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Potential immunocompetence of proteolytic fragments produced by proteasomes before evolution of the vertebrate immune system.

Niedermann G, Grimm R, Geier E, Maurer M, Realini C, Gartmann C, Soll J, Omura S, Rechsteiner MC, Baumeister W, Eichmann K - J. Exp. Med. (1997)

Bottom Line: Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes.However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis.The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Immunbiologie, 79108 Freiburg, Germany.

ABSTRACT
To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes, phylogenetically ancient multicatalytic high molecular weight endoproteases. We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have structural features in common with peptides eluted from MHC class I molecules, suggesting that many MHC class I ligands are direct products of proteasomal proteolysis. Here, we report that the processing of polypeptides by proteasomes is conserved in evolution, not only among vertebrate species, but including invertebrate eukaryotes such as insects and yeast. Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes. Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length and the NH2 and COOH termini preferred by MHC class I. Thus, the ability of proteasomes to generate potentially immunocompetent peptides evolved well before the vertebrate immune system. We demonstrate with polypeptide substrates that interferon gamma induction in vivo or addition of recombinant proteasome activator 28alpha in vitro alters proteasomal proteolysis in such a way that the generation of peptides with the structural features of MHC class I ligands is optimized. However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis. The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

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Most peptides efficiently produced by 20S proteasomes from  invertebrate eukaryotes fall into the size range of MHC class I ligands.  The 44-mer peptide Ova239-281 (A and B) and the 123-aa SSU of ribulose  1,5 bisphosphate carboxylase (C, D, and E) were digested with isolated  proteasomes. After substrate consumption, the resulting peptide mixtures  were subjected to MALDI-Tof-MS. (B) Mass spectrum of the peptide  mixture obtained upon digestion of Ova239-281 with D. melanogaster proteasomes. The mass peak representing the immunodominant OVA  epitope SIINFEKL (theoretical mass: 964.3) is indicated. (D and E) Mass  spectra of the peptides mixtures obtained upon digestion of SSU with  proteasomes isolated from the murine cell line EL4 (D) and from yeast  (E). The shaded areas in B, D and E mark the mass range of 8–10-mer  peptides, i.e., the typical size range of MHC class I ligands. The mass  ranges of all overlapping peptides of Ova239-281 and SSU are indicated in A  and C, respectively. In the SSU digests with proteasomes isolated from  EL4 cells (D) or from yeast (E ), 56.7 and 58.6%, respectively, of all discernible masses fell into the size range of 8–10-mer peptides.
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Figure 4: Most peptides efficiently produced by 20S proteasomes from invertebrate eukaryotes fall into the size range of MHC class I ligands. The 44-mer peptide Ova239-281 (A and B) and the 123-aa SSU of ribulose 1,5 bisphosphate carboxylase (C, D, and E) were digested with isolated proteasomes. After substrate consumption, the resulting peptide mixtures were subjected to MALDI-Tof-MS. (B) Mass spectrum of the peptide mixture obtained upon digestion of Ova239-281 with D. melanogaster proteasomes. The mass peak representing the immunodominant OVA epitope SIINFEKL (theoretical mass: 964.3) is indicated. (D and E) Mass spectra of the peptides mixtures obtained upon digestion of SSU with proteasomes isolated from the murine cell line EL4 (D) and from yeast (E). The shaded areas in B, D and E mark the mass range of 8–10-mer peptides, i.e., the typical size range of MHC class I ligands. The mass ranges of all overlapping peptides of Ova239-281 and SSU are indicated in A and C, respectively. In the SSU digests with proteasomes isolated from EL4 cells (D) or from yeast (E ), 56.7 and 58.6%, respectively, of all discernible masses fell into the size range of 8–10-mer peptides.

Mentions: We have previously shown that the immunodominant ovalbumin epitope Ova257-264 (SIINFEKL; reference 30) is the major stable product generated by mouse 20S proteasomes from the 22-mer OvaY249-269 as well as from the 44-mer Ova239-281 (12). Here we show that this octamer is also a dominant product of digestion of Ova239-281 by 20S proteasomes isolated from D. melanogaster Schneider cells (Fig. 3 A and Fig. 4 A). As a second example, we studied the generation of the nonamer TLWVDPYEV, an endogenous peptide derived from the product of the antiproliferative B cell translocation gene 1 (BTG1) and eluted as a major self-epitope from the human class I molecule HLA-A2.1 (31). Fig. 3, B and C show that this nonamer peptide is the major dual cleavage product generated by yeast (S. cerevisiae) proteasomes of the synthetic 24 mer encompassing this peptide in the sequence of BTG1. Moreover, we studied a 21-mer sequence derived from the tyrosine kinase JAK1 containing the nonamer SYFPEITHI, the most abundant self-peptide presented by mouse H-2Kd molecules of P815 cells (32, 33), and previously shown to be generated by digestion with mouse 20S proteasomes (34). We detected the epitope as the predominant dual cleavage product of the 21 mer with Drosophila proteasomes (Fig. 3 D). Thus, proteasomes from invertebrate eukaryotes have a high potency to generate proteolytic fragments that have been proven to serve as ligands of MHC molecules in the vertebrate immune system.


