MHC class I epitopes are generated through a complex pathway that is influenced by proteasomes, peptidases, TAP, and tapasin as well as physical binding of mature epitopes to MHC class I alleles. The efficiencies by which potential epitopes are generated from precursor proteins influences epitope abundance and recognition, which is important in vaccine design and the development of immunodominance hierarchies. While some steps in the pathway are relatively well understood (e.g. TAP translocation, proteasome processing) the importance and direction of some other steps (e.g. peptidase processing) have not been as well studied.
N-terminally-extended epitopes, generated by the proteasome (16
), can be trimmed by aminopeptidases to generate peptides of the correct length for MHC class-I binding (24
). However, only a small fraction of the peptide pool bind MHC class-I molecules and even fewer trigger a significant T-cell response. This immunodominance can be a result of antigen processing and ultimately epitope abundance at the cell (9
). In cells lacking the ER-resident aminopeptidase ERAP1, ER-targeted precursors are presented poorly if at all and ERAP1-deficient mice have markedly different presentation of many MHC class I epitopes (11
). Previous studies have also shown that this aminopeptidase can contribute to the pattern of immunodominance (11
). ERAP1 is a unique aminopeptidase in that its substrate preferences are guided in part by the C-terminus of the recognized peptide (28
). However, its specificity is not otherwise well characterized.
Most previous analyses of the N-terminal sequence specificity of ERAP1 have been done with non-physiological substrates (such as dipeptides) and it has been clear that in some cases such substrates don’t model ERAP1’s behavior with longer “physiological” epitope precursors (43
) Moreover, these analyses have been primarily performed in cell free systems with purified enzymes or microsomes and it is not clear whether these assays replicate what occurs in living cells. Since ERAP1 is such a key player in the antigen presentation pathway we set out to understand its specificity for N-terminal amino acids on N-extended epitopes and how this specificity could influence peptide delivery to MHC class-I molecules.
Using purified ERAP1 we showed that this aminopeptidase was capable of removing many different amino acids from the N-terminus of an epitope precursor. This broad activity is of obvious biological importance in allowing ERAP1 to trim the panoply of MHC class I-presented epitopes. However, the rates at which N-terminal residues were cleaved varied considerably and reproducibly between different amino acids. This suggests that ERAP1s specificity could limit peptide supply from ER precursors if epitopes are extended on the N-terminus by unfavorable amino acids. Conversely, more efficient trimming by ERAP1 of other amino acids could lead to greater epitope production for competition and presentation to the immune system. Previous studies characterizing ERAP1 or microsomal extracts, using a limited set of simple single amino acid fluorogenic substrates (X-AMC) (38
) have shown activity primarily for cleaving Leu and Met residues. This is consistent with our results that show that these amino acids are the most rapidly removed from the XS-L peptides and presumably reflects the higher affinity of Leu and Met for the ERAP1 active site. However, it is clear from our work that ERAP1 can remove many other amino acids, although more slowly. Our results are largely consistent with an analysis of the trimming of a limited number of peptide precursors by microsomes in vitro (38
); the few variations between these studies, particularly for charged amino acids, may reflect differences between our using a pure enzyme versus the earlier study using crude microsome preparations (potentially contaminated with cytosolic peptidases).
Another finding of interest was that regardless of the N-terminal flanking residues, purified ERAP1 virtually stopped trimming when the mature S-L epitope was generated. This is consistent with our previous findings, that ERAP1 trims with a molecular ruler down to a peptide of 8 or 9 residues in length (27
), and indicates that this ruler is not influenced by the identity of the P1 residue. This unique function may contribute to the dominant role ERAP1 plays in antigen presentation and epitope generation due to the fact that trimming ceases once peptides capable of MHC class-I binding are generated. Kanaseki et al
recently suggested that ERAP1 does not have a molecular ruler because it hydrolyzed and destroyed a mature antigenic epitope (45
); however this epitope was a 9mer and we have previously established that ERAP hydrolyzes about 50% of 9 mers to 8mers (27
). In other words, the concept of a molecular ruler is that ERAP1 trims down to a final core size (which can be 8 or 9 residues) and this core may or may not result in a mature epitope. This explains why ERAP1 can help to both create and destroy epitopes (11
). Importantly, the specificity of ERAP1 for N-terminal residues was not just seen at the 9mer to 8mer conversion but also at sequences further upstream of the epitope (e.g. the conversion of 12mers to 11mers to 10mers). To determine the relevance of these findings to the in vivo
situation we transfected cells with minigenes encoding ER-targeted (signal sequence) epitope precursors. The amino acid in the P1 or P2 positions strongly influenced the amount of MHC class I presentation from precursors in the ER. The same trends were seen whether the residue upstream of the epitope was present as a single amino acid or as a doublet, when the residue was preceded by a longer sequence (e.g. LEQLX
S-L), when the precursor peptide was a 10mer or a 12mer, and when different unrelated epitopes were used including 8mers and 9mers (S-L, F-L, or R-Y). When residues that were efficiently or poorly removed were paired in either orientation, the poor residue usually dominated and led to low presentation, as expected since trimming of the N-terminal extension would be affected whether it is the first or second residue that is removed slowly. Taken together these results demonstrate that in vivo
the amino acids in the P1 or P2 positions of both long and short precursors are critical determinants of the amount of epitope presented on MHC class I molecules.
The specificity of trimming of the ER-targeted precursors was similar in both HeLa and COS7 cells. This suggests that our findings are not cell type specific but more general, although additional cell types need to be examined to fully evaluate this issue. Another point is that since COS7 cells express ERAP1 and ERAP2, our findings suggest that ERAP1 is the dominant ER-trimming enzyme; of note this is true even for charged residues that ERAP2 could potentially trim. Nevertheless, it is possible that the few differences in presentation that were observed between HeLa and COS7 cells were due to the presence or absence of ERAP2. Moreover, it remains possible that ERAP2 plays a more important role for other sequences and/or in different cells.
