By
From the Howard Hughes Medical Institute, Yale University School of Medicine, Section of Immunobiology, New Haven, Connecticut 06510
Major histocompatibility complex (MHC) class II-positive cell lines which lack HLA-DM expression accumulate class II molecules associated with residual invariant (I) chain fragments
(class II-associated invariant chain peptides [CLIP]). In vitro, HLA-DM catalyzes CLIP dissociation from class II-CLIP complexes, promoting binding of antigenic peptides. Here the physical interaction of HLA-DM with HLA-DR molecules was investigated. HLA-DM complexes with class II molecules were detectable transiently in cells, peaking at the time when the class II
molecules entered the MHC class II compartment. HLA-DR dimers newly released from I
chain, and those associated with I chain fragments, were found to associate with HLA-DM in
vivo. Mature, peptide-loaded DR molecules also associated at a low level. These same species,
but not DR-I chain complexes, were also shown to bind to purified HLA-DM molecules in
vitro. HLA-DM interaction was quantitatively superior with DR molecules isolated in association with CLIP. DM-DR complexes generated by incubating HLA-DM with purified DR
CLIP contained virtually no associated CLIP, suggesting that this superior interaction reflects a prolonged HLA-DM association with empty class II dimers after CLIP dissociation. Incubation of peptide-free
dimers in the presence of HLA-DM was found to prolong their
ability to bind subsequently added antigenic peptides. Stabilization of empty class II molecules
may be an important property of HLA-DM in facilitating antigen processing.
The recognition of antigen by CD4+ T helper cells requires the presentation of short peptides displayed in
the binding groove of MHC class II molecules (1, 2).
MHC class II molecules are cell surface expressed heterodimeric ( The expression of HLA-DM, encoded by MHC-linked
genes (18, 19), is required for class II-restricted processing
and presentation of most protein antigens but not for the
presentation of exogenously added antigenic peptides (reviewed in reference 20). Class II molecules in DM-negative B-lymphoblastoid cell lines (B-LCL) are expressed on
the cell surface at wild-type levels but are associated with a
set of peptides derived from residues 81-104 of the I chain (class II-associated invariant chain peptides, or CLIP) instead of antigenic peptides (21, 22). Similar complexes accumulate in class II-positive cell lineages in mice in which
the Ma (DMA) gene is disrupted (23). The ability to
isolate Recently, we and others have shown that CLIP removal
and peptide loading are directly catalyzed by DM in vitro
(29), suggesting that DM also removes CLIP from Cell Lines.
The EBV transformed B-lymphoblastoid cell line
(B-LCL) Pala (HLA-DR3.Dw17) and the T × B hybrid cell line
.174 × CEM.T2 (33) transfected with HLA-DR3 (T2.DR3; 34)
were maintained in Iscove's DMEM (GIBCO BRL, Gaithersburg, MD) with 5% calf serum (GIBCO BRL) in the presence of
5% CO2 at 37°C. The derivation of the mutant B-LCL 10.24.6 (HLA-DR3, -DRw52a, -DQ2, -DP4.1) and the progenitor BLCL 8.1.6 have been described (35). These cells were maintained in RPMI (GIBCO BRL) supplemented with 1% glutamine (GIBCO BRL) and 10% calf serum.
Antibodies and Peptide.
The hybridoma cell lines L243 (antiHLA-DR ) glycoproteins. Newly synthesized class II
and
chains assemble in the ER with a third transmembrane glycoprotein, the invariant (I) chain (3, 4). With the
aid of the molecular chaperone calnexin, three
dimers
bind sequentially to a trimer of the I chain in the ER (5, 6).
The nonameric complex moves through the Golgi apparatus and is sorted by signals in the cytoplasmic domain of the
I chain (7) and the class II
chain (8) to endosomal compartments with late endosomal (class II-containing vesicles, CIIV; 9) or lysosome-like characteristics (MHC class II compartment [MIIC]1; 10-13) where the I chain is proteolytically cleaved by aspartic and cysteine proteases (14). The
released
dimers are loaded with peptides derived from
internalized pathogen-derived or endogenous proteins present
in the endocytic system and transported to the cell surface.
CLIP complexes from wild-type APCs (2), their
transient appearance during pulse-chase analysis of class II
transport (26), and the proteolytic generation of
CLIP complexes from
I in vitro (27), all indicate that
CLIP complexes are an intermediate in the class II processing pathway and that CLIP probably represents the end
product of I chain proteolysis. Recent x-ray crystallographic analysis of HLA-DR3-CLIP complexes has demonstrated that CLIP binds in the antigen binding groove of
the class II molecules (28) indicating that CLIP must first be
removed before endocytically generated peptides can bind.
CLIP complexes in vivo. For DM to catalyze the release of
CLIP, it seemed likely that a DM-class II interaction must
occur and, indeed, Sanderson et al. (32) have shown by coprecipitation that DM and DR associate under steady state
conditions in vivo. The association is favored by low pH in
the nonionic detergent digitonin, and occurs in dense compartments, probably in MIICs. In this study, using biosynthetic labeling and coprecipitation of DR with DM, we
show that in vivo the DM-DR association is transient, and
that DM associates with peptide loaded
dimers, extending the initial observations reported by Sanderson et al.
