By
From the * Department of Pathology, and Committees on Immunology and Cancer Biology,
University of Chicago, Illinois 60637; and the § Department of Biochemistry, Molecular Biology, and
Cell Biology, Northwestern University, Evanston, Illinois 60208
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Abstract |
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To characterize the importance of a highly conserved region of the class II chain, we introduced an amino acid substitution that is predicted to eliminate a hydrogen bond formed between the class II molecule and peptide. We expressed the mutated
chain with a wild-type
chain in a murine L cell by gene transfection. The mutant class II molecule (81
H
) assembles
normally in the endoplasmic reticulum and transits the Golgi complex. When invariant chain (Ii)
is coexpressed with 81
H
, the class II-Ii complex is degraded in the endosomes. Expression of
81
H
in the absence of Ii results in a cell surface expressed molecule that is susceptible to proteolysis, a condition reversed by incubation with a peptide known to associate with 81
H
.
We propose that 81
H
is protease sensitive because it is unable to productively associate with
most peptides, including classII-associated invariant chain peptides. This model is supported by
our data demonstrating protease sensitivity of peptide-free wild-type I-Ad molecules. Collectively, our results suggest both that the hydrogen bonds formed between the class II molecule
and peptide are important for the integrity and stability of the complex, and that empty class II
molecules are protease sensitive and degraded in endosomes. One function of DM may be to
insure continuous groove occupancy of the class II molecule.
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Introduction |
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Occupancy of the MHC-encoded class II molecule
plays a key role in its fate. Association of the surrogate
peptide CLIP,1 derived from invariant chain (Ii), with class
II molecules enhances assembly and export from the endoplasmic reticulum (ER) (1). In the absence of Ii, class II
molecules can bind other proteins in the ER via the peptide
binding pocket (7, 8), thus suggesting a drive for binding
site occupancy. In addition, when Zhong and co-workers
engineered a class II chain to express a covalently linked,
antigenic peptide at its amino terminus, it assembled more
efficiently with
chain and egressed more quickly from the ER than wild-type (WT) class II molecules (9). Hence,
occupancy of the class II binding site in the ER by a tethered peptide promotes rapid transport of class II-peptide
complexes into the Golgi.
The association of peptide with class II molecules also has consequences late in biosynthesis, i.e., in post-Golgi compartments. Several groups have shown that the exogenous provision of peptide increases the half-life and yield of class II molecules in both the endosomal compartments and at the cell surface (10). In addition, the work of Germain and co-workers suggests that in the absence of peptide, empty class II molecules aggregate in endosomes (12). Hence, peptide plays an important role in the entire life cycle of the class II molecule, from facilitating its assembly in the ER to determining its longevity in the endosomal compartments and at the cell surface.
Solving the three-dimensional crystal structures of both a
class I-peptide complex and a class II-peptide complex directly demonstrated that the MHC-encoded and
chains formed a groove occupied by peptide (13, 14). The
crystal structures also provided insight into exactly how the
class II molecule associates with peptide (14), identifying both the pockets that form stable interactions with the
peptide side chains, as well as the hydrogen bonds that
form between amino acid (aa) side chains of the class II
molecule and the main chain atoms of the peptide (Fig. 1,
top). The majority of these hydrogen bonds involve aa's
that are conserved in mouse and human class II molecules
(14).
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One highly conserved region of the class II chain lies
at the periphery of its antigen-binding pocket at residues
79-83. Amino acids 81 and 82 form hydrogen bonds to the
peptide main chain. To characterize the importance of the
histidine at position 81, we created a transfected murine fibroblast L cell line that expressed the mutant class II molecule formed by association of a WT Ad
chain with an Ad
chain in which a conservative aa substitution was introduced at residue 81, changing a histidine to an asparagine
(His
Asn) (19). The mutant Ad molecule lacking the potential for a single hydrogen bond to the peptide main
chain will hereafter be referred to as 81m or as 81
H
.
