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Concerted in vitro trimming of viral HLA-B27-restricted ligands by human ERAP1 and ERAP2 aminopeptidases.

Lorente E, Barriga A, Johnstone C, Mir C, Jiménez M, López D - PLoS ONE (2013)

Bottom Line: In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity.In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors.These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.

ABSTRACT
In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.

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Stacked area charts of M163-177 and NS231-45 synthetic peptides digested with purified ERAPs.M163-177 (panels A-D) and NS231-45 (panels E-H) (sequences are indicated at the top of the figure and the respective HLA ligands identified by MS are bolded) synthetic peptides were digested at different times with ERAP enzymes as follows: ERAP1 at an E/S ratio of 1:800 (panels A and E), ERAP2 at an E/S ratio of 1:800 (panels B and F), both ERAP1 and 2 at an E/S ratio of 1:800 (panels C and G), and both ERAP1 and 2 at an E/S ratio of 1:1600 (panels D and H). The intensity peaks obtained by MALDI-TOF analysis for all peptides at each time point were added and taken as 100% for each time point and depicted. The different N-end trimming products detected are named in their respective region. The results depicted are the mean values of three or four independent experiments.
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pone-0079596-g002: Stacked area charts of M163-177 and NS231-45 synthetic peptides digested with purified ERAPs.M163-177 (panels A-D) and NS231-45 (panels E-H) (sequences are indicated at the top of the figure and the respective HLA ligands identified by MS are bolded) synthetic peptides were digested at different times with ERAP enzymes as follows: ERAP1 at an E/S ratio of 1:800 (panels A and E), ERAP2 at an E/S ratio of 1:800 (panels B and F), both ERAP1 and 2 at an E/S ratio of 1:800 (panels C and G), and both ERAP1 and 2 at an E/S ratio of 1:1600 (panels D and H). The intensity peaks obtained by MALDI-TOF analysis for all peptides at each time point were added and taken as 100% for each time point and depicted. The different N-end trimming products detected are named in their respective region. The results depicted are the mean values of three or four independent experiments.

Mentions: The results shown in Figure 1 and other intermediate points are summarized and depicted in Figure 2A for further clarification. After only 5 minutes in the presence of the enzyme, major substrate cleavage was observed and trimmed 14-mer, 13-mer, and 11-mer products with a predominant 12-mer signal were detected (Figure 2A). After 15 minutes, traces of the 10-mer peptide were also found. After 45 minutes, the signal of the 9-mer, which is the natural ligand identified in HRSV-infected cells, was detected. Peptides shorter than the 9-mer were not detected in the experiments.


Concerted in vitro trimming of viral HLA-B27-restricted ligands by human ERAP1 and ERAP2 aminopeptidases.

Lorente E, Barriga A, Johnstone C, Mir C, Jiménez M, López D - PLoS ONE (2013)

Stacked area charts of M163-177 and NS231-45 synthetic peptides digested with purified ERAPs.M163-177 (panels A-D) and NS231-45 (panels E-H) (sequences are indicated at the top of the figure and the respective HLA ligands identified by MS are bolded) synthetic peptides were digested at different times with ERAP enzymes as follows: ERAP1 at an E/S ratio of 1:800 (panels A and E), ERAP2 at an E/S ratio of 1:800 (panels B and F), both ERAP1 and 2 at an E/S ratio of 1:800 (panels C and G), and both ERAP1 and 2 at an E/S ratio of 1:1600 (panels D and H). The intensity peaks obtained by MALDI-TOF analysis for all peptides at each time point were added and taken as 100% for each time point and depicted. The different N-end trimming products detected are named in their respective region. The results depicted are the mean values of three or four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815102&req=5

pone-0079596-g002: Stacked area charts of M163-177 and NS231-45 synthetic peptides digested with purified ERAPs.M163-177 (panels A-D) and NS231-45 (panels E-H) (sequences are indicated at the top of the figure and the respective HLA ligands identified by MS are bolded) synthetic peptides were digested at different times with ERAP enzymes as follows: ERAP1 at an E/S ratio of 1:800 (panels A and E), ERAP2 at an E/S ratio of 1:800 (panels B and F), both ERAP1 and 2 at an E/S ratio of 1:800 (panels C and G), and both ERAP1 and 2 at an E/S ratio of 1:1600 (panels D and H). The intensity peaks obtained by MALDI-TOF analysis for all peptides at each time point were added and taken as 100% for each time point and depicted. The different N-end trimming products detected are named in their respective region. The results depicted are the mean values of three or four independent experiments.
Mentions: The results shown in Figure 1 and other intermediate points are summarized and depicted in Figure 2A for further clarification. After only 5 minutes in the presence of the enzyme, major substrate cleavage was observed and trimmed 14-mer, 13-mer, and 11-mer products with a predominant 12-mer signal were detected (Figure 2A). After 15 minutes, traces of the 10-mer peptide were also found. After 45 minutes, the signal of the 9-mer, which is the natural ligand identified in HRSV-infected cells, was detected. Peptides shorter than the 9-mer were not detected in the experiments.

Bottom Line: In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity.In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors.These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.

ABSTRACT
In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.

Show MeSH