the failure to present an antigen in the presence of the drug is due to the inability to generate the determinant or due to interference with Ii degradation.
Cat S is a key enzyme involved in complete destruction
of Ii both in human (37) and in mouse cells (Fig. 2), and is
expressed mostly in professional APC (38). Specific inhibitors of this cysteine protease, such as LHVS, are good candidates for preventing presentation of specific combinations
of class II plus peptide. First, inhibition of Cat S with
LHVS, or even of the other cysteine proteases with leupeptin, does not prevent the formation of many antigenic peptides, as deduced from the abundance of peptide-loaded I-Ak or I-As complexes in APC treated with these inhibitors. However, in cells expressing both I-Ab and I-Ak, treatment with leupeptin or LHVS should prevent presentation of many determinants by I-Ab without hindering presentation by I-Ak. Second, Ii is degraded gradually along the endocytic route (50, 51), as is likely the case for endocytosed
antigens. This results in heterodimers bind peptides generated
by degradation of endocytosed antigens and display
them as MHC class II-peptide complexes at the cell surface
for recognition by CD4+ T cells (reviewed in reference 1).
Antigens, taken up by the APC, negotiate the entire trajectory of the endocytic pathway, culminating in arrival in lysosomal compartments. This process subjects the antigens
to a range of conditions that differ in pH, redox potential,
as well as protease content and concentration. Gradual proteolysis and generation of discrete sets of breakdown products at the different stations of the endocytic route is the likely result. To encounter the antigenic determinants, newly synthesized MHC class II molecules associate in the endoplasmic reticulum (ER)1 with an accessory molecule, invariant chain (Ii), which carries the information necessary
to direct the
Ii trimer to the endocytic route (2, 3). In
forming this complex, the CLIP region of Ii (from class II-
associated Ii peptide; reference 4) is inserted into the peptide binding site of the
heterodimer similarly to antigenic peptides (5). This interaction protects the site from
binding to ER-resident proteins (8) and preserves its peptide-binding capacity (9, 10). However, proteolysis of Ii
and dislodging of CLIP are required to allow association of antigenic peptides with the binding site of
dimers upon
entry of the
Ii complex into the endocytic route (1).
-CLIP complexes at the
cell surface (11). These observations provided the basis
for a model in which proteolysis of Ii results in formation
of
-CLIP, which is converted to its antigenic peptide-associated counterpart in a reaction mediated by H-2M/
HLA-DM (18). Indeed, displacement of CLIP is catalyzed by HLA-DM in vitro (19). In this model, formation of
-CLIP would be a prerequisite for presentation of antigenic peptides. However, HLA-DM can also interact with
HLA-DR associated with larger fragments of Ii both in
vivo and in vitro (22), although in vitro its preferred substrate is the DR-CLIP complex (23).
-peptide complexes. However, formation of these complexes in cells treated with inhibitors is strongly dependent on the MHC class II allelic
product considered. Consequently, inhibition of the proteases that degrade Ii could result in prevention of antigen
presentation in an allele-dependent manner. Correct identification of these proteases, and of those that participate in
the generation of antigenic peptides, therefore, is important
because these enzymes represent potential targets for selective inhibition of immune responses.
dimers.
rabbit serum was the gift of Dr.
R.N. Germain (National Institutes of Health, Bethesda, MD) (44). Rabbit antiserum specific for mouse Cat D has been described elsewhere (45).
/
mice has been described (45).
Because Cat D-deficient mice die around 24 d of age, heterozygous couples were crossed, and Cat D
/
and Cat D+/
littermates selected for further experiments. Newborns were genotyped by PCR using as template genomic DNA isolated from tail
biopsies (46). Two PCR reactions were performed with each DNA.
