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

Related in: MedlinePlus

Trimming of the N-terminal leucine of N- and C- extended versions of LSIINFEKL peptide.Panels A–C, typical C18 chromatograms comparing the trimming for LSIINFEKL and the C- and N-terminal extended versions LSIINFEKLAAAL and LAAALSIINFEKL. In each panel two chromatograms are shown, the top one in the absence of enzyme and the bottom in the presence of 40 ng ERAP1. All incubations were done for 1 hr at 37°C. Black arrows indicate new peaks detected after incubation with the enzyme. Under these conditions LSIINFEKL and LSIINFEKLAAAL are trimmed about to the same extent and LAAALSIINFEKL is resistant to any trimming. Panel D, ERAP1 trimming rates calculated as the average of 3 to 5 experiments. Because of the large difference between trimming rates the y-axis is logarithmic for better visualization.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2573961&req=5

pone-0003658-g008: Trimming of the N-terminal leucine of N- and C- extended versions of LSIINFEKL peptide.Panels A–C, typical C18 chromatograms comparing the trimming for LSIINFEKL and the C- and N-terminal extended versions LSIINFEKLAAAL and LAAALSIINFEKL. In each panel two chromatograms are shown, the top one in the absence of enzyme and the bottom in the presence of 40 ng ERAP1. All incubations were done for 1 hr at 37°C. Black arrows indicate new peaks detected after incubation with the enzyme. Under these conditions LSIINFEKL and LSIINFEKLAAAL are trimmed about to the same extent and LAAALSIINFEKL is resistant to any trimming. Panel D, ERAP1 trimming rates calculated as the average of 3 to 5 experiments. Because of the large difference between trimming rates the y-axis is logarithmic for better visualization.

Mentions: Many of the antigenic peptide precursors that are sent to the ER through the action of TAP are peptides up to 13–15 amino acid [39]. To test whether strong internal sequence related effects are also evident when ERAP1 is trimming peptides of such lengths and also to gain insight on the mode of molecular recognition of peptides by ERAP1 we measured the N-terminal trimming of 3 peptides based on the sequence of the ovalbumin antigenic epitope SIINFEKL (Figure 8). A control peptide with the sequence LSIINFEKL was used that we expected (based on previously published results on closely related sequences[19]) to be trimmed efficiently by ERAP1 leading to the removal of the N-terminal leucine residue. Two more peptides were tested: a peptide with the sequence LSIINFEKLAAAL and one with the sequence LAAALSIINFEKL, both based on the same internal template LSIINFEKL but with C- and N-terminal alanine extensions respectively. We designed the extensions to contain alanine residues since alanine lacks an extended side chain that may offer extra interactions with the binding site of the enzyme and complicate this analysis. Since it has been established that both the N- and C- termini of the peptide are important for determining the rate of trimming by ERAP1, we designed all 3 peptides to carry the same N and C terminal residue.


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 leucine of N- and C- extended versions of LSIINFEKL peptide.Panels A–C, typical C18 chromatograms comparing the trimming for LSIINFEKL and the C- and N-terminal extended versions LSIINFEKLAAAL and LAAALSIINFEKL. In each panel two chromatograms are shown, the top one in the absence of enzyme and the bottom in the presence of 40 ng ERAP1. All incubations were done for 1 hr at 37°C. Black arrows indicate new peaks detected after incubation with the enzyme. Under these conditions LSIINFEKL and LSIINFEKLAAAL are trimmed about to the same extent and LAAALSIINFEKL is resistant to any trimming. Panel D, ERAP1 trimming rates calculated as the average of 3 to 5 experiments. Because of the large difference between trimming rates the y-axis is logarithmic for better visualization.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003658-g008: Trimming of the N-terminal leucine of N- and C- extended versions of LSIINFEKL peptide.Panels A–C, typical C18 chromatograms comparing the trimming for LSIINFEKL and the C- and N-terminal extended versions LSIINFEKLAAAL and LAAALSIINFEKL. In each panel two chromatograms are shown, the top one in the absence of enzyme and the bottom in the presence of 40 ng ERAP1. All incubations were done for 1 hr at 37°C. Black arrows indicate new peaks detected after incubation with the enzyme. Under these conditions LSIINFEKL and LSIINFEKLAAAL are trimmed about to the same extent and LAAALSIINFEKL is resistant to any trimming. Panel D, ERAP1 trimming rates calculated as the average of 3 to 5 experiments. Because of the large difference between trimming rates the y-axis is logarithmic for better visualization.
Mentions: Many of the antigenic peptide precursors that are sent to the ER through the action of TAP are peptides up to 13–15 amino acid [39]. To test whether strong internal sequence related effects are also evident when ERAP1 is trimming peptides of such lengths and also to gain insight on the mode of molecular recognition of peptides by ERAP1 we measured the N-terminal trimming of 3 peptides based on the sequence of the ovalbumin antigenic epitope SIINFEKL (Figure 8). A control peptide with the sequence LSIINFEKL was used that we expected (based on previously published results on closely related sequences[19]) to be trimmed efficiently by ERAP1 leading to the removal of the N-terminal leucine residue. Two more peptides were tested: a peptide with the sequence LSIINFEKLAAAL and one with the sequence LAAALSIINFEKL, both based on the same internal template LSIINFEKL but with C- and N-terminal alanine extensions respectively. We designed the extensions to contain alanine residues since alanine lacks an extended side chain that may offer extra interactions with the binding site of the enzyme and complicate this analysis. Since it has been established that both the N- and C- termini of the peptide are important for determining the rate of trimming by ERAP1, we designed all 3 peptides to carry the same N and C terminal residue.

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