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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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PA28α enhances the rate of accumulation of dual cleavage  products without changing the cleavage site specificity of the proteasome.  The substrate OvaY249-269 (for the sequence, see Fig. 5 A) was incubated  with 20S proteasomes from EL4 cells in the absence (A and C) or presence (B and D) of recombinant PA28α. At the time points indicated, the  mixtures were separated by reverse phase HPLC. The peptides in the  peaks marked with numbers are: TEWTS (1), YVSGLEQLE (2), YVSGLEQL (3), ESIINFEKL and the Kb ligand SIINFEKL (4), SIINFEKLTEWTS and ESIINFEKLTEWTS (5), YVSGLE (6), SIINFE (7), and  SIINF (8). The large peak at the right of A and B is undigested substrate.  Peptides were identified by MALDI-Tof-MS and Edman degradation.
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Figure 7: PA28α enhances the rate of accumulation of dual cleavage products without changing the cleavage site specificity of the proteasome. The substrate OvaY249-269 (for the sequence, see Fig. 5 A) was incubated with 20S proteasomes from EL4 cells in the absence (A and C) or presence (B and D) of recombinant PA28α. At the time points indicated, the mixtures were separated by reverse phase HPLC. The peptides in the peaks marked with numbers are: TEWTS (1), YVSGLEQLE (2), YVSGLEQL (3), ESIINFEKL and the Kb ligand SIINFEKL (4), SIINFEKLTEWTS and ESIINFEKLTEWTS (5), YVSGLE (6), SIINFE (7), and SIINF (8). The large peak at the right of A and B is undigested substrate. Peptides were identified by MALDI-Tof-MS and Edman degradation.

Mentions: Digestions of synthetic peptides (6 μg) and of the small subunit of ribulose 1,5 bisphosphate carboxylase (10 μg) with isolated proteasomes (1 μg) were performed at 37°C, except in the case of Thermoplasma proteasomes at 60°C, in a total volume of 300 μl buffer (20 mM Hepes/KOH [pH 7.0] 1 mM EGTA, 0.5 mM EDTA, 5 mM MgCl2, 0.5 mM 2-mercaptoethanol, 0.02% sodium azide). Digestions of ovalbumin fragments Ova37-77 and Ova239-281, and of the protein substrate were done in the presence of 0.004% SDS. In the experiments shown in Fig. 7, recombinant PA28α was added in a fivefold molar excess over the proteasome. Aliquots of the reaction mixture were separated by reverse phase HPLC on a SMART system equipped with a μRPC C2/C18 SC 2.1/10 column (Pharmacia). Eluent A was 0.1% (vol/vol) TFA/water; eluent B was 80% (vol/ vol) acetonitril/water (0.081% TFA). The identity of peptides in individual HPLC fractions was established by MALDI-Tof-MS and aa sequence analysis by Edman degradation.


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)

PA28α enhances the rate of accumulation of dual cleavage  products without changing the cleavage site specificity of the proteasome.  The substrate OvaY249-269 (for the sequence, see Fig. 5 A) was incubated  with 20S proteasomes from EL4 cells in the absence (A and C) or presence (B and D) of recombinant PA28α. At the time points indicated, the  mixtures were separated by reverse phase HPLC. The peptides in the  peaks marked with numbers are: TEWTS (1), YVSGLEQLE (2), YVSGLEQL (3), ESIINFEKL and the Kb ligand SIINFEKL (4), SIINFEKLTEWTS and ESIINFEKLTEWTS (5), YVSGLE (6), SIINFE (7), and  SIINF (8). The large peak at the right of A and B is undigested substrate.  Peptides were identified by MALDI-Tof-MS and Edman degradation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198974&req=5

Figure 7: PA28α enhances the rate of accumulation of dual cleavage products without changing the cleavage site specificity of the proteasome. The substrate OvaY249-269 (for the sequence, see Fig. 5 A) was incubated with 20S proteasomes from EL4 cells in the absence (A and C) or presence (B and D) of recombinant PA28α. At the time points indicated, the mixtures were separated by reverse phase HPLC. The peptides in the peaks marked with numbers are: TEWTS (1), YVSGLEQLE (2), YVSGLEQL (3), ESIINFEKL and the Kb ligand SIINFEKL (4), SIINFEKLTEWTS and ESIINFEKLTEWTS (5), YVSGLE (6), SIINFE (7), and SIINF (8). The large peak at the right of A and B is undigested substrate. Peptides were identified by MALDI-Tof-MS and Edman degradation.
Mentions: Digestions of synthetic peptides (6 μg) and of the small subunit of ribulose 1,5 bisphosphate carboxylase (10 μg) with isolated proteasomes (1 μg) were performed at 37°C, except in the case of Thermoplasma proteasomes at 60°C, in a total volume of 300 μl buffer (20 mM Hepes/KOH [pH 7.0] 1 mM EGTA, 0.5 mM EDTA, 5 mM MgCl2, 0.5 mM 2-mercaptoethanol, 0.02% sodium azide). Digestions of ovalbumin fragments Ova37-77 and Ova239-281, and of the protein substrate were done in the presence of 0.004% SDS. In the experiments shown in Fig. 7, recombinant PA28α was added in a fivefold molar excess over the proteasome. Aliquots of the reaction mixture were separated by reverse phase HPLC on a SMART system equipped with a μRPC C2/C18 SC 2.1/10 column (Pharmacia). Eluent A was 0.1% (vol/vol) TFA/water; eluent B was 80% (vol/ vol) acetonitril/water (0.081% TFA). The identity of peptides in individual HPLC fractions was established by MALDI-Tof-MS and aa sequence analysis by Edman degradation.

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
Related in: MedlinePlus