(32). Additionally, we examine the in vitro interaction of
affinity-purified DM with DR molecules associated with I
chain, fragments of I chain, CLIP, or the normal complement of peptides expressed in wild-type cells. The results
indicate a degree of specificity for
CLIP in the interaction, and suggest that one function of HLA-DM is to prolong the survival of empty class II molecules in the MIIC, a
chaperone-like function which is likely to be important in
antigen processing.
; 36), DA6.147 (anti-HLA-DR
chain; 37),
GAP.A3 (anti-HLA-A3; 38), W6/32 (anti-HLA-class I; 39),
HB10A (anti-HLA-DR
chain; 40), PIN.1 (anti-I chain NH2terminal; 41), and CerCLIP.1 (anti-CLIP24; 42), have been previously described. Rabbit anti-DM serum was a generous gift of
Dr. Hans Zweerink (Merck Research Laboratories, Rahway, NJ)
and was generated against recombinant, soluble DM, (31). Rabbit
anti-C23V serum, specific for an influenza hemagglutinin peptide,
was generated in our laboratory and has been described (42).
Metabolic Labeling.
Cells for pulse labeling were washed once
with methionine and cysteine-free DMEM supplemented with
3% dialyzed FBS, 15 mM Hepes, 2 mM glutamine, and 1 mM
sodium pyruvate (labeling medium; all from GIBCO BRL) and
preincubated at 3 × 106 cells/ml in labeling medium for 60 min
at 37°C. Cells were pelleted and resuspended at 1 × 107 cells/ml
in fresh labeling medium plus 1 mCi/ml of L-[35S]methionine in
vitro cell labeling mix (Amersham Corp., Arlington Heights, IL).
After incubation at 37°C for 15 min to 6 h, the pulsed cells were
pelleted, resuspended at 1 × 106 cells/ml with IDMEM/5% calf
serum/5% FBS containing a 15-fold excess of cold methionine
and cysteine, and then chased for up to 18 h at 37°C. At each
chase point, the cells were diluted into ice-cold serum-free IDMEM, pelleted by centrifugation, and either lysed immediately
for immunoprecipitation and affinity purification or stored at
20°C until ready for use.
Immunoprecipitations and Endo H Digestion.
Radiolabeled cell pellets were extracted for 30 min on ice in 20 mM Tris, 130 mM
NaCl (pH 7.4) (TBS), containing 1% CHAPS (Pierce, Rockford,
IL), or 1% CHAPS plus 1% Triton X-100 (Sigma Chem. Co., St.
Louis, MO), 0.5 mM PMSF, 0.1 mM TLCK, and 5 mM iodoacetamide (Sigma) at 5 × 105-2 × 106 cells/ml. After the removal
of nuclear material by centrifugation, lysates were precleared with
2 µl normal rabbit serum and 75 µl Zysorbin (Zymed Laboratories, South San Francisco, CA) per ml of cell lysate for 1 h at 4°C
before incubation with specific antibody (2-3 µl ascites or rabbit antiserum, 100 µl tissue culture supernatant) and protein A-Sepharose
(Sigma) or protein G-Sepharose (Pharmacia, Piscataway, NJ). For
anti class II or I chain immunoprecipitations 5 × 105 cell equivalents were used. For anti DM immunoprecipitation 2 × 106 cell
equivalents were used. Sepharose pellets were washed three times
with TBS plus 1% CHAPS or 10 mM Tris, pH 8.0, 300 mM
NaCl, 0.1% SDS, 0.05% Triton X-100, or TBS plus 0.1% Triton-100, and stored at 20°C. For reprecipitation experiments, 1 ml of TBS containing 0.5% deoxycholate (DOC; Sigma) was
added to the DM precipitates and they were incubated for 3 h at
4°C. After centrifugation, the released DR was immunoprecipitated from the DOC supernatants as described above. For Endo H
digestion, beads were boiled for 5 min in 20 µl of 0.06 M NaPO4,
0.3% SDS, 0.06% NaN3, diluted with 40 µl H2O, and the supernatants were divided into two aliquots. To one aliquot, 2 mU recombinant Endo H (Boehringer Mannheim, Indianapolis, Indiana)
was added and both aliquots were incubated overnight at 37°C.
Affinity Purification of Radiolabeled Class II Complexes and HLADM.
Affinity purification of I,
LIP,
CLIP, and
peptide complexes from radiolabeled cell lysates were as previously described (14, 29, 43). The affinity purification of HLADM from wild-type B-LCLs has also been described (29).
SDS Stability Assay.
Affinity-purified HLA-DM (2 ng) was
incubated with 500 nM MOMP peptide and ~10,000 CPM of
radiolabeled mutant (10.24.6-derived) or wild-type CLIP
complexes in TBS (pH 8), 1% CHAPS. The pH was adjusted to
5.0 by the addition of 1 M acetic acid and the samples were incubated at 37°C for 0-120 min. After neutralization by the addition
of 1 M Tris, 10× non-reducing Laemmli sample buffer was
added and the samples were separated by 10.5% SDS-PAGE.
In Vitro Association Assay.
