26 S proteasome-mediated production of an authentic major histocompatibility class I-restricted epitope from an intact protein substrate

Sary Ben-Shahar, Arthur Komlosh, Eran Nadav, Isabella Shaked, Tamar Ziv, Arie Admon, George N. DeMartino, Yuval Reiss

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

Peptides displayed on the cell surface by major histocompatibility class I molecules (MHC class I) are generated by proteolytic processing of protein- antigens in the cytoplasm. Initially, antigens are degraded by the 26 S proteasome, most probably following ubiquitination. However, it is unclear whether this proteolysis results in the generation of MHC class I ligands or if further processing is required. To investigate the role of the 26 S proteasome in antigen presentation, we analyzed the processing of an intact antigen by purified 26 S proteasome. A recombinant ornithine decarboxylase was produced harboring the H-2Kb-restricted peptide epitope, derived from ovalbumin SIINFEKL (termed ODC-ova). Utilizing recombinant antizyme to target the antigen to the 26 S proteasome, we found that proteolysis of ODC-ova by the 26 S proteasome resulted in the generation of the Kb-ligand. Mass spectrometry analysis indicated that in addition to SIINFEKL, the N- terminally extended ligand, HSIINFEKL, was also generated. Production of SIINFEKL was linear with time and directly proportional to the rate of ODC- ova degradation. The overall yield of SIINFEKL was approximately 5% of the amount of ODC-ova degraded. The addition of PA28, the 20 S, or the 20 S-PA28 complex to the 26 S proteasome did not significantly affect the yield of the antigenic peptide. These findings demonstrate that the 26 S proteasome can efficiently digest an intact physiological substrate and generate an authentic MHC class I-restricted epitope.

Original languageEnglish (US)
Pages (from-to)21963-21972
Number of pages10
JournalJournal of Biological Chemistry
Volume274
Issue number31
DOIs
StatePublished - Jul 30 1999

Fingerprint

Histocompatibility
Proteasome Endopeptidase Complex
Epitopes
Ovum
Antigen Presentation
Substrates
Antigens
Proteins
Proteolysis
Ligands
Peptides
Molecules
Processing
Ornithine Decarboxylase
Ubiquitination
Ovalbumin
Mass Spectrometry
Cytoplasm
Mass spectrometry
Degradation

ASJC Scopus subject areas

  • Biochemistry

Cite this

26 S proteasome-mediated production of an authentic major histocompatibility class I-restricted epitope from an intact protein substrate. / Ben-Shahar, Sary; Komlosh, Arthur; Nadav, Eran; Shaked, Isabella; Ziv, Tamar; Admon, Arie; DeMartino, George N.; Reiss, Yuval.

In: Journal of Biological Chemistry, Vol. 274, No. 31, 30.07.1999, p. 21963-21972.

Research output: Contribution to journalArticle

Ben-Shahar, Sary ; Komlosh, Arthur ; Nadav, Eran ; Shaked, Isabella ; Ziv, Tamar ; Admon, Arie ; DeMartino, George N. ; Reiss, Yuval. / 26 S proteasome-mediated production of an authentic major histocompatibility class I-restricted epitope from an intact protein substrate. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 31. pp. 21963-21972.
@article{661bf64b27854055a19521de4c50e775,
title = "26 S proteasome-mediated production of an authentic major histocompatibility class I-restricted epitope from an intact protein substrate",
abstract = "Peptides displayed on the cell surface by major histocompatibility class I molecules (MHC class I) are generated by proteolytic processing of protein- antigens in the cytoplasm. Initially, antigens are degraded by the 26 S proteasome, most probably following ubiquitination. However, it is unclear whether this proteolysis results in the generation of MHC class I ligands or if further processing is required. To investigate the role of the 26 S proteasome in antigen presentation, we analyzed the processing of an intact antigen by purified 26 S proteasome. A recombinant ornithine decarboxylase was produced harboring the H-2Kb-restricted peptide epitope, derived from ovalbumin SIINFEKL (termed ODC-ova). Utilizing recombinant antizyme to target the antigen to the 26 S proteasome, we found that proteolysis of ODC-ova by the 26 S proteasome resulted in the generation of the Kb-ligand. Mass spectrometry analysis indicated that in addition to SIINFEKL, the N- terminally extended ligand, HSIINFEKL, was also generated. Production of SIINFEKL was linear with time and directly proportional to the rate of ODC- ova degradation. The overall yield of SIINFEKL was approximately 5{\%} of the amount of ODC-ova degraded. The addition of PA28, the 20 S, or the 20 S-PA28 complex to the 26 S proteasome did not significantly affect the yield of the antigenic peptide. These findings demonstrate that the 26 S proteasome can efficiently digest an intact physiological substrate and generate an authentic MHC class I-restricted epitope.",
author = "Sary Ben-Shahar and Arthur Komlosh and Eran Nadav and Isabella Shaked and Tamar Ziv and Arie Admon and DeMartino, {George N.} and Yuval Reiss",
year = "1999",
month = "7",
day = "30",
doi = "10.1074/jbc.274.31.21963",
language = "English (US)",
volume = "274",
pages = "21963--21972",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "31",