The first was aimed to amplify the neo-resistance gene contained
in the targeting vector. To distinguish between +/
and
/
mice, a second PCR was performed using oligonucleotides that
hybridized at the flanking regions of the fourth exon of the Cat D
gene. The presence or absence of Cat D was confirmed in some
animals by immunoprecipitation of pulse-labeled splenocytes using an anti-Cat D rabbit serum. Mice 15-20 d of age were used
in experiments involving Cat D-deficient animals. Animals used
in this study were maintained at the animal facilities of the Massachusetts Institute of Technology in compliance with institutional
guidelines.
80°C.
dimers relative to the total amount of
chain in immunoprecipitates from
control, leupeptin, and LHVS-treated cells was calculated as follows: the intensity of the region of the gel containing the SDS-stable dimers in the nonboiled sample minus the intensity in the
equivalent region of its boiled counterpart was first determined.
The resulting value was then divided by the intensity of the band
corresponding to the
chain in the boiled sample.
Role of Cysteine Proteases on the Maturation of MHC Class
II Molecules in Freshly Isolated Mouse Splenocytes.
dimers (Fig. 1 A). The mAb
N22 reacts with both I-Ad and I-Ed molecules, and allowed
recovery of roughly similar amounts of
and
subunits at
all time points of the pulse-chase experiment, indicating
that N22 recognizes with similar efficiency Ii- and peptide-associated
dimers, but not any of the three individual subunits. This reactivity was confirmed by immunoprecipitation of in vitro translated I-Ad
and
subunits and of
mouse Ii (Wolf, P., and H.L. Ploegh, unpublished observations). Pulse-labeled samples showed MHC class II
and
subunits, the p33 form of Ii, and a small amount of the p41
form of Ii (47). Maturation of MHC class II in control cells
during the chase was accompanied by addition of sialic acids to both
and
chains, which is responsible for the increased complexity in the pattern of bands observed in the
region and for the reduced mobility of
. Furthermore,
degradation of Ii occurred; the apparent increase in the
amount of full-length Ii at 60 min of chase is due to association of excess labeled Ii to unlabeled
dimers. In addition, we observed the transient accumulation of a polypeptide of 10 kD at 60 min (P10) and the generation of
-peptide complexes, a fraction of which is resistant to
dissociation in SDS at room temperature (48).
Fig. 1.
Maturation of mouse MHC class II molecules in the
absence or the presence of leupeptin. (A) Fresh H-2d splenocytes
were pulse labeled for 30 min and chased for the times indicated in
the absence (control) or the presence of 1 mM leupeptin. MHC
class II molecules were immunoprecipitated with mAb N22, and
loaded on 12.5% reducing SDS-PAGE without (NB) or after (B)
boiling. The position of the MHC class II and
subunits, SDS-stable mature
heterodimers, full-length Ii, and intermediate degradation products of Ii detected in the abscence (P10) or the presence of leupeptin (LIP25, LIP22, LIP18, and LIP10) are indicated. Leupeptin-treated pulsed splenocytes showed the same pattern and intensity of bands as the control sample (data not shown). (B) Reimmunoprecipitation of the leupeptin-induced polypeptides LIP22 and LIP10. H-2d splenocytes were pulse labeled for 30 min and
chased 240 min in the presence of 1 mM leupeptin. After immunoprecipitation with mAb N22, MHC class II molecules were
fully denatured by boiling in 1% SDS and the released polypeptides immunoprecipitated in parallel with rabbit sera for the NH2-terminal or the CLIP region of Ii, or with mAb P4H5. Samples
were loaded on 10% reducing SDS-PAGE with a fraction of the
boiled N22 immunoprecipitate. The lower part of the panel corresponds to a longer exposure of the same gel shown in the upper half. (C) Structure of mouse Ii and the degradation intermediates that accumulate in
mouse splenocytes treated with leupeptin (LIP22 and LIP10), as deduced from reimmunoprecipitations. The N-linked carbohydrates at positions 113 and
119, the region against which mAb P4H5 was raised, and the transmembrane, CLIP, and trimerization regions of Ii are indicated according to references 6, 7. The enzymes involved in each stage of degradation of Ii and the protease inhibitors that block those steps are indicated.