We were not able to identify any simple chemical feature that determined whether an amino acid is a good substrate or not. In general, charged residues are processed slowly; but the large hydrophobic Trp, the polar non-charged Asn, and the small non-polar Gly are each also processed slowly. Thus while there is clear specificity in the trimming process it is not simply defined by the chemical class of amino acid side chains.
In almost all cases when two amino acids from either end of the presentation hierarchy were combined the inefficient residue was dominant and led to poor presentation. One exception suggests that context (i.e. adjacent amino acids, or peptide length) may also influence processing of some residues. When valine was immediately adjacent to S-L or F-L, the precursors were slowly trimmed, whereas when Val was separated from S-L by one residue (e.g. VLS-L) or in the peptide VR-Y, processing was efficient. It is also interesting that even when the presentation of epitopes preceded by an efficient/inefficient or inefficient/efficient pair was low, it was better than was observed with the inefficient residue alone, suggesting that the rate of trimming in vivo is also influenced to some extent by adjacent amino acids or peptide length. Similarly, presentation of the long XXYYS-L peptides was generally higher than for the same residue as an XXS-L peptide. Nevertheless, it is important to emphasize that while context may influence trimming (particularly of valine), there clearly are definable rules of efficiency and these rules apply with different epitopes.
Knocking down ERAP1 strongly inhibited presentation from the vast majority of ER targeted epitope precursors, suggesting a major role in trimming. We interpret the difference in presentation between the different precursors as reflecting the specificity of ERAP1 trimming. This conclusion makes the assumption that the various XX residues do not influence cleavage by the signal peptidase (which liberates the epitope precursor from the signal peptide). Two pieces of data support this assumption. The first is that we have observed essentially the same results for sequences that are adjacent (ss X-S-L) or 6 residues away (ss LEQLXS-L) from the signal sequence cleavage site. The second is that the results with the presentation from minigenes and trimming of the same sequences by purified ERAP1 correlates well with one another. Nevertheless, it should be noted that this in vitro to in vivo correlation is not 100%. Where concordance is not perfect there must be other factors that influence peptide trimming in vivo. In these situations we can’t exclude a minor contribution from the signal peptidase although the difference could equally well reflect the participation of other ER molecules (chaperones, other peptidases) or that the conditions in the ER and cell free buffer system are not identical and this somehow influences ERAP1’s properties. A complete understanding of ERAP1’s specificity will require further study and may be aided when its crystal structure is solved.
We believe our results are potentially contributory to helping to define some of the specificity of antigen processing. The power of current algorithms to predict presented peptides from the sequence of an antigen is limited presumably because they do not take into account all of the events, such as peptide trimming, that are needed to generate (or destroy) epitopes. Consistent with these idea that peptide trimming is an important factor in determining the repertoire of MHC class I-presented peptides (the presented “peptidome”) we find that the residues that empirically lead to high-level antigen presentation in our system (Tyr, Met, Leu, Ala, and Cys) are all over-represented N-terminal to natural epitopes, by at least 2 standard deviations above background frequency; while residues that are underrepresented adjacent to natural epitopes (Pro and Val, and charged residues as a group) are poorly processed in our system. Overall these results are mostly similar qualitatively to the earlier analysis of a smaller set of sequences by Schatz et al (38
). It should be noted that one limitation of the databases used for these analyses is that the majority of the epitopes were defined based of their ability to optimally stimulate specific CD8 T cells and because of this some may not be identical to the naturally processed peptides. However, it is thought that only a small minority of epitopes are incorrectly assigned and therefore this should have a minimal effect on such analyses. Therefore, nonrandom frequencies of various amino acids are almost certainly due to differences in antigen processing.
While it is possible that the upstream residues also affect proteasome cleavage or TAP transport, the residues that are consistently associated with high-level presentation of our model epitopes are not particularly preferred for proteasomal cleavage (46
) or TAP translocation (23
). This implies that aminopeptidase specificities, specifically ERAP1, play an important role in vivo
in determining the generation of presented peptides, and that our system accurately defines at least some of these specificities. It is in fact remarkable that correlations are seen between ERAP1’s specificity and the presented peptidome because there are other peptidases (e.g. cytosolic ones) that can contribute to trimming N-terminal residues.
Presumably other peptidases do contribute to the trimming of the sequences that are poorly trimmed by ERAP1; this may especially be the case for epitopes flanked by charged residues. Such flanking residues show only a modest reduction in frequency in the databases of presented peptides yet are very poorly trimmed by ERAP1 in vitro and in vivo
. This might be because cytosolic aminopeptidases such as leucine aminopeptidase can remove charged residues (15
). Alternatively, the proteasome’s tryptic active site, which cleaves preferentially on the carboxlylic side of basic residues and its caspase-like site, which cleaves after acid residues, might remove these charged residues. Consistent with this later idea, the presentation from ovalbumin of SIINFEKL (which is flanked on its N-terminus with a charged E residue) is not affected in ERAP1 deficient cells. Interestingly, although ERAP2 can hydrolyze basic residues (40
), we find that precursors with charged flanking residues are still poorly presented in ERAP2-expressing cells.
In summary our findings reveal that the amount of MHC-peptide complex presented to the immune system is determined in part by the amino acids upstream of epitope precursors and the ability of ERAP1 to remove these. The specificity of this trimming process for ERAP1 in vivo and in vitro for multiple epitopes is defined and reveals that that ERAP1 influences the extent of presentation in predictable ways.