Departments of 1 Immunology and 2 Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
Correspondence to: D. B. Williams; E-mail: david.williams{at}utoronto.ca
Transmitting editor: H. Ploegh
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: antigen presentation, calreticulin, endoplasmic reticulum, H-2Dd, histocompatibility, tapasin, transporter associated with antigen processing
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The importance of tapasin in the peptide-loading complex was first elucidated in studies involving the human tapasin-negative cell line LCL721.220 (4,710) and subsequently confirmed in tapasin-deficient mice (11,12). In both systems, the surface expression of class I molecules is substantially reduced, but levels can be up-regulated by the addition of class I-binding peptides. Both intracellular and cell-surface class I molecules are more thermolabile than in wild-type cells, and they can be stabilized by peptide addition. Furthermore, presentation of viral antigens to cytotoxic T lymphocytes (CTL) is impaired and, in tapasin-deficient mice, significant defects are apparent in both positive and negative selection of CD8+ T cells (11). Collectively, these findings suggest a role for tapasin in the loading of peptides onto class I molecules. In addition, the association between class I molecules and the TAP transporter is lost in the absence of tapasin (4,13,14). This latter observation, combined with the finding that tapasin binds to class I heterodimers in the absence of TAP and to TAP in the absence of class I, led to the suggestion that tapasin forms a bridge between the TAP transporter and class I heterodimers (4). However, this bridging role does not appear to be crucial to tapasins functions since a soluble version of tapasin that binds to class I but not to TAP, and hence cannot serve as a bridge, is fully capable of restoring normal peptide loading to tapasin-deficient 721.220 cells (15).
Although tapasin facilitates peptide loading, it is not absolutely essential. Cells from tapasin-deficient mice could be recognized by and could stimulate alloreactive CTL (11,12), and furthermore such mice were tolerant to immunization with cells expressing syngeneic class I molecules (12). Thus, there appears to be substantial overlap between the self peptides expressed in tapasin-containing versus tapasin-deficient cells. In addition, cell-surface class I molecules in the absence of tapasin interaction are less stable than in wild-type cells, but are more stable than class I molecules that completely lack peptides. This suggests the presence of an altered peptide repertoire in class I molecules loaded in the absence of tapasin rather than a complete lack of peptides (8,12). Direct analysis of peptides bound to mouse H-2Kb and human HLA-B*2705 molecules in the absence or presence of tapasin revealed a reduction in the overall yield of peptides from tapasin-deficient cells (8,10). However, those peptides that could be recovered exhibited considerable (but not complete) overlap with the repertoire of peptides recovered from class I molecules expressed in tapasin-containing cells. Therefore, there appear to be both quantitative and qualitative influences of tapasin on the loading of peptides onto class I molecules.
How tapasin effects its functions in peptide loading remains unclear. Several recent studies have suggested functions that include the ER retention of empty class I molecules (8,11,16), modulation of TAP expression levels (15), promotion of peptide binding and translocation by TAP (11,17), and stabilization of peptide-deficient H chainß2m heterodimers (15). Furthermore, some of tapasins effects may be indirect since tapasin binding to class I molecules is believed to occur in collaboration with CRT. It has been reported that class I molecules that do not bind tapasin also do not associate with CRT, leading to a model of cooperative binding between tapasin and CRT (18,19). A similar model has been suggested for the association of ERp57 with class I (19,20). To add to the complexity of tapasin functions, various human class I allotypes have been shown to possess different levels of tapasin dependence. For example, HLA-B*2705 expressed in tapasin-negative 721.220 cells is expressed on the cell surface at levels similar to that observed in tapasin-expressing cells and can present viral antigens to CTL. In contrast, HLA-B*4402 surface expression and antigen presentation are highly dependent on the presence of tapasin, whereas HLA-B8 exhibits an intermediate dependence (9). In the murine system as well, Myers et al. showed that on the surface of tapasin-deficient cells the H-2Kb, -Kd and -Ld molecules differ in their ratios of peptide-receptive versus conformationally mature forms (7).
