Several studies have addressed the role of MHC class I glycans in MHC class I folding and assembly (5
) but the role of the glycan of tapasin in the functional activity of tapasin has not been investigated. We previously observed that glycan and ERp57-dependent interactions contribute to calreticulin recruitment to the PLCs of murine fibroblasts (12
). The current findings () support the model that tapasin-associated ERp57 and tapasin’s glycan are the relevant sites that mediate calreticulin recruitment. PLCs from cells expressing the tapasin(C95A) mutant showed the presence of calreticulin, heavy chains and β2m, although at reduced levels compared to that those present in the context of wild-type tapasin (, lanes 3–4). Mutation to tapasin(CANQ) further reduced the efficiency of heavy chain recruitment and rendered β2m and calreticulin binding undetectable (, lanes 3–4 compared to 7–8). These findings indicate the presence of PLCs in which MHC class I recruitment to TAP is stabilized by tapasin’s glycan, likely via the binding of calreticulin (). The observation that the tapasin(N233Q) mutation has a stronger impact upon PLC formation in the context of tapasin(C95A) compared to the wild-type tapasin context () is important, as it suggests that tapasin’s glycan may in fact be relevant for stabilizing PLCs containing tapasin molecules that are not in heterodimeric association with ERp57.
Within the PLC, calreticulin equilibrates between tapasin glycan- and tapasin(C95)-associated forms, and intermediate heavy chain-depleted PLCs are detectable
The tapasin(N233Q) mutation reduced the extent of ERp57 recruitment to tapasin and TAP compared to wild-type tapasin (, ERp57 blots, lanes 1–2 compared to 5–6). Furthermore β2m deficiency, which strongly impaired calreticulin recruitment to the PLC (), or calreticulin-deficiency itself, also impacted the recruitment efficiencies of ERp57 into the PLC (data not shown). Calreticulin bound to tapasin’s glycan could transiently initiate ERp57 recruitment via a calreticulin P-domain-ERp57 interaction. This interaction could position ERp57 in appropriate proximity for conjugation to C95 of tapasin. Molecular modeling studies (using calnexin’s structure (2
) as a model for calreticulin structure and the tapasin-ERp57 complex structure (36
)) suggest that a calreticulin bound to tapasin’s glycan cannot simultaneously contact the ERp57 molecule present within the same tapasin-ERp57 conjugate (Wijeyesakare and Raghavan, unpublished observations). Thus, we postulate that conjugation between C57 of ERp57 and C95 of tapasin may induce a conformational change within the complex depicted in , that disengages calreticulin from tapasin’s glycan, re-positioning calreticulin for binding to tapasin-conjugated ERp57, via a P domain-based interaction (). Thus, calreticulin may equilibrate between conformational states in which its recruitment to the PLC is stabilized by tapasin’s glycan and tapasin-associated ERp57 (
7B). In turn, calreticulin could stabilize the recruitment of both ERp57 and MHC class I molecules via cooperative binding interactions.
Within the same cell type, differences were noted in the compositions and functional activities of tapasin complexes in IFN-γ treated cells compared to untreated cells ( compared to 2C). Calreticulin recruitment to tapasin was more strongly tapasin(N233)-dependent in cells that were not treated with IFN-γ compared to 2C, calreticulin blots, lanes 1–6). Nonetheless, tapasin(N233)-mediated stabilization of MHC class I recruitment was not readily detectable in the absence of IFN-γ treatment (, MHC class I blot, lanes 3–4 compared to 7–8). Since MHC class I heavy chain and β2m expression levels are both low in M553 cells in the absence of IFN-γ treatment, these findings are consistent with the possibility that binding of MHC class I molecules to tapasin in an interaction stabilized by calreticulin, shifts the 7A
7B binding equilibrium towards 7B. Thus, sub-complexes of 7A (for example, those involving just tapasin and calreticulin) may accumulate under conditions where MHC class I expression is low (). Furthermore, complexes of the 7A type might also accumulate under conditions where ERp57 levels become limiting relative to those of tapasin and MHC class I molecules, a scenario possible in IFN-γ-treated cells, when tapasin is under endogenous IFN-γ control. Together, the findings of suggest a coupling between tapasin(N233)-mediated complexes and tapasin(C95)-mediated complexes (
7B), and that cellular conditions determine the particular complex that predominates in the steady-state. The data also indicate that, in human cells, tapasin(C95)-mediated complexes are more stabilizing for MHC class I recruitment. Tapasin from species that lack C95 but which have N233, such as Grass carp (Ctenopharyngodon idellus
), Zebra fish (Danio rerio
) and Atlantic salmon (Salmo salar
), may stabilize their interactions with MHC class I molecules solely via the tapasin glycan-dependent mechanism.