Affinity-purified, radiolabeled I,
LIP/SLIP,
CLIP,
peptide, or 10.24.6
CLIP complexes (~40,000 CPM) in TBS (pH 8), 1% CHAPS were incubated in the absence or presence of either 50 ng affinity-purified
DM or decreasing amounts of DM (50 ng to 0.156 ng) and the
pH was adjusted to 5.0 by the addition of 1 M acetic acid. After
an incubation at 37°C for 20-30 min, the samples were placed on
ice and 500 µl of 50 mM sodium acetate, 150 mM NaCl (pH 6.0),
1% CHAPS and 2 µl anti-DM or anti-C23V and 25 µl protein
A-Sepharose were added. The samples were incubated for 30 min at 4°C with rotating, washed three times with 50 mM sodium acetate, 150 mM NaCl (pH 6.0), 1% CHAPS, analyzed by
SDS-PAGE and quantitated by image analysis.
In Vitro Stabilization Assay.
Radiolabeled DR3 CLIP complexes (10,000 cpm; 25 nM) were incubated in the presence or
absence of affinity-purified HLA-DM (25 nM) in TBS (pH 8.0),
1% octyl glucoside. The pH was adjusted to pH 4.5 and the samples were incubated at 37°C for 0 to 10 h. At various time points,
MOMP peptide was added to a concentration of 1 µM and the
samples were incubated for an additional 45 min at 37°C. After
neutralization by the addition of 1 M Tris, 10× non-reducing
Laemmli sample buffer was added and the samples were separated
by 10.5% SDS-PAGE and analyzed by image analysis.
HLA-DM has been
shown to mediate the exchange of CLIP for antigenic peptides under acidic conditions in vitro (29). For DM to
catalyze the release of CLIP, it seemed likely that a direct physical interaction between DM and class II molecules
must occur. HLA-DM is a resident of the MIIC (44),
and class II molecules traffic through this compartment to
be loaded with antigenic peptides by DM before subsequent arrival at the cell surface. Therefore, any interaction
between DM and DR molecules should occur in the MIIC
and be transient in nature. Sanderson et. al. (32), have demonstrated direct DM-DR association under steady-state
conditions in vivo. To examine the kinetics of DM-DR association, wild-type Pala cells were pulsed for 15 min with
[35S]methionine and chased in the presence of 15-fold excess methionine and cysteine for up to 12 h (Fig. 1 A). At
each time point, radiolabeled cells were lysed in 1% CHAPS
and split into four aliquots. One aliquot was immunoprecipitated with a polyclonal rabbit antiserum to the DM heterodimer (anti-DM) and another with a negative control rabbit antiserum to an influenza hemagglutinin peptide
(anti-C23V). DM and DR have similar mobilities on SDSPAGE, however their processing patterns are somewhat
different. DM-DR association has been shown to be sensitive to the detergent used for solubilization of the cells (32;
unpublished data). To determine the exact position and
processing pattern of DM and DR on SDS-PAGE, 1% Triton X-100 was added to the other two aliquots of cell
lysate to dissociate the DM-DR complexes. Immunoprecipitation was performed with anti-DM or with the DRspecific mAb, L243, followed by extensive washing with a
buffer containing Triton X-100 and SDS (see Materials and Methods) to ensure any remaining DM-DR complexes
were dissociated. SDS-PAGE analysis (Fig. 1 A, lower right)
shows that in Pala cells L243-reactive dimers were initially generated between 1 and 2 h of chase and that probable invariant chain fragments (26; indicated on the right
hand side with an arrow) were coprecipitated with the DR
molecules early in the chase period (1 to 4 h). Immunoprecipitation with anti-DM serum (Fig. 1 A; upper right) revealed the position of the DM
and DM
chains in the
absence of any associated DR molecules. DM
dimers
were observed immediately after the pulse and remained
throughout the chase period, consistent with previous reports (42). Comparison of the anti-DM and L243 immunoprecipitates in Triton X-100 clearly demonstrates that
the DM and DR processing patterns on SDS-PAGE are
very different. SDS-PAGE analysis of an anti-DM immunoprecipitate in the detergent CHAPS (Fig. 1A; upper left)
showed that after the pulse point, only the DM
dimer
was precipitated. However, after 1 h of chase additional species with the same mobilities as DR
and
subunits
coprecipitated with the DM dimers. The coprecipitated
species peaked at 2 h and then decreased with time, with
some remaining associated even at the 12 h time point. Additionally, fragments of the invariant chain (marked with an
arrow) were coprecipitated with the DM complex at 2 h
indicating that DM can associate with putative DR molecules still complexed with these fragments, suggesting that
HLA-DM is associated with the class II-I chain complex
during degradation of the I chain.
DM-positive 10.24.6 cells express a mutant DR chain
containing a proline to serine substitution at position 96 that results in the addition of an extra N-linked glycan. In
this cell line DR3
dimers accumulate as
CLIP complexes (35). Purified
CLIP complexes from 10.24.6 cannot be loaded with peptide by recombinant soluble DM in
vitro (31) or by affinity-purified native DM (see below),
and steady-state DM-DR association is greatly reduced in
this cell line (32). To confirm that the putative DR-DM
interaction seen in wild-type Pala cells was specific, we repeated the pulse-chase experiment with the 10.24.6 cell
line and the matched parental cell line, 8.1.6 (35). The results (Fig. 1 B, upper left) showed that in the wild-type cell
line 8.1.6, DR was coprecipitated with DM and that this
association was strongest at 1-2 h of chase, similar to what
was observed in Pala cells (Fig. 1 A). In 10.24.6 cells, antiDM and L243 (anti-DR) precipitations in Triton X-100
clearly showed that this cell line expresses both DM and
DR (Fig. 1 B lower right). The reduced mobility of the mutant DR
chain (
) caused by the additional N-linked glycan is readily apparent. None of the mutant DR was coprecipitated with DM in CHAPS (Fig. 1 B, upper right)
even upon prolonged autoradiographic exposure (data not
shown).