Given the diversity of functions attributed to tapasin and its apparent cooperation with CRT and ERp57 in associating with the peptide-loading complex, there is considerable interest in understanding how the components of this complex are organized. Cresswell and co-workers demonstrated that up to four class Itapasin complexes are associated with each TAP heterodimer (14). Several subsequent studies focused on defining the nature of the class Itapasin association. Using truncation mutants, the N-terminal 50 residues of tapasin were shown to mediate its association with class I (21). Conversely, site-directed mutagenesis identified several residues within the class I H chain that are important for binding to tapasin (18,2225). These include the glycosylation site at residue 86 in the 1 domain of the H-2Ld molecule (18), residues 115, 122 and 134 in the
2 domain of HLA-A2 (2325) or residues 133 and 134 in the
2 domain of H-2Ld (18) and, within the
3 domain, residue 222 in H-2Dd (22) or 227 in H-2Ld (18). For some of these mutants, peptide deficiency phenotypes similar to those observed in tapasin-deficient cells were observed (18,2224), whereas others did not appear to be impaired in peptide binding (25).
Since previous studies on the nature of the tapasinclass I interaction focused almost entirely on the H-2Ld and HLA-A2 molecules, and various phenotypes were observed with different non-tapasin-binding mutants, we decided to undertake a systematic examination of a third molecule, H-2Dd, in an effort to identify residues involved in tapasin association and to characterize phenotypes associated with a loss of tapasin binding. Significant differences were observed in the sites of tapasin interaction with H-2Dd and in the association of Dd mutants with CRT indicating isotype-specific differences in the organization of the peptide-loading complex.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
R111A: 5'-CTG CCA GTA CCC Ggc GAG GAG GCG CCC-3'
D122A: 5'-CAG GGC GAT GTA tgC GCA GCC GTC GTA GG-3'
N127A: 5'-CGT TTT CAG GTC cTG Ggc CAG GGC GAT GTA ATC-3'
E128A: 5'-CCA CGT TTT CAG GTC TgC GTT CAG GGC G-3'
D129A: 5'-CGT CCA CGT TTT CAG GgC TTC GTT CAG G-3'
T132A: 5'-CCG CCG TCC ACG ccT TaA GGT CTT CGT TCA GG-3'
N127A, E128
A: 5'-CCA CGT TTT CAG GTC TgC Ggc CAG GGC GAT GTA ATC-3'
D129A, T132
A: 5'-CCG CCG TCC ACG cTT TCA GGg CTT CGT TCA GG-3'
A135Q, A136
Q: 5'-GCG CCG CCA TGT CCt gCt gCG TCC ACG TTT TCA GG-3'
N220A: 5'-GGT CAG CTC CTC gcc GGC CAA CTG CCA GGT CAG-3'
D212A, T214
A: 5'-CTG CCA GGT CAG ctg GAT GgC AGC AGG GTA GAA GC-3'
T225A, Q226
A: 5'-CAA GCT CCA TTT CCg cGG cCA GCT CCT CCC C-3'
All mutations were confirmed by DNA sequencing before the mutated fragment was inserted back into pSV2-neo containing the rest of the H-2Dd gene.
PSV2-neo plasmids containing mutant Dd genes were transfected into mouse L cells (H-2k) using the Superfect reagent (Qiagen, Hilden, Germany). Stable transfectants were obtained by geneticin selection (Life Technologies, Gaithersburg, MD) and cells positive for surface Dd expression were separated from negative cells by FACS. Mutant cells expressing D129A/T132A could not be sorted due to low expression, thus clones were obtained by limiting dilution.
Cells, antibodies and other reagents
Transfected mouse L cells were grown in DMEM (Life Technologies) supplemented with 2 mM glutamine, 10% FCS, antibiotics and 500 µg/ml geneticin. For detection of H-2Dd, mAb 34-2-12S and 34-5-8S were used, the latter being specific for ß2m-associated Dd (26). mAb 16-3-1N, which recognizes ß2m-associated H-2Kk (27), was used as a positive control in flow cytometry experiments. Rabbit anti-TAP2 antiserum was provided by Drs Y. Yang and P. Peterson (R. W. Johnson, La Jolla CA). Anti-tapasin antiserum was raised in rabbits against the C-terminal 20 amino acids of murine tapasin (28). Rabbit anti-CNX antiserum directed against the ER luminal domain of CNX has been described previously (29). Anti-CRT antiserum, SPA-600, was purchased from StressGen (Victoria, BC, Canada). The H-2Dd-binding peptide, designated Tum (sequence NGPPHSNNF), was synthesized by the Alberta Peptide Institute (Edmonton) and was used in peptide occupancy experiments.