The mutant class II
chain assembles normally with the
WT
chain in the ER. The assembled mutant class II molecule then transits the Golgi, obtains mature glycosylation,
and has a half-life comparable to that of WT Ad expressed in
L cells (20). However, 81
H
does not form detectable
SDS-stable dimers, suggesting that 81
H
does not stably
associate with peptide. In addition, 81
H
is not detected
in the endosomes, unlike WT class II where 15% of the
steady-state pool is localized to these compartments (20).
In this paper, we characterize the fate of the mutant class
II molecule, 81H
. Although coexpression of Ii redistributes 81
H
to the endosomes (19), we show here that Ii
also dramatically reduces the level of 81
H
expressed at
the cell surface and changes its fate within the cell. Upon
reaching the endosomes, 81
H
in association with Ii is
rapidly degraded. Based on our experiments examining the
susceptibility of 81
H
to proteases as well as its ability to
bind peptides, we hypothesize that when 81
H
accesses
the endosomes and class II-associated invariant chain peptide (CLIP) dissociates, 81
H
is unable to productively
associate with available peptides. These empty class II molecules are then susceptible to degradation. We conclude
that peptide is key to the endosomal survival of both mutant and WT class II molecules and propose that a principle role of CLIP and DM is to insure continuous groove occupancy by peptide.
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Materials and Methods |
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Cell Lines, DNA Construct Cell Lines, and Protease Treatment.
Cell lines were maintained at 37°C and 5% CO2 in DMEM containing 5% FCS and 5% BCS supplemented with 5 mM Hepes, 2 mM glutamine, and 1 mM nonessential amino acids (complete medium). All media and supplements were purchased from GIBCO BRL (Gaithersburg, MD) unless otherwise noted. The derivation of L cell transfectants expressing 81Monoclonal Antibodies.
The hybridomas producing mAb reactive with I-Ad (MKD6, M5/114) and with class I Kk (16-1-11N) were obtained from American Type Culture Collection (Rockville, MD). The specificity of the anti-class II mAbs has been described previously (22). ID4B is a rat mAb that recognizes murine LAMP-1 and was provided by Dr. Thomas August (Dept. of Pharmacology and Molecular Sciences, Johns Hopkins, Baltimore, MD). The rabbit anti-I-AdPeptides.
The AB and cystatin-C (cys) peptides were synthesized by Dr. Giri Reddy of the University of Chicago Amino Acid and Protein Core Labs., Chicago, IL. The cys derived peptide is DAYHSRAIQVVRARKQ (aa 40-55), and the AB peptide is CQKGPRGPPPAGLLQ, which corresponds to the cytoplasmic tail of I-AFlow Cytometry Analysis.
MHC cell surface expression was measured by staining with mAb followed by a secondary staining reagent FITC-labeled goat anti-mouse Ig (FITC-GAM) (Cappel Laboratories, Cochranville, PA) as described previously (24). The samples were analyzed on a FACScan® cytofluorometer (Becton Dickinson, Mountain View, CA).Metabolic Labeling, Immunoprecipitation, and SDS-PAGE.
For the pulse-chase experiments, 5 × 105 cells per time point were plated out the night before in complete media in 60-mm tissue culture dishes. The next day, the cells were prelabeled for 1.5 h in leucine-free DMEM supplemented with 5% dialyzed FCS. The cells were then labeled in the same medium containing 300-350 µCi/ml of [3H]leucine (Amersham Corp., Arlington Heights, IL), at 37°C, 5% CO2 for 30-45 min. The pulse plates were washed once in cold complete media and lysed in 0.5% NP-40 lysis buffer with the protease inhibitors TPCK (50 µg/ml), PMSF (200 µg/ml), leupeptin (0.5 µg/ml) (Boehringer Mannheim Biochemicals, Indianapolis, IN), and 20 mM iodo-acetamide. The chase point plates were washed and incubated in prewarmed medium containing a twofold excess of unlabeled leucine for the indicated chase times. The radiolabeled cells were lysed on the dishes on ice. Postnuclear supernatants were precleared for 1 h with 60 µl of packed PAS (Pharmacia Biotech AB, Uppsala, Sweden) for the mouse mAbs and rabbit antisera or with PGS (Pharmacia Biotech AB) for the rat mAbs. The lysates were then incubated with PAS or PGS prebound with the appropriate antibody for at least 2 h. The immunoprecipitated material was washed three times in lysis buffer, resuspended in sample buffer counting 2% 2-ME, boiled, and analyzed by SDS-10% PAGE. Gels were treated with En3Hance (NEN Research Products, Boston, MA) or Fluor-Hance (Research Products International, Mount Prospect, IL) and subjected to autoradiography atCLIP Analysis.
