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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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Proteasomes from IFN-γ–treated cells generate more peptides with hydrophobic and fewer peptides with acidic COOH termini  from polypeptides. The substrates OvaY249-269 (A and B) and BTG197-120  (C and D) were incubated in the presence of 20S proteasomes from untreated EL4 cells (A and C) or EL4 cells treated with IFN-γ (B and D).  The sequences of the substrates are given in Fig. 5, A and B, respectively.  After consumption of the substrates, the peptide mixtures were separated  by reverse phase HPLC. Peptides contained in the peaks marked with  numbers are: (A and B) TEWTS (1), YVSGLE (2), YVSGLEQLE (3),  YVSGLEQL (4), ESIINFEKL and the Kb ligand SIINFEKL (5), and SIINFEKLTEWTS/ESIINFEKLTEWTS (6); (C and D) LLPSEL (1),  TLWVDPYE (2), TLWVDPYEVS (3), the HLA-A2.1 ligand TLWVDPYEV (4), and TLWVDPYEVSY (5). Peptides were identified by MALDI-Tof-MS and Edman degradation.
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Figure 6: Proteasomes from IFN-γ–treated cells generate more peptides with hydrophobic and fewer peptides with acidic COOH termini from polypeptides. The substrates OvaY249-269 (A and B) and BTG197-120 (C and D) were incubated in the presence of 20S proteasomes from untreated EL4 cells (A and C) or EL4 cells treated with IFN-γ (B and D). The sequences of the substrates are given in Fig. 5, A and B, respectively. After consumption of the substrates, the peptide mixtures were separated by reverse phase HPLC. Peptides contained in the peaks marked with numbers are: (A and B) TEWTS (1), YVSGLE (2), YVSGLEQLE (3), YVSGLEQL (4), ESIINFEKL and the Kb ligand SIINFEKL (5), and SIINFEKLTEWTS/ESIINFEKLTEWTS (6); (C and D) LLPSEL (1), TLWVDPYE (2), TLWVDPYEVS (3), the HLA-A2.1 ligand TLWVDPYEV (4), and TLWVDPYEVSY (5). Peptides were identified by MALDI-Tof-MS and Edman degradation.

Mentions: The existence of IFN-γ–inducible proteasomal elements in vertebrates indicates that the proteasomes themselves have evolved by adapting to their novel immunological role. If the specificity of proteasomal proteolysis was drastically and qualitatively altered by the IFN-γ–inducible elements, a significant restricting role of proteasomes in the evolution of MHC would be less likely. Most previous studies on the functional effects of the IFN-γ–inducible β subunits LMP2, LMP7, and MECL1 used short (3–4 aa) fluorogenic substrates, and inconsistent changes in peptidase activities have been reported by different investigators. By digesting polypeptide sequences more likely to resemble physiological proteasome substrates, we compared proteasomes from untreated with that of IFN-γ–treated EL4 cells. Enhanced expression of LMP2 and LMP7 in induced compared to uninduced cells was monitored by Western blot analyses (not shown). Fig. 6, A and B show the HPLC patterns obtained upon digestion of OvaY249-269, Fig. 6, C and D that of the BTG1-derived 24 mer. In both cases, we observe that proteasomes from IFN-γ–treated cells generate increased amounts of fragments with hydrophobic COOH termini (YVSGLEQL is peak 4 in Fig. 6, A and B, and TLWVDPYEV is peak 4 in Fig. 6, C and D) and decreased amounts of fragments with acidic COOH termini (YVSGLE is peak 2, and YVSGLEQLE is peak 3 in Fig. 6, A and B; TLWVDPYE is peak 2 in Fig. 6, C and D). This is in line with general preferences of MHC class I molecules, although it does not necessarily result in improved production of each individual epitope; production of BTG1103-111 (TLWVYPDEV, peak 4 in Fig. 6, C and D) is improved, whereas production of Ova257-264 (SIINFEKL, contained in peak 5, theoretical mass: 963.14, in Fig. 6, A and B) is impaired. In spite of these quantitative changes, however, the same set of major proteolytic fragments is produced by proteasomes isolated from uninduced and from IFN-γ–induced cells.