[View Larger Versions of these Images (73 + 55 + 16K GIF file)]
complexes was severely decreased
in fresh splenocytes treated with leupeptin (Fig. 1 A). The
amount of the P10 polypeptide recovered at 60 min of
chase was reduced in comparison to the control sample, but
showed an increase at 240 min (LIP10, from leupeptin-induced-polypeptide) (32, 33). Analysis in Tris-tricine gels
revealed that LIP10 actually consists of two distinct polypeptides of similar molecular weight (data not shown). Another polypeptide of 22 kD (LIP22) likewise accumulated,
as did two minor polypeptides of about 25 kD (at 60 min
chase) and 18 kD (at 240 min). Both LIP10 and LIP22 were
recognized by rabbit antisera directed against the NH2-terminal end and the CLIP region of mouse Ii (residues 87-
100) upon reimmunoprecipitation. By contrast, the mAb
P4H5, directed against the 99-116 region of Ii (43), failed
to precipitate LIP10 (Fig. 1 B). Therefore, both LIP10 and
LIP22 are degradation intermediates of Ii that remain associated with MHC class II molecules. LIP10 spans approximately residues 1-100, where CLIP ends, whereas LIP22
roughly extends to residue 160, based on its mobility in
SDS-PAGE (Fig. 1 C). LIP22 was also observed in control
samples (Fig. 1 A) upon long exposure of the autoradiogram, indicating that, like LIP10, it represents a normal degradation intermediate in the maturation of mouse MHC
class II molecules. The relative amounts of LIP10/LIP22 at
60 min and 240 min of chase suggests that LIP22 is the precursor of LIP10. Similarly, LIP25 and LIP18 likely correspond to precursors of LIP22 and LIP10, respectively. These
results suggest that mouse Ii is degraded following a staged
pattern, in which COOH-terminal segments are removed
first (Fig. 1 C) (50, 51). The enzyme(s) involved in the initial stages of degradation of Ii is a noncysteine protease, because the rates of disappearance of full-length Ii during the
chase in the absence or presence of leupeptin are comparable (Fig. 1 A). It is the further degradation of the LIP22 and
LIP10 intermediates that appear to require one or more
cysteine proteases.
Fig. 2.
Role of Cat S on degradation
of mouse Ii. (A) LHVS is a specific inhibitor
of Cat S at the 1-10 nM range. Mouse splenocytes were incubated with the indicated
concentrations of LHVS or leupeptin (1 mM)
followed by addition of Cbz-[125I]-Tyr-
Ala-CN2. The bands corresponding to Cat
S and to the high and low molecular weight
forms of Cat B are indicated. (B) A cys protease different from Cat S converts LIP22
into LIP10. H-2d splenocytes were pulse labeled for 30 min and chased for 240 min
without (control) or with 1 mM leupeptin or
3 nM LHVS. N22 immunoprecipitates
were loaded without boiling in 12.5% SDS-PAGE. (C) Cat S cleaves Ii NH2 terminally
of CLIP. H-2d splenocytes were pulse
chased in the presence of LHVS and immunoprecipitated with N22. The precipitate
was resuspended in Cat S buffer and incubated with or without Cat S for 1 h at 37°C.
After incubation, samples were boiled in 1%
SDS, 1/5 loaded directly on gel, and the remainder diluted in lysis buffer and reimmunoprecipitated as in Fig. 1 B. The arrow at
the right of the figure indicates the position
of CLIP-containing fragments devoid of the
NH2-terminal region of Ii.