CLIP analysis was performed essentially as described above except for the following changes. The cells were labeled for 0.5 h in 250 µCi/ml of [35S]methionine (Amersham Corp.) in methionine-free DMEM. The immunoprecipitated proteins were resolved on a 12.5% SDS polyacrylamide gel which was run until the dye front was ~2 inches from the bottom of the glass plates. The gels were treated sequentially, for 20 min each, with fixative (30% methanol, 10% acetic acid), water, and then Fluor-Hance (Research Products International) before drying at 75°C for 2 h.Cell Surface Biotinylation and Western Blot Analysis.
The cells were plated the night before at a density of 5 × 105/2 ml on 60-mm tissue culture dishes. The next day the plates were rinsed six times with cold PBS-CM (PBS with 1 µM CaCl and 250 µM MgCl) and then incubated 30 min with 1.5 µg/ml NHS-SS-biotin (Pierce Chemical Co., Rockford, IL) in PBS-CM on ice with gentle rocking. The biotinylation reaction was stopped by washing the cells six times with 50 mM glycine in PBS-CM. Cells were lysed on the dishes on ice in lysis buffer containing 0.5% CHAPS with the protease inhibitors and iodo-acetamide as described above. The postnuclear supernatants were precleared with PAS as described above and then sequentially immunoprecipitated with PAS prebound to 16-1-11N, followed by immunoprecipitation with PAS prebound to the rabbit antiserum (10 µl/ sample). The immunoprecipitates were washed two to three times with lysis buffer, resuspended, boiled in sample buffer lacking 2-ME, and resolved by SDS-PAGE. The proteins were transferred on to nitrocellulose membranes at 250 milliamps for 2 h. The membranes were blocked with 3% BSA in a buffer composed of 0.5% Tween 20, 1 M D-glucose, and 10% glycerol. The membranes were then incubated for 1 h in a 50-ml solution containing 10% of the solution described above plus 15 µl of streptavidin conjugated to HRP (GIBCO BRL) and then washed extensively with Tris-buffered saline containing 0.05% Tween 20 (TBST) and developed by chemiluminescence using ECL (Amersham Corp.). Western blot analysis of the p12 fragment was carried out as described elsewhere (25). In brief, the nitrocellulose containing the immobilized proteins was blocked for 1 h in water containing 5% powdered nonfat milk, 0.1% sodium azide, and 0.028% antifoam (Sigma Chemical Co.). The membranes were then probed sequentially for class II and Ii with M5114 and In-1, respectively, for at least 3 h. A 1:5,000 dilution of a species-specific secondary antibody coupled to horseradish peroxidase was added to the blot for 1 h before extensive washes in TBST. The blots were then developed using chemiluminescence using LumiGLOTM (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). ![]() |
Results |
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Although the His
Asn substitution at position 81 of the class II
chain is
considered a conservative aa substitution, this mutation is
predicted to disrupt a hydrogen bond between class II and
the peptide backbone (Fig. 1). When the gene encoding
the substituted
chain is introduced into L cells, the mutated
chain assembles with WT Ad
chain and is expressed at the cell surface at levels comparable to WT (19).
Surprisingly, coexpression of Ii causes a dramatic decrease
in the amount of 81
H
expressed at the cell surface (Fig.
2 A, right). These data are in contrast to studies done in our
lab and others (2, 4) where coexpression of Ii with WT
class II molecules facilitates transport and cell surface expression of class II.
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The low levels of class II surface expression observed on
81H
Ii-expressing cells are not caused by the loss of class
II gene expression, but rather correlate with rapid degradation (Fig. 2 B). At time 0,
, Ii, and
chains are present.
