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Hydrogen Bond Integrity Between MHC
Class II Molecules and Bound Peptide
Determines the Intracellular Fate of MHC
Class II Molecules
Lynne S. Arneson, John F. Katz, Michael Liu and Andrea J.
Sant
J Immunol 2001; 167:6939-6946; ;
doi: 10.4049/jimmunol.167.12.6939
http://www.jimmunol.org/content/167/12/6939
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Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Hydrogen Bond Integrity Between MHC Class II Molecules
and Bound Peptide Determines the Intracellular Fate of MHC
Class II Molecules1
Lynne S. Arneson,2 John F. Katz,3 Michael Liu, and Andrea J. Sant4
M
ajor histocompatibility complex class II molecules are
associated with bound peptides for most of their lifetime. Peptide association has been shown to affect
both the structure and the fate of the class II molecule (1, 2). MHC
class II molecules primarily acquire peptides within endosomal
compartments of APCs. Thus, although MHC class II molecules
are synthesized in the endoplasmic reticulum (ER),5 they must
gain access to the endocytic pathway to engage peptides. Efficient
targeting of class II molecules to endosomes is facilitated by their
association with the invariant chain glycoprotein. Immediately following synthesis in the ER, class II ␣␤ heterodimers associate with
invariant chain and the class II-invariant chain complex transits
through the Golgi apparatus to the trans-Golgi network, where
dileucine-based signals in the cytosolic domain of invariant chain
mediates trafficking of the complex to endosomal compartments
(1, 2). Following arrival in the endocytic pathway, invariant chain
is degraded by resident proteases. Invariant chain proteolysis proceeds in a stepwise fashion, but its release from class II is incom-
Department of Pathology, University of Chicago, Chicago, IL 60637
Received for publication May 22, 2001. Accepted for publication October 12, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the National Institutes of Health (RO1
AI34359 and PO1 DK49799), by the Juvenile Diabetes Foundation International (to
A.J.S.), and by a Postdoctoral Fellowship award from the American Heart Association, Midwest Affiliate, Inc. (to L.S.A.).
2
Current address: Department of Biology, American University, Washington, DC.
3
Current address: Department of Pathology, Harvard Medical School, Boston, MA.
4
Address correspondence and reprint requests to Dr. Andrea J. Sant, Department of
Pathology, Committees on Immunology and Cancer Biology, University of Chicago,
5841 South Maryland Avenue, MC 1089, Chicago, IL 60637. E-mail address: asant@
midway.uchicago.edu
5
Abbreviations used in this paper: ER, endoplasmic reticulum; CLIP, class II-associated invariant chain peptide; HEL, hen egg lysosome.
Copyright © 2001 by The American Association of Immunologists
plete, as a peptide derived from invariant chain, termed class IIassociated invariant chain peptide (CLIP; amino acids 89 –104),
remains associated in the peptide-binding groove of class II molecules (3, 4). The accessory molecule DM (H-2M in the mouse)
facilitates the removal of CLIP and subsequent loading of class II
with peptide derived from internalized proteolyzed Ag (5, 6). Having bound a peptide in the endocytic pathway, the class II peptide
complex is sorted to the cell surface.
Structurally, class II can be considered to exist as a three-chain
molecule composed of the ␣- and ␤-chains of class II plus either
invariant chain or antigenic peptide associated with the peptide
binding groove. Class II molecules not associated with peptide
have variously been shown to aggregate (7) and to be sensitive to
proteolytic degradation (8), indicating that the association of class
II with peptide is required for structural integrity and functional
stability.
The trafficking pathway of class II molecules indicates that although class II is associated with peptide for most of its lifetime,
the identity of the bound peptide changes as the class II molecule
moves through the cell. Thus, a requisite event in class II biogenesis is the exchange of bound peptide, for example, the release of
CLIP and acquisition of antigenic peptide in endosomal compartments. The mechanism by which class II associates with peptides
likely reflects this requirement for peptide exchange. The crystal
structures of class II peptide complexes have provided insight into
how peptides interact with class II in the binding groove (9 –11).
Two different types of interactions contribute to stable class II
peptide binding. The amino acid side chains of the bound peptide
associate with pockets formed in the class II peptide binding
groove. These pocket interactions are generally hydrophobic in
nature, although some class II alleles contain pockets that favor a
polar or charged side group (10). In the current paradigm of MHCpeptide binding, pocket interactions result not only in peptide-specific class II binding, but also in the formation of stable class II
peptide complexes (reviewed in Ref. 2). In addition to these pocket
0022-1767/01/$02.00
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MHC class II molecules associate with peptides through pocket interactions and the formation of hydrogen bonds. The current
paradigm suggests that the interaction of side chains of the peptide with pockets in the class II molecule is responsible for the
formation of stable class II-peptide complexes. However, recent evidence has shown that the formation of hydrogen bonds between
genetically conserved residues of the class II molecule and the main chain of the peptide contributes profoundly to peptide stability.
In this study, we have used I-Ak, a class II molecule known to form strong pocket interactions with bound peptides, to probe the
general importance of hydrogen bond integrity in peptide acquisition. Our studies have revealed that abolishing hydrogen bonds
contributed by positions 81 or 82 in the ␤-chain of I-Ak results in class II molecules that are internally degraded when trafficked
through proteolytic endosomal compartments. The presence of high-affinity peptides derived from either endogenous or exogenous
sources protects the hydrogen bond-deficient variant from intracellular degradation. Together, these data indicate that disruption
of the potential to form a complete hydrogen bond network between MHC class II molecules and bound peptides greatly diminishes the ability of class II molecules to bind peptides. The subsequent failure to stably acquire peptides leads to protease sensitivity
of empty class II molecules, and thus to proteolytic degradation before export to the surface of APCs. The Journal of Immunology, 2001, 167: 6939 – 6946.