[View Larger Versions of these Images (43 + 34 + 76K GIF file)]
-associated
LIP10 fragment was degraded by Cat S in vitro, yielding smaller polypeptides that could be reimmunoprecipitated
with antibodies against CLIP, but not against the NH2-terminal region of Ii (Fig. 2 C). These results indicate that Cat
S is capable of cleaving mouse Ii NH2 terminally of CLIP
(see Fig. 1 C). As in human B cells, the inhibition of formation of peptide-bound class II complexes was less complete than with leupeptin (37; Fig. 2 B) suggesting that
when Cat S is inactivated other cysteine proteases can also degrade LIP10, albeit less efficiently.
region of the N22 immunoprecipitates obtained from these mice, as compared
with the H-2d samples used for the experiments in Figs. 1
and 2. Most I-Ab molecules complexed with antigenic peptides are SDS resistant (48), as shown in the nonboiled precipitate recovered after 4 h of chase, in which no free
is
detectable (Fig. 3). This property of I-Ab allows a clear assessment of the lack of involvement of Cat D in the generation of the bulk of peptide-bound class II molecules. The
effects of leupeptin on class II maturation were similar for Cat D+/
and Cat D
/
splenocytes (Fig. 3). The lack of effect of the Cat D
/
mutation, therefore, is not masked by
any of the cysteine proteases inhibited by leupeptin. We
conclude that Cat D is not required for the degradation of
Ii or for the generation of the bulk of peptides bound by
MHC class II molecules.
Fig. 3.
Maturation of I-Ab proceeds normally in Cat D-deficient
mice. Splenocytes from Cat D+/ (top) or Cat D
/
(bottom) littermates
were pulse-chased in the absence or presence of leupeptin as indicated
and I-Ab molecules immunoprecipitated with mAb N22 and analyzed on
12.5% SDS-PAGE as in Fig. 1 B. The position of the I-Ab
-LIP10
SDS-stable complex is indicated as
l.
[View Larger Version of this Image (56K GIF file)]
l) of ~70 kD (Fig. 3) (40, 54). This complex most
likely corresponds to
-LIP10 trimers, because (a) the
amount of LIP10 increased dramatically when this complex
was dissociated by boiling, (b)
l could be immunoprecipitated with a rabbit serum directed against the NH2-terminus of Ii (data not shown), and (c) both I-Ab
and the
NH2-terminal region of Ii were detected in this complex by immunoblot (data not shown). The resistance of the
LIP10-I-Ab complex to dissociation in SDS is not surprising, because stable I-Ab-CLIP complexes can also be resolved by SDS-PAGE (15). This finding emphasizes the
remarkable stability of the I-Ab dimer associated with antigenic peptides or with Ii fragments.
-
peptide complexes induced by leupeptin appeared higher
for I-Ab than for H-2d (40). This observation prompted the
investigation of the effect of cysteine protease inhibitors on
the maturation of different MHC class II allelic products.
and
subunits
in the ER (55), and for HLA-DM to ensure proper removal of CLIP and peptide loading (27, 56), varies among
class II specificities. We blocked degradation of Ii with either leupeptin or with LHVS and analyzed their effect on
maturation of various MHC class II allelic products.
chain (44) (Fig. 4 A). The amount of the I-A
subunit in the pulsed samples was larger than in their chased counterparts. This is probably due to the presence of excess
free
chains in the pulsed samples that fail to assemble
with
subunits and are degraded during the chase. Because
the anti-I-A
serum can recognize free
chains (44; see
Fig 1 B), these can be recovered along with the
dimers.
Fig. 4.
The effect of cysteine protease inhibition on maturation of
MHC class II molecules varies widely among allelic products. (A) Spleen
cells of mice of the haplotypes indicated were pulse-labeled and chased for
4 h in the absence or presence of 1 mM leupeptin or 3 nM LHVS, and
their I-A molecules immunoprecipitated with an anti-I-A rabbit serum
(44). Immunoprecipitates were run on 12.5% SDS-PAGE without (top
half) or after (bottom half) boiling. (B) Amount of I-A SDS-stable dimers
generated in leupeptin-treated splenocytes of different haplotypes relative
to their control counterparts. The amount of SDS-stable I-Ad complexes
in control cells was too small to perform a reliable comparison to the
drug-treated samples. (C) Same as in B, for the LHVS-treated samples.
(D) Cbz-[125I]-Tyr-Ala-CN2 labeling of H-2d, H-2b, and H-2k splenocytes.
