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2 Domain Loop Influences Interaction with the Assembly Complex1
*
Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110;
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198; and
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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2
domain on interaction with TAP, tapasin, and calreticulin. In this
report, we present evidence indicating that many residues surrounding
position 134 in H-2Ld influence interaction with assembly
complex components. Most mutations decreased association, but one
(LdK131D) strongly increased it. The Ld
mutants, with the exception of LdK131D, exhibited
characteristics suggesting suboptimal intracellular peptide loading,
similar to the phenotype of Ld expressed in a
tapasin-deficient cell line. Notably, K131D was less peptide inducible
than wild-type Ld, which is consistent with its unusually
strong association with the endoplasmic reticulum assembly
complex. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Although the selective roles of the members of the ER peptide-loading complex are unknown, these proteins facilitate assembly and surface expression of functional class I. In addition to class I, calreticulin is known to chaperone other glycoproteins in the ER (4). It has been suggested that tapasin and/or TAP may also serve class I as chaperones (5), and recent evidence supports this theory. Tapasin facilitates MHC class I surface expression, even apart from its interactions with TAP (6). Furthermore, it has been shown that TAP is either an active chaperone or a necessary accessory to a molecule that chaperones class I (7).
Recent evidence suggests that ER chaperones may interact differently with various allelic forms of MHC class I H-chain. In one study, TAP association has been reported to vary among HLA alleles. Specifically, several HLA alleles were evaluated for TAP interaction, and the majority of HLA-B alleles examined were found to associate very inefficiently with TAP (8). This observation might be due to competition among class I H-chains for TAP (9), or to differential association of class I H-chains with tapasin, which is required for class I/TAP association (2). Consistent with this latter explanation, allelic variation in tapasin dependence for assembly has been shown for class I molecules. Cotransfection of tapasin, along with B*2705, B8, or B*4402, into the tapasin-negative 721.220 cell line did not boost B*2705 surface expression; however, it did increase B8 expression by 5-fold and B*4402 expression by 25-fold (10). At this time, species or allelic variations in class I/calreticulin association have not yet been reported.
Three spatially distinct sites on the MHC class I H-chain have been
shown biochemically to influence interaction with both TAP and
calreticulin. These sites are indicated on the Ld
three-dimensional structure (11) in Fig. 1
. The sites are the N-linked
carbohydrates at position 86 in the 1 domain (3),
position 134 in the 2 domain (12, 13, 14), and an acidic
residue at position 227 in the 3 domain (3). (We have
also found that an Ld mutant with changes at both
positions 227 and 229 loses TAP association (data not shown)). Of these
three sites, position 134 would seem the most likely to be highly
influenced by class I/peptide binding. Because calreticulin has been
shown to bind glycosyl groups on other proteins (4), it is
likely that calreticulin binds the carbohydrate at position 86 on class
I. In a recent study, data were presented that suggested TAP binding
was influenced, directly or indirectly, by residues in the 3 domain.
In this case, amino acids 219–233 of the H-2Db
3 domain were exchanged for the same number of amino acids from the
MHC class II chain. This mutant was not expressed at the cell surface,
and its interaction with TAP was sharply reduced (15).
Also, a recent publication suggested that a mutation at position 222 in
the 3 domain could prevent association with tapasin and TAP
(16). However, any influence of this amino acid
substitution on calreticulin association cannot be clearly ascertained
from this report. In total, several residues within a certain area of
the 3 domain have been implicated in assembly complex interaction;
however, at this time only one 2 domain residue (position 134) has
been identified as important to such associations.