6940
Materials and Methods
Reagents and cell lines
Mutations in I-Ak class II molecules were made at positions 81 or 82 in the
␤-chain by site-directed PCR mutagenesis (14). Amino acid 81 was mutated from His to Asn, whereas 82 was mutated from Asn to Ser. Following
sequencing to verify that only the intended mutations were introduced, Ltk
cells were stably transfected with I-Ak␣ and I-Ak␤81His ⬎ Asn or
I-Ak␤82Asn ⬎ Ser cDNAs with the selectable marker pSV2neo. Cells that
survived drug selection were subcloned, and clones expressing the hydrogen bond-deficient class II molecules were supertransfected with either the
p31 form of murine invariant chain cDNA or a p31 invariant chain construct in which the CLIP region has been replaced with amino acids 48 – 61
from hen egg lysosome (HEL; Ref. 15) with the selectable marker blastocidin. Cells stably expressing class II and invariant chain were then additionally transfected with DM, with the selectable marker zeocin. Expression of these proteins was monitored by either flow cytometry or Western
blot analysis. B cell hybridomas secreting the anti-I-Ak mAbs 10.2–16, 40L
or 40M (16), or 16-1-11N, which binds H-2Kk class I molecules (17), were
used as a source of culture supernatant. The anti-HEL45– 61/I-Ak-specific
mAb C4H3 was generously provided by Dr. R. N. Germain (National
Institutes of Health, Bethesda, MD) (18). The anti-DM␤ chain antisera was
made in rabbits immunized with a peptide corresponding to the cytoplasmic region of the murine DM␤ chain conjugated to keyhole limpet hemocyanin in CFA, followed by subsequent immunization in IFA.
Metabolic labeling
Cells were plated on 100-mm tissue culture plates at a density of 2 ⫻ 106
cells/plate and were allowed to adhere overnight. The cells were prelabeled
in leucine-free medium for 1 h at 37°C to deplete intracellular stores of
leucine. The medium was aspirated and fresh leucine-free medium containing 0.25 mCi of [3H]leucine/ml medium (New England Nuclear, Bos-
ton, MA) was added and the plates were incubated at 37°C for 1 h. The
medium was then aspirated, complete DMEM plus 10% FCS was added to
each plate, and the cells were incubated at 37°C for increasing times. Following the chase, detergent lysates of the labeled cells were prepared by
lysing cells on ice for 20 min in 0.5% Nonidet P-40, 150 mM NaCl, 50 mM
Tris, pH 7.6, 5 mM EDTA, and 5 mM iodoacetimide plus protease inhibitors. The presence of iodoacetimide prevents disulfide bond formation
between invariant chain molecules by air oxidation following cell lysis.
The samples were precleared overnight with protein A-Sepharose (Pharmacia, Peapack, NJ), then class I and class II molecules were serially
isolated by immunoprecipitation using anti-class I or anti-class II Abs prebound to protein A-Sepharose (Pharmacia). Class I molecules are used to
quantify any sample loss through the detergent lysis and immunoprecipitation, and these typically are within 10 –15% of each other when comparing different cell lines in individual experiments. Samples were dissociated
in SDS-sample buffer with 2% 2-ME, electrophoresed on SDS 10% PAGE,
and the gel was processed for autoradiography using Flouro-Hance (Research Products, Madison, WI) for 30 min. The treated gel was dried and
exposed to film at ⫺70°C. Experiments shown have been repeated typically two to three times and representative data are shown.
SDS stable dimer analysis
Cells were harvested by brief exposure to trypsin, and they were counted,
pelleted, and lysed in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonic acid lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.6, and
5 mM EDTA) on ice for 30 min. Postnuclear supernatants were incubated
with 10.2-16 prebound to protein A-Sepharose (100 ␮l of culture supernatant per immunoprecipitation) and were rotated at 4°C for 4 h. Material
bound to protein A-Sepharose was eluted with SDS sample buffer for 30
min at room temperature with frequent mixing. The protein A-Sepharose
beads were pelleted and the supernatant was split evenly into two tubes.
One tube was boiled for 3 min, whereas the other tube was not. The supernatant was fractionated on a 10% acrylamide gel overnight, with the
current never exceeding 20 mAmps per gel. Protein was transferred onto
nitrocellulose and the nitrocellulose was incubated with 5% milk in TBST
and NaN3 (“Blotto”) for 1 h. The blot was then incubated with 10.2-16 in
Blotto overnight, washed, then incubated in peroxidase-linked goat antimouse at 1/10,000 for 1 h. Following extensive washing, the blot was
exposed to film with chemiluminescence.
Results
Previous work from our laboratory used a mutation at position 81
in the ␤-chain of I-Ad to determine the effect of the loss of a single
hydrogen bond on stable peptide acquisition (8, 12, 13). Although
these studies determined that the loss of a single hydrogen bond
resulted in dramatically decreased peptide binding efficiency,
which in turn leads to endosomal degradation of the hydrogen
bond-deficient variant, the class II molecule used has been shown
to bind peptide in the absence of strong pocket interactions (11)
and thus may rely to an unusual degree on the hydrogen bond
network for stable peptide binding. Therefore, we extended these
studies to gain a more generalized understanding of the relative
importance of hydrogen bond interactions between peptides and
MHC. To do this, we have examined the effects of the formation
of the hydrogen bond network on peptide acquisition by I-Ak, an
allelic form of class II molecule that uses strong pocket interactions to facilitate stable peptide binding (12, 16). We mutated I-Ak
such that one or more hydrogen bonds formed between class II and
bound peptides were lost. The solved crystal structure of I-Ak:
HEL48 – 61 complex indicates that 12 of the 26 nitrogen atoms
present in the bound peptide directly form hydrogen bonds with
I-Ak, whereas nine associate with the class II molecule by forming
hydrogen bonds with water, which in turn form extended hydrogen
bond networks with I-Ak. Both amino acids 81 and 82 in the
␤-chain of I-Ak directly form hydrogen bonds with the bound peptide (10). PCR site-directed mutagenesis was used to mutate either
␤81 from His to Asn or ␤82 from Asn to Ser to abrogate the
formation of either one (Asn␤81) or two (Ser␤82) hydrogen bonds
(Fig. 1), and the hydrogen bond-deficient class II molecules and
wild-type I-Ak were expressed in L cell fibroblasts (Table I).