By 2 h, the
chain has increased in size, as has the
chain,
indicating that the
Ii complex has left the ER and has
undergone additional glycosylation in the Golgi complex.
At 3 h, there is noticeable attrition of the entire
Ii complex, which is nearly gone by the 4-h chase point. We conclude that the deficiency in cell surface class II expression
on 81
H
Ii-expressing cells cannot be accounted for by
low class II protein synthesis; rather, its loss occurs relatively late in its biogenesis.
We compared the fate of 81H
Ii with that of 81
H
,
WT, and WTIi in a pulse-chase experiment (Fig. 3). All of
the cell lines have similar maturation kinetics early in the
pulse-chase experiment where mature
and
chains are
present after 2 h. Cells expressing WT class II with Ii lose Ii
between 2 and 3 h, indicating that the class II-Ii complex has
accessed the endosomal compartments. A strikingly different
pattern is observed in cells expressing 81
H
Ii (Fig. 3, D and
B). In Fig. 3 B, the mature 81
H
Ii complex begins to disappear at 3 h and is undetectable by 4 h. In contrast, mature
class II molecules persist in cells expressing 81
H
, WT, or
WT class II and Ii after 5 h of chase (Fig. 3, A, C, and D). We
conclude that the loss of 81
H
in the presence of Ii is
unique to the mutant class II molecule, as it is not seen in cells
expressing 81
H
, WT class II, or WT class II and Ii.
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Because degradation of 81H
occurs late in biosynthesis
after addition of N-linked glycans in the Golgi and because
our previous intracellular staining data indicates that 81
H
Ii
accesses the endosomes (19), we hypothesized that the loss of class II molecules occurs when the 81
H
Ii complex
reaches the endosomes. To test this, we used the weak base, NH4Cl, to raise the pH of the endosomal compartments. In addition, because sulfhydryl proteases have been
implicated in Ii degradation (26), we tested the effects
of a sulfhydryl protease inhibitor, Z-phe-ala, on 81
H
Ii.
Cells expressing 81
H
Ii were biosynthetically labeled
overnight in the presence or absence of either NH4Cl or
Z-phe-ala, and class II immunoprecipitates were prepared.
In Fig. 4 A, the short exposure clearly shows that the
81
H
Ii complex is preserved by NH4Cl treatment. The
same experiment (Fig. 4 A; right) shows that mature class II
molecules in 81
H
Ii-expressing cells can also be preserved by a sulfhydryl protease inhibitor. Our results
suggest that when 81
H
has been transported with Ii into
the endocytic compartments, the complex is degraded by
endosomal proteases.
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In Fig. 4 B, we compared the fate of 81H
Ii with that
of WTIi in the presence or absence of NH4Cl. Ammonium
chloride treatment profoundly preserved Ii in both cell
lines, indicating successful neutralization of the endosomal
compartment. In addition, the amounts of mature
and
chains are greatly increased in 81
H
Ii in the presence of
NH4Cl. We also observed some preservation of WT class II
molecules by NH4Cl. We conclude that the entire 81
H
Ii
complex is degraded, whereas only a small amount of WTIi
is degraded in the endocytic compartments upon proteolysis and release of Ii.
Finally, to show that the class II-Ii complexes in cells expressing either WTIi or 81H
Ii access the same endosomal environment, we assayed for the Ii-derived p12 fragment in association with class II (Fig. 4 C, p12) (33, 34).
This 12 kD degradation intermediate is derived from the amino terminus of Ii (aa 1~102) and contains the transmembrane and CLIP region. Fig. 4 C shows that when similar
amounts of class II are immunoprecipitated from 81
H
Ii
and WTIi, comparable amounts of p12 are present. This
result suggests that the pool of p12-associated class II molecules is the same in cells expressing either WTIi or 81
H
Ii.
Taken together, the data in Fig. 4 suggest that the class II
attrition observed in 81
H
Ii occurs endosomally but at a
point after the Ii-p12-81
H
complex is generated.