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To improve our understanding of the protein interactions vital to MHC
assembly, we have examined the effect of multiple, individual point
mutations in the MHC class I 2 domain on association with TAP,
tapasin, and calreticulin. To be able to assess the relationship
between class I H-chain folding status and the function of the assembly
complex, we have used as our experimental model
H2-Ld, for which Abs specific for both open and
folded conformations are available. Our results show that not only
position 134 but also a large number of amino acid residues around it
are involved in interactions between the assembly complex and the open
form of Ld. Within the 128–136 sequence,
individual mutations at all but one position caused decreased
association. Surprisingly, a Lys to Asp substitution at position 131
greatly strengthened the interaction. Furthermore, the lack of assembly
complex binding was correlated with functional consequences, as
exhibited by altered accessibility to exogenous peptides.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The 721.220 cell line is a human B lymphoblastoid cell line that does not express tapasin (2). The 721.221 cell line is a closely related cell line that does express tapasin (17, 18). L-Ld cells were made by introduction of the Ld gene into Ltk- DAP-3 (H-2k) fibroblast cells (19). All Ld mutants are named as follows: original amino acid residue, position, and substituted amino acid residue. The construction of the LdN86Q and LdD227K mutant cDNAs and their transfection into DAP-3 has been described previously (3, 20). For the generation of Ld mutants with amino acid substitutions in the 128–137 loop, the following approach was taken to make nonconservative mutations in such a way as to require the minimal DNA sequence changes. First, two unique blunt-end restriction sites, SmaI and EcoRV, were sequentially introduced into an Ld cDNA (21) in the vector RSV.5neo (22) using the Quik Change Mutagenesis Kit from Stratagene Cloning Systems (La Jolla, CA). The synthetic oligodeoxynucleotides that were used for mutagenesis to insert the SmaI site were as follows: 5'-GGCTGCGATTACATCGCCCGGGACCTGAAAACGTGG-3' and 5'-CCACGTTTTCAGGTCTTCGTCCCGGATGTAATCGCAGCC-3'. For the mutagenesis required for insertion of the EcoRV restriction endonuclease site, the synthetic oligonucleotides used were 5'-GCGGCGGACATGGCGGCGGATATCACCCGACGCAAGTGG-3' and 5'-CCACTTGCGTCGGGTGATATCCGCCGCCATGTCCGCCGC-3'. These two mutations would result in the following codon changes. For the SmaI mutation, residues 126 and 127 would be changed from Leu and Asn to Arg and Asp (GCC CTG AAC to GCC CGG GAC). For the EcoRV mutation, residue 141 would be changed from Gln to Asp (CAG ATC to GAT ATC). The unique SacII/Sse 8387I fragment encompassing these two mutations was sequenced completely and swapped with the corresponding fragment from RSV.5neo-Ld. The resultant mutant Ld construct was then digested with SmaI and EcoRV sequentially and ligated with pairs of annealed and phosphorylated oligodeoxynucleotides that would include each individual mutation on the loop while reverting the changes at residues 126, 127, and 141. The recombinants were first screened by restriction mapping and then confirmed by DNA sequencing for the correct orientation. For example, the pairs of oligodeoxynucleotides used to create the LdE128R mutation were 5'-TGAACAGAGACCTGAAAACGTGGACGGCGGCGGACATGGCGGCGCAG-3' and 5'-CTGCGCCGCCATGTCCGCCGCCGTCCACGTTTTCAGGTCTCTGTTCA-3'. In the correct orientation, the nucleotide sequence corresponding to codons 126–142 would be CTG AAC AGA ... . . CAG ATC.
Cells were maintained at 37°C, 5% CO2 in RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 10% bovine calf serum (HyClone Laboratories, Logan, UT), glutamine, pyruvate, and penicillin/streptomycin, or were cultured at 37°C, 7% CO2 in DMEM (Life Technologies) containing 10% FCS (HyClone Laboratories), glutamine, pyruvate, and penicillin/streptomycin.
Ab, immunoprecipitations, and Western blotting
The mAb 64-3-7 is an IgG2 Ab specific for the 1 domain of
open Ld (19, 23, 24, 25); mAb 30-5-7 is
an IgG2 Ab that binds the 2 domain of folded
Ld (24, 25, 26).
Ld forms with the 64-3-7 epitope are not
recognized by several conformation-sensitive mAbs (e.g., 30-5-7 and
B22/249), which have epitopes that surround the peptide-binding groove.