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interactions between MHC and peptide, hydrogen bonds are
formed between conserved amino acids in the class II molecule
and the main chain of the bound peptide. In both human and
mouse, the class II residues that participate in this hydrogen bond
network are generally highly conserved across class II alleles, suggesting the importance of the resulting hydrogen bond network
formed between the class II molecule and the bound peptide.
Previous studies from our laboratory suggest that the loss of a
single hydrogen bond in the hydrogen bond network formed between class II and bound peptides can have a profound effect on
stable peptide acquisition (8, 12, 13). However, these studies used
the I-Ad class II molecule, which has been shown to bind peptides
without apparently strong pocket interactions (11). Thus, I-Ad may
rely to an atypically high degree on the formation of the hydrogen
bond network to achieve stable peptide binding. To understand in
a general way the importance of the full integrity of the hydrogen
bond network, we initiated a set of studies using a different allelic
form of class II molecule, I-Ak, that engages peptides through
strong pocket interactions (reviewed in Ref. 16). We found that the
loss of one or more hydrogen bonds results in a severely decreased
ability to acquire peptides, resulting in intracellular degradation of
the class II molecule. Although the addition of a high-affinity peptide, by either endogenous or exogenous routes, can protect hydrogen bond-deficient class II molecules from degradation, the
presence of the chaperone molecule DM does not prevent degradation, suggesting that high-affinity peptides that can sustain binding in the absence of hydrogen bond integrity may be rare within
the normal peptide milieu of APCs. Together, these data indicate
that disruption of the hydrogen bond network formed between
MHC class II and bound peptides greatly diminishes the ability of
the class II molecule to bind peptides, and that such failure to
stably acquire peptides leads to increased protease sensitivity of
empty class II molecules, and thus to proteolytic degradation before export to the surface of APCs. Thus, it appears that hydrogen
bond integrity is a critical structural element for stable peptide
binding by class II molecules.
HYDROGEN BOND-DEFICIENT CLASS II MOLECULES
The Journal of Immunology
6941
To study the impact of hydrogen bond loss on the structural
integrity and peptide binding ability of the class II molecule, pulsechase analysis was used to assess the folding, assembly, and intracellular fate of the wild-type and hydrogen bond-deficient class
II molecules. Cells expressing wild-type I-Ak or I-Ak␤81H⫺ were
pulsed for 1 h with medium containing [3H]leucine to label newly
synthesized protein, and were then chased in complete medium for
0, 1, or 4 h to allow the labeled protein to transit through the
secretory pathway. At the end of each chase point, the cells were
lysed and class I and class II molecules were serially immunoprecipitated from the postnuclear lysate. Fig. 2 shows that the mature
hydrogen bond-deficient class II molecule (I-Ak␤81H⫺) persisting
following a 4-h chase was similar to that seen with wild-type I-Ak,
indicating that the trafficking and half-life of these molecules is
similar. Coprecipitation of class II ␣-chain by the ␤-chain-specific
Ab indicated that both wild-type I-Ak and I-Ak␤81H⫺ assembled
into correctly folded class II heterodimers that transited through
the Golgi apparatus as indicated by the apparent increase in m.w.
due to N-linked sugar maturation. Similar results were seen with
I-Ak␤82H⫺ in the absence of coexpressed invariant chain (Fig. 3).
Previous work has indicated that the hydrogen bond network has
a profound effect on class II peptide binding, which occurs in endosomal compartments. Therefore, we assessed the effect of the
loss of one or more hydrogen bonds on the fate of class II molecules directed to endosomal compartments by coexpression of in-
FIGURE 2. Coexpression of invariant chain results in premature degradation of hydrogen bond-deficient class II molecules. L cells stably expressing either I-Ak (A) or I-Ak␤81H⫺ (B) class II in the absence or presence of coexpressed invariant chain were pulsed with [3H]leucine for 1 h
and were then chased in unlabeled complete medium for 0, 1, or 4 h. After
each chase point, an aliquot of cells was lysed and class I and class II
molecules were serially immunoprecipitated from the postnuclear lysate
with 16-1-11N and 10.2-16, respectively. Mature ␣ and ␤ class II chains
are indicated, as are p31 invariant chain and the class I H chain.
variant chain. Cells expressing either wild-type I-Ak or
I-Ak␤81H⫺ plus invariant chain were pulsed with [3H]leucine for
1 h and chased in the absence of radiolabel for 0, 1, or 4 h. Following each chase point, the cells were lysed, and class I and class
II were serially immunoprecipitated from the detergent lysate.
Wild-type I-Ak that is directed to endosomal compartments by
association with invariant chain persists in a mature state after a
4-h chase, as indicated by the presence of mature ␤-chain (Fig. 2).
Mature wild-type I-Ak ␣-chain is also present in the 4-h chase
point, but is less visible due to the formation of multiple glycosylation states. In contrast, both I-Ak␤81H⫺ (Fig. 2) and
Table I. Flow cytometry analysis of surface class II expressiona
Class II Molecule
Ab
k
A wt
Ak81
Ak82
14.4.4S (neg)b
10.2.16
40L
40M
11
542
505
407
12
305
246
213
11
165
111
104
a
Cells expressing wild-type or the hydrogen bond-deficient class II molecules
were stained with the Abs indicated in the left column. Shown are the mean fluorescence intensity obtained with each Ab.
b
Negative Ab 14.4.4S.
FIGURE 3. Engineering a high-affinity peptide into the class II binding
site prevents premature degradation. Cells expressing I-Ak␤82H⫺ alone or
coexpressing either p31 invariant chain or an invariant chain construct in
which the CLIP segment has been replaced a peptide corresponding to
HEL48 – 61 were pulsed with [3H]leucine for 1 h and were chased in unlabeled complete medium for 0, 1, or 4 h. After each chase point, an aliquot
of cells was lysed and the class I (B) and class II (A) molecules were
serially immunoprecipitated from the postnuclear lysate with 16-1-11N or
10.2-16, respectively. Mature ␣ and ␤ class II chains are indicated, as are
p31 and p31HEL invariant chain and the class I H chain.