Flow cytometry
To determine the cell-surface levels of the various H-2Dd mutants, transfected L cells (3.5 x 105) were removed from plates by trypsinization and incubated on ice for 1520 min in 0.1 ml of FACS buffer (HBSS with 1% BSA and 0.01% NaN3) containing 1.5 µg of either mAb 34-5-8S or mAb 16-3-1N. Cells were washed once and incubated for 1520 min on ice with 0.4 µg of fluorescein-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) in 0.1 ml FACS buffer. Cells were washed twice and resuspended in 0.3 ml of FACS buffer containing 20 µg/ml propidium iodide for the detection of live cells. Analysis followed within 30 min using an Epics Elite flow cytometer (Beckman Coulter, Fullerton, CA). For experiments measuring the turnover of cell-surface Dd molecules, cells were removed from plates by treatment with 2.5 mM EDTA in PBS and, following antibody incubations, were fixed in 0.5% paraformaldehyde prior to analysis.
Metabolic radiolabeling and immunoisolation
Typically, 5 x 106 transfected L cells in a single 100-mm plate were starved for 30 min with Met-free RPMI and radiolabeled for 30 min in 1 ml of medium containing 0.3 mCi of [35S]Met (>1000 Ci/mmol; Amersham, Little Chalfont, UK). Cells were lysed at 4°C for 30 min in PBS, pH 7.4, containing 1% digitonin, 10 mM iodoacetamide, 60 µg/ml Pefabloc (Roche, Basel, Switzerland), and 10 µg/ml each of leupeptin, antipain and pepstatin. Lysates were centrifuged at 11,000 g to pellet nuclei and cell debris. To isolate Dd molecules associated with TAP, tapasin or CRT, supernatant fractions were subjected to a first round of immunoisolation by incubating with anti-TAP, anti-tapasin or anti-CRT antisera for 2 h. Immune complexes were recovered over a period of 1 h with Protein Aagarose beads. The beads were then heated at 40°C in PBS containing 0.2% SDS for 1 h to disrupt protein complexes. Eluted material was adjusted to 5% skim milk and 2% Nonidet P-40, and a second round of immunoisolation was performed at 4°C for 2 h with the anti-Dd mAb 34-2-12S or an isotype-matched control mAb, MKD6. Immune complexes recovered with Protein A beads were analyzed by SDSPAGE (10% gels).
For pulsechase experiments, 1 x 106 transfected L cells were radiolabeled for 10 min as above except that 0.1 mCi of [35S]Met was used. Cells were washed and then chased for various periods in DMEM containing 1 mM Met. Lysis was conducted in PBS containing 1% Nonidet P-40, 2 mM iodoacetamide and protease inhibitors, and then lysates were centrifuged as above. In the case of peptide occupancy experiments, supernatant fractions were incubated for 1 h at 4°C in the presence or absence of 50 µM Tum peptide and then incubated at 37°C for an additional h. Peptide-containing Dd molecules were isolated with mAb 34-5-8S and immune complexes were collected with Protein Aagarose beads. Dd molecules were eluted from the beads with 0.1 M citrate buffer, pH 6, containing 0.1% SDS and digested with endo-ß-N-acetylglucosaminidase H (Endo H; New England Biolabs, Beverly, MA) prior to analysis by SDSPAGE. Radioactive bands were quantified using a STORM Phosphorimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). In all cases, band intensities were corrected by subtracting a background value determined by quantifying a blank area of the gel corresponding in size to the radioactive band of interest.
Turnover kinetics of cell-surface H-2Dd
L cell transfectants (1.5 x 106) were incubated for 18 h at 26°C in serum-free medium containing 10 µg/ml of human ß2m (Sigma, St Louis, MO). Cells were then washed with cold medium and resuspended in 4 ml of pre-warmed DMEM containing 10 µg/ml of Brefeldin A (Sigma). A 1-ml aliquot of cells was immediately transferred to a tube containing 2 ml of FACS buffer while the rest was transferred to a 37°C water bath. Aliquots were collected at the indicated time points and analyzed by FACS as described above using the conformation-dependent H-2Dd mAb 34-5-8S.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Mutant Dd molecules with reduced surface expression are deficient in tapasin/TAP interactions
To determine if the mutations introduced in the Dd molecule had an effect on its interaction with tapasin/TAP, we assayed for mutant association with tapasin or TAP by sequential immunoisolation. Initially, TAP or tapasin was isolated from digitonin lysates of radiolabeled cells and then immune complexes were disrupted in SDS and any associated Dd molecules were recovered in a second round of immune isolation with the anti-Dd mAb, 34-2-12S (Fig. 3A). As expected, based on our previous work (22), the wild-type Dd H chain could be recovered from TAP or tapasin immune isolates indicating that the wild-type Dd molecule associates with both tapasin and TAP (Fig. 3A, lanes 4 and 5). In contrast, mutant E222K, which we previously showed was incapable of tapasin/TAP association (22), could not be recovered from tapasin or TAP immune complexes (Fig. 3A, lanes 6 and 7). Note that in these experiments, TAP and tapasin could be detected in the second round of isolation with the anti-Dd mAb (Fig. 3A, lanes 47). Since these proteins were also present in control experiments wherein an isotype-matched mAb (MKD6) was used in place of anti-Dd mAb (Fig. 3A, lanes 2 and 3), this indicates either incomplete dissociation of initial anti-tapasin or anti-TAP immune complexes in SDS or renaturation of some of the anti-tapasin or anti-TAP antibodies during the second round of isolation. This is a consequence of the low temperature employed in the first round SDS dissociation step that was required to preserve the mAb 34-2-12S epitope for isolation of Dd in the second round.