}

TY - JOUR

T1 - 26 S proteasome-mediated production of an authentic major histocompatibility class I-restricted epitope from an intact protein substrate

AU - Ben-Shahar, Sary

AU - Komlosh, Arthur

AU - Nadav, Eran

AU - Shaked, Isabella

AU - Ziv, Tamar

AU - Admon, Arie

AU - DeMartino, George N.

AU - Reiss, Yuval

PY - 1999/7/30

Y1 - 1999/7/30

N2 - Peptides displayed on the cell surface by major histocompatibility class I molecules (MHC class I) are generated by proteolytic processing of protein- antigens in the cytoplasm. Initially, antigens are degraded by the 26 S proteasome, most probably following ubiquitination. However, it is unclear whether this proteolysis results in the generation of MHC class I ligands or if further processing is required. To investigate the role of the 26 S proteasome in antigen presentation, we analyzed the processing of an intact antigen by purified 26 S proteasome. A recombinant ornithine decarboxylase was produced harboring the H-2Kb-restricted peptide epitope, derived from ovalbumin SIINFEKL (termed ODC-ova). Utilizing recombinant antizyme to target the antigen to the 26 S proteasome, we found that proteolysis of ODC-ova by the 26 S proteasome resulted in the generation of the Kb-ligand. Mass spectrometry analysis indicated that in addition to SIINFEKL, the N- terminally extended ligand, HSIINFEKL, was also generated. Production of SIINFEKL was linear with time and directly proportional to the rate of ODC- ova degradation. The overall yield of SIINFEKL was approximately 5% of the amount of ODC-ova degraded. The addition of PA28, the 20 S, or the 20 S-PA28 complex to the 26 S proteasome did not significantly affect the yield of the antigenic peptide. These findings demonstrate that the 26 S proteasome can efficiently digest an intact physiological substrate and generate an authentic MHC class I-restricted epitope.

AB - Peptides displayed on the cell surface by major histocompatibility class I molecules (MHC class I) are generated by proteolytic processing of protein- antigens in the cytoplasm. Initially, antigens are degraded by the 26 S proteasome, most probably following ubiquitination. However, it is unclear whether this proteolysis results in the generation of MHC class I ligands or if further processing is required. To investigate the role of the 26 S proteasome in antigen presentation, we analyzed the processing of an intact antigen by purified 26 S proteasome. A recombinant ornithine decarboxylase was produced harboring the H-2Kb-restricted peptide epitope, derived from ovalbumin SIINFEKL (termed ODC-ova). Utilizing recombinant antizyme to target the antigen to the 26 S proteasome, we found that proteolysis of ODC-ova by the 26 S proteasome resulted in the generation of the Kb-ligand. Mass spectrometry analysis indicated that in addition to SIINFEKL, the N- terminally extended ligand, HSIINFEKL, was also generated. Production of SIINFEKL was linear with time and directly proportional to the rate of ODC- ova degradation. The overall yield of SIINFEKL was approximately 5% of the amount of ODC-ova degraded. The addition of PA28, the 20 S, or the 20 S-PA28 complex to the 26 S proteasome did not significantly affect the yield of the antigenic peptide. These findings demonstrate that the 26 S proteasome can efficiently digest an intact physiological substrate and generate an authentic MHC class I-restricted epitope.

UR - http://www.scopus.com/inward/record.url?scp=0033618399&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033618399&partnerID=8YFLogxK

U2 - 10.1074/jbc.274.31.21963

DO - 10.1074/jbc.274.31.21963

M3 - Article

VL - 274

SP - 21963

EP - 21972

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 31

ER -