Pulsechase analysis (Fig. 1 A) showed that DM-DR association
was maximal after 1-2 h of chase, the time at which the
I complex is delivered into the MIIC and the invariant
chain is being degraded in this cell line (46; unpublished results). Therefore, the interaction must occur in a post-Golgi
compartment and the DM-associated DR should be resistant
to Endo H digestion. To determine this, wild-type Pala
cells were pulse-labeled for 15 min with [35S]methionine
and chased in the presence of an excess of methionine and
cysteine. After lysis of the cells in CHAPS, the DM-class II
complexes were immunoprecipitated with anti-DM. The
coprecipitated DR was eluted from the DM immunoprecipitates with 0.5% DOC and reprecipitated from supernatants with a DR
chain-specific mAb, HB10A or with a
negative control antibody to HLA class I molecules, W6/
32. Duplicate HB10A and W6/32 precipitates were treated
with Endo H or mock-treated overnight and analyzed by
SDS-PAGE. No DM associated DR was detected early in
transport since the faint Endo H-sensitive bands present
immediately after the pulse in the HB10A precipitates (Fig.
2 A) were also present in the negative control precipitates
(Fig. 2 B). As observed in Fig. 1, DM-associated DR was
first detected after 1 h of chase and the associated DR was
resistant to Endo H digestion (Fig. 2 A, note that one of the DR
chain N-linked glycans remained in the high
mannose form upon maturation; 48). These data indicate
that DM and DR are not associated in the ER and that
DM-DR must interact after the DR molecules have traversed the medial Golgi. No HLA class I molecules were
coprecipitated with DM, verifying the specificity of the DR-DM association (Fig. 2 B).
DM can be coprecipitated by the DR3 conformational
specific mAb 16.23 (32). This antibody is thought to recognize only peptide loaded DR3 molecules and does not
bind I or
CLIP complexes (49). Sanderson et al. (32),
suggest that because DM can be efficiently coprecipitated
by mAb 16.23, that the DM is able to associate with mature, peptide-loaded DR3 molecules. Since the exact specificity of mAb 16.23 is not known, the DM associated DR
they observed may actually be empty DR3 molecules.
SDS-stability of class II molecules has been shown to correlate with peptide binding (50). To determine if DM can associate with peptide-loaded DR molecules, Pala cells were
pulsed with [35S]methionine, chased for various times, extracted in CHAPS, and immunoprecipitated with antiDM. After release of the DM-associated DR by 0.5%
DOC, the DR was reprecipitated with HB10A as described above. The HB10A immunoprecipitates were incubated in SDS-PAGE sample buffer at room temperature
for 30 min and analyzed by SDS-PAGE for SDS-stable,
peptide-loaded
dimers. The peak of class II association
with DM was again at 2 h, and at this time the majority is
clearly unstable. However, stable
dimers were clearly
present, indicating that DM can associate with mature,
peptide-loaded
dimers (Fig. 2 C ). This result is reproducible and does not reflect an artefactual, post-solubilization association of DM and mature DR molecules, because
coextraction under the same conditions of labeled T2.DR3
cells mixed with unlabeled T2.DM cells did not generate
such complexes (data not shown). Although the relative amount of the total DR that was SDS-stable increased with
time, the majority of the DM-associated DR was not stable
in SDS. This suggests that most of the peptide loaded DR
dissociated from DM during the chase.
We have shown that DM is able to catalyze in vitro
the removal of LIP, a 21-kD NH2-terminal fragment of the
I chain that accumulates in the presence of the protease inhibitor leupeptin, from LIP complexes (29). This suggested that DM can interact with class II complexed with
fragments of the I chain. Additionally, pulse-chase analysis
of DM in the detergent CHAPS (Fig. 1 A) demonstrated a
band at 2 h that coprecipitated with DM and probably is a
fragment of the I chain. To directly examine if DM can interact with DR complexed with I chain fragments, Pala cells
were pulsed with [35S]methionine for 15 min and chased
for 2 h in the presence of the protease inhibitor leupeptin
to accumulate
LIP (14) and
SLIP (15) complexes.
Radiolabeled cells were lysed in CHAPS, immunoprecipitated with anti-DM or anti-C23V (negative control), or
lysed in CHAPS plus Triton X-100 and immunoprecipitated with anti-DM, the DR specific mAb L243, the I chain
specific mAb PIN.1, or the HLA-A3 specific mAb, GAP.A3
(negative control) and analyzed by SDS-PAGE. Immediately after the pulse, as expected, no DM-associated DR
was observed and no
LIP and
SLIP had yet been
generated (Fig. 3 A). After a 2-h chase, the mAb L243 precipitated DR
in addition to lower molecular weight
fragments of the I chain, LIP and SLIP (which runs on the
dye front of the gel). Immunoprecipitation with anti-DM
showed that
LIP and
SLIP complexes were coprecipitated with DM in the detergent CHAPS but not in Triton
X-100 (Fig. 3 A), as observed in Fig. 1. These results show
that DM can associate with DR complexed with I chain
fragments.