|
The sequential immunoisolation technique was also used to test the capability of the single Dd mutants to interact with tapasin. As shown in Fig. 3(B), mutants N127A, E128A and T132A exhibited very low but detectable association with tapasin (10% relative to wild-type Dd), whereas mutant D129A failed to bind to tapasin.
To ensure that the mutations introduced in Dd did not cause substantial misfolding of the molecule, we isolated the various mutants and showed that they all are capable of association with ß2m (Fig. 3C). Furthermore, all mutants retained the ability to bind exogenous peptide in cell lysates (see Fig. 5 below). Therefore, in addition to our previous identification of residue 222 in the 3 domain (22), we have identified a cluster of residues (residues 122136) within the
2 domain of H-2Dd involved in its interaction with tapasin and TAP.
|
|
Most H-2Dd mutants are occupied with peptides
Tapasin has been shown to influence the amount and sequence of peptides binding to class I molecules (714). To determine if an impaired tapasin interaction affects the peptide occupancy status of our mutants, we performed two independent assays. In the first experiment, we compared the thermal stability of different mutants in cell lysates to that of the wild-type molecule. It is well established that class I molecules that lack peptides or contain low-affinity peptides will dissociate upon incubation at 37°C and will not be recognized by conformation-sensitive mAb (33). Transfected cells were radiolabeled for 10 min with [35S]Met and chased for 1 h. Following lysis, they were incubated at 4°C for 1 h in the absence or presence of a stabilizing Dd-binding peptide and then shifted to 37°C for an additional 1 h prior to immune isolation with the conformation-sensitive mAb 34-5-8S. As shown in Fig. 5(A), the bulk (60%) of Endo H-resistant, wild-type Dd molecules could be recovered whether or not stabilizing exogenous peptides were present, indicating that most wild-type Dd molecules had been loaded with peptides and acquired thermal stability. In our hands, Endo H-sensitive class I molecules rarely exhibited thermal stability suggesting that peptide loading is followed rapidly by export to the Golgi. As a control for the peptide-deficient phenotype, we examined the E222K mutant that does not associate with tapasin/TAP and acquires peptides inefficiently (22). Accordingly, only 29% of Endo H-resistant E222K molecules exhibited thermal stability. None of the other Dd mutants that were converted to Endo H-resistant forms exhibited the same degree of thermal sensitivity as E222K. Rather, E128A and D122A exhibited an intermediate level of thermal stability (44 and 48% respectively), and mutants N127A/E128A, N127A and T132A were indistinguishable from wild-type Dd (ranging from 60 to 70% thermal stability) (Fig. 5A). Thus, all of these mutants had been loaded to a substantial degree with stabilizing peptides.