Tapasin-MHC class I interactions were generally stronger in IFN-γ-treated cells compared to untreated cells. For example, whereas β2m recruitment to tapasin was readily detectable in IFN-γ-treated cells, this interaction was not detectable in untreated cells even in the context of wild-type tapasin, and heavy chain recruitment was strongly diminished by the tapasin(C95A) mutation alone in cells that were not treated with IFN-γ (). IFN-γ-treatment markedly enhances steady-state levels of TAP, tapasin, MHC class I heavy chains and β2m, and the increase in these protein concentrations could directly contribute to stronger PLC assembly in IFN-γ-treated M553 cells compared to untreated cells. Somewhat paradoxically, the assembly-promoting functions of tapasin were much less significant in IFN-γ-treated M553 cells than in untreated cells ( compared to 1C). However, it is to be noted that under the experimental conditions used in this study, tapasin expression is not under endogenous IFN-γ-control. Thus, tapasin’s activity may become limited (saturated) under conditions in which MHC class I heavy chains or β2m are expressed in stoichiometric excess relative to tapasin. Further investigations are needed to understand whether, in tapasin-sufficient cells, IFN-γ treatments generally induce stoichiometric increments in TAP, tapasin and MHC class I heavy chain components. Regardless of tapasin expression, when TAP activity is high following IFN-γ–treatment, there may be increased probability for tapasin-independent assembly arising from increased peptide availability, resulting in a reduction in the stringency of quality control. Such a scenario may be advantageous to the host in an infectious setting, in order to present repertoires of both optimal and sub-optimal peptide-MHC complexes, and increase the diversity of pathogenic peptides that can be presented.
PLCs of 721.221 cells are able to incorporate β2
m, calreticulin and ERp57 under conditions of heavy chain deficiency, and enhanced recruitment of heavy chains into the PLC of 721.221 cells does not result in a parallel increase in the recruitment of β2
m, calreticulin or ERp57 (). The latter findings are supported by previous studies that have also shown binding of β2m and calreticulin to TAP in the absence of classical heavy chains in 721.221 cells (37
), and the lack of significant induction of β2m or calreticulin recruitment into the TAP complex by over-expression of HLA-B27 heavy chains (38
). The analyses described here confirm that associations of β2
m and calreticulin with the PLC are relatively unaffected by an eight-fold or higher enhancement in heavy chain incorporation. ERp57 incorporation into the PLC was slightly induced by the presence of HLA-B*3503 heavy chains. While it is possible that non-classical MHC class I heavy chains facilitate β2
m, calreticulin and ERp57 recruitment, non-classical heavy chains are estimated to be present at low levels (≤ 1%) in 721.221 cells compared to classical MHC class I heavy chain (39
). Together, these findings support the existence of intermediate PLCs containing tapasin, ERp57, β2m and calreticulin (). It is presently unclear whether the observed binding of calreticulin to TAP in the 721.221 cells is dominated by N233-dependent interactions, C95-dependent interactions, or both. Thus, while intermediate heavy chain-depleted complexes are depicted as being tapasin(N233)- and tapasin(C95)-linked (), further investigations will be needed to verify both possibilities.