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FIGURE 1. The crystal structure of the I-Ak class II molecule adapted
from Ref. 10. Shown is a top view of the ribbon diagram of the I-Ak class
molecule (in blue) bound to the HEL48 – 61 peptide. Hydrogen bonds contributed directly by class II amino acids are shown by dotted lines and
amino acids 81(His) and 82(Asn) in the ␤-chain of I-Ak, which contribute
one and two hydrogen bonds, are indicated in red and yellow, respectively.
6942
binding groove of I-Ak␤82H⫺ class II molecules. The CLIP region
of invariant chain was replaced with amino acids 48 – 61 from HEL
(15), a peptide previously shown to bind to wild-type I-Ak with
high affinity (Refs. 16 and 21). Cells expressing I-Ak␤82H⫺ were
supertransfected with the mutated invariant chain construct (IiHEL) and the intracellular fate of class II in the presence of the
high-affinity HEL48 – 61 peptide was assessed by pulse-chase analysis. Cells were pulsed for 1 h with [3H]leucine and were chased
for 0, 1, or 4 h in the absence of radiolabeled amino acids. Following each chase point, the cells were lysed and class I and class
II molecules were immunoprecipitated from the lysate. As indicated above, I-Ak␤82H⫺ class II molecules targeted to endosomes
are degraded rapidly, as shown by the very low amount of mature
class II following a 4-h chase. However, the coexpression of the
invariant chain construct containing the HEL peptide results in the
presence of mature I-Ak␤82H⫺ class II molecules after 4 h of
chase (Fig. 3), suggesting that the high-affinity interaction of
HEL48 – 61 and I-Ak␤82H⫺ results in formation of class II peptide
complexes that persist, leading the molecule to maintain protease
resistance.
FIGURE 4. Expression of Ii-HEL construct leads to egress of Ak-[HEL48 – 61] at the cell surface, but does not confer SDS stability to the hydrogen
bond-deficient variant of I-Ak. A, Expression of the I-Ak/HEL48 – 61 complex at the cell surface. Wild-type Ak or Ak␤82H⫺ cells expressed without invariant
chain (left two panels) or with the Ii-HEL construct (right two panels) were stained with the class II-specific Ab 10.2-16 (solid line) or its negative control
mouse Ab 14-4-4S (dotted line), shown in the left-most panels of each group. Alternatively, the cells were stained with the rat mAb C4H3 that detects the
I-Ak[HEL48 – 61] complex (solid line) or its rat Ab control 2.43 (dotted line) shown in the right panels of each group. B, SDS stability of I-Ak wild type vs
I-Ak␤82H⫺. Shown is SDS-stable dimer analyses of Ak wild type or Ak82 expressed either without invariant chain (—), with normal p31 invariant chain
(p31), or the invariant chain construct with HEL48 – 61 cassetted into the CLIP region of Ii (HEL). Cells were lysed and samples were immunoprecipitated
with the class II mAb 10.2-16. Immunoprecipitated material was eluted at room temperature, split into two equal aliquots, either boiled (⫹) or left at room
temperature (⫺), and fractionated by SDS-PAGE. Proteins were transferred to nitrocellulose and were probed in a Western Blot for class II ␤-chains using
10.2-16. Indicated on the left of the figure are the positions of SDS-stable ␣␤ dimers or free ␤-chains.
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I-Ak␤82H⫺ (Fig. 3) class II molecules are greatly diminished and
almost undetectable after 4 h of chase when targeted to endosomes.
Thus, the hydrogen bond-deficient class II molecules have a decreased half-life as compared with wild-type I-Ak (Fig. 2). These
data suggest the loss of one or more hydrogen bonds formed between class II and bound peptide results in premature intracellular
degradation of the class II molecule when targeted to endosomal
compartments by association with invariant chain.
Studies using class II molecules engineered to be transited to the
cell surface in a peptide-free state indicated that empty class II
molecules are protease sensitive, a property reversible by stable
peptide binding (8). We have observed a similar protease sensitivity of the Ak hydrogen bond variants (not shown). These results,
together with our studies of the decreased intracellular half-life of
the hydrogen bond-deficient class II molecules, led us to hypothesize that the hydrogen bond-deficient class II molecules are unable to efficiently bind peptide, resulting in an increased population
of empty class II molecules that undergo intracellular proteolytic
degradation. To test this hypothesis, we used the invariant chain
molecule to efficiently load a high-affinity peptide into the peptide-
HYDROGEN BOND-DEFICIENT CLASS II MOLECULES
The Journal of Immunology
6943
ments of the transfectants. In contrast, both I-Ak␤81H⫺ and
I-Ak␤82H⫺ undergo rapid degradation during the pulse due to
targeting to endosomal compartments by coexpressed invariant
chain, resulting in very low levels of mature class II molecules
remaining at the end of the pulse. Incubation of cells expressing
these hydrogen bond-deficient class II molecules with exogenous
HEL results in an increase in the mature class II present in the cells
following the 8-h pulse (Fig. 5). These results indicate that highaffinity peptides from exogenous sources can associate with hydrogen bond-deficient class II molecules, an event that leads to
protection of the class II molecules from proteolytic degradation.