Based on
Figs. 1-4, we concluded that when the 81H
Ii complex
reaches the endocytic compartments, it is degraded. To explain why 81
H
in association with Ii is susceptible to endosomal degradation, we hypothesized that after Ii is removed, 81
H
is unable to productively bind peptides
available in the endocytic compartment. In support of this
hypothesis, we showed earlier that 81
H
was unable to
form SDS-stable dimers (20). In addition, when we compared the peptide binding capacity of the 81
H
molecule
to the WT Ad molecule in an in vitro translation system,
we found that 81
H
binds very poorly to most peptides.
An exception was found, however, in a cys derived peptide
(aa 40-55), which did bind 81
H
(Wolf Bryant, P., H. Ploegh, and A.J. Sant, manuscript in preparation). This same
cys derived peptide was one of the five predominant peptides
eluted from I-Ad molecules purified from A20 cells (35).
To test whether the 81H
molecule is inherently more
protease sensitive than WT class II molecules, we treated
intact cells with either the broadly reactive protease, PK or
with a more restricted enzyme, trypsin, which cleaves after
arginines and lysines (Fig. 5). Treatment with PK reduces
surface 81
H
class II expression approximately twofold as
detected by the monoclonal antibody MKD6 (Fig. 5 A,
panel 1), but had no effect on WT expression (Fig. 5 A,
panel 3). Treatment of 81
H
and WT Ad-expressing cells
with trypsin gave a similar result (Fig. 5 A, panels 5 and 7).
These experiments support our hypothesis that the 81
H
molecule is protease sensitive compared with WT. Our
data are in contrast to the inherent protease resistance of
the class II molecule demonstrated by a number of groups
in the early studies of Ii association with class II (36).
These workers showed that protease treatment of class II-Ii
complexes to release Ii left the class II molecules intact.
One possible explanation for the observed protease sensitivity of 81
H
compared with WT, which we proposed
earlier, is that 81
H
fails to acquire peptide, leading to
enhanced protease sensitivity. We hypothesize that empty
class II molecules are protease sensitive.
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To determine if 81H
is protease sensitive because it is
empty, we asked whether peptide loading could protect
81
H
from proteolysis. L cells expressing 81
H
were
incubated overnight with the cys derived peptide and then treated with proteases. Strikingly, addition of cys peptide to 81
H
-expressing cells not only protected the cells from
protease treatment (Fig. 5 A, panels 2 and 6), but also increased the amount of surface 81
H
expression (Fig. 5 B).
Fig. 5 B, panel 1 shows that incubation of 81
H
with cys
peptide increases the cell surface expression of 81
H
nearly fourfold, increasing the mean channel fluorescence
from 130 to 530. This increase in surface expression of
81
H
after culture with peptide was detected by a panel
of different mAbs (data not shown) indicating an overall increase in cell surface class II expression. In contrast, WT
class II expression is increased only 1.3-fold by incubation
with cys peptide (Fig. 5 B, panel 2). These data support our
model that the primary mechanism underlying the protease
sensitivity of 81
H
is its underoccupancy by peptide. Because exogenous addition of a peptide able to bind 81
H
,
i.e., cys, confers protease resistance, we conclude that the 81
H
molecule is protease sensitive when it is underoccupied by peptide.
To show that the increase in cell surface 81H
expression by peptide treatment is due to an increase in the yield
of class II molecules rather than the restoration of mAb
epitopes, we used a biochemical assay that did not rely on
reactivity with mAbs. Cell surface molecules were biotinylated after three different culture conditions: cells expressing 81
H
incubated alone, with irrelevant peptide, AB,
or with cys derived peptide. Immunoprecipitation with an
anti-class I mAb shows that equal numbers of cells were biotinylated (Fig. 6 A). Class II molecules were isolated from
the same lysates with a rabbit antiserum reactive with the
cytoplasmic tail of the I-A class II
chain. When we compare 81
H
molecules on cells incubated alone (or with a
control peptide) with those incubated with cys peptide, the
amount of recovered 81
H
dimers increases approximately three- to fourfold in the presence of cys peptide.