This serologic pattern suggests that the Ld
64-3-7+ form has an open and unfolded cleft
(23). Peptide binding to 64-3-7+
Ld causes a conversion to the folded
(30-5-7+) form, as demonstrated by titration of
radioiodinated peptide ligand into cell lysates and immunoprecipitation
of the Ld molecules. With increasing
concentrations of peptide ligand, the amount of
64-3-7+ Ld decreased and
the amount of 30-5-7+ Ld increased
(25). Furthermore, the labeled peptide was coprecipitated
in a dose-dependent fashion with the 30-5-7+
Ld form and could be simultaneously visualized on
the autoradiograph (25). In vivo, a precursor-product
relationship exists between 64-3-7+
Ld and 30-5-7+
Ld, as indicated by pulse-chase analysis. As the
chase time increases, 30-5-7+
Ld levels were shown to rise and
64-3-7+ Ld levels to drop
(25).
The anti-calnexin serum specific for a C-terminal segment from mouse and human calnexin (27) was donated by Dr. David McKean (Mayo Clinic, Rochester, MN). Anti-calreticulin serum (28) was purchased from Stressgen (Victoria, British Columbia, Canada). The murine anti-TAP-1 serum was generated against a TAP-1 peptide coupled to keyhole limpet hemocyanin (20). The rabbit anti-mouse tapasin serum was also made against a synthetic peptide (GPAIECWFVEDAGGGGLSKC) coupled to keyhole limpet hemocyanin.
For radiolabeling, cells were preincubated for 30 min at 37°C in culture media without methionine. Next, [35S]methionine (100 µCi/ml) was added, and the cells were radiolabeled for 30 min. The cells were then washed three times in PBS containing iodoacetamide (Sigma, St. Louis, MO) and lysed in 1% 3-([cholamidopropyl]dimethylammonio)-1-propanesulfonate (CHAPS) (Boehringer-Mannheim, Indianapolis, IN) in Tris-buffered saline (pH 7.4) with freshly added 0.2 mM PMSF (Sigma) and 20 mM iodoacetamide. The lysis buffer was supplemented with a saturating volume of mAb before its addition to pelleted cells. After incubation for 1 h on ice, nuclei were removed by centrifugation, and lysates were incubated with protein A-Sepharose beads (Pharmacia, Piscataway, NJ). The beads were washed four times with 0.1% CHAPS in Tris-buffered saline (pH 7.4), and the samples were eluted by boiling in 0.125 M Tris (pH 6.8)/2% SDS/12% glycerol/2% bromophenol blue. All immunoprecipitates were electrophoresed on 4–20% or 8% acrylamide/Tris-glycine or 7% acrylamide/NuPage gels (Novex, San Diego, CA). Gels were soaked in Amplify (Amersham, Boston, MA) plus 2% glycerol, dried, and exposed to BioMax MR film (Eastman Kodak, Rochester, NY) at -70°C for varied lengths of time.
For Western blotting, immunoprecipitates electrophoresed by SDS-PAGE as described above were transferred to Immobilon P membranes (Millipore, Bedford, MA). After overnight blocking, membranes were incubated in a dilution of Ab for 2 h, washed three times with PBS/0.5% Tween 20, and incubated for 1 h with biotin-conjugated goat anti-mouse or anti-rabbit IgG (Caltag Laboratories, San Francisco, CA). Following three washes with PBS/0.05% Tween 20, membranes were incubated for 1 h with streptavidin-conjugated HRP (Zymed, San Francisco, CA), washed three times with PBS/0.3% Tween 20, and incubated with enhanced chemiluminescence Western blot developing reagents (Amersham). Membranes were exposed to BioMax MR or ML film for varied lengths of time.