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)

Proteasomes from IFN-γ–treated cells generate more peptides with hydrophobic and fewer peptides with acidic COOH termini  from polypeptides. The substrates OvaY249-269 (A and B) and BTG197-120  (C and D) were incubated in the presence of 20S proteasomes from untreated EL4 cells (A and C) or EL4 cells treated with IFN-γ (B and D).  The sequences of the substrates are given in Fig. 5, A and B, respectively.  After consumption of the substrates, the peptide mixtures were separated  by reverse phase HPLC. Peptides contained in the peaks marked with  numbers are: (A and B) TEWTS (1), YVSGLE (2), YVSGLEQLE (3),  YVSGLEQL (4), ESIINFEKL and the Kb ligand SIINFEKL (5), and SIINFEKLTEWTS/ESIINFEKLTEWTS (6); (C and D) LLPSEL (1),  TLWVDPYE (2), TLWVDPYEVS (3), the HLA-A2.1 ligand TLWVDPYEV (4), and TLWVDPYEVSY (5). Peptides were identified by MALDI-Tof-MS and Edman degradation.
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Related In: Results  -  Collection

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Figure 6: Proteasomes from IFN-γ–treated cells generate more peptides with hydrophobic and fewer peptides with acidic COOH termini from polypeptides. The substrates OvaY249-269 (A and B) and BTG197-120 (C and D) were incubated in the presence of 20S proteasomes from untreated EL4 cells (A and C) or EL4 cells treated with IFN-γ (B and D). The sequences of the substrates are given in Fig. 5, A and B, respectively. After consumption of the substrates, the peptide mixtures were separated by reverse phase HPLC. Peptides contained in the peaks marked with numbers are: (A and B) TEWTS (1), YVSGLE (2), YVSGLEQLE (3), YVSGLEQL (4), ESIINFEKL and the Kb ligand SIINFEKL (5), and SIINFEKLTEWTS/ESIINFEKLTEWTS (6); (C and D) LLPSEL (1), TLWVDPYE (2), TLWVDPYEVS (3), the HLA-A2.1 ligand TLWVDPYEV (4), and TLWVDPYEVSY (5). Peptides were identified by MALDI-Tof-MS and Edman degradation.
Mentions: The existence of IFN-γ–inducible proteasomal elements in vertebrates indicates that the proteasomes themselves have evolved by adapting to their novel immunological role. If the specificity of proteasomal proteolysis was drastically and qualitatively altered by the IFN-γ–inducible elements, a significant restricting role of proteasomes in the evolution of MHC would be less likely. Most previous studies on the functional effects of the IFN-γ–inducible β subunits LMP2, LMP7, and MECL1 used short (3–4 aa) fluorogenic substrates, and inconsistent changes in peptidase activities have been reported by different investigators. By digesting polypeptide sequences more likely to resemble physiological proteasome substrates, we compared proteasomes from untreated with that of IFN-γ–treated EL4 cells. Enhanced expression of LMP2 and LMP7 in induced compared to uninduced cells was monitored by Western blot analyses (not shown). Fig. 6, A and B show the HPLC patterns obtained upon digestion of OvaY249-269, Fig. 6, C and D that of the BTG1-derived 24 mer. In both cases, we observe that proteasomes from IFN-γ–treated cells generate increased amounts of fragments with hydrophobic COOH termini (YVSGLEQL is peak 4 in Fig. 6, A and B, and TLWVDPYEV is peak 4 in Fig. 6, C and D) and decreased amounts of fragments with acidic COOH termini (YVSGLE is peak 2, and YVSGLEQLE is peak 3 in Fig. 6, A and B; TLWVDPYE is peak 2 in Fig. 6, C and D). This is in line with general preferences of MHC class I molecules, although it does not necessarily result in improved production of each individual epitope; production of BTG1103-111 (TLWVYPDEV, peak 4 in Fig. 6, C and D) is improved, whereas production of Ova257-264 (SIINFEKL, contained in peak 5, theoretical mass: 963.14, in Fig. 6, A and B) is impaired. In spite of these quantitative changes, however, the same set of major proteolytic fragments is produced by proteasomes isolated from uninduced and from IFN-γ–induced cells.

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