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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 vertebrates and from eukaryotic invertebrates show highly conserved cleavage patterns in polypeptides. Synthetic  peptides OvaY249-269 (A), Ova239-281 (B), and Ova37-77 (C) were incubated  in the presence of 20S proteasomes isolated from the indicated cell lines  or organisms. After substrate consumption, the mixtures were subjected  to pool sequencing by Edman degradation. Proteasome cleavage sites  were determined and quantitatively estimated by the sequence cycle numbers and the yields of unique amino acids. For example, the strong signal  for asparagine (N) in sequence cycle 4 (panel A) indicates a strong cleavage site four residues towards the NH2 terminus of N between E-S. Although isoleucine (I) is not unique, these signals are necessary for the interpretation of the phenylalanine (F) signal, and are therefore included in  panel B. I undergoes racemization to iso- and alloleucine, the latter representing 30-40% and coeluting with F. The phenylalanine signals in cycles  2 and 3 are therefore caused by isoleucine (e.g., EL4 digest). Cleavage  sites are indicated by arrows, with the sizes reflecting estimates of the relative efficiency of cleavage. The CTL epitopes SIINFEKL and KVVRFDKL are underlined.
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Figure 5: Proteasomes from vertebrates and from eukaryotic invertebrates show highly conserved cleavage patterns in polypeptides. Synthetic peptides OvaY249-269 (A), Ova239-281 (B), and Ova37-77 (C) were incubated in the presence of 20S proteasomes isolated from the indicated cell lines or organisms. After substrate consumption, the mixtures were subjected to pool sequencing by Edman degradation. Proteasome cleavage sites were determined and quantitatively estimated by the sequence cycle numbers and the yields of unique amino acids. For example, the strong signal for asparagine (N) in sequence cycle 4 (panel A) indicates a strong cleavage site four residues towards the NH2 terminus of N between E-S. Although isoleucine (I) is not unique, these signals are necessary for the interpretation of the phenylalanine (F) signal, and are therefore included in panel B. I undergoes racemization to iso- and alloleucine, the latter representing 30-40% and coeluting with F. The phenylalanine signals in cycles 2 and 3 are therefore caused by isoleucine (e.g., EL4 digest). Cleavage sites are indicated by arrows, with the sizes reflecting estimates of the relative efficiency of cleavage. The CTL epitopes SIINFEKL and KVVRFDKL are underlined.

Mentions: We studied the cleavage site (...P3P2P1 − P1′P2′P3′...) preferences in polypeptides of 20S proteasomes isolated from a variety of organisms including archaebacteria, eubacteria, and nonvertebrate and vertebrate eukaryotes (see Fig. 2). Fig. 5 A shows the results obtained by pool sequencing of digests of the 22-mer OvaY249-269 containing the immunodominant Ova257-264 (SIINFEKL) epitope. The cleavage patterns of all proteasomes of eukaryotic origin, including the murine cell line EL4, the human cell lines T1 and K562, as well as of insects and of yeast are remarkably similar; the predominant cleavage sites reside after the same hydrophobic (L264-T265) and acidic (E256-S257) aa. These cleavage sites precisely coincide with the NH2- and COOH-terminal epitope boundaries. In contrast, the cleavage pattern of archaebacterial proteasomes is clearly different; they prefer to cleave after aromatic (F261-E262) and aliphatic aa (L264-T265, L255-E256), no cleavage after acidic aa is seen, and the major cleavage site destroys the epitope. Analyses of the degradation of longer substrates is shown in Fig. 5, B and C. The 44-mer Ova239-281 (Fig. 5 B) represents a longer fragment containing the immunodominant SIINFEKL; the 41-mer Ova37-77 (Fig. 5 C) contains the poorly immunogenic epitope Ova55-62 (KVVRFDKL), also presented by Kb (22, 35). For bacterial proteasomes, the data in Fig. 5 C highlight the preference for aromatic and aliphatic aa in P1, whereas cleavage after charged aa is rare but not impossible (Fig. 5 B). Cleavage patterns of all eukaryotic examples, although not fully identical, reflect the same broad but characteristic P1 specificity spectrum. About 60–65% of the peptide bonds hydrolyzed (e.g., 11/17 in Ova239-281 by mouse EL4-proteasomes; 11/18 by yeast proteasomes) have an aromatic or a hydrophobic aliphatic aa in the P1 position. Most of the remaining peptide bonds have either a positively (R) or negatively charged (E, D) aa in the P1 position. In addition, together with results in Fig. 3 (see above), these data extend to invertebrate eukaryotes our previous finding that proteasomes have a preference for small or polar aa in the P1′ position of the scissile bond (12). Major cleavage sites are: E256-S257 and L264-T265 in OvaY249-269 (Fig. 5 A), L102-T103 and V111-S112 in BTG197-120 (see Fig. 3, B and C), and F354-S355 in JAK1348-368 (see Fig. 3 D).