In the second approach, we monitored the stability of mutant molecules that were expressed at the cell surface. To ensure that the entire spectrum of surface-expressed molecules was examined, cells were incubated overnight at 26°C in the presence of exogenous ß2m to preserve any labile molecules that might lack peptide or contain suboptimal peptides. The temperature was then shifted to 37°C and the turnover of surface Dd molecules was monitored over time by flow cytometry. As expected, wild-type Dd molecules remained largely stable over the 2 h duration of the experiment (Fig. 5B). Conversely, empty Dd molecules expressed in TAP-deficient LKD8c cells (34) were extremely unstable with most mAb reactivity disappearing within 30 min at 37°C. Of the Dd mutants created in this study, only those that were severely impaired in their export from the ER exhibited thermal stabilities substantially less than wild-type Dd. As shown in Fig. 5(B), mutants D129A/T132A, A135Q/A136Q and D129A lost roughly half of their reactivity with mAb 34-5-8S over the 2 h period, indicating the presence of empty molecules or molecules loaded with readily dissociable peptides in addition to peptide-stabilized molecules. The results for the remaining mutants were consistent with the findings of the thermal stability assays in cell lysates (compare Fig. 5A and B). Mutants D122A, N127A/E128A, N127A, E128A and T132A all exhibited surface decay kinetics that were not significantly different from wild-type Dd, indicating that they were loaded with high-affinity, stabilizing peptides despite their impaired associations with tapasin/TAP.
H-2Dd mutants bind CRT and CNX
Several previous studies using various mouse or human class I molecules have correlated a lack of association with tapasin with a loss of CRT binding, suggesting that tapasin and CRT associate with the peptide-loading complex in a cooperative fashion (18,19). To determine if this observation also holds true for the H-2Dd molecule, we immunoisolated CRT from cells transfected with wild-type or mutant Dd molecules and assessed the presence of Dd among the CRT-associated proteins by subsequent immunoisolation with mAb 34-2-12S (Fig. 6A, right panel). A direct immunoisolation with mAb 34-2-12S was also performed to evaluate the synthetic rate of each mutant. The latter experiment revealed that mutants E222K and D122A were synthesized at lower rates than the other mutants (Fig. 6A, left panel). Remarkably, despite the complete absence of tapasin association for each of the mutants tested (see Fig. 3A), all could be recovered from anti-CRT immune precipitates. This was not simply due to non-specific adsorption of Dd to immune complexes since none could be recovered when mAb 34-2-12S was replaced by the isotype-matched control mAb, MKD6. Furthermore, with the exception of E222K, the various mutants were recovered from the pool of CRT-associated proteins as readily as wild-type Dd. Given this unexpected finding, it was important to determine if association of the mutant molecules with CNX was also maintained since it is conceivable that CRT could be substituting for some loss of CNX interaction. Using the same type of sequential immunoisolation approach used to characterize the DdCRT interaction, we established that all of the mutant proteins tested retained the ability to associate with CNX (Fig. 6B). The finding that wild-type Dd and various Dd mutants that fail to associate with tapasin remain capable of binding CRT suggests that the cooperative binding model cannot be extended to all class I molecules.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As summarized in Fig. 7, we found that of the residues mutated within the 3 domain CD8 binding loop (residues 222229), only E222 that we studied previously (22) was involved in Dd binding to tapasin. Mutations at N220, E223 [studied previously (22)], T225 or Q226 had no effect. We did not mutate residue E227 or residue E229 since previous mutagenesis studies at these positions in the Dd molecule failed to reveal the reduction in cell-surface expression that would be expected if association with tapasin was impaired (35). Additional
3 domain residues located on the same face of the Dd molecule, D212 and T214, were also not involved in interactions with tapasin. Consistent with the involvement of the CD8 binding loop in tapasin interaction, mutation of residue E227 in the Ld molecule has been shown to prevent tapasin binding (18) (Fig. 7). Analogous mutations in the HLA-A2 molecule have not been tested for tapasin interaction. However, in a recent report, HLA-B27 possessing the double D227K/E229K mutation was shown to exhibit impaired association with tapasin (36).