The hydrogen bond-deficient class II molecules undergo premature degradation when targeted to an endosomal compartment by
association with invariant chain, suggesting that degradation is occurring in endosomes. To test this hypothesis, we incubated cells
with ammonium chloride, a lysosomotropic agent that neutralizes
the pH in endosomes, thus decreasing the proteolytic capacity of
these organelles. Cells expressing either wild-type I-Ak,
I-Ak␤81H⫺, or I-Ak␤82H⫺ and invariant chain were pulsed for
8 h with [3H]leucine and were incubated in the presence or absence
of 20 mM ammonium chloride. Following the pulse, the cells were
lysed and class I and class II were immunoprecipitated from the
cell lysate and analyzed by SDS-PAGE for levels of mature class
II remaining at the end of the 8-h pulse (Fig. 5). Incubation in the
presence of ammonium chloride resulted in increased levels of
labeled class II molecules following the overnight pulse, indicating
that degradation of the hydrogen bond-deficient class II molecules
indeed occurred in acidic proteolytic compartments, most likely
endosomes.
We next determined the effect of DM coexpression on the fate
of the hydrogen bond-deficient class II molecules directed to endosomal compartments. If high-affinity peptides reside in endosomal compartments in the fibroblasts transfected with class II, yet
the hydrogen bond-deficient class II molecules are rapidly degraded before loading can occur, the presence of DM might rescue
the class II molecules by increasing the efficiency of peptide loading. However, if peptides of sufficiently high affinity are rare in the
APC, the addition of DM would have no effect on the degradation
of the hydrogen bond-deficient class II molecules. Such a prediction, of course, assumes that the hydrogen bond-deficient variants
FIGURE 5. High-affinity peptides derived from exogenous Ag rescues
the hydrogen bond-deficient class II molecules from endosomal degradation. Cells expressing invariant chain and either I-Ak, I-Ak␤81H⫺, or
I-Ak␤82H⫺ were pulsed with [3H]leucine for 8 h. During the pulse, the
cells were incubated in either complete medium, or medium modified by
the addition of either 8 mg/ml HEL or 20 mM ammonium chloride. Following the pulse, the cells were lysed and class II (A) and class I (B)
molecules were serially immunoprecipitated from the postnuclear lysate
with 16-1-11N or 10.2-16, respectively. Mature ␣ and ␤ class II chains and
the class I H chain are indicated.
FIGURE 6. Coexpression of DM does not result in efficient peptide
loading of hydrogen bond-deficient class II molecules. Cells expressing
I-Ak␤82H⫺ class II were supertransfected with either p31 invariant chain,
p31HEL, or p31 and DM ␣- and ␤-chains. These cells were pulsed with
[3H]leucine for 1 h and were then chased in unlabeled complete medium
for either 0 or 4 h. After each chase point, one- half of the cells were lysed,
and class I (B) and class II (A) molecules were serially immunoprecipitated
with 16-1-11N or 10.2-16, respectively. Mature ␣ and ␤ class II chains are
indicated, as are p31, p31HEL, and the class I H chain.
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To show that the I-Ak wild-type and ␤82H⫺ class II molecules
that emerged to the cell surface were carrying the HEL48 – 61 peptide within their peptide binding pockets, we examined the cells for
reactivity with a mAb (C4H3) that detects the I-Ak/HEL48 – 61 (18).
Fig. 4A shows that upon expression of the Ii-HEL gene construct,
both wild-type I-Ak and the I-Ak␤82H⫺ class II molecules gain
readily detectable expression of this mAb epitope, indicating that
a significant fraction of the class II molecules displayed by the
transfectants bear HEL48 – 61 within the class II peptide binding
pocket. Shown in Fig. 4B is an analysis of SDS-stable dimers
within the wild-type and I-Ak␤82H⫺ cells. These analyses indicated that despite the presence of the HEL48 – 61 peptide within its
Ag-binding pocket, the I-Ak␤82H⫺ class II molecules do not gain
SDS stability. This result is consistent with the data suggesting that
accessibility to SDS at the amino-terminal end of the MHC class
II peptide binding pocket, rather than peptide binding per se, determine the phenotype of SDS stability (19).
The Ii-HEL construct used in the previous experiment enhanced
the efficiency of HEL peptide loading onto the hydrogen bonddeficient class II molecules before arrival in endosomal compartments. In a more physiological setting, the class II molecules
would encounter the high-affinity HEL peptides within endosomal
compartments in the presence of other peptide Ags. It has been
shown that coculture of H-2k splenocytes with HEL Ag increases
life span and the steady-state expression of the I-Ak molecule (20,
21). To determine whether the high-affinity HEL peptide can be
loaded onto the hydrogen bond-deficient class II molecules following invariant chain proteolysis and CLIP removal, cells expressing either wild-type I-Ak, I-Ak␤81H⫺, or I-Ak␤82H⫺ and
that coexpressed invariant chain were pulsed for 8 h with
[3H]leucine in the presence or absence of 8 mg/ml exogenous HEL
protein. Following the pulse, the cells were lysed and class I and
class II molecules were serially immunoprecipitated from the cell
lysate. During the 8-h pulse, class II molecules were continually
being synthesized and transported through the cell. Fig. 5 shows
that at the end of the pulse, significant levels of wild-type I-Ak
were present in the cell, and these levels were not altered appreciably by incubation with exogenous HEL. This result suggests
that the quantity of peptides able to bind to the wild-type Ak molecule spontaneously are not limiting in the endosomal compart-
6944
can interact productively with DM, an issue that we currently have
no data on. Nevertheless, we sought to determine what effect DM
has on degradation of these class II molecules. Therefore, we supertransfected cells expressing I-Ak␤82H⫺ and invariant chain
with genes encoding murine DM ␣- and ␤-chains. Cells expressing
DM were pulsed for 1 h, and they were then lysed immediately or
chased for 4 h to assess the half-life of the class II molecule. Class
I and class II molecules were then serially immunoprecipitated
from postnuclear cell lysates. Coexpression of DM had no appreciable effect on the apparent half-life of the I-Ak␤82H⫺ class II
molecules (Fig. 6), thus supporting the conclusion that the ability
of the hydrogen bond-deficient class II molecule to stably acquire
peptide is severely compromised.