Flow cytometry
To analyze peptide-pulsed cells by FACS, the cells were incubated overnight at a density of 1 x 106 cells/ml with defined concentrations of peptide in DMEM supplemented with glutamine, penicillin, streptomycin, and 10% bovine calf serum. After incubation, the cells were centrifuged and resuspended at 5 x 106/ml in PBS with 0.2% BSA and 0.1% sodium azide. The cell suspension, in 0.1-ml aliquots, was distributed to wells of a 96-well plate. The cells were incubated with saturating concentrations of Ab or PBS/BSA/azide alone for 30 min at 4°C, washed twice, and incubated with a fluorescein-conjugated, Fc-specific F(ab')2 fragment of goat anti-mouse IgG for 30 min at 4°C. The cells were then washed twice, resuspended, and analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Statistical analyses were performed with Cell Quest software.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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2 domain and
the assembly complex. Mutations over a considerable region surrounding
position 134, as well as mutations at positions 86 and 227, were
discovered to alter the interaction between Ld
and TAP/tapasin/calreticulin. These individual amino acid changes were
found to have consequences for class I/assembly complex interaction as
measured by both biochemical and functional assays. Individual substitutions between position 128 and 136 of Ld disrupt TAP and calreticulin interaction
It has been reported that a Lys for Thr substitution in HLA-A2 at
position 134 (T134K) abrogates association with TAP and calreticulin
(12, 13, 14). For the purpose of examining 1) whether this
position influences association of a different MHC class I molecule,
H-2Ld, with the assembly complex, 2) whether
residues flanking 134 also affect interaction, and 3) what the
consequences of any loss of interaction would be, a series of
Ld mutants with individual amino acid changes
between position 128 and position 136 were made (see footnote
4 and Fig. 2).
The choice of Ld permits the use of mAbs specific
for precursor (open) and product (folded) class I assembly states. As
shown in Fig. 3, these
Ld mutants were all expressed at similar levels
within the cell. When interactions of the open form of
Ld and Ld mutants with TAP
and calreticulin were assessed, it was found that (unlike wild-type
(wt) Ld) none of these mutants associated with
either TAP or calreticulin (Fig. 3). Thus H-2Ld,
like HLA-A2, requires the wt residue at position 134 for interaction
with TAP and calreticulin. This indicates that position 134 is
important for these interactions in murine as well as human MHC class I
molecules. Furthermore, the whole length of the 128–136 region around
this position contributes to these interactions in
Ld. These results demarcate a sizable area of the
class I 2 domain, rather than a single MHC class I amino acid
residue, as interacting with the assembly complex. The size of the
region on the 2 domain that is involved suggests that a component of
the assembly complex shares a large area of its surface with class I.
Also, the structural nature of some of the class I amino acids involved
suggests potential biochemical interactions. For example, because an E
to R mutation at position 128 prevents association, it can be
postulated that there may be binding of a basic residue in the assembly
complex to this glutamic acid on class I.
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Alterations in three widely separated areas (1, 2, and 3
domains) of the MHC class I molecule have been shown to abrogate
association with both TAP and calreticulin (Refs. 3, 14, 20 and this report). Furthermore, a mutation in the 3 domain
has been suggested to abrogate the binding of tapasin
(16). In Fig. 4, mutants
with substitutions in each of those three areas are directly compared
with wt Ld and to an additional mutant,
LdD137H. The LdD137H mutant
has an amino acid substitution just carboxy-terminal to the area
(positions 128–136) that is shown in Fig. 3
to affect TAP and
calreticulin association. As can be seen in Fig. 4, TAP and
calreticulin interaction is maintained by
LdD137H, defining the C-terminal boundary of the
chaperone interaction site as position 136. Mutants
LdW133T and LdT134K, like
LdN86Q and LdD227K, were
found to lack TAP, tapasin, and calreticulin association, although
interaction with calnexin was not impaired (Figs. 4 and 5). Thus, mutations at sites in each of
the three domains of Ld disrupt interaction with
tapasin, as well as TAP and calreticulin. These results are consistent
with a model in which calreticulin and tapasin exhibit cooperativity in
their binding. Possibly the 2 and 3 sites (which lie in the same
plane) both interact with tapasin, and the 1 domain oligosaccharide
interacts with calreticulin.
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Surprisingly, a substitution at position 131, unlike the other
individual site-directed mutations in this loop,4
was found to strengthen association with TAP, calreticulin, and
tapasin. Fig. 5 compares the LdK131D mutant with
wt Ld, E128R, W133T, and T134K. The E128R, W133T,
and T134K mutants fail to bind to TAP, calreticulin, and tapasin. In
contrast, the K131D mutant binds much more strongly to these proteins
than even wt Ld. This result was verified with
the use of a separate K131D transfectant that, despite having low
intracellular expression, still exhibited very strong interaction with
the assembly complex (data not shown). Therefore, introduction of an
acidic amino acid residue at this position facilitates interaction with
assembly complex components, suggesting a basic residue in the assembly
complex may be juxtaposed to this class I position.