|
|
Examination of the H-2Dd mutants deficient in tapasin interaction revealed a complex array of phenotypes. With respect to export from the ER, a lack of tapasin interaction has been documented to either increase (8,16,21,2325) or decrease (22,28) the rate of class I transport from the ER to Golgi. Both phenotypes were observed for the non-tapasin binding Dd mutants (Table 1). Mutants D122A and E128A were shown to leave the ER faster than wild-type Dd but most of the Dd mutants displayed retarded transport. Mutants N127A/E128A, N127A and T132A were slightly delayed, whereas mutants D129A, D129A/T132A and A135Q/A136Q were extremely slow to leave the ER. Thus there was no correlation between tapasin interaction and ER to Golgi transport rate. Furthermore, although all non-tapasin binding mutants exhibited reduced surface expression compared to wild-type Dd, there was also no clear correlation between tapasin interaction and degree of peptide occupancy as assessed by thermal stability (Table 1). Most non-tapasin-binding mutants were indistinguishable from wild-type Dd in terms of thermal stability. Only the D129A, D129A/T132A and A135Q/A136Q mutants exhibited decreased peptide occupancy. Given the well-documented effects of tapasin on increasing peptide-binding efficiency of most class I molecules including Dd (4,714), it seems likely that the diverse phenotypes we observe are the result of more complex influences than a simple binding or non-binding to tapasin. It is possible that mutants with near-normal ER export rates and thermal stabilities retain some interaction with tapasin in living cells that is disrupted upon immune isolation, even when the ability to detect association is enhanced through the use of a proteasome inhibitor (data not shown). Such residual interaction may be sufficient to provide near normal peptide loading. For the very slowly transported mutants that do exhibit peptide deficiency, it may be that in addition to a loss of tapasin association, the mutations induce some degree of misfolding that is detected by ER chaperones, thereby leading to retention. Such misfolding would likely be quite localized since these mutants retained the ability to associate with ß2m and bind exogenous peptide. Despite these caveats, the mutants remain informative as to the residues of the Dd H chain that contribute to tapasin binding.
In the early steps of class I H chain folding, before any association with the peptide-loading complex, the dominant interaction of human and mouse H chains is with the chaperone CNX (3739). CNX promotes H chain folding and retains free H chains in the ER (13). CNX also binds ERp57 and the TAP/tapasin complex independently of class I, where it is thought to stabilize the proteins until the recruitment of H chainß2m heterodimers (38). Upon H chain association with ß2m and subsequent formation of the peptide-loading complex, CRT largely replaces CNX, although in mouse CNX can still be a major component (4,5,30). Investigations into the interplay between constituents of the peptide-loading complex, which consists of H chainß2m, CRT, ERp57, tapasin and TAP, have indicated that the presence of tapasin is required for the inclusion of CRT and ERp57 in the complex. Neither of the latter components has been found associated with class I molecules in the absence of tapasin (1820). This raises the question of whether CRT binds directly to class I molecules that are somehow made receptive by their simultaneous association with tapasin (CRTclass Itapasin) or if CRT binds to tapasin which in turn associates with class I (CRTtapasinclass I). In tapasin-negative insect cells, it has been shown that CRT can substitute for CNX and promote the folding of free mouse Db H chains (39), which implies a physical contact between CRT and the H chain. Furthermore, in the current study, Dd mutants that failed to bind detectably to tapasin retained the ability to bind to CRT. Thus, at least for H-2Dd, CRT appears to bind directly to Ddß2m heterodimers. Unfortunately, we were unable to extend our examination of the interactions of non-tapasin-binding Dd mutants to include ERp57. Extensive co-immunoisolation experiments with anti-ERp57 antiserum or anti-Dd mAb failed to detect an interaction of this enzyme even with the wild-type Dd molecule (data not shown).
It is not clear why we can readily detect CRT interactions with Ddß2m heterodimers in the absence of apparent tapasin association in contrast to several other studies using different human or mouse class I molecules. We cannot exclude the possibility of a weak tapasin interaction for certain Dd mutants prior to cell lysis despite their lack of detectable association even under conditions of proteasome inhibition. However, given the strong co-isolation of CRT we observed with the various mutants, it seems unlikely that the binding to CRT can be explained solely by the existence of a putative residual tapasin interaction. It is well established that CRT and CNX can interact with non-native protein substrates via proteinprotein interactions as well as via a lectin site (40,41). Therefore, one possibility is that polypeptide-based binding of CRT to Dd in the absence of tapasin is stronger than with other human or mouse class I molecules due to differences in H chain sequences. Another possibility may be that the organization of CRT in the peptide-loading complex may not be the same for all class I molecules. Indeed, while this manuscript was under review, Hansen and co-workers described another example of this unusual behavior in which an S132K mutation in the HLA-B27 molecule disrupted tapasin/TAP interactions but association with CRT was maintained (36). The functions of CRT remain one of the more enigmatic aspects of the peptide-loading complex. The determination of whether CRT acts as a chaperone directly for class I in this complex or indirectly via tapasin will fundamentally influence subsequent experimentation on the functional aspects of CRT in class I biogenesis.
![]() |
Acknowledgements |
---|
![]() |
Abbreviations |
---|
CRTcalreticulin
CTLcytotoxic T lymphocyte
Endo Hendo-ß-N-acetylglucosaminidase H
ERendoplasmic reticulum
TAPtransporter associated with antigen processing
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|