Discussion
CLIP, occupies the peptide binding groove. In endosomes, CLIP is
removed from the class II molecule and is replaced with a peptide
from an endogenous or internalized protein. The class II molecule
presents these peptides at the cell surface, and may exchange
bound peptides later following internalization from the cell surface. We speculate that features in the class II molecule responsible for stable peptide binding may respond to changes in pH found
in endosomes, allowing exchange of associated peptides and may
rely most extensively on cooperative binding interactions that allow stable yet reversible peptide binding.
Similar to class II peptide association, the formation of class I
peptide complexes is mediated both by pocket interactions and the
formation of hydrogen bonds between the class I molecule and the
main chain of the bound peptide. However, unlike class II peptide
complexes in which hydrogen bonds form throughout the length of
the bound peptide, the hydrogen bonds formed in class I peptide
complexes are clustered near the amino and carboxyl termini of the
bound peptide. In fact, the amino and carboxyl groups at the first
and last peptide residues participate in extensive hydrogen bond
networks, which likely contributes substantially to the stability of
the class I peptide complex (23) and to the closing of the peptide
binding pocket at the periphery. Substitution of the terminal amino
or carboxyl peptide groups with methyl groups (24), or rearrangement of the MHC molecule to facilitate binding of a 10-mer peptide (25), resulting in the loss of one or more hydrogen bonds
between the class I molecule and the bound peptide, results in
destabilization of the class I peptide complex. Thus, although both
class I and class II molecules have important hydrogen bonding
interactions with peptides, the precise role of the hydrogen bonds
is likely distinct in these two molecules.
Our data indicates that the loss of a single hydrogen bond between a class II molecule and the bound peptide results in inefficient peptide binding and thus susceptibility to endosomal degradation. DM has previously been shown to enhance the loading of
peptide onto class II molecules, in addition to removing CLIP from
the class II peptide binding site (5). Therefore, DM may additionally function to protect class II molecules from premature degradation in endosomes by maintaining class II in a continuously
peptide loaded state. Based on this theory, we would expect the
coexpression of DM with the hydrogen bond-deficient class II
molecules trafficked to endosomes to enhance peptide loading, resulting in protection from endosomal degradation. However, the
coexpression of DM is not sufficient to promote peptide binding
within the APC, suggesting that the hydrogen bond loss variants
have severely compromised ability to stably acquire peptides. Although the model cell system used is not a professional APC,
previous data have shown that this cell type is capable of presenting most, if not all, Ags used (reviewed in Ref 5). Therefore, it
seems unlikely that the repertoire of peptides generated in L cells
is more limited in abundance or diversity than would be found in
B cells or freshly isolated APCs, and therefore, that the type of
peptide that can bind to the hydrogen deficient class II molecules
is very rare. This in turn suggests that the integrity of the hydrogen
bond network is essential for stable association of most peptides
with class II molecules.
An alternative or additional explanation for the inability of DM
coexpression to result in promotion of peptide binding is that DM
may be unable to associate with the hydrogen bond-deficient class
II molecules. Random mutagenesis of HLA-DR molecules has
identified a region of class II in close proximity to the amino terminus of the bound peptide that is required for productive interaction with DM (26). The class II molecules used in our studies
lack one or more hydrogen bonds near the amino terminus of the
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MHC class II molecules bind peptides through two types of interactions, the insertion of peptide side chains into pockets in the
class II binding groove, and the formation of hydrogen bonds between conserved class II residues and the main chain of the bound
peptide. Although the current paradigm indicates that pocket interactions are responsible both for peptide specificity and the formation of stable class II peptide complexes, recent work and this
paper suggest that the hydrogen bond network formed between
class II and bound peptides is crucial for efficient peptide binding
(8, 12, 13, 22). In this study, we demonstrate that a type of class
II molecule that generally associates with bound peptides through
strong peptide side chain-class II pocket interactions also requires
the formation of the hydrogen bond network for efficient peptide
binding. In the absence of complete hydrogen bond network formation, these class II molecules are susceptible to degradation in
endosomal/lysosomal compartments. The addition of a high-affinity peptide results in peptide binding, despite the disruption of the
hydrogen bond network, resulting in stabilization of the class IIpeptide complex and resistance to internal degradation. However,
coexpression of DM does not result in decreased class II proteolysis, indicating that the loss of one or more hydrogen bonds results
in class II molecules that are profoundly compromised in their
ability to stably acquire peptides.
MHC class II molecules undergo peptide exchange during their
lifetime, for instance, associating with CLIP throughout the secretory pathway, which is released in endosomal compartments and
exchanged for a peptide derived from either self- or foreign-internalized protein. Following expression at the cell surface and internalization, class II may exchange a previously bound peptide for
a new one and return to the cell surface to display the new peptide
for T cell surveillance. For peptide exchange to occur, the interactions resulting in the formation of stable class II-peptide complexes must be reversible. Thus, it seems likely that class II molecules have evolved to rely less on static pocket interactions and
more on a cooperative interaction between pocket association and
hydrogen bond formation in which these interactions are dependent on the integrity of different but nearby bonds. This cooperative interaction of nearby hydrogen bonds thus allows these bonds
to be formed and dissolved relatively easily, allowing reversible
peptide binding by class II molecules.
Although both class I and class II molecules bind and present
peptides at the cell surface, their intracellular trafficking differs.
Class I molecules associate with peptide in the ER shortly after
synthesis, and peptide association is required for egress from the
ER. In general, class I molecules are not typically thought to exchange bound peptides during their lifetime in APCs. Class II molecules do not bind antigenic peptide until late in their intracellular
trafficking pathway. To prevent polypeptide binding in the ER,
class II molecules associate with invariant chain, a part of which,
HYDROGEN BOND-DEFICIENT CLASS II MOLECULES
The Journal of Immunology
T cells in the thymus and the presence of autoreactive T cells in the
periphery (35–38).
Acknowledgments
We thank Dr. Craig Beeson for contributing Fig. 1 and Chris Lazarski for
critical reading of the manuscript.
References
1. Bakke, O., and B. Dobberstein. 1990. MHC class II-associated invariant chain
contains a sorting signal for endosomal compartments. Cell 63:707.