The phenotype of LdK131D may have some similarities to a reported HLA-A2 mutant, S132C (13). This A2 mutant presents endogenous peptides well, but not exogenous peptides. Furthermore, S132C has an extended period of association with TAP. Notably, in all murine MHC class I molecules except one, Lys is found at position 131 (29). The sole exception is Q1k, which has Glu at that position. Because our substitution of an Asp for the natural Lys in Ld greatly strengthened assembly complex association, it could be hypothesized that Q1k has unusually strong association with these ER proteins.
ß2m association with the open form of MHC class I correlates with chaperone association
In their folded forms, all of the mutant Ld
H-chains (listed in footnote 4) were found to be associated with
ß2m (data not shown). As demonstrated in Fig. 6, ß2m can be
detected by autoradiography coprecipitated with the folded form of wt
Ld, E128R, K131D, and T134K. Very little W133T is
present in the folded form (Fig. 6), and detection of
ß2m with the 30-5-7+ form
of W133T required a long exposure of a Western blot probed with
anti-mouse ß2m serum (data not shown). The
ability of the mutants other than W133T to adopt the folded form
verifies that the mutations do not cause gross conformational changes.
Also, association of ß2m with the folded form
of the Ld mutants indicates that the lack of
interaction with the assembly complex is not secondary to the complete
inability of these Ld mutants to bind
ß2m. ß2m interacts
weakly with the open form of mutants E128R, D129R, L130D, T132K, W133T,
T134K, A135H, and A136H compared with wt Ld (Fig. 6 and data not shown). The observation that ß2m
is poorly associated with the open form of these 2 domain mutants
suggests that stable assembly of H-chains with
ß2m before peptide binding requires association
with the assembly complex.
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2 domain, and lack of
ß2m association with the form precipitated by
an anti-3 domain Ab. Furthermore, dm6 cells were not lysed by
Dd-specific alloreactive T cells
(31), which is consistent with the original detection of
Ddm6 as a loss mutation (30).
Similarly, the LdW133T mutant characterized here
was found to have very low surface expression after transfection into L
cells. Indeed, of all the mutants characterized in this study,
LdW133T was the most impaired in its surface
expression (data not shown). Furthermore, a significant pool of
intracellular LdW133T molecules were detected in
our L cell transfectants, but very few mutant
LdW133T molecules were detected in a folded
(30-5-7+) conformation (Fig. 6). In addition,
surface LdW133T molecules were found not to be
inducible with exogenous peptide (data not shown), unlike all other
mutants in this study, which were specifically inducible with
Ld peptide ligands. These cumulative findings
clearly indicate that substitution of the Trp at position 133 ablates
H-chain interaction with peptide. It should also be noted that the W133
amino acid residue is conserved among all MHC class I H-chains
(including the class Ib proteins), and that its side chain points
inward (i.e., it lacks solvent accessibility) in the
Ld three-dimensional structure (Ref.
11 and Fig. 2). Based on the more dramatic phenotype of
the 133 mutation compared with other mutations in this region and based
on the inward projection of the W133 side chain, we propose that the
W133 residue may be critically involved in the overall folding of the
class I H-chain. If this is the case, the severe phenotype of the
substitutions at 133 extends beyond a lack of interaction with the
assembly complex.
Quite interestingly, there is strong positive correlation between
ß2m association and assembly complex
association for the open (64-3-7+) forms of the
mutants (Figs. 3, 5, and 6). The open forms of mutants
LdE128R, LdW133T, and
LdT134K (all of which do not interact with the
assembly complex) have extremely little coprecipitated
ß2m. In contrast, the amount of
ß2m coprecipitated with
LdK131D (which strongly binds the assembly
complex) exceeds the amount coprecipitated with
Ld. Thus, assembly complex binding and
ß2m binding to class I H-chain are
coordinated.
