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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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N-terminal specificity of decapeptide trimming by ERAP1.All peptides are based on the same template varying in their N-terminus (indicated as X in the sequence below the graph). 100 µM of each peptide was incubated with 40 ng of ERAP1 at 37°C and the reaction products analyzed as in figure 1. A representative experiment is shown here. Peptides carrying hydrophobic residues at their N-termini were trimmed fastest, whereas peptides carrying charged (R, K or E) or hydrophilic (T) residues in their N-termini were more resistant to cleavage by ERAP1.
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pone-0003658-g002: N-terminal specificity of decapeptide trimming by ERAP1.All peptides are based on the same template varying in their N-terminus (indicated as X in the sequence below the graph). 100 µM of each peptide was incubated with 40 ng of ERAP1 at 37°C and the reaction products analyzed as in figure 1. A representative experiment is shown here. Peptides carrying hydrophobic residues at their N-termini were trimmed fastest, whereas peptides carrying charged (R, K or E) or hydrophilic (T) residues in their N-termini were more resistant to cleavage by ERAP1.

Mentions: ERAP1 has been characterized before as a leucine aminopeptidase because it preferably degrades dipeptide fluorigenic substrates that have a leucine at their N-terminus [22]. However this strong preference conflicts, to a certain extent, with the role of ERAP1 in antigen processing, where it must trim peptides with a vast range of sequences. To address the N-terminal specificity of ERAP1 when degrading peptides we over-expressed human recombinant full-length ERAP1 in insect cell suspension culture and used the purified active enzyme in degradation assays in which we measured the amount of trimming of the N-terminal residue of a panel of peptides that varied only on their N-terminus (Figure 1 and 2). We found that in agreement with the dipeptide digestion results, leucine was the preferred N-terminal residue. Other hydrophobic residues, such as methionine, phenylalanine and alanine were also digested reasonably fast. Non-optimal residues such as charged or hydrophilic in nature, required larger (at least 10 times) amounts of ERAP1 for their removal (data not shown). Overall, ERAP1 appears to indeed act as a leucine aminopeptidase when degrading model peptides although the digestion proceeds, albeit at a much slower rate, for other residues as well.


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)

N-terminal specificity of decapeptide trimming by ERAP1.All peptides are based on the same template varying in their N-terminus (indicated as X in the sequence below the graph). 100 µM of each peptide was incubated with 40 ng of ERAP1 at 37°C and the reaction products analyzed as in figure 1. A representative experiment is shown here. Peptides carrying hydrophobic residues at their N-termini were trimmed fastest, whereas peptides carrying charged (R, K or E) or hydrophilic (T) residues in their N-termini were more resistant to cleavage by ERAP1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2573961&req=5

pone-0003658-g002: N-terminal specificity of decapeptide trimming by ERAP1.All peptides are based on the same template varying in their N-terminus (indicated as X in the sequence below the graph). 100 µM of each peptide was incubated with 40 ng of ERAP1 at 37°C and the reaction products analyzed as in figure 1. A representative experiment is shown here. Peptides carrying hydrophobic residues at their N-termini were trimmed fastest, whereas peptides carrying charged (R, K or E) or hydrophilic (T) residues in their N-termini were more resistant to cleavage by ERAP1.
Mentions: ERAP1 has been characterized before as a leucine aminopeptidase because it preferably degrades dipeptide fluorigenic substrates that have a leucine at their N-terminus [22]. However this strong preference conflicts, to a certain extent, with the role of ERAP1 in antigen processing, where it must trim peptides with a vast range of sequences. To address the N-terminal specificity of ERAP1 when degrading peptides we over-expressed human recombinant full-length ERAP1 in insect cell suspension culture and used the purified active enzyme in degradation assays in which we measured the amount of trimming of the N-terminal residue of a panel of peptides that varied only on their N-terminus (Figure 1 and 2). We found that in agreement with the dipeptide digestion results, leucine was the preferred N-terminal residue. Other hydrophobic residues, such as methionine, phenylalanine and alanine were also digested reasonably fast. Non-optimal residues such as charged or hydrophilic in nature, required larger (at least 10 times) amounts of ERAP1 for their removal (data not shown). Overall, ERAP1 appears to indeed act as a leucine aminopeptidase when degrading model peptides although the digestion proceeds, albeit at a much slower rate, for other residues as well.

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