Previous in vivo analysis has shown that DM coprecipitated with full-length I chain in the ER and with I chain
fragments (including LIP) in dense Percoll gradient fractions (32). However, these experiments were performed
using immunoprecipitation with an antibody specific for
the NH2-terminal region of the I chain (VICY1) followed by Western blotting, and thus it was impossible to distinguish between direct DM interaction with the I chain or
association with the I chain via its interaction with class II
molecules. A similar problem exists in the experiment
shown in Fig. 3 A. An experiment was designed to determine whether the DM-associated I chain fragments seen in
Fig. 3 A were free or complexed to class II molecules. Pala
cells were pulsed for 15 min with [35S]methionine, chased
for 2 h in the presence of leupeptin, lysed in CHAPS and
immunoprecipitated with anti-DM. The DM-associated material was eluted by the addition of 0.5% DOC, which
should not dissociate I or
complexed to fragments of
the I chain. The eluted complexes were reprecipitated
from the DOC supernatant with mAb PIN.1 (anti-I chain),
mAb HB10A (anti-DR
chain) or a negative control mAb
W6/32 (anti-class I). SDS-PAGE analysis of the immunoprecipitates is shown in Fig. 3 B. Immunoprecipitation with the class II specific mAb HB10A showed that after a
15-min pulse, no class II was associated with DM and reprecipitation with an antibody specific for the I chain
showed that very little, if any, I chain was associated with
DM. After 2 h of chase, re-precipitation of DM-associated
class II with mAb HB10A showed that LIP was clearly present, demonstrating that DM interacts with
LIP
complexes. Reprecipitation with the I chain specific mAb
PIN.1 also precipitated
LIP complexes. Because HLADM can bind to
CLIP and
peptide complexes (see
below), it seems likely that DM associates with LIP via an
interaction with the DR molecule and not directly with the LIP fragment itself.
Class II molecules containing a mutant
DR chain in the DM positive 10.24.6 cell line are unable
to associate with DM and therefore arrive at the cell surface
complexed with CLIP (Fig. 1 B; 32, 35). To determine if
10.24.6-derived
CLIP complexes could be loaded in
vitro by native DM we compared the rates at which mutant and wild-type
CLIP complexes could be converted to SDS-stable dimers. T2.DR3 and 10.24.6 cells were
pulsed with [35S]methionine for 4 h and chased in the presence of a 15-fold excess of methionine and cysteine for 16 h
to ensure degradation of the I chain and maturation of the
CLIP complexes. Mutant and wild-type
CLIP complexes were affinity-purified from radiolabeled cell lysates
using a CerCLIP.1 antibody column. Radiolabeled wildtype and mutant DR3
CLIP complexes and the DR3specific peptide MOMP were added to affinity-purified
DM and incubated at pH 5 for periods up to 120 min at
37°C (29). After neutralization, the samples were analyzed
by SDS-PAGE. The kinetics of DM-induced dimer formation for wild-type
CLIP complexes showed that SDSstable
dimers were detected after only 5 min of incubation at 37°C and the formation of dimers proceeded rapidly
for the first 30-45 min of incubation before leveling off
(Fig. 4, A and B). In contrast,
CLIP complexes purified
from 10.24.6 molecules were inefficiently loaded by affinity-purified DM. After 120 min only 18% of the mutant
CLIP complexes were converted to SDS-stable dimers
compared to 95% dimers for wild-type
CLIP. These results show that native DM is unable to efficiently load mutant
CLIP complexes from 10.24.6 cells and confirm the results of Sloan et al. (31), who demonstrated that the mutant
CLIP complexes cannot be loaded in vitro by recombinant, soluble DM.
DM Associates with
DM is
able to catalyze the removal of CLIP from CLIP complexes and exchange it for antigenic peptides in vitro (29).
This suggests that DM and DR must associate during the in
vitro reaction. To look for such an interaction, radiolabeled, CerCLIP.1-purified normal
CLIP from T2.DR3
or mutant
CLIP from 10.24.6 were added to affinitypurified DM and incubated at pH 5.0 for 20-30 min at 37°C. After adjusting the pH to 6.0, the reaction mixtures
were immunoprecipitated with anti-DM or anti-C23V,
which served as a negative control antibody, and analyzed
by SDS-PAGE. When affinity-purified DM and wild-type
DR
CLIP complexes were incubated together, antiDM but not anti-C23V coprecipitated the radiolabeled
DR (Fig. 4 C). However, DM failed to form a detectable
complex with mutant
CLIP, since immunoprecipitation
with anti-DM did not coprecipitate radiolabeled mutant
DR. Image analysis (Fig. 4 D) indicated that 14% of the
and
chains from wild-type
CLIP was coprecipitated with DM, whereas less than 4% of those of the mutant
CLIP complexes were coprecipitated (Fig 4 D). These
results show that DM and DR
CLIP complexes do interact in vitro and that no additional components are necessary for the interaction. CLIP, which runs below the dye
front on SDS-PAGE (29), is conspicuously absent from the
class II complex coprecipitated by anti-DM in Fig. 4 C,
even though it can readily be seen in the initial
CLIP
complex (second lane from the right). DM appears to be
associated with empty class II
dimers in this experiment, because no peptides were added to the reaction mixture. CLIP is consistently absent from or severely reduced
in complexes formed by incubating DM with
CLIP.
