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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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A. Alanine scan of N-terminal trimming of the same 10mer peptide by ERAP1. Peptide variants of the sequence LYWANATRSG designed so that one internal position at a time is sequentially replaced by alanine were analyzed for their susceptibility to N-terminus trimming by 40 ng of ERAP1. Error bars represent the variability between three separate experiments performed in parallel. Less than 1% trimming was detected for peptide LYWANATASG under the conditions of the experiment presented. B. Rates of N-terminus trimming by ERAP1 for peptides LYWANATRSG and LYWANATASG. Substitution of the arginine residue at position 8 by alanine leads to reduction of trimming rates by almost 100 fold. Rates are plotted in logarithmic scale for clarity.
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pone-0003658-g003: A. Alanine scan of N-terminal trimming of the same 10mer peptide by ERAP1. Peptide variants of the sequence LYWANATRSG designed so that one internal position at a time is sequentially replaced by alanine were analyzed for their susceptibility to N-terminus trimming by 40 ng of ERAP1. Error bars represent the variability between three separate experiments performed in parallel. Less than 1% trimming was detected for peptide LYWANATASG under the conditions of the experiment presented. B. Rates of N-terminus trimming by ERAP1 for peptides LYWANATRSG and LYWANATASG. Substitution of the arginine residue at position 8 by alanine leads to reduction of trimming rates by almost 100 fold. Rates are plotted in logarithmic scale for clarity.

Mentions: To investigate whether internal residues of the peptide are important for N-terminal trimming by ERAP1 we used a collection of 10mer peptides based on the sequence LYWANATRSG, where one internal residue at a time is replaced by alanine. In every case the removal of the optimal N-terminal residue type (leucine) by ERAP1 was followed by reverse-phase HPLC. Substituting alanine in most positions affected N-terminal trimming by ERAP1 to a moderate degree. Replacement of the arginine residue at position 8 (relative to the N-terminus of the peptide) resulted to a peptide that was surprisingly resistant to N-terminal trimming by ERAP1 (Figure 3A). Specifically, the N-terminus of the peptide LYWANATASG was trimmed with a rate of 0.35±0.02 pmol/µg ERAP1×sec, whereas the control peptide LYWANATRSG was trimmed with a rate 30.4±7.1 pmol/µg ERAP1×sec, a rate almost 100 fold higher (Figure 3B). This finding indicates that internal positions of the peptide substrate can be just as important in determining N-terminal trimming as the nature of the N-terminus.


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)

A. Alanine scan of N-terminal trimming of the same 10mer peptide by ERAP1. Peptide variants of the sequence LYWANATRSG designed so that one internal position at a time is sequentially replaced by alanine were analyzed for their susceptibility to N-terminus trimming by 40 ng of ERAP1. Error bars represent the variability between three separate experiments performed in parallel. Less than 1% trimming was detected for peptide LYWANATASG under the conditions of the experiment presented. B. Rates of N-terminus trimming by ERAP1 for peptides LYWANATRSG and LYWANATASG. Substitution of the arginine residue at position 8 by alanine leads to reduction of trimming rates by almost 100 fold. Rates are plotted in logarithmic scale for clarity.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2573961&req=5

pone-0003658-g003: A. Alanine scan of N-terminal trimming of the same 10mer peptide by ERAP1. Peptide variants of the sequence LYWANATRSG designed so that one internal position at a time is sequentially replaced by alanine were analyzed for their susceptibility to N-terminus trimming by 40 ng of ERAP1. Error bars represent the variability between three separate experiments performed in parallel. Less than 1% trimming was detected for peptide LYWANATASG under the conditions of the experiment presented. B. Rates of N-terminus trimming by ERAP1 for peptides LYWANATRSG and LYWANATASG. Substitution of the arginine residue at position 8 by alanine leads to reduction of trimming rates by almost 100 fold. Rates are plotted in logarithmic scale for clarity.
Mentions: To investigate whether internal residues of the peptide are important for N-terminal trimming by ERAP1 we used a collection of 10mer peptides based on the sequence LYWANATRSG, where one internal residue at a time is replaced by alanine. In every case the removal of the optimal N-terminal residue type (leucine) by ERAP1 was followed by reverse-phase HPLC. Substituting alanine in most positions affected N-terminal trimming by ERAP1 to a moderate degree. Replacement of the arginine residue at position 8 (relative to the N-terminus of the peptide) resulted to a peptide that was surprisingly resistant to N-terminal trimming by ERAP1 (Figure 3A). Specifically, the N-terminus of the peptide LYWANATASG was trimmed with a rate of 0.35±0.02 pmol/µg ERAP1×sec, whereas the control peptide LYWANATRSG was trimmed with a rate 30.4±7.1 pmol/µg ERAP1×sec, a rate almost 100 fold higher (Figure 3B). This finding indicates that internal positions of the peptide substrate can be just as important in determining N-terminal trimming as the nature of the N-terminus.

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
Related in: MedlinePlus