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The internal sequence of the peptide-substrate determines its N-terminus trimming by ERAP1.

Evnouchidou I, Momburg F, Papakyriakou A, Chroni A, Leondiadis L, Chang SC, Goldberg AL, Stratikos E - PLoS ONE (2008)

Bottom Line: Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini.Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide's length and C-terminus.It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Scientific Research Demokritos, IRRP, Aghia Paraskevi, Greece.

ABSTRACT

Background: Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims N-terminally extended antigenic peptide precursors down to mature antigenic peptides for presentation by major histocompatibility complex (MHC) class I molecules. ERAP1 has unique properties for an aminopeptidase being able to trim peptides in vitro based on their length and the nature of their C-termini.

Methodology/principal findings: In an effort to better understand the molecular mechanism that ERAP1 uses to trim peptides, we systematically analyzed the enzyme's substrate preferences using collections of peptide substrates. We discovered strong internal sequence preferences of peptide N-terminus trimming by ERAP1. Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini. Molecular modelling of ERAP1 revealed a large internal cavity that carries a strong negative electrostatic potential and is large enough to accommodate peptides adjacent to the enzyme's active site. This model can readily account for the strong preference for positively charged side chains.

Conclusions/significance: To our knowledge no other aminopeptidase has been described to have such strong preferences for internal residues so distal to the N-terminus. Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide's length and C-terminus. We therefore propose that ERAP1 recognizes the full length of its peptide-substrate and not just the N- and C- termini. It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.

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Trimming of the N-terminal residue of a library of 9mer peptides by ERAP1.Peptide series vary in one position per collection (indicated on the top of each panel; varying amino acid is shown as X) and are presented from left to right and from top to bottom for positions 2 to 9 from the N-terminus. The y-axis indicates percentage of substrate (peptide) depleted based on analysis by HPLC. The x-axis indicates the amino acid in the particular peptide in each collection, ordered by hydrophobicity (from hydrophilic amino-acids to hydrophobic). Note that for some collections the effect of substituting for particular amino acids is much higher than in others. One representative experiment is presented for each peptide set.
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pone-0003658-g004: Trimming of the N-terminal residue of a library of 9mer peptides by ERAP1.Peptide series vary in one position per collection (indicated on the top of each panel; varying amino acid is shown as X) and are presented from left to right and from top to bottom for positions 2 to 9 from the N-terminus. The y-axis indicates percentage of substrate (peptide) depleted based on analysis by HPLC. The x-axis indicates the amino acid in the particular peptide in each collection, ordered by hydrophobicity (from hydrophilic amino-acids to hydrophobic). Note that for some collections the effect of substituting for particular amino acids is much higher than in others. One representative experiment is presented for each peptide set.

Mentions: To systematically test the role of internal residues of peptide substrates on the rate of N-terminus trimming by ERAP1 we used the active recombinant enzyme in degradation assays with an already available collection of more than 70 synthetic model peptides (Figure 4). This peptide collection has been used before in the investigation of the specificity of the peptide transporter TAP [11]. All peptides were 9mers and had a threonine residue at their N-termini. Degradation of the N-terminus of each peptide was followed by HPLC as described in the experimental section. Each peptide series (varying at one position) was analyzed in parallel to account for variability in enzyme activity between preparations and during storage. Several experiments were performed for each peptide series to fine-tune the reaction conditions (reaction time and amount of enzyme used) and to test reproducibility of results. One representative set is shown in Figure 4. The efficiency of ERAP1 trimming was strongly affected by the nature of the residue at several positions in the peptide sequence (Figure 4). Specifically, positions 2, 5 and 7 (with position 1 defined as the N-terminal residue of the peptide) were found to be most important for the sensitivity of the peptide to ERAP1 degradation. Some degree of residue preference was also evident for positions 4, 8 and 9. Positions 3 and 6 showed the least specificity although some small effects were present. Residue preferences were only seen for hydrophobic and positively charged residues. No preference was seen for negative or hydrophilic residues in any of the positions. The presence of a negatively charged residue (glutamate) anywhere in the peptide sequence seemed to negatively affect the peptide's degradation by ERAP1 regardless of its location in the peptide sequence (to a lesser extent for position 3). The same general observation seems to apply for glycine and proline residues. Certain positions showed a very strong preference for particular amino-acid side-chains. Position 2 for example, exhibited a strong preference for a methionine residue whereas position 7 showed a very strong preference for positively charged residues (lysine or arginine). Interestingly, position 5 showed a strong preference for either a positive charge or an aromatic residue (phenylalanine). This “dual” preference was observed in other positions also (position 9) and may indicate alternative binding configurations for the two peptides (refer to the molecular modeling section below). Overall, strong sequence preferences were clearly evident from this library screen even without a more detailed kinetic study. To simplify screening, a single time point analysis was used in Figure 4 and as a result some of the differences seen there could be under-estimations of the kinetic differences, especially for reactions where the substrate consumption is over 50%. However, several of the preferences are so strong that are clearly evident even from a single time-point analysis.