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 vertebrates and from eukaryotic invertebrates show highly conserved cleavage patterns in polypeptides. Synthetic  peptides OvaY249-269 (A), Ova239-281 (B), and Ova37-77 (C) were incubated  in the presence of 20S proteasomes isolated from the indicated cell lines  or organisms. After substrate consumption, the mixtures were subjected  to pool sequencing by Edman degradation. Proteasome cleavage sites  were determined and quantitatively estimated by the sequence cycle numbers and the yields of unique amino acids. For example, the strong signal  for asparagine (N) in sequence cycle 4 (panel A) indicates a strong cleavage site four residues towards the NH2 terminus of N between E-S. Although isoleucine (I) is not unique, these signals are necessary for the interpretation of the phenylalanine (F) signal, and are therefore included in  panel B. I undergoes racemization to iso- and alloleucine, the latter representing 30-40% and coeluting with F. The phenylalanine signals in cycles  2 and 3 are therefore caused by isoleucine (e.g., EL4 digest). Cleavage  sites are indicated by arrows, with the sizes reflecting estimates of the relative efficiency of cleavage. The CTL epitopes SIINFEKL and KVVRFDKL are underlined.
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Related In: Results  -  Collection

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Figure 5: Proteasomes from vertebrates and from eukaryotic invertebrates show highly conserved cleavage patterns in polypeptides. Synthetic peptides OvaY249-269 (A), Ova239-281 (B), and Ova37-77 (C) were incubated in the presence of 20S proteasomes isolated from the indicated cell lines or organisms. After substrate consumption, the mixtures were subjected to pool sequencing by Edman degradation. Proteasome cleavage sites were determined and quantitatively estimated by the sequence cycle numbers and the yields of unique amino acids. For example, the strong signal for asparagine (N) in sequence cycle 4 (panel A) indicates a strong cleavage site four residues towards the NH2 terminus of N between E-S. Although isoleucine (I) is not unique, these signals are necessary for the interpretation of the phenylalanine (F) signal, and are therefore included in panel B. I undergoes racemization to iso- and alloleucine, the latter representing 30-40% and coeluting with F. The phenylalanine signals in cycles 2 and 3 are therefore caused by isoleucine (e.g., EL4 digest). Cleavage sites are indicated by arrows, with the sizes reflecting estimates of the relative efficiency of cleavage. The CTL epitopes SIINFEKL and KVVRFDKL are underlined.
Mentions: We studied the cleavage site (...P3P2P1 − P1′P2′P3′...) preferences in polypeptides of 20S proteasomes isolated from a variety of organisms including archaebacteria, eubacteria, and nonvertebrate and vertebrate eukaryotes (see Fig. 2). Fig. 5 A shows the results obtained by pool sequencing of digests of the 22-mer OvaY249-269 containing the immunodominant Ova257-264 (SIINFEKL) epitope. The cleavage patterns of all proteasomes of eukaryotic origin, including the murine cell line EL4, the human cell lines T1 and K562, as well as of insects and of yeast are remarkably similar; the predominant cleavage sites reside after the same hydrophobic (L264-T265) and acidic (E256-S257) aa. These cleavage sites precisely coincide with the NH2- and COOH-terminal epitope boundaries. In contrast, the cleavage pattern of archaebacterial proteasomes is clearly different; they prefer to cleave after aromatic (F261-E262) and aliphatic aa (L264-T265, L255-E256), no cleavage after acidic aa is seen, and the major cleavage site destroys the epitope. Analyses of the degradation of longer substrates is shown in Fig. 5, B and C. The 44-mer Ova239-281 (Fig. 5 B) represents a longer fragment containing the immunodominant SIINFEKL; the 41-mer Ova37-77 (Fig. 5 C) contains the poorly immunogenic epitope Ova55-62 (KVVRFDKL), also presented by Kb (22, 35). For bacterial proteasomes, the data in Fig. 5 C highlight the preference for aromatic and aliphatic aa in P1, whereas cleavage after charged aa is rare but not impossible (Fig. 5 B). Cleavage patterns of all eukaryotic examples, although not fully identical, reflect the same broad but characteristic P1 specificity spectrum. About 60–65% of the peptide bonds hydrolyzed (e.g., 11/17 in Ova239-281 by mouse EL4-proteasomes; 11/18 by yeast proteasomes) have an aromatic or a hydrophobic aliphatic aa in the P1 position. Most of the remaining peptide bonds have either a positively (R) or negatively charged (E, D) aa in the P1 position. In addition, together with results in Fig. 3 (see above), these data extend to invertebrate eukaryotes our previous finding that proteasomes have a preference for small or polar aa in the P1′ position of the scissile bond (12). Major cleavage sites are: E256-S257 and L264-T265 in OvaY249-269 (Fig. 5 A), L102-T103 and V111-S112 in BTG197-120 (see Fig. 3, B and C), and F354-S355 in JAK1348-368 (see Fig. 3 D).

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