Lack of interaction with the assembly complex does not completely
prevent adoption of the folded class I conformation in the ER (Fig. 6).
This is consistent with the previous observation that folded, although
not heat-stable, class I H-chain/ß2m
heterodimers are present in lysates of RMA-S cells (25).
These folded forms are presumably occupied by low-affinity peptides.
Despite the presence of some folded forms in the ER, the lack of
association between these 2 domain mutants and the assembly complex
has perceptible functional consequences, because it causes the class I
molecule to become loaded with suboptimal peptides that are unusually
susceptible to replacement (see below).
Cell surface Ld 2 domain loop mutants are more
peptide accessible than wt Ld
The degree of cell surface stabilization by exogenous peptide is
inversely correlated with the efficiency of intracellular loading. We
postulated that inability to associate with the assembly complex might
lead to production of class I molecules that have suboptimal peptides
or that are functionally empty. To test the relevance of our
observation that positions other than 134 in the 2 domain prevent
assembly complex binding, we have examined the ability of exogenous
peptide to induce folding of some of the 2 domain
Ld mutants. In our comparison, we have also
included mutants with substitutions in the other two areas that affect
assembly complex binding (i.e., position 86 and amino acid 227). As
shown in Fig. 7, Ld
mutants unable to bind assembly complex proteins are more peptide
inducible than wt Ld. After culture with either
of two peptides, LdT134K surface expression rose
by >7-fold, compared with <5-fold for Ld (Fig. 7A and data not shown). Similarly, surface expression of wt
Ld was induced only 
4-fold by murine CMV
(MCMV) peptide, but surface expression of the
LdE128R, LdN86Q, and
LdD227K mutants was induced 5- to 6-fold (Fig. 7B and data not shown). Therefore, Ld
mutants unable to bind tapasin and other assembly complex components
obtain relatively low-affinity peptides in the ER. This suggests that
the absence of direct binding to the complex denies the MHC class I
H-chain access to the optimal selection of peptides. In line with our
observation that LdK131D has unusually strong
binding to TAP/calreticulin/tapasin, this mutant exhibited low peptide
inducibility (only 2.8-fold, Fig. 7B).
LdK131D likely binds peptides with unusually good
affinity due to its strong association with the assembly complex.
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, peptide induces
Ld expression on 721.221 by 3.7-fold, but it
induces Ld expression on 721.220 by 7-fold. Thus,
complete absence of cellular tapasin also causes class I to be
suboptimally loaded or functionally empty, and to a similar (although
perhaps slightly greater) extent than prevention of tapasin/class I
interaction by mutation of the class I H-chain.
Ld expressed on a tapasin-negative cell could be
slightly more peptide inducible than an Ld mutant
unable to bind tapasin due to additional activities of tapasin that do
not involve direct binding to class I. For example, tapasin has been
shown to increase the stabilization of TAP (6), which
likely improves the pool of available peptides. Notably, the influence
of tapasin on Ld expression is relative and not
absolute. For example, tapasin is not required for functional Ag
presentation by Ld (data not shown). However,
Ld expressed in the absence of tapasin is more
peptide accessible at the cell surface; thus, tapasin must contribute
to optimization of peptide binding in the ER. Our
721.220-Ld results suggest that the phenotype of
the 2 domain loop mutations could possibly be due to a deficiency in
tapasin interaction.
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-helix extending above the peptide-binding groove. Thus, the
128–136 loop could be a hinge region susceptible to conformational
change when a peptide binds. If true, chaperones could monitor the
conformational change in the 128–136 loop such that they would only
bind to class I when the loop is in the open conformation. Tapasin may
be the most likely candidate to monitor peptide binding because it is a
specific chaperone for class I. Interestingly, others have speculated
that tapasin interacts with the 3 domain (16). Indeed,
as shown in Fig. 1, residues 128–136 in the 2 domain and residues
227–229 in the 3 domain are in the same plane along the side of the
H-chain. Thus tapasin could interact with both of these determinants.