2. Lotteau, V., L. Teyton, A. Peleraux, T. Nilsson, L. Karlsson, S. Schmid,
V. Quaranta, and P. Peterson. 1990. Intracellular transport of class II MHC molecules directed by invariant chain. Nature 348:600.
3. Ceman, S., and A. J. Sant. 1995. The function of invariant chain in class IIrestricted antigen presentation. Semin. Immunol. 7:373.
4. Cresswell, P. 1996. Invariant chain structure and MHC class II function. Cell
84:505.
5. Denzin, L. K., and P. Cresswell. 1995. HLA-DM induces CLIP dissociation from
MHC class II ␣␤ dimers and facilitates peptide loading. Cell 82:155.
6. Morris, P., J. Shaman, M. Attaya, M. Amaya, S. Goodman, C. Bergman, M. J.J.,
and E. Mellins. 1994. An essential role for HLA-DM in antigen presentation by
class II major histocompatibility molecules. Nature 368:551.
7. Germain, R. N., and A. G. J. Rinker. 1993. Peptide binding inhibits protein
aggregation of invariant-chain free class II dimers and promotes surface expression of occupied molecules. Nature 363:725.
8. Ceman, S., S. H. Wu, T. S. Jardetzky, and A. J. Sant. 1998. Alteration of a single
hydrogen bond between class II molecules and peptide results in rapid degradation of class II molecules after invariant chain removal. J. Exp. Med. 188:2139.
9. Stern, L., J. Brown, T. S. Jardetzky, J. Gorga, R. Urban, J. Strominger, and
D. Wiley. 1994. Crystal structure of the human class II MHC protein HLA-DR1
complexed with an influenza virus peptide. Nature 368:215.
10. Fremont, D. H., D. Monnaie, C. A. Nelson, W. A. Hendrickson, and
E. R. Unanue. 1998. Crystal structure of I-Ak in complex with a dominant epitope
of lysozyme. Immunity 8:305.
11. Scott, C. A., P. A. Peterson, L. Teyton, and I. A. Wilson. 1998. Crystal structures
of two I-Ad-peptide complexes reveal that high affinity can be achieved without
large anchor residues. Immunity 8:319.
12. McFarland, B. J., C. Beeson, and A. J. Sant. 1999. A single, essential hydrogen
bond controls the stability of peptide-MHC class II complexes. J. Immunol. 163:
3567.
13. Bryant, P. W., P. Roos, H. L. Ploegh, and A. J. Sant. 1999. Deviant trafficking of
I-Ad mutant molecules is reflected in their peptide binding properties. Eur. J. Immunol. 29:2729.
14. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989.
Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51.
15. Arneson, L. S., M. Peterson, and A. J. Sant. 2000. The MHC class II molecule
I-Ag7 exists in alternate conformations that are peptide dependent. J. Immunol.
165:2059.
16. Braunstein, N.S. Germain, R. N., Loney, K., and N. Berkowitz. 1990. Structurally
interdependent and independent regions of allelic polymorphism in MHC class II
molecules: implications for Ia function and evolution. J. Immunol. 145:1635.
17. Ozato, K., N. Mayer, and D. Sachs. 1980. Hybridoma cell lines secreting monoclonal antibodies to mouse H-2 and Ia antigens. J. Immunol. 124:533.
18. Zhong, G., C. Reis e Sousa, and R. N. Germain. 1997. Production, specificity, and
functionality of monoclonal antibodies to specific peptide-major histocompatibility complex class II complexes formed by processing of exogenous protein.
Proc. Natl. Acad. Sci. USA 94:13856.
19. Natarajan, S. K., L. J. Stern, and S. Sadegh-Nasseri. 1999. Sodium dodecyl sulfate stability of HLA-DR1 complexes correlates with burial of hydrophobic residues in pocket 1. J. Immunol. 162:3463.
20. Germain, R. N., and L. R. Hendrix. 1991. MHC class II structure, occupancy and
surface expression determined by post-endoplasmic reticulum antigen binding.
Nature 353:134.
21. Nelson, C. A., S. J. Petzold, and E. R. Unanue. 1994. Peptides determine the life
span of MHC class II molecules in the antigen-presenting cell. Nature 371:250.
22. Sant, A. J., C. Beeson, B. J. McFarland, J. Cao, S. Ceman, P. W. Bryant, and
S. H. Wu. 1999. Individual hydrogen bonds play a critical role in MHC class
II:peptide interactions: implications for the dynamic aspects of class II trafficking
and DM-mediated exchange. Immunol. Rev. 172:239.
23. Jardetzky, T. S. 1996. Crystal structures of MHC class I and class II molecules.
In HLA and MHC Genes, Molecules, and Function. M. Browning and A. McMichael, eds. BIOS Scientific, Oxford, UK., p. 249.
24. Bouvier, M., and D. Wiley. 1994. Importance of peptide amino and carboxyl
termini to the stability of MHC class I molecules. Science 265:398.
25. Collins, E. J., D. N. Garboczi, and D. C. Wiley. 1994. Three-dimensional structure of a peptide extending from one end of a class I MHC binding site. Nature
371:626.
26. Doebele, C. R., R. Busch, M. H. Scott, A. Pashine, and E. D. Mellins. 2000.
Determination of the HLA-DM interaction site on HLA-DR molecules. Immunity
13:517.
27. Guerra, C. B., R. Busch, R. C. Doebele, W. Liu, T. Sawada, W. W. Kwok,
M. D. Chang, and E. D. Mellins. 1998. Novel glycosylation of HLA-DR␣ disrupts antigen presentation without altering endosomal localization. J. Immunol.
160:4289.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
bound peptide, and therefore, any conformational changes resulting from the loss of these hydrogen bonds may affect the efficiency
or consequences of DM interaction. We do not yet have any biochemical evidence that the hydrogen bond-deficient variant can
interact productively with DM. Finally, it is conceivable that the
levels of DM expressed within the transfectants are insufficient for
promoting peptide binding.