The internal sequence of the peptide-substrate determines its N-terminus trimming by ERAP1.

Evnouchidou I, Momburg F, Papakyriakou A, Chroni A, Leondiadis L, Chang SC, Goldberg AL, Stratikos E - PLoS ONE (2008)

Trimming of the N-terminal residue of a library of 9mer peptides by ERAP1.Peptide series vary in one position per collection (indicated on the top of each panel; varying amino acid is shown as X) and are presented from left to right and from top to bottom for positions 2 to 9 from the N-terminus. The y-axis indicates percentage of substrate (peptide) depleted based on analysis by HPLC. The x-axis indicates the amino acid in the particular peptide in each collection, ordered by hydrophobicity (from hydrophilic amino-acids to hydrophobic). Note that for some collections the effect of substituting for particular amino acids is much higher than in others. One representative experiment is presented for each peptide set.
© Copyright Policy
Related In: Results  -  Collection

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pone-0003658-g004: Trimming of the N-terminal residue of a library of 9mer peptides by ERAP1.Peptide series vary in one position per collection (indicated on the top of each panel; varying amino acid is shown as X) and are presented from left to right and from top to bottom for positions 2 to 9 from the N-terminus. The y-axis indicates percentage of substrate (peptide) depleted based on analysis by HPLC. The x-axis indicates the amino acid in the particular peptide in each collection, ordered by hydrophobicity (from hydrophilic amino-acids to hydrophobic). Note that for some collections the effect of substituting for particular amino acids is much higher than in others. One representative experiment is presented for each peptide set.
Mentions: To systematically test the role of internal residues of peptide substrates on the rate of N-terminus trimming by ERAP1 we used the active recombinant enzyme in degradation assays with an already available collection of more than 70 synthetic model peptides (Figure 4). This peptide collection has been used before in the investigation of the specificity of the peptide transporter TAP [11]. All peptides were 9mers and had a threonine residue at their N-termini. Degradation of the N-terminus of each peptide was followed by HPLC as described in the experimental section. Each peptide series (varying at one position) was analyzed in parallel to account for variability in enzyme activity between preparations and during storage. Several experiments were performed for each peptide series to fine-tune the reaction conditions (reaction time and amount of enzyme used) and to test reproducibility of results. One representative set is shown in Figure 4. The efficiency of ERAP1 trimming was strongly affected by the nature of the residue at several positions in the peptide sequence (Figure 4). Specifically, positions 2, 5 and 7 (with position 1 defined as the N-terminal residue of the peptide) were found to be most important for the sensitivity of the peptide to ERAP1 degradation. Some degree of residue preference was also evident for positions 4, 8 and 9. Positions 3 and 6 showed the least specificity although some small effects were present. Residue preferences were only seen for hydrophobic and positively charged residues. No preference was seen for negative or hydrophilic residues in any of the positions. The presence of a negatively charged residue (glutamate) anywhere in the peptide sequence seemed to negatively affect the peptide's degradation by ERAP1 regardless of its location in the peptide sequence (to a lesser extent for position 3). The same general observation seems to apply for glycine and proline residues. Certain positions showed a very strong preference for particular amino-acid side-chains. Position 2 for example, exhibited a strong preference for a methionine residue whereas position 7 showed a very strong preference for positively charged residues (lysine or arginine). Interestingly, position 5 showed a strong preference for either a positive charge or an aromatic residue (phenylalanine). This “dual” preference was observed in other positions also (position 9) and may indicate alternative binding configurations for the two peptides (refer to the molecular modeling section below). Overall, strong sequence preferences were clearly evident from this library screen even without a more detailed kinetic study. To simplify screening, a single time point analysis was used in Figure 4 and as a result some of the differences seen there could be under-estimations of the kinetic differences, especially for reactions where the substrate consumption is over 50%. However, several of the preferences are so strong that are clearly evident even from a single time-point analysis.

Bottom Line: Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini.Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide's length and C-terminus.It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Scientific Research Demokritos, IRRP, Aghia Paraskevi, Greece.

ABSTRACT

Background: Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims N-terminally extended antigenic peptide precursors down to mature antigenic peptides for presentation by major histocompatibility complex (MHC) class I molecules. ERAP1 has unique properties for an aminopeptidase being able to trim peptides in vitro based on their length and the nature of their C-termini.

Methodology/principal findings: In an effort to better understand the molecular mechanism that ERAP1 uses to trim peptides, we systematically analyzed the enzyme's substrate preferences using collections of peptide substrates. We discovered strong internal sequence preferences of peptide N-terminus trimming by ERAP1. Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini. Molecular modelling of ERAP1 revealed a large internal cavity that carries a strong negative electrostatic potential and is large enough to accommodate peptides adjacent to the enzyme's active site. This model can readily account for the strong preference for positively charged side chains.

Conclusions/significance: To our knowledge no other aminopeptidase has been described to have such strong preferences for internal residues so distal to the N-terminus. Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide's length and C-terminus. We therefore propose that ERAP1 recognizes the full length of its peptide-substrate and not just the N- and C- termini. It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.

Show MeSH