In this model, the interaction between the membrane proximal Ig fold of
tapasin (32) and the 3 domain of the H-chain could be
similar to the interaction of the complementarity-determining regions
of CD8 with H-chain residue 227 (33), and the C terminal
portion of tapasin could interact with the 2 loop region of the
H-chain. The other site on the H-chain shown to influence association
with the assembly complex is N86, the glycosylation site in the 1
domain of all mouse and human class I molecules. Based on its lectin
properties, calreticulin likely binds to the 1 domain
oligosaccharide (2, 3). Consistent with this model, the
location of N86 is spatially separated from the aforementioned,
putative tapasin interaction sites (residues 128–136, and around 227)
(Fig. 1). Thus tapasin and calreticulin could both simultaneously bind
H-chain. The fact that mutation at each of these sites ablates TAP
association could then be explained if calreticulin, like tapasin
(2, 34), is required for class I to associate stably
with TAP.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Joyce C. Solheim, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. E-mail address:
3 Abbreviations used in this paper: ER, endoplasmic reticulum; H-chain, heavy chain; ß2m, ß2-microglobulin; MCMV, murine CMV; CHAPS, 3-([cholamidopropyl]dimethylammonio)-1-propanesulfonate; wt, wild type.
4 Ld mutant cell lines: L-LdN86Q, L-LdE128R, L-LdD129R, L-LdL130D, L-LdK131D, L-LdT132K, L-LdW133T, L-LdT134K, L-LdA135H, L-LdA136H, L-LdD137H, and L-LdD227K.
Received for publication April 19, 1999. Accepted for publication July 30, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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2 domain of HLA-A.1 define a functionally relevant interaction with TAP. Curr. Biol. 6:873.[Medline]
ík, J. J. Monaco, S. Vukmanovi
. 1998. Physical and functional association of the major histocompatibility complex class I heavy chain 3 domain with the transporter associated with antigen processing. J. Exp. Med. 187:865.3 domain. J. Immunol. 162:1530. and HLA-A2. Nature 387:630.[Medline]
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 L. Sesma, B. Galocha, M. Vazquez, A. W. Purcell, M. Marcilla, J. McCluskey, and J. A. L. de Castro Qualitative and Quantitative Differences in Peptides Bound to HLA-B27 in the Presence of Mouse versus Human Tapasin Define a Role for Tapasin as a Size-Dependent Peptide Editor J. Immunol., June 15, 2005; 174(12): 7833 - 7844. [Abstract] [Full Text] [PDF]
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 X. Wang, R. Connors, M. R. Harris, T. H. Hansen, and L. Lybarger Requirements for the Selective Degradation of Endoplasmic Reticulum-Resident Major Histocompatibility Complex Class I Proteins by the Viral Immune Evasion Molecule mK3 J. Virol., April 1, 2005; 79(7): 4099 - 4108. [Abstract] [Full Text] [PDF]
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S. Tourdot, M. Nejmeddine, S. J. Powis, and K. G. Gould Different MHC Class I Heavy Chains Compete with Each Other for Folding Independently of {beta}2-Microglobulin and Peptide J. Immunol., January 15, 2005; 174(2): 925 - 933. [Abstract] [Full Text] [PDF]
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J. L. Petersen, H. D. Hickman-Miller, M. M. McIlhaney, S. E. Vargas, A. W. Purcell, W. H. Hildebrand, and J. C. Solheim A Charged Amino Acid Residue in the Transmembrane/Cytoplasmic Region of Tapasin Influences MHC Class I Assembly and Maturation J. Immunol., January 15, 2005; 174(2): 962 - 969. [Abstract] [Full Text] [PDF]
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X. Wang, L. Lybarger, R. Connors, M. R. Harris, and T. H. Hansen Model for the Interaction of Gammaherpesvirus 68 RING-CH Finger Protein mK3 with Major Histocompatibility Complex Class I and the Peptide-Loading Complex J. Virol., August 15, 2004; 78(16): 8673 - 8686. [Abstract] [Full Text] [PDF]