Loading of the hydrogen bond-deficient class II molecules with
either endogenous or exogenous HEL peptide protects the class II
molecule from proteolysis. This change in susceptibility to proteolysis may result from a conformational change in the class II
molecule induced by peptide binding. Previous studies have shown
that class II molecules undergo a conformational change when
they associate with peptide (28, 29). The formation of a compact
class II dimer associated with a peptide that is resistant to SDS
denaturation is also consistent with a peptide-induced conformational change in the class II molecule that results in inability of
other molecules, either proteases or SDS, to act on the complex
(30, 31). Finally, this data suggests that the initial proteolytic sites
are sequestered by this conformational change, and then become
accessible in the absence of peptide binding.
The finding that the hydrogen bond-deficient class II molecules
are degraded is consistent with the view that full integrity of the
hydrogen bond network is required for stable peptide binding, an
event that allows escape from the proteolytic environment of endosomal compartments. This view is supported by our own in vitro
peptide binding studies, which have shown that the loss of a single
hydrogen bond between class II molecules and associated peptide
results in accelerated dissociation of peptides (12). Hydrogen
bonds are formed between the class II molecule and the main chain
of the bound peptide along its full length, with sets of symmetrical
hydrogen bonds formed at opposite ends of the peptide
(␤His81Asn82 vs ␣His68Asn69). Recent work has demonstrated
that in I-Ad class II molecules, these different sets of hydrogen
bonds contribute disproportionately to peptide-MHC stability. The
loss of hydrogen bonds near the amino terminus of the bound
peptide, as previously demonstrated, resulted in substantial acceleration in peptide dissociation, whereas the loss of hydrogen bonds
near the peptide’s carboxyl terminus were shown to have significantly less effect, indicating that they contribute less to complex
stability (32). We have not yet explored the relative importance of
individual hydrogen bonds in human class II molecules, although
the hydrogen bonding network was first noted in these crystal
structures (9, 33). Based on the high degree of genetic conservation in the residues that contribute hydrogen bonds to the bound
peptide, we expect that most class II peptide complexes will rely
on these interactions for complex stability.
It is interesting to note that although the positions in the class II
molecule that form hydrogen bonds with the bound peptide are
highly conserved, natural polymorphisms at these positions do
rarely occur. Interestingly, class II alleles that do contain natural
polymorphisms at these positions show susceptibility to autoimmune disease. For instance, in I-Au, ␤81His is changed to a tyrosine, an alteration that would either alter the hydrogen bond or
result in a substantial shift in the peptide backbone around the P1
(34). I-As also contains polymorphisms at positions ␣68 and ␣69,
both of which are implicated in the hydrogen bond network formed
in I-Ad and I-Ak (12, 13). Despite the apparent lesser importance
of these hydrogen bonds on the stability of the MHC-peptide complex (32), the loss of these hydrogen bonds at the carboxyl end of
the bound peptide does accelerate peptide dissociation, and therefore could affect the stabilization of peptide binding. Thus, these
class II molecules may generally bind peptides with decreased affinity, which in turn could result in decreased negative selection of
6945
6946
28. Chervonsky, A. V., R. M. Medzhitov, L. K. Denzin, A. K. Barlow,
A. Y. Rudensky, and C. A. J. Janeway. 1998. Subtle conformational changes in
major histocompatibility complex class II molecules by binding peptides. Proc.
Natl. Acad. Sci. USA 95:10094.
29. Rohren, E. M., D. J. McCormick, and L. R. Pease. 1994. Peptide-induced conformational changes in class I molecules: direct detection by flow cytometry.
J. Immunol. 152:5337.
30. Sadegh-Nasseri, S., and R. N. Germain. 1991. A role for peptide in determining
MHC class II structure. Nature 353:167.
31. Sadegh-Nasseri, S., L. J. Stern, D. C. Wiley, and R. N. Germain. 1994. MHC
class II function preserved by low-affinity peptide interactions preceding stable
binding. Nature 370:647.
32. McFarland, B. J., J. F. Katz, C. Beeson, and, and A. J. Sant. 2001. Energetic
asymmetry among hydrogen bonds in MHC class II:peptide interactions. Proc.
Natl. Acad. Sci. USA 98:9231.
33. Jardetzky, T. S., J. H. Brown, J. C. Gorga, L. J. Stern, R. G. Urban,
J. L. Strominger, and D. C. Wiley. 1996. Crystallographic analysis of endogenous
HYDROGEN BOND-DEFICIENT CLASS II MOLECULES
34.
35.
36.
37.
38.
peptides associated with HLA-DR1 suggests a common, polyproline II-like conformation for bound peptides. Proc. Natl. Acad. Sci. USA 93:734.
Lee, C., and H. M. McConnell. 1995. A general model of invariant chain association with class II major histocompatibility complex proteins. Proc. Natl. Acad.
Sci. USA 92:8269.
Cibotti, R., J. M. Kanellopoulos, J. P. Cabaniols, O. Halle-Panenko,
K. Kosmatopoulos, E. Sercarz, and P. Kourilsky. 1992. Tolerance to a self-protein involves its immunodominant but does not involve its subdominant determinants. Proc. Natl. Acad. Sci. USA 89:416.
Harrington, C. J., A. Paez, T. Hunkapiller, V. Mannikko, T. Brabb, M. Ahearn,
C. Beeson, and J. Goverman. 1998. Differential tolerance is induced in T cells
recognizing distinct epitopes of myelin basic protein. Immunity 8:571.
Liu, G. Y., and D. C. Wraith. 1995. Affinity for class II MHC determines the
extent to which soluble peptides tolerize autoreactive T cells in naive and primed
adult mice—implications for autoimmunity. Int. Immunol. 5:1159.
Liu, G. Y., P. J. Fairchild, R. M. Smith, J. R. Prowle, D. Kioussis, and
D. C. Wraith. 1995. Low avidity recognition of self-antigen by T cells permits
escape from central tolerance. Immunity 3:407.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017