[View Larger Versions of these Images (91 + 39 + 40 + 57K GIF file)]
dimers (Fig. 4 A). I-Ad
was the only specificity accumulating a substantial amount
of LIP22 in the presence of leupeptin, suggesting that the
conversion of LIP22 into LIP10 is also influenced by allelic
polymorphism. Similar results were observed after 18 h of
chase (data not shown).
-LIP10 complexes were converted to
-peptide with different efficiency in a postlysis
reaction. However, this is unlikely because it would imply
that virtually all the I-Ak or I-As
-LIP10 complexes accumulated in LHVS-treated cells would be converted to
SDS-stable
-peptide complexes in the cell lysate without dissociation, and loss from the immunoprecipitate, of a
significant amount of dimers.
dimers that accumulate in the presence of leupeptin or LHVS
into
-peptide without previous conversion of LIP10
into CLIP. I-Ab and I-Ad cannot bind antigenic peptides
unless LIP10 is converted into CLIP, which is then displaced in a DM-dependent fashion (19). I-Au would
represent an intermediate situation. These results also indicate that inhibition of cysteine proteases with leupeptin or
LHVS does not result in a significant reduction in the generation, or survival of, the bulk of antigenic peptides that
are required to generate I-Ak or I-As SDS-stable complexes.
dimers receptive to
antigenic peptides is strongly dependent on the allelic product considered, and suggest that the specificity of H-2M in
vivo can be broader than was established in vitro (23).
-CLIP (Fig. 1
C). In this model, different proteases of the endocytic route would be responsible for specific cleavages of Ii. However,
because Cat S alone can also degrade full-length Ii in vitro
(Fig. 2 B) (37), it is possible that this enzyme is sufficient
also in vivo. In this case, accumulation of LIP10 and LIP22
in the presence of leupeptin or LHVS would be a consequence of other proteases taking over the role of Cat S
when the latter is blocked.
-Ii complexes would be activated sequentially. The compartmentalization of the stages of Ii degradation is crucial
because
-CLIP can escape to the cell surface (15, 60). Therefore, cleavage of LIP10 NH2 terminally of CLIP
by Cat S probably takes place in compartments rich in antigenic peptides and H-2M such as CIIV/MIIC, in which
formation of most mature
-peptide complexes occurs
(39, 40, 61). We emphasize the importance of degrading Ii in a staged fashion as a means to ensure proper loading of antigenic peptides, because the enzymes that cleave
Ii and the intermediates that they generate have been conserved during evolution, and appear similar in mouse splenocytes and in human B cells (32, 37, 39, 40, 67).
Riese et al. (37) proposed two potential cleavage sites for
Cat S in human Ii NH2 terminally of CLIP, after residues
arg78 and lys80, both of which are conserved in mouse Ii
(Fig. 5). Crystallography of HLA-DR1-CLIP complexes
has shown that the NH2-terminal end of CLIP protrudes
from the peptide binding region of the
heterodimer (5).
Therefore, the region of Ii that contains the potential cleavage sites for Cat S may be located in an exposed loop
in the
Ii trimer.
Fig. 5.
Conservation of potential cleavage sites for Cat S in human
and mouse Ii. Residues 77-111 of human Ii (single-letter code), with the
corresponding mouse sequence. The bars indicate the CLIP regions (15,
78). The shaded bar indicates the residues of human CLIP that interact directly with the peptide-binding cleft of HLA-DR3 (5). The arrows indicate the potential target sites for Cat S as proposed in reference 37.
[View Larger Version of this Image (14K GIF file)]
and Cat D
/
cells.
We conclude that Cat D is not involved in the mechanism for degrading Ii that we have described, and is most likely
not involved in the production of the bulk of antigenic determinants. Analysis of activation of class II-restricted T
cells by Cat D
/
APC will be required to determine
whether generation of certain antigenic determinants requires Cat D in vivo.
-CLIP the Only Precursor of
-Peptide Complexes In
Vivo?