Empty class II
dimers have been shown to aggregate and lose their ability
to bind antigenic peptides (51). The absence of CLIP
from class II complexes coprecipitated with DM (Fig. 4 C)
indicated that DM continues to interact with empty class II molecules after CLIP dissociation. Such an association
might stabilize the
dimers and prevent them from aggregating and losing the capacity to bind antigenic peptides. To test this hypothesis, we took advantage of the
ability of the detergent octyl glucoside to induce CLIP release from DR3
CLIP complexes (21, 27). Radiolabeled
DR3
CLIP complexes were incubated in 1% octyl glucoside at pH 4.5 with the DR3-specific MOMP peptide in
the presence and absence of affinity-purified DM. After
acidification and incubation for 0 to 10 hr at 37°C, the
samples were neutralized, analyzed by SDS-PAGE and
quantitated by image analysis. The results showed that the
conversion of class II
CLIP complexes to
peptide complexes in the detergent octyl glucoside was almost as
efficient in the absence of DM as in its presence. By 6 h,
the percentage of SDS-stable dimers was virtually identical
(~90%; Fig. 5, A and B). This demonstrates that octyl glucoside can induce CLIP release from class II
CLIP complexes almost as efficiently as DM under these experimental
conditions. To determine whether DM could stabilize
empty class II molecules, radiolabeled
CLIP complexes
were incubated without peptide in 1% octyl glucoside in
the presence and absence of DM at pH 4.5 for 0-10 h. At
each time point, MOMP peptide was added to 1 µM and
the samples were incubated for an additional 45 min at
37°C to allow surviving, functional class II molecules to
bind peptide. After neutralization, the samples were analyzed by SDS-PAGE and quantitated by image analysis.
The results showed that after only 1 h of incubation in the
absence of peptide more peptide-receptive class II molecules survived in the presence of DM (Fig. 5 C). With increasing incubation time, the amount of peptide-receptive
class II progressively decreased both with and without DM.
However, at all time points the presence of HLA-DM resulted in increased peptide loading. After 10 h of incubation no peptide-receptive class II molecules were present in
the absence of DM, whereas ~35% of class II molecules retained their peptide binding function when DM was
present (Fig. 5, C and D). Control proteins at the same or
10-fold higher concentrations failed to preserve peptidereceptive class II molecules demonstrating that this function
is a specific property of HLA-DM (data not shown).
DM Interacts In Vitro with
During transport, maturing class II complexes have
many opportunities to interact with DM. In vivo pulsechase (Figs. 1-3) and steady-state Western blotting experiments (32) showed that DM does not coprecipitate with
I complexes, but can be coprecipitated with
complexed to I chain fragments (LIP) and with peptide-loaded
dimers. Lack of association with
I, particularly,
could either be a reflection of limiting amounts of DM in
the ER and later
I-containing compartments or of an
inability of DM to interact. To examine this question,
[35S]methionine-labeled
I,
LIP/SLIP and mature
dimers were purified from wild type cells. SDS-PAGE of
the purified
I,
LIP/SLIP,
CLIP, and
peptide
complexes used for the in vitro association assays are shown
in Fig. 6 A. The loaded samples represent one-tenth the
amount of each complex used for the association assay. In this particular experiment, the SLIP fragment (15) was unusually well represented in the class II molecules purified
from leupeptin-treated Pala cells. For association experiments, the various complexes were incubated in the presence and absence of affinity-purified DM at pH 5.0 for 30 min at 37°C, immunoprecipitated with anti-DM or antiC23V (negative control) and analyzed by SDS-PAGE (Fig. 6 B). The results showed that DM interacted detectably
with
LIP/SLIP,
CLIP, and
peptide complexes,
but not with
I. In other experiments we were unable to
detect association of DM with affinity-purified I chain trimers (data not shown). Image analysis of the co-association experiment (Fig. 6 C) suggested that DM interacted better
with
CLIP complexes than with
LIP/SLIP or
peptide complexes. To better quantitate the interactions,
titrated amounts of purified DM were added to each of the
purified class II complexes and the percentage of each class
II complex coprecipitated with DM was determined for
each concentration. The results were quantitated by image
analysis after SDS-PAGE (Fig. 6 D). DM clearly associated better with class II molecules when
CLIP was used than
when
dimers complexed with larger I chain fragments
or
peptide complexes were used. These results probably
reflect prolonged association with empty
dimers after
CLIP dissociation (see Fig. 5). Interaction with
I was essentially undetectable.
