Limits...
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.

Show MeSH
Typical chromatogram of peptide product analysis after ERAP1 digestion.Samples were analyzed by HPLC reverse phase chromatography. 100 µM peptide with sequence FYWANATRSG (written from N-terminus to C-terminus) was mixed with 40 ng of ERAP1 and the mixture was incubated at 37°C. At different time points a sample of the reaction was extracted and mixed with an equal volume of 0.6% Trifluoroacetic acid (to stop the enzymatic reaction) and kept at −20°C until analysis. Solid line: Sample at zero time point, indicating the elution of the undigested peptide (peak 1, confirmed by control runs of peptide alone). Gray line: Sample after 1 hr of incubation. The surface area of peak 1 is reduced indicating partial digestion of peptide. A new peak can be seen (peak 2) corresponding to the peptide product of the reaction YWANATRSG. The reduction of the surface area of peak 1 is equal to the surface of peak 2 and is typically used to calculate the percent consumption of the peptide substrate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2573961&req=5

pone-0003658-g001: Typical chromatogram of peptide product analysis after ERAP1 digestion.Samples were analyzed by HPLC reverse phase chromatography. 100 µM peptide with sequence FYWANATRSG (written from N-terminus to C-terminus) was mixed with 40 ng of ERAP1 and the mixture was incubated at 37°C. At different time points a sample of the reaction was extracted and mixed with an equal volume of 0.6% Trifluoroacetic acid (to stop the enzymatic reaction) and kept at −20°C until analysis. Solid line: Sample at zero time point, indicating the elution of the undigested peptide (peak 1, confirmed by control runs of peptide alone). Gray line: Sample after 1 hr of incubation. The surface area of peak 1 is reduced indicating partial digestion of peptide. A new peak can be seen (peak 2) corresponding to the peptide product of the reaction YWANATRSG. The reduction of the surface area of peak 1 is equal to the surface of peak 2 and is typically used to calculate the percent consumption of the peptide substrate.

Mentions: The digestion of model peptides by ERAP1 was followed by analysis of peptide products of the digestion on a reverse phase C18 column (Higgins Analytical 0546-C183) [16]. Briefly, 100 µM peptide was mixed in 50 µL total volume with 40 to 400 ng purified recombinant enzyme in 20 mM Tris pH 8 buffer containing 100 mM NaCl. The mixture was incubated at 37°C for 30 min to 4 hrs. After incubation the reaction was stopped by the addition of 50 µL of 0.6% TFA and the sample centrifuged for 15 min at 15 000 g. 50 µL of the supernatant were subjected to HPLC analysis. The reverse phase column was equilibrated in either 0.05% trifluoroacetic acid and 5% acetonitrile or 10 mM Sodium Phosphate pH 6.8, 5% acetonitrile before the sample was injected. The elution was done with a 5% to 40% acetonitrile gradient at 1 ml/min, while following the absorbance at 214 nm or 280 nm. Typically, the decrease in peak surface area for a specific peptide (identified by running control experiments or by LC-MS experiments) upon digestion with ERAP1 was used to estimate the amount of peptide that was digested. In all cases, the decrease of the initial peak resulted in the appearance of a new single product peak that had a surface area equal to the surface area decrease of the substrate peak (measured from a control experiment in the absence of enzyme). A typical chromatogram of peptide product analysis after ERAP1 digestion is shown in Figure 1. Several experiments were performed for each peptide tested to fine-tune the reaction conditions (reaction time and amount of enzyme used) and to test reproducibility of results. Each peptide series (varying at one position) was tested in parallel to account for possible variability in the reactions due to changes in enzyme activity upon storage and from preparation to preparation.


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)

Typical chromatogram of peptide product analysis after ERAP1 digestion.Samples were analyzed by HPLC reverse phase chromatography. 100 µM peptide with sequence FYWANATRSG (written from N-terminus to C-terminus) was mixed with 40 ng of ERAP1 and the mixture was incubated at 37°C. At different time points a sample of the reaction was extracted and mixed with an equal volume of 0.6% Trifluoroacetic acid (to stop the enzymatic reaction) and kept at −20°C until analysis. Solid line: Sample at zero time point, indicating the elution of the undigested peptide (peak 1, confirmed by control runs of peptide alone). Gray line: Sample after 1 hr of incubation. The surface area of peak 1 is reduced indicating partial digestion of peptide. A new peak can be seen (peak 2) corresponding to the peptide product of the reaction YWANATRSG. The reduction of the surface area of peak 1 is equal to the surface of peak 2 and is typically used to calculate the percent consumption of the peptide substrate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2573961&req=5

pone-0003658-g001: Typical chromatogram of peptide product analysis after ERAP1 digestion.Samples were analyzed by HPLC reverse phase chromatography. 100 µM peptide with sequence FYWANATRSG (written from N-terminus to C-terminus) was mixed with 40 ng of ERAP1 and the mixture was incubated at 37°C. At different time points a sample of the reaction was extracted and mixed with an equal volume of 0.6% Trifluoroacetic acid (to stop the enzymatic reaction) and kept at −20°C until analysis. Solid line: Sample at zero time point, indicating the elution of the undigested peptide (peak 1, confirmed by control runs of peptide alone). Gray line: Sample after 1 hr of incubation. The surface area of peak 1 is reduced indicating partial digestion of peptide. A new peak can be seen (peak 2) corresponding to the peptide product of the reaction YWANATRSG. The reduction of the surface area of peak 1 is equal to the surface of peak 2 and is typically used to calculate the percent consumption of the peptide substrate.
Mentions: The digestion of model peptides by ERAP1 was followed by analysis of peptide products of the digestion on a reverse phase C18 column (Higgins Analytical 0546-C183) [16]. Briefly, 100 µM peptide was mixed in 50 µL total volume with 40 to 400 ng purified recombinant enzyme in 20 mM Tris pH 8 buffer containing 100 mM NaCl. The mixture was incubated at 37°C for 30 min to 4 hrs. After incubation the reaction was stopped by the addition of 50 µL of 0.6% TFA and the sample centrifuged for 15 min at 15 000 g. 50 µL of the supernatant were subjected to HPLC analysis. The reverse phase column was equilibrated in either 0.05% trifluoroacetic acid and 5% acetonitrile or 10 mM Sodium Phosphate pH 6.8, 5% acetonitrile before the sample was injected. The elution was done with a 5% to 40% acetonitrile gradient at 1 ml/min, while following the absorbance at 214 nm or 280 nm. Typically, the decrease in peak surface area for a specific peptide (identified by running control experiments or by LC-MS experiments) upon digestion with ERAP1 was used to estimate the amount of peptide that was digested. In all cases, the decrease of the initial peak resulted in the appearance of a new single product peak that had a surface area equal to the surface area decrease of the substrate peak (measured from a control experiment in the absence of enzyme). A typical chromatogram of peptide product analysis after ERAP1 digestion is shown in Figure 1. Several experiments were performed for each peptide tested to fine-tune the reaction conditions (reaction time and amount of enzyme used) and to test reproducibility of results. Each peptide series (varying at one position) was tested in parallel to account for possible variability in the reactions due to changes in enzyme activity upon storage and from preparation to preparation.

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