We conclude that the increase in 81
H
detected both by
several different mAbs (Fig. 5 and data not shown) and by
cell surface biotinylation is not due simply to a subtle conformational change, but rather reflects an increase in the
amount of 81
H
expressed at the cell surface.
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The rapid degradation of 81H
Ii complexes in the endosomes predicts
that either Ii digestion products such as CLIP do not remain associated with 81
H
or that CLIP binding does not
confer protease resistance to 81
H
. To explore the first
possibility, we isolated class II molecules from WTIi and
81
H
Ii expressing cells in a pulse-chase experiment and
looked for the appearance of the p12 fragment and CLIP
(Fig. 7). After 2 h of chase, abundant levels of p12 are
found associated with class II molecules in both WTIi and
81
H
Ii-expressing cells. After 3 h of chase, we can detect
CLIP ahead of the dye front in cells expressing WTIi:
however, there is no CLIP found associated with class II at
any time points in 81
H
Ii-expressing cells. Since we cannot detect CLIP associated with 81
H
either in a pulse-
chase (Fig. 7) or in a continuous label (data not shown), we
conclude that loss of the hydrogen bond at His 81 causes
CLIP to rapidly dissociate from 81
H
. Exogenous addition of either human or murine CLIP peptides to 81
H
-expressing cells does not increase cell surface class II expression (data not shown). These data are consistent with our
in vitro data indicating a failure of this molecule to stably
associate with CLIP. Thus, we conclude that when 81
H
reaches the endocytic pathway in association with Ii, the Ii molecule is removed by proteolysis, leaving a p12-class II
complex. When this complex is further processed to CLIP-
class II, CLIP rapidly dissociates, and the empty 81
H
molecule is degraded.
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One issue raised by our preceding studies was whether the
protease sensitivity of 81H
was due exclusively to its underoccupancy by peptide. The finding that peptide occupancy by a high affinity peptide confers protease resistance
argues in favor of this conclusion. However, we wished to
evaluate the protease sensitivity of WT I-Ad molecules to
be able to generalize the conclusions we made. Empty soluble class II molecules have been shown by other workers
to aggregate when they are in solution. This behavior complicates attempts to assess their protease sensitivity. Thus,
we adopted an alternate strategy to examine empty WT Ad
molecules. We constructed class II I-Ad molecules that
would be tethered to the cell surface by glycan-phosphatidylinositol (GPI) linkage, contributed by the carboxy-
terminal segment of the GPI-linked dimer HPAP. Davis and
co-workers (21) have characterized I-Ek molecules constructed in such a way and conclude that these molecules exist free of peptide at the cell surface. Recombinant I-Ad-
GPI-linked dimers were engineered from I-Ek-HPAP by
PCR and introduced into CHO cells. These molecules are released from the cell surface by phospholipase treatment.
They do not allow presentation of antigen, but do present
peptide (data not shown). Thus, we conclude that like their
I-E counterparts, GPI-linked I-Ad molecules exist at the
cell surface primarily in a form devoid of peptide.
When tested for their protease sensitivity (Fig. 8), GPI-linked I-Ad molecules were found to be sensitive to trypsin
treatment (Fig. 8, C), a property protected by prebinding
of peptide during overnight incubation (Fig. 8, D). Their
protease sensitivity is comparable to 81H
(Fig. 8, B).
WT I-Ad with intact transmembrane segments expressed in
CHO cells that presumably access peptide en route to the
cell surface are not protease sensitive (Fig. 8, A). We conclude from these studies that peptide occupancy by class II
is an essential feature to its well-known protease resistance.
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Discussion |
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In this paper, we provide evidence that a class II molecule with a compromised ability to bind peptides is susceptible to proteolysis. A number of observations support this
model. First, we observed that 81H
in association with
Ii is degraded in the endosomes. Then, we showed that the
81
H
molecule itself is protease sensitive and that addition of a cys derived peptide restores the protease-resistant
character observed with WT Ad. These observations led us
to propose that 81
H
is protease sensitive because it is
empty. In support, earlier work in our lab showed that
81
H
was unable to form SDS-stable dimers, suggesting
an inability to productively associate with peptide (20) and
our more recent work showing that 81
H
displays greatly
enhanced dissociation rates from peptides (McFarland, B.,
C. Beeson, and A.J. Sant, manuscript in preparation).
An important question raised by these studies is why
changing a histidine to an asparagine at position 81 affects
the ability of the class II molecule to associate with peptide.