Potential immunocompetence of proteolytic fragments produced by proteasomes before evolution of the vertebrate immune system.

Niedermann G, Grimm R, Geier E, Maurer M, Realini C, Gartmann C, Soll J, Omura S, Rechsteiner MC, Baumeister W, Eichmann K - J. Exp. Med. (1997)

Most peptides efficiently produced by 20S proteasomes from  invertebrate eukaryotes fall into the size range of MHC class I ligands.  The 44-mer peptide Ova239-281 (A and B) and the 123-aa SSU of ribulose  1,5 bisphosphate carboxylase (C, D, and E) were digested with isolated  proteasomes. After substrate consumption, the resulting peptide mixtures  were subjected to MALDI-Tof-MS. (B) Mass spectrum of the peptide  mixture obtained upon digestion of Ova239-281 with D. melanogaster proteasomes. The mass peak representing the immunodominant OVA  epitope SIINFEKL (theoretical mass: 964.3) is indicated. (D and E) Mass  spectra of the peptides mixtures obtained upon digestion of SSU with  proteasomes isolated from the murine cell line EL4 (D) and from yeast  (E). The shaded areas in B, D and E mark the mass range of 8–10-mer  peptides, i.e., the typical size range of MHC class I ligands. The mass  ranges of all overlapping peptides of Ova239-281 and SSU are indicated in A  and C, respectively. In the SSU digests with proteasomes isolated from  EL4 cells (D) or from yeast (E ), 56.7 and 58.6%, respectively, of all discernible masses fell into the size range of 8–10-mer peptides.
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Related In: Results  -  Collection

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Figure 4: Most peptides efficiently produced by 20S proteasomes from invertebrate eukaryotes fall into the size range of MHC class I ligands. The 44-mer peptide Ova239-281 (A and B) and the 123-aa SSU of ribulose 1,5 bisphosphate carboxylase (C, D, and E) were digested with isolated proteasomes. After substrate consumption, the resulting peptide mixtures were subjected to MALDI-Tof-MS. (B) Mass spectrum of the peptide mixture obtained upon digestion of Ova239-281 with D. melanogaster proteasomes. The mass peak representing the immunodominant OVA epitope SIINFEKL (theoretical mass: 964.3) is indicated. (D and E) Mass spectra of the peptides mixtures obtained upon digestion of SSU with proteasomes isolated from the murine cell line EL4 (D) and from yeast (E). The shaded areas in B, D and E mark the mass range of 8–10-mer peptides, i.e., the typical size range of MHC class I ligands. The mass ranges of all overlapping peptides of Ova239-281 and SSU are indicated in A and C, respectively. In the SSU digests with proteasomes isolated from EL4 cells (D) or from yeast (E ), 56.7 and 58.6%, respectively, of all discernible masses fell into the size range of 8–10-mer peptides.
Mentions: We have previously shown that the immunodominant ovalbumin epitope Ova257-264 (SIINFEKL; reference 30) is the major stable product generated by mouse 20S proteasomes from the 22-mer OvaY249-269 as well as from the 44-mer Ova239-281 (12). Here we show that this octamer is also a dominant product of digestion of Ova239-281 by 20S proteasomes isolated from D. melanogaster Schneider cells (Fig. 3 A and Fig. 4 A). As a second example, we studied the generation of the nonamer TLWVDPYEV, an endogenous peptide derived from the product of the antiproliferative B cell translocation gene 1 (BTG1) and eluted as a major self-epitope from the human class I molecule HLA-A2.1 (31). Fig. 3, B and C show that this nonamer peptide is the major dual cleavage product generated by yeast (S. cerevisiae) proteasomes of the synthetic 24 mer encompassing this peptide in the sequence of BTG1. Moreover, we studied a 21-mer sequence derived from the tyrosine kinase JAK1 containing the nonamer SYFPEITHI, the most abundant self-peptide presented by mouse H-2Kd molecules of P815 cells (32, 33), and previously shown to be generated by digestion with mouse 20S proteasomes (34). We detected the epitope as the predominant dual cleavage product of the 21 mer with Drosophila proteasomes (Fig. 3 D). Thus, proteasomes from invertebrate eukaryotes have a high potency to generate proteolytic fragments that have been proven to serve as ligands of MHC molecules in the vertebrate immune system.

Bottom Line: Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes.However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis.The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Immunbiologie, 79108 Freiburg, Germany.

ABSTRACT
To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes, phylogenetically ancient multicatalytic high molecular weight endoproteases. We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have structural features in common with peptides eluted from MHC class I molecules, suggesting that many MHC class I ligands are direct products of proteasomal proteolysis. Here, we report that the processing of polypeptides by proteasomes is conserved in evolution, not only among vertebrate species, but including invertebrate eukaryotes such as insects and yeast. Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes. Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length and the NH2 and COOH termini preferred by MHC class I. Thus, the ability of proteasomes to generate potentially immunocompetent peptides evolved well before the vertebrate immune system. We demonstrate with polypeptide substrates that interferon gamma induction in vivo or addition of recombinant proteasome activator 28alpha in vitro alters proteasomal proteolysis in such a way that the generation of peptides with the structural features of MHC class I ligands is optimized. However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis. The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

Show MeSH