Despite recruitment of β2m into intermediate PLCs in a heavy chain-independent manner, the PLC does not appear to be the site for initial heavy chain-β2
m assembly, as PLCs deficient in β2
m recruitment (via the tapasin(C95A) mutation) were competent for early assembly of heterodimers (). Rather, the intermediate PLCs that are enriched in β2
m (relative to heavy chains) may serve as a platform for peptide exchange that is coupled to β2
m exchange from unstable heavy chain-β2
m heterodimers that are recruited into the PLC. Indeed, the presence of excess exogenous β2
m has been shown to facilitate peptide exchange from MHC class I molecules in vitro
). Interactions of sub-optimally assembled heavy chain-β2
m heterodimers with intermediate β2
m-enriched PLC could result in peptide and β2
m exchange from the pre-assembled heterodimers, simultaneously recruiting heavy chains into the PLC. Previous in vitro
studies have demonstrated interactions between heavy chains and tapasin with sub-stoichiometric levels of β2m (unless β2m is added in excess (41
)). Additionally, β2m-deficient cells are able to recruit heavy chains with low efficiency ( and (42
)). These findings, taken together with the findings that β2m can be recruited to the PLC independently of heavy chains (), lead to the postulate that ERp57- and/or calreticulin-dependent recruitment of β2m into the PLC can stabilize weaker binding between heavy chains and tapasin, and serve as a point of contact for stable heavy chain recruitment to the PLC. Thus, distinct interactions may be important for stabilizing heavy chain and β2m recruitment to the PLC.
The findings described here also show that the conserved heavy chain glycan is not central to calreticulin recruitment into the PLC. The finding that the heavy chain glycan impacts the efficiency with which heavy chain is recruited into the PLC () raises the possibility that the heavy chain glycan is engaged by the glycan binding site of calreticulin within the tapasin(C95)-dependent mode of calreticulin recruitment (). However, if β2m is an important point of contact between heavy chain and the PLC as suggested above, an alternative possibility is that the heavy chain glycan mutant has disrupted interactions with β2m within the PLC, also suggested by the finding of reduced efficiency of early heterodimer assembly in cells in the context of HA-HLA-A2(N86Q) compared to HA-HLA-A2 (). Further studies are needed to resolve these two possibilities. Thus, while structural studies of the tapasin-ERp57 heterodimers (36
) provide a valuable starting point for the construction of model for a mature PLC, whether calreticulin in fact interacts with the MHC class I glycan within the PLC as suggested (36
) remains to be established, as well as the nature of interactions between tapasin-ERp57, β2m and calreticulin.
Tapasin facilitated an early step in the assembly of heterodimers, and this step did not require tapasin-associated ERp57 or calreticulin (). Tapasin stabilizes TAP and increases peptide transport by TAP (43
), and by this mechanism, tapasin could impact an early step in heterodimer assembly. However, we have previously shown that the tapasin(C95A) mutant induces MHC class I assembly via a mechanism independent of its effect on TAP stabilization and peptide transport (16
). It is possible that tapasin facilitates heterodimer formation simply by localizing heavy chains in the vicinity of the peptide source (TAP). Thus, there are at least two distinct levels of tapasin-assisted assembly, an early step that impacts heterodimer formation, and a later step involving calreticulin and ERp57, that could impact the generation of optimally-assembled heterodimers. The conserved MHC class I glycan also facilitates an early step in the assembly of heterodimers (). The MHC class I glycan likely facilitates heterodimer formation by directly stabilizing heavy chain-β2
m complexes, and/or by facilitating calreticulin recruitment, which could enhance the formation or stability of heterodimers.
In summary, the studies described here provide evidence for distinct and important roles for different glycans within the assembly complex. Furthermore, evidence is presented for the presence of intermediate complexes that are heavy chain-depleted. These studies provide new insights into the sequence and nature of assembly events within the MHC class I assembly pathway.