The histidine at position 81 of the chain forms a hydrogen bond to the main chain of bound peptides (Fig. 1, top).
This hydrogen bond is observed in three human MHC
class II structures (14) and in the structures of murine
MHC class II molecules (17, 18). Although in principle, asparagine also has a nitrogen that could form a similar hydrogen bond, the side chain of asparagine is predicted to be
too short to reach the peptide backbone. All of the commonly observed rotamers of asparagine were compared to
histidine in the modeling of the mutant. The asparagine
rotamer with a similar orientation to the histidine was chosen to show that the shorter asparagine side chain cannot
form a hydrogen bond with peptides without a large reorientation of the MHC alpha helix (Fig. 1, bottom). Our
modeling shows that the asparagine would be too far from
the peptide (~4.2 Å) to form a strong hydrogen bond. The
simplest explanation for the structural consequences of the
histidine to asparagine mutation is the loss of a specific peptide, class II hydrogen bond. The absence of this hydrogen
bond may destabilize the binding of many peptides. Additionally, the hydrogen bond formed at position 81 may be
an important first step for initiating peptide binding to the
class II molecule, acting to position or dock the peptide before stable binding to the class II molecule occurs.
Additional data from both our lab and another lab support our conclusion that the hydrogen bonds between the
class II molecule and peptide are important for the overall
stability and integrity of the class II-peptide complex.
Glimcher and co-workers demonstrated the importance of
the aa's at the periphery of the class II peptide binding
pocket for class II surface expression (41). They described a
mutagenized murine B lymphoma that had lost cell surface
Ad expression. They cloned and sequenced both the and
chain genes and found only a single aa substitution at residue 82, changing an asparagine to a serine in the class II
chain. This single aa substitution resulted in a class II molecule (82m) which associated with Ii but was unable to access the cell surface, i.e., it was retained intracellularly. Using an L cell transfection system, we also found that
coexpression of Ii reduces 82m expression at the cell surface (data not shown). In addition, 82m expressed at the
cell surface in the absence of Ii is even more protease sensitive than 81
H
(data not shown). Our preliminary data
also suggest very rapid endosomal degradation of 82m
when it is expressed in association with Ii. Fig. 1 shows that
the aa at position 82 of the
chain forms two hydrogen
bonds with the peptide backbone. The Asn
Ser substitution at position 82 is predicted to eliminate both hydrogen bonds. Such a molecule may be even more impaired in its
ability to bind or remain stably associated with peptide,
which might explain why 82m is even more protease sensitive than 81
H
.
The importance of these hydrogen bonds between conserved MHC residues and main chain atoms of the peptide
has been examined for a class I-peptide complex. By substituting methyl groups for charged aa's in the peptide,
Bouvier and Wiley showed that more energy was contributed by the hydrogen bonds than by the anchor residues of
the peptide, suggesting that the hydrogen bonds play an
important role in the stability of the complex (42). In
agreement with this work, Hill and co-workers demonstrated that a significant amount of free energy of binding
arises from the hydrogen bonds formed between the class II
binding site and the amide bonds of the ligand (43). In Fig.
1, aa's 53, 62, 68, 69, and 76 of the class II chain are
shown to form hydrogen bonds with the peptide backbone. Peccoud and co-workers substituted alanine at each
of these positions in a class II Ak molecule and found that
the ability to present peptides to a number of T cell hybridomas was detectable, although impaired. The most striking loss was observed when aa 62 was substituted (44). This
work supports the idea that the hydrogen bonds formed between the class II molecule and peptide are very important and that some hydrogen bonds may be particularly
critical for formation of stable peptide-class II complexes.