Upon arrival in the MIIC, the I chain component of I
complexes is degraded, leaving CLIP as a residual fragment
bound to class II
dimers. Through a process mediated
by HLA-DM, CLIP is exchanged for endocytically generated peptides and the mature class II molecules are transported to the cell surface (29). This process implies a
physical interaction between class II molecules and HLADM. In this paper, we have confirmed that such an interaction, first described by Sanderson et al. (32), occurs in vivo,
and have complemented this finding by analyzing the specificity of the interaction for different class II complexes in
vitro. In addition, we have shown that HLA-DM associates
with empty class II molecules after CLIP dissociation, and
that this association increases the stability of the peptidefree
dimers, prolonging their ability to bind peptides.
HLA-DM is a resident protein of MIICs (44). Not
surprisingly, therefore, the interaction between class II molecules and HLA-DM is readily detectable at steady state in
dense Percoll fractions (32). Examination of the kinetics of
the interaction by pulse-chase analysis (Fig. 1), shows that it
is transient, peaking at the time when, based on our previous experiments, HLA-DR molecules arrive in MIICs (46;
unpublished data). Consistent with this, DM association is
not seen until the class II molecules are resistant to Endo
H digestion (Fig. 2). Additionally, in MIICs purified by a
combination of Percoll gradient separation and immunoisolation using an antibody to the DM cytoplasmic domain, we have also been able to observe DM-DR association
after CHAPS solubilization (Hammond, C., unpublished observations).
There are two possible, non-mutually exclusive, explanations for the peak of DM-DR association occurring in
MIICs. First, this is believed to be where CLIP is generated and
CLIP could be the optimal form of class II for
DM binding. Second, the interaction could peak in MIICs
because this is the organelle where DM is maximally concentrated. At one extreme, all the forms of class II (
I
through
with endocytically acquired peptides) could bind equally well to DM, but because DM is encountered
primarily in MIICs, only the later forms would be found to
associate in vivo. Some evidence for this is seen in Fig. 1,
where what appears to be an I chain fragment is coprecipitated with DM, consistent with the idea that
dimers
with residual I chain fragments can bind. If complexes of
class II with I chain fragments are deliberately induced by
leupeptin treatment, their in vivo association with DM is
even more dramatically evident (Fig. 3; 32). Under such conditions, these complexes accumulate in MIICs. In addition, it is clear that SDS-stable dimers, presumably loaded
with peptides other than CLIP, can associate with DM
(Fig. 2 C). At later times in the chase these may represent
an internalized subset of surface class II molecules. They do
not reflect an artefactual, post-solubilization association of
DM and mature DR molecules, because coextraction under the same conditions of labeled T2.DR3 cells mixed
with unlabeled T2.DM cells did not result in the formation
of such complexes (data not shown).
To properly determine the specificity of DM for the different forms of maturing class II molecules, the development of an in vitro binding assay was essential. Such an
assay also allows one to differentiate between a binding interaction between class II and DM and the functional activity of DM. We previously found that LIP and
CLIP
were substrates for DM-mediated peptide loading, whereas
I and mature
peptide complexes from wild-type DR3-positive Pala cells were not (29). The experiments in
Figs. 4 and 5 show that DR3
CLIP,
LIP/SLIP, and
peptide complexes can all associate with HLA-DM under the in vitro conditions used, whereas
I complexes
do not. The
CLIP complexes associate best although
mutant
CLIP complexes from the 10.24.6 cell line,
which fail to bind antigenic peptides when incubated with
DM (31; Fig. 4, A and B), fail to associate with HLA-DM
both in vivo (32; Fig. 1 B) and in vitro (Fig. 4, C and D).
Thus, binding and functional susceptibility correlate well,
with the exception of mature
peptide complexes which
bind to HLA-DM but exchange peptides poorly (29).
CLIP-containing
dimers from the cell line 10.24.6 presumably do not bind DM because of steric interference by
the additional N-linked glycan on the mutant
chain (35).
Similarly,
I complexes may not bind to DM because the
COOH-terminal region of the I chain, which has been
postulated to interact directly with the
dimer (54, 55)
also sterically interferes with the interaction.
The in vitro association of DM with class II molecules is
relatively inefficient. In the experiments shown in Figs. 4
and 6, DM was added in a large excess over DR and in the
best experiments only ~15% of the class II (in this case,
CLIP) added is recoverable as DM-associated material.
This inefficiency is probably attributable to several limitations of the in vitro system. First, it has been shown that
DM-DR complexes are more efficiently isolated from intact cells at pH 5.0 than at pH 7.0 (32). The use of immunoprecipitation to recover DM-DR complexes required
increasing the pH from 5.0 to 6.0, a pH at which the DM-DR
complex may not be particularly stable. Second, DM and
DR molecules are present in vivo in a membrane-bound form and are concentrated in MIICs. These conditions are
not met in vitro. Additionally, there could be other proteins that stabilize the DM-DR interaction in vivo which
are absent from our assay. However, we think this is unlikely since purified, soluble recombinant DM appears to
be fully functional in vitro. DM-DR association in vitro
may be inefficient simply because the complex is in equilibrium with free class II and DM, so that any time only a
fraction of the DM and DR are associated.