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S. Ford, A. Antoniou, G. W. Butcher, and S. J. Powis Competition for Access to the Rat Major Histocompatibility Complex Class I Peptide-loading Complex Reveals Optimization of Peptide Cargo in the Absence of Transporter Associated with Antigen Processing (TAP) Association J. Biol. Chem., April 16, 2004; 279(16): 16077 - 16082. [Abstract] [Full Text] [PDF]
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B. Park and K. Ahn An Essential Function of Tapasin in Quality Control of HLA-G Molecules J. Biol. Chem., April 11, 2003; 278(16): 14337 - 14345. [Abstract] [Full Text] [PDF]
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C. R. Morris, J. L. Petersen, S. E. Vargas, H. R. Turnquist, M. M. McIlhaney, S. D. Sanderson, J. T. Bruder, Y. Y. L. Yu, H.-G. Burgert, and J. C. Solheim The Amyloid Precursor-like Protein 2 and the Adenoviral E3/19K Protein Both Bind to a Conformational Site on H-2Kd and Regulate H-2Kd Expression J. Biol. Chem., April 4, 2003; 278(15): 12618 - 12623. [Abstract] [Full Text] [PDF]
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M.-E. Paquet and D. B. Williams Mutant MHC class I molecules define interactions between components of the peptide-loading complex Int. Immunol., April 1, 2002; 14(4): 347 - 358. [Abstract] [Full Text] [PDF]
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P. Tan, H. Kropshofer, O. Mandelboim, N. Bulbuc, G. J. Hammerling, and F. Momburg Recruitment of MHC Class I Molecules by Tapasin into the Transporter Associated with Antigen Processing-Associated Complex Is Essential for Optimal Peptide Loading J. Immunol., February 15, 2002; 168(4): 1950 - 1960. [Abstract] [Full Text] [PDF]
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H. R. Turnquist, S. E. Vargas, A. J. Reber, M. M. McIlhaney, S. Li, P. Wang, S. D. Sanderson, B. Gubler, P. van Endert, and J. C. Solheim A Region of Tapasin That Affects Ld Binding and Assembly J. Immunol., October 15, 2001; 167(8): 4443 - 4449. [Abstract] [Full Text] [PDF]
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M. R. Harris, L. Lybarger, N. B. Myers, C. Hilbert, J. C. Solheim, T. H. Hansen, and Y. Y. L. Yu Interactions of HLA-B27 with the peptide loading complex as revealed by heavy chain mutations Int. Immunol., October 1, 2001; 13(10): 1275 - 1282. [Abstract] [Full Text] [PDF]
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T. Chun, A. G. Grandea III, L. Lybarger, J. Forman, L. Van Kaer, and C.-R. Wang Functional Roles of TAP and Tapasin in the Assembly of M3-N-Formylated Peptide Complexes J. Immunol., August 1, 2001; 167(3): 1507 - 1514. [Abstract] [Full Text] [PDF]
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M. R. Harris, L. Lybarger, Y. Y. L. Yu, N. B. Myers, and T. H. Hansen Association of ERp57 with Mouse MHC Class I Molecules Is Tapasin Dependent and Mimics That of Calreticulin and not Calnexin J. Immunol., June 1, 2001; 166(11): 6686 - 6692. [Abstract] [Full Text] [PDF]
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B. A. L. Owen and L. R. Pease Thermal Stability of MHC Class I-{{beta}}2-Microglobulin Peptide Complexes in the Endoplasmic Reticulum Is Determined by the Peptide Occupancy of the Transporter Associated with Antigen Processing Complex J. Immunol., February 1, 2001; 166(3): 1740 - 1747. [Abstract] [Full Text] [PDF]
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A. W. Purcell, J. J. Gorman, M. Garcia-Peydro, A. Paradela, S. R. Burrows, G. H. Talbo, N. Laham, C. A. Peh, E. C. Reynolds, J. A. Lopez de Castro, et al. Quantitative and Qualitative Influences of Tapasin on the Class I Peptide Repertoire J. Immunol., January 15, 2001; 166(2): 1016 - 1027. [Abstract] [Full Text] [PDF]
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M. J. Barnden, A. W. Purcell, J. J. Gorman, and J. McCluskey Tapasin-Mediated Retention and Optimization of Peptide Ligands During the Assembly of Class I Molecules J. Immunol., July 1, 2000; 165(1): 322 - 330. [Abstract] [Full Text] [PDF]
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