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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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Substitution of a lysine residue at position 8 of two model 9mer H2-Kb epitope precursors leads to a marked decrease of N-terminus trimming by ERAP1 in vitro.Panels A and B, peptides LSIINFEKL and the variant LSIINFEAL (both at 50 µM) were incubated with 7.8 nM of ERAP1 for 1 hr at 37°C and the products analyzed by RP-HPLC. The product of the digestions, either SIINFEKL or SIINFEAL is marked on the chromatogram by an arrow. Panel C, trimming rates for excision of the N-terminus of peptide LSIINFEKL and LSIINFEAL by ERAP1, average of 3 separate experiments. Panels D and E, peptides LVNVDYSKL and the variant LVNVDYSAL (both at 50 µM) were incubated with 15.6 nM of ERAP1 for 1 hr at 37°C and the products analyzed by RP-HPLC. The product of the digestions, either VNVDYSKL or VNVDYSAL is marked on the chromatogram by an arrow. Panel F, trimming rates for excision of the N-terminus of peptides LVNVDYSKL and LVNVDYSAL by ERAP1, average of 3 separate experiments. Note that in both cases, substituting an alanine for the lysine at position 8, leads to a decrease of N-terminus trimming rate by about 8–10 fold.
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pone-0003658-g009: Substitution of a lysine residue at position 8 of two model 9mer H2-Kb epitope precursors leads to a marked decrease of N-terminus trimming by ERAP1 in vitro.Panels A and B, peptides LSIINFEKL and the variant LSIINFEAL (both at 50 µM) were incubated with 7.8 nM of ERAP1 for 1 hr at 37°C and the products analyzed by RP-HPLC. The product of the digestions, either SIINFEKL or SIINFEAL is marked on the chromatogram by an arrow. Panel C, trimming rates for excision of the N-terminus of peptide LSIINFEKL and LSIINFEAL by ERAP1, average of 3 separate experiments. Panels D and E, peptides LVNVDYSKL and the variant LVNVDYSAL (both at 50 µM) were incubated with 15.6 nM of ERAP1 for 1 hr at 37°C and the products analyzed by RP-HPLC. The product of the digestions, either VNVDYSKL or VNVDYSAL is marked on the chromatogram by an arrow. Panel F, trimming rates for excision of the N-terminus of peptides LVNVDYSKL and LVNVDYSAL by ERAP1, average of 3 separate experiments. Note that in both cases, substituting an alanine for the lysine at position 8, leads to a decrease of N-terminus trimming rate by about 8–10 fold.

Mentions: Both the alanine scan in Figure 3 and the library screen in Figure 4 revealed a strong preference for a positively charged residue at position 8 relative to the N-terminus of the peptide. To test whether such a preference is more general we measured the trimming rate of two model antigenic peptide precursors based on epitopes of the MHCI molecule H-Kb, namely LSIINFEKL and LVNVDYSKL (Figure 9). Both peptides carry identical N- and C- termini, as well as a lysine residue at position 8 relative to the N-terminus. Both peptides were efficiently trimmed by ERAP1, resulting to the mature antigenic epitope. In both cases substitution of the position 8 lysine residue by an alanine lead to a marked decrease in trimming rate by ERAP1 by 8–10 fold (Figure 9). This rate change is consistent with the decrease in trimming for the series TVDKNTRXY in Figure 4 when a lysine is compared to an alanine. Furthermore, it is consistent with the effect seen for the 10mer in Figure 3, although the change in rate was more pronounced in that case. Overall, we demonstrate that eradication of a positive charge at position 8 of four unrelated peptide sequences leads to a marked reduction in trimming by ERAP1, suggesting that this effect might be more general.


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)

Substitution of a lysine residue at position 8 of two model 9mer H2-Kb epitope precursors leads to a marked decrease of N-terminus trimming by ERAP1 in vitro.Panels A and B, peptides LSIINFEKL and the variant LSIINFEAL (both at 50 µM) were incubated with 7.8 nM of ERAP1 for 1 hr at 37°C and the products analyzed by RP-HPLC. The product of the digestions, either SIINFEKL or SIINFEAL is marked on the chromatogram by an arrow. Panel C, trimming rates for excision of the N-terminus of peptide LSIINFEKL and LSIINFEAL by ERAP1, average of 3 separate experiments. Panels D and E, peptides LVNVDYSKL and the variant LVNVDYSAL (both at 50 µM) were incubated with 15.6 nM of ERAP1 for 1 hr at 37°C and the products analyzed by RP-HPLC. The product of the digestions, either VNVDYSKL or VNVDYSAL is marked on the chromatogram by an arrow. Panel F, trimming rates for excision of the N-terminus of peptides LVNVDYSKL and LVNVDYSAL by ERAP1, average of 3 separate experiments. Note that in both cases, substituting an alanine for the lysine at position 8, leads to a decrease of N-terminus trimming rate by about 8–10 fold.
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Related In: Results  -  Collection

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pone-0003658-g009: Substitution of a lysine residue at position 8 of two model 9mer H2-Kb epitope precursors leads to a marked decrease of N-terminus trimming by ERAP1 in vitro.Panels A and B, peptides LSIINFEKL and the variant LSIINFEAL (both at 50 µM) were incubated with 7.8 nM of ERAP1 for 1 hr at 37°C and the products analyzed by RP-HPLC. The product of the digestions, either SIINFEKL or SIINFEAL is marked on the chromatogram by an arrow. Panel C, trimming rates for excision of the N-terminus of peptide LSIINFEKL and LSIINFEAL by ERAP1, average of 3 separate experiments. Panels D and E, peptides LVNVDYSKL and the variant LVNVDYSAL (both at 50 µM) were incubated with 15.6 nM of ERAP1 for 1 hr at 37°C and the products analyzed by RP-HPLC. The product of the digestions, either VNVDYSKL or VNVDYSAL is marked on the chromatogram by an arrow. Panel F, trimming rates for excision of the N-terminus of peptides LVNVDYSKL and LVNVDYSAL by ERAP1, average of 3 separate experiments. Note that in both cases, substituting an alanine for the lysine at position 8, leads to a decrease of N-terminus trimming rate by about 8–10 fold.
Mentions: Both the alanine scan in Figure 3 and the library screen in Figure 4 revealed a strong preference for a positively charged residue at position 8 relative to the N-terminus of the peptide. To test whether such a preference is more general we measured the trimming rate of two model antigenic peptide precursors based on epitopes of the MHCI molecule H-Kb, namely LSIINFEKL and LVNVDYSKL (Figure 9). Both peptides carry identical N- and C- termini, as well as a lysine residue at position 8 relative to the N-terminus. Both peptides were efficiently trimmed by ERAP1, resulting to the mature antigenic epitope. In both cases substitution of the position 8 lysine residue by an alanine lead to a marked decrease in trimming rate by ERAP1 by 8–10 fold (Figure 9). This rate change is consistent with the decrease in trimming for the series TVDKNTRXY in Figure 4 when a lysine is compared to an alanine. Furthermore, it is consistent with the effect seen for the 10mer in Figure 3, although the change in rate was more pronounced in that case. Overall, we demonstrate that eradication of a positive charge at position 8 of four unrelated peptide sequences leads to a marked reduction in trimming by ERAP1, suggesting that this effect might be more general.

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