Proteasomes generate many N-terminally-extended precursors. For these precursors to be presented on an MHC class I molecule they must be trimmed by peptidases to mature epitopes. Previous studies had shown that the removal of amino terminal flanking residues does indeed occur in living cells. This N-terminal trimming was “downstream” (independent) of proteasomes and mediated by aminopeptidases. It was clear that this peptide editing could in principle occur either in the cytosol, where peptides were first generated by proteasomes, and/or after transport into the ER by TAP. However, the relative contribution of these two compartments to trimming and their specificity had been incompletely understood.
Using fluorescent substrates and purified cytosol or ER the specificity of trimming in these compartments in vitro
has been studied (31
). However, in vivo
the effect sequences N-terminal to an epitope have on processing and presentation on MHC class I molecules has only been systematically studied in the ER (32
). This is important to define for cytosolic trimming because extracts may not faithfully reproduce the conditions in living cells (concentrations and ionic conditions are changed, enzymes may be activated or inactivated, metabolic pathways are inhibited, etc.) and presumably because of this the specificity of trimming we and Reits et al
) observe in vivo
is not identical to that reported by Shatz et al
). Defining what is occurring in vivo
is important biologically because the specificity of trimming can clearly influence the magnitude of responses and overall immunodominance hierarchies. Here we analyze the trimming of precursors in the cytosol of living cells and compare it to the trimming of the same precursors in the ER.
Our experimental approach was to express, in living cells, N-extended precursors in which we systematically varied the amino acids at the P2 and/or P1 position N-terminal to the SL8 epitope. Our findings clearly demonstrate that: (1) Trimming of these precursors can occur both in the cytosol and the ER; (2) The efficiency of cytosolic trimming process, like that of ER trimming (32
) is affected by the N-terminal residues, i.e. it has specificity; (3) The specificity of cytosolic trimming is distinct from that in the ER; (4) Recycling of peptides from the ER to the cytosol may occur, potentially allowing sequential trimming of peptides in both compartments in either order; and (5) The net effect of cytosolic trimming is to broaden the repertoire of peptides that can be presented on MHC class I molecules.
Our experimental approach makes certain assumptions that are worth discussing. We expressed a series of peptides from minigenes that were transfected into antigen presenting cells. Our interpretation of the results assumes that the transcription, translation and, for Ub-X constructs, post-translational ubiquitin cleavage, are similar for all constructs. Because the ubiquitin construct bicistronically expresses GFP, we are able to gate on cells expressing similar levels of GFP that should also be expressing similar levels of the ubiquitin fusion proteins. To further test this assumption, we compared presentation from minigene constructs that are processed very differently and obtained very similar results using MAXXSL, Ub-XXS-L and Ub-XS-L constructs. This rules out the possibility that differences in presentation arising from differential ubiquitin-X cleavage and makes it highly unlikely that 1 (X) or 2 (XX) residues placed at different locations (2 or 76 residues) from the translational start site would affect translation (or transcription) and do so in the exact same way. Therefore, differences in translation, transcription and or Ub cleavage are unlikely to account for the differences in presentation that were seen with different specific sequences. While it is possible that these upstream residues may also affect TAP transport of cytosolic precursors, the residues that are consistently associated with high-level presentation of our model epitope are not particularly preferred for TAP translocation (40
). Moreover, we saw very similar results in mouse and human cells, although mouse and human TAP have somewhat different preferences for translocation (42
). We interpret therefore, that presentation observed with the various epitope precursors reflects aminopeptidase specificity within the cell. However, it should be further pointed out that even if TAP selectivity contributes to some of the observed differences, our results still define the overall specificity of antigen presentation within the cell (i.e. cytosolic trimming, TAP transport and ER trimming). In addition, for the ER-targeted constructs we assume that XX residues do not influence cleavage by the signal peptidase (which liberates the epitope precursor from the signal peptide). This is supported by two pieces of data which show that the same results are obtained for sequences that are adjacent (ss X-S-L) or 6 residues away (ss LEQLXS-L) from the signal sequence cleavage site and that presentation from minigenes and trimming of the same sequences by purified ERAP1 correlates well with one another (32
Our data show that much of the trimming of precursor peptides occurs in the ER, even when the peptides are originally generated in the cytosol, but the relative importance of ER vs. cytosolic trimming depends on the specific N-terminal residues. In one case, extensive trimming of a cytosolic precursor can occur in the cytosol (WWS-L). This N-terminal flanking sequence must be very efficiently removed in the cytosol, because when this same construct was targeted into the ER directly it was efficiently trimmed by ERAP1. In general, cytosolic trimming is more important for those sequences that are more poorly trimmed in the ER, broadening the sequences that can be efficiently processed and presented. As a result, the difference between the best and the worst presented constructs was smaller when N-extended peptides were expressed in the cytosol, compared to the ER. Only N-terminal glycine was associated with relatively poor presentation in both cytosolic and ER-targeted constructs. Nevertheless, the trimming that does occur in the cytosol does have specificity. There is a reproducible hierarchy, observed in both human and mouse cells, in the trimming and presentation of the various constructs based on the identity of their N-terminal residues.