Our results showing the profound effects that follow from the loss in potential for a single hydrogen bond between peptide and MHC class II molecules are particularly interesting in light of the recent successful crystallization of the I-Ad molecule bound to antigenic peptides (18). The structure obtained shows that I-Ad achieves stable peptide binding with minimal pocket interactions between the class II molecule and the R groups of the peptide. The pockets within the binding groove of I-Ad appear to be either empty (P1 and P9) or only partially filled (P4). Thus, strong pocket interactions are not essential for stable peptide interactions to the class II molecules. In contrast, our results suggest that loss in potential for a single hydrogen bond can profoundly diminish the capacity of class II molecules to acquire peptide. It is not yet clear if the contribution of hydrogen bonds will be similarly great for those MHC class II peptide interactions that are characterized by strong pocket interactions. We are currently examining this issue.
Although the histidine at position 81 is highly conserved,
there are some chains which do not have a histidine at
residue 81: H-2 A
u (I-Au) and HLA DRw53. Both have a
tyrosine at position 81, suggesting that the histidine is not
required for a viable class II molecule. In describing the association of I-Au and peptide, McConnell and Lee proposed that the tyrosine substitution at 81 would either delete the hydrogen bond or force a substantial shift in the
peptide backbone around the P1 pocket (45). Based on our
model that residue 81 is key for stable peptide association with class II, one might predict that class II molecules with a tyrosine at position 81 have evolved compensatory mechanisms for stable association with peptide. In support of this
hypothesis, there are aa substitutions found at positions in
the I-Au molecule that are not present in other known I-A
alleles.
Alternatively, it is interesting to consider the possibility that a higher proportion of I-Au molecules may be empty. It is well documented that I-Au has a low affinity for the immunodominant epitope of myelin basic protein, Ac1-9, which is encephalitogenic in H-2u mice (46). The low affinity of I-Au for Ac1-9 is thought to contribute to disease onset because autoreactive T cells escape self-tolerance in the thymus. One might predict that substituting a histidine at position 81 of I-Au would result in a higher affinity for Ac1-9, and perhaps other peptides that interact with this class II molecule.
In conclusion, we describe here a class II molecule,
81H
, that is unable to remain associated with peptide
because a hydrogen bond has been altered. When 81
H
Ii
complexes access the endosomes in the presence of Ii, they are degraded
presumably because the CLIP peptide immediately dissociates after removal of Ii and no other self-peptides are able to bind 81
H
. From our data, as well as
the work of others (10), we propose that empty class II
molecules in the endocytic pathway are degraded. For this
reason, the cell has two chaperones to orchestrate continuous groove occupancy of the class II molecule, Ii and DM.
Ii directs the class II-Ii complex to the late endosomal
compartments via the strong sorting signal in its tail. Under
the acidic, proteolytic conditions of the endosomes, Ii is
degraded whereas the class II molecule bound by CLIP is
protected. At this point, the second chaperone, DM, facilitates the exchange of CLIP for antigenic peptides. However, there are some class II molecules on which CLIP has
a very fast off-rate, specifically I-Ak, I-Ed, and I-Ek (49).
The existence and survival of such molecules suggest that
the critical function of DM is not to remove CLIP, but
rather to load peptide onto empty class II molecules which
would be susceptible to proteolysis. Hence, we propose
that the cooperative function between Ii and DM may be
to insure continuous groove occupancy of the class II molecule by peptide.
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Footnotes |
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Address correspondence to Dr. Andrea J. Sant, University of Chicago, Dept. of Pathology and Committee on Immunology, 5841 S. Maryland Ave. MC1089, Chicago, IL 60637. Phone: 773-702-3990; Fax: 773-702-3701; E-mail: asant{at}flowcity.bsd.uchicago.edu
Received for publication 14 August 1998.
S. Ceman is supported by National Institutes of Health grant F32 AI09218 and the Weber Fellowship Fund. T.S. Jardetzky is supported by the International Frontier Science Program and the Pew Memorial Trust. A.J. Sant is supported by National Institutes of Health grant R01 AI34359.We would like to thank L.J. Tan for making the L cell transfectants. We would also like to thank John Katz, Chris Stebbins, Yair Argon, and Jim Miller for critically reading this manuscript and for helpful comments and suggestions.
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