-LIP10 accumulates
and to which generation of
-peptide complexes is inhibited by leupepin or LHVS is dramatically affected by allelic variation (40). The basis for these differences is in the
structure of the MHC class II molecules themselves, because the active protease content in cells of the different haplotypes treated with the inhibitors, conditions in which the
allele-dependent differences are revealed, appears similar.
dimer (5), the structural differences created by polymorphism in or around the binding site may affect the accessibility of the target sequences to the corresponding protease(s). A similar argument could apply to the conversion
of LIP10 into CLIP. However, it is unlikely that LIP10 can
be degraded in cysteine protease-depleted H-2k or H-2s
splenocytes as efficiently as in control cells, whereas the
same conditions completely block LIP10 degradation in
H-2b or H-2d cells. We favor the alternative explanation
that
-LIP10 can be directly converted into
-peptide
complexes without prior formation of CLIP, and that the
efficiency of this reaction varies among class II allelic products. I-Ak, but not I-Ad, can bind antigenic peptides in
HLA-DM-deficient T2-transfected cells (56), suggesting
that the affinity of CLIP for I-Ak is low enough to allow its
displacement without the assistance of HLA-DM. In vitro
studies have also shown that the affinity of CLIP for different allelic products varies (72, 73). Similarly, LIP10 could
dissociate from I-Ak or I-As much more efficiently than
from I-Ab or I-Ad, with I-Au being intermediate. Moreover, if displacement of LIP10 is also facilitated by H-2M/
HLA-DM (22), the differences observed here might
depend not only on the affinity of LIP10 for the
dimer, but also of
-LIP10 for H-2M.
-Ii
complexes (74). Our results show that the identity of the
MHC class II molecule is an important factor that must be
considered when interpreting those experiments: an antigenic determinant may be poorly presented by I-Ab or I-Ad
and efficiently presented by I-Ak or I-As in leupeptin-treated cells without recycling of preexisting
-peptide complexes from the cell surface (75). Comparing different
determinants presented by the same class II molecule, therefore, is necessary to establish the dependence on de novo
synthesis of class II molecules (76).
-Ii (fragments) complexes unevenly distributed in compartments whose antigenic content is probably also heterogeneous (40). Therefore, many
peptides available in the compartments where
-LIP10 is
predominant may be absent in the compartments enriched
for
-CLIP, and vice versa. Altering the fraction of
-
LIP10 and
-CLIP complexes by inhibiting Cat S might
result in increased presentation of some determinants and
reduction of others. Thus, specific inhibition of Cat S could
provide the basis for treatment of autoimmune diseases triggered by specific MHC class II-peptide combinations without ablating the capacity to present other epitopes.
Address correspondence to Hidde L. Ploegh, Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, 40 Ames St., Cambridge, MA 02139. Phone: 617-253-0520; FAX: 617-253-9891; E-mail: ploegh{at}mit.edu
Received for publication 1 May 1997 and in revised form 20 June 1997.
1 Abbreviations used in this paper: Cat B, cathepsin B; Cat D, cathepsin D; Cat S, cathepsin S; CLIP, class II-associated invariant chain peptides; ER, endoplasmic reticulum; Ii, invariant chain; LHVS, N-morpholinurea leucine-homophenylalanine-vinylsulfone-phenyl; LIP, leupeptin-induced polypeptide.We wish to thank Drs. J. Davoust, R. Germain, J. Miller, and R.M Steinman for providing antibodies and Dr. D. Brömme for the LHVS inhibitor. We also thank A. Arroyo, B. Galocha, and E. Wang for comments on the manuscript.
This work was supported by National Institutes of Health grants 5-RO-AI34893 (H.L. Ploegh), 2-P30-CA14051-24 (H.L. Ploegh), T32-HL07633 (R.J. Riese), and HL48261 (H.A. Chapman). J.A. Villadangos was supported by fellowships from the Ministerio de Educacion y Ciencia (Spain) and the Lady Tata Foundation (England).
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