Many of the precursors that were targeted by a signal sequence through SEC61 into the ER that were efficiently trimmed were presented equally well in control cells and cells in which TAP was inhibited with ICP47, as expected. However, a remarkable finding was that the presentation of some ER-targeted precursors was strongly inhibited by ICP47. It is formally possible that these were ones that failed to translocate through SEC61. However, this explanation seems unlikely because SEC61 can clearly transport the particular residues, when they are in other locations (e.g. E in S-L), or even when the residue is present as a singlet (ss XS-L) and SEC61 obviously transports proteins that have all 20 amino acids. Another possibility is that the signal peptidase failed to cleave the signal peptide from certain precursors, and these had to be retrotranslocated to the cytosol in order for the signal peptide to be removed. However, whether cleavage does occur depends mainly on features of the signal peptide that remain unchanged in all of the constructs used throughout this study, particularly the amino acids at positions −3 and −1 N-terminal of the cleavage site (reviewed in (46
)). It seems more plausible that these sequences need export and trimming in the cytosol because they are very poorly trimmed in the ER. Indeed, the residues that are ICP47-inhibitable are the ones that are poorly removed by ERAP1 (32
) and are particularly poorly removed when they are present in tandem.
Several previous studies have raised the possibility of export followed by re-import of peptides into the ER for presentation. For example, in vivo,
epitopes are generated in a TAP-dependent mechanism from the signal sequence of LCMV gp33 protein (37
), as are HLA-E-binding epitopes generated from the signal sequences of MHC class I molecules (38
). Such TAP dependent presentation could be due to failed translocation of the signal sequence into the ER or retrotranslocation of peptide precursors from the ER into the cytosol. An elegant study by Altrich-VanLith et al
has shown this to be the case for an epitope derived from tyrosinase (39
). In this system, TAP inhibition significantly diminished presentation from ER targeted precursors with two-residue extensions containing histidine (HX). His is one of the amino acids poorly removed in the ER, and also required cytosolic trimming in our experimental system (ss HHS-L). In addition, earlier studies of isolated microsomes also demonstrated that ER-luminal peptides can be retrotranslocated out of the ER and recycle back in a TAP-dependent fashion (47
). Thus there is growing evidence that ER-to-cytosol peptide translocation can occur, although the biological importance of this phenomenon was unknown. In these earlier studies why retrotranslocation might be important for presentation was not clear. Our findings suggest that ER-to-cytosol recycling may be important in situations where ERAP1 does not trim particular sequences down to mature epitopes. Our data further suggest that recycling of peptides between the ER and cytosol is likely to be a general phenomenon that occurs physiologically and contributes to MHC class I antigen presentation.
In conclusion our findings reveal that the amount of MHC-peptide complex presented to the immune system is determined by the identity of amino acids upstream of the epitope precursor and the ability of aminopeptidases to remove them. In many cases mature epitope generation is a combination of processing which has occurred in the cytosol and the ER. Taken together this demonstrates an important role of aminopeptidases in influencing the specificity of CD8+ T cell responses.