Effects of major-histocompatibility-complex-encoded subunits on the peptidase and proteolytic activities of human 20S proteasomes. Cleavage of proteins and antigenic peptides.
The proteasome is responsible for the non-lysosomal degradation of misfolded, transient, or ubiquitin-tagged proteins. This fact and the identification of two major-histocompatibility-complex-(MHC)-encoded proteasomal subunits, LMP2/7, suggest an important role of the proteasome in antigen processing. Using purified 20S proteasomes from a wild-type and a LMP2/7-deletion T lymphoblastoid cell line, we analyzed the effect of LMP2/7 on the peptidase and proteolytic activities of the complex in the context of various purification and activation methods. The incorporation of LMP2/7 alters the peptidase activity against fluorogenic substrates, but these effects are not reflected in the time-dependent degradation pattern of oxidized insulin B chain or of peptide epitopes of an antigenic protein. No effect of LMP2/7 on the degradation pattern of these substrates was observed by either reverse-phase chromatography, pool sequencing, or mass spectrometry. The 20S proteasome can cleave insulin B chain at nearly every position, showing that the P1 position alone does not determine the cleavage sites. The maximum of the length distribution of the end products, makes these ideal candidates for MHC display; yet we find that a natural epitope derived from human histone H3 is further degraded by 20S proteasomes. Alanine scans and substitutions with related amino acids of this epitope indicate that, as in insulin B chain, the cleavage sites are not determined by the P1 position alone.
Ehring
B
B
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Meyer
T H
TH
Eckerskorn
C
C
Lottspeich
F
F
Tampé
R
R
D000595:Amino Acid Sequence; D000941:Antigens / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D010450:Endopeptidases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D008285:Major Histocompatibility Complex; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013379:Substrate Specificity
D000941:Antigens; D007328:Insulin; D009097:Multienzyme Complexes; D010455:Peptides; D011506:Proteins; C071313:LMP-2 protein; D007371:Interferon-gamma; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C077436:LMP7 protein; D046988:Proteasome Endopeptidase Complex