The substoichiometric levels of DM compared to class II
in vivo (29, 42) suggest that DM acts catalytically on multiple class II molecules. One way to ensure that DM interacts
with many class II molecules would be to regulate DMclass II association by equilibrium. It has been demonstrated
that in clones of wild-type human Swei cells transfected
with I-Ab, low DM expression results in more CLIP and
less
peptide being expressed at the cell surface (56). Ramachandra et al. (56), use this data to argue that there must
be a quasi-stoichiometric ratio of class II to DM for efficient
peptide loading, and that DM may not be functioning catalytically. However, if class II moves through the MIIC at
the same rate, less DM could still result in less peptide loading of
CLIP complexes.
Previously, we reported that DM does not associate with
I chain in the detergent Triton X-100 (42), and in vitro association experiments in which affinity-purified I chain trimers were incubated with affinity-purified DM in the detergent CHAPS also failed to demonstrate an interaction
(data not shown). However, Karlsson and co-workers have
provided good evidence in the mouse system that DM and
the I chain can associate in vivo (57). Removal of the cytoplasmic tail of the mouse DM chain and coexpression with the
chain resulted in cell surface expression of
mouse DM (57, 58). When the transfected molecule was
coexpressed with I chain, it was redistributed to MIIC,
showing that the two proteins interact (57). Additionally,
association between DM and I chain has been detected by
coprecipitation experiments in the detergent digitonin (32,
47, 57). However, Sanderson et al. (32), demonstrated that
the DM-associated I chain complex never became Endo
H-resistant, indicating that the complex failed to leave the
ER, a result which we have confirmed (data not shown). Such a complex may result from mis-assembly in the ER.
Pulse-chase analysis of DM in CHAPS (Fig. 1 A) showed
that fragments of the I chain are coprecipitated with DM.
However, elution of the DM associated proteins followed
by reprecipitation indicated that the majority, and perhaps
all, of the I chain fragments are still complexed to class II
molecules (Fig 3; unpublished results). Overall, the data argue against a functionally important direct interaction between DM and the I chain, at least in the human system.
What is the physiological substrate for DM in wild-type
cells? Recent in vitro experiments have implicated CLIP as the DM substrate although some other peptides
can also be removed from
dimers by DM (31), and
LIP can serve as an in vitro substrate (29). The data presented here are consistent with the hypothesis that
CLIP
is the preferred substrate. First, the level of class II association with DM in vitro is much higher when
CLIP complexes, rather than
LIP/SLIP or
peptide complexes,
are used (Fig. 6). Second, when
CLIP is incubated with
DM, virtually no CLIP is visible in the DM-class II complex formed (e.g., Fig. 4 C). This is also true for DM-DR
complexes isolated from intact cells. For example, in Fig. 1 A,
upper left, no CLIP is visible below the dye front where it
normally runs, even at the peak of DM-DR association and
even upon over-exposure of the gels (data not shown). A
reasonable hypothesis to explain this is that CLIP release is
so readily mediated by DM that it does not remain associated for any significant period after the complex forms.
Thus, the higher level of
and
subunit association with
DM seen when
CLIP is added, compared to when
LIP/SLIP or
peptide is added, could be because DM
actually has a higher affinity for empty class II molecules
than for class II molecules occupied with I chain fragments
or peptides.
Preferred association of HLA-DM with empty class II
molecules could in part account for its ability to catalyze
peptide loading. HLA-DM molecules may bind with
dimers associated with CLIP, other peptides or I chain
fragments. This interaction might favor a conformation (or
stabilize a transition state; 28) which has a significantly reduced affinity for CLIP and certain other peptides, but not
for the majority of bound peptides. After dissociation of
CLIP, stabilization of the empty molecules by DM association (Fig. 5) could enhance their ability to associate with
other, endocytically generated, peptides. The COOH-terminal region of the I chain has been reported to stabilize
empty recombinant DR1 molecules, which normally tend
to aggregate (51), thus enhancing in vitro peptide binding
(54). We previously observed that DM was superior to octyl glucoside in facilitating peptide binding to DR3
CLIP complexes, even though octyl glucoside was better at
inducing CLIP dissociation (29), a result now explained by the stabilization of empty
dimers by DM association
(Fig. 5). This chaperone-like function of HLA-DM could
also explain the finding that it can enhance antigen processing involving class II alleles with a low affinity for CLIP,
for example, I-Ak (34). After spontaneous release of CLIP
from I-Ak molecules in vivo, empty I-Ak molecules may be
better preserved in MIICs by interaction with DM, enhancing their ability to generate functional I-Ak-peptide
complexes. Ultimately, these ideas can best be tested by
isolating and characterizing DM-DR complexes. Such experiments are the next challenge in understanding the catalytic function of HLA-DM.
Address correspondence to Peter Cresswell, Howard Hughes Medical Institute, Yale University School of Medicine, Section of Immunobiology, 310 Cedar Street, 415 FMB, New Haven, CT 06510.
Received for publication 28 June 1996
This work was supported by National Institutes of Health grant AI 23081, a gift from Pfizer Inc., and the Howard Hughes Medical Research Institute. L.K. Denzin is supported by a fellowship from the Patrick and Catherine Weldon Donaghue Medical Research Foundation.We would like to thank Dr. Hans Zweerink who kindly supplied the anti-DM antiserum, Dr. Betsy Mellins for providing us with the 10.24.6 and 8.1.6 cell lines, Dr. Derek Sant'Angelo for critical reading of the manuscript, and N. Dometios for expert preparation of the manuscript.
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