Differences in the production of spliced antigenic peptides by the standard proteasome and the immunoproteasome

Abstract

Peptide splicing allows the production of antigenic peptides composed of two fragments initially non-contiguous in the parental protein. The proposed mechanism of splicing is a transpeptidation occurring within the proteasome. Three spliced peptides, derived from FGF-5, melanoma protein gp100 and nuclear protein SP110, have been described. Here, we compared the production of these spliced peptides by the standard proteasome and the immunoproteasome. Differential isotope labelling was used to quantify (by mass spectrometry) the fragments contained in digests obtained with precursor peptides and purified proteasomes. The results show that both the standard and the immunoproteasomes can produce spliced peptides although they differ in their efficiency of production of each peptide. The FGF-5 and gp100 peptides are more efficiently produced by the standard proteasome, whereas the SP110 peptide is more efficiently produced by the immunoproteasome. This seems to result from differences in the production of the two splicing partners, which depends on a balance between cleavages liberating or destroying those fragments. By showing that splicing depends on the efficiency of production of the splicing partners, these results also support the transpeptidation model of peptide splicing. Furthermore, given the presence of immunoproteasomes in dendritic cells and cells exposed to IFN-c, the findings may be relevant for vaccine design.Key words: Mass Spectrometry . Peptide splicing . Proteasome IntroductionSpliced peptides comprise two non-contiguous fragments of the parental protein that are linked together after the excision of an intervening segment. Three spliced antigenic peptides have been described to date. The first is a 9-mer (NTYASPRFK) derived from FGF-5 and presented by HLA-A3 molecules [1]. It was identified using CTL raised against a human renal carcinoma. Another spliced peptide (RTKQLYPEW) is produced from the melanosomal protein gp100 and is recognized, in association with HLA-A32, by CTL raised against a human melanoma [2]. The third example of spliced peptide (SLPRGTSTPK) is a human minor histocompatibility antigen created by a polymorphism in the SP110 gene [3]. This peptide is recognized, in association with HLA-A3, by CTL isolated from a myeloma patient after MHCmatched hematopoietic cell transplantation.We previously showed that the three spliced peptides were produced by transpeptidation inside the catalytic chamber of the proteasome [2][3][4]. In the course of proteolysis, the hydroxyl group of the N-terminal threonine of a catalytic b-subunit produces a nucleophilic attack on the peptide bond, leading to the formation of an acyl-enzyme intermediate between the SHORT COMMUNICATIONCorrespondence: Dr. Benoît J. Van In this study, we compared the production of the three spliced antigenic peptides by the standard proteasome (SP) and its IFN-ginducible counterpart, the immunoproteasome (IP), using a cellular approach and an in vitro approach. We used differential isotope labelling to perform a direct and ...

An antigenic peptide produced by reverse splicing and double asparagine deamidation

“…Hence, the better production of the spliced peptide by the standard proteasome seems to result from a better production of the fragments to splice. This conclusion is in line with our recent study of the differential processing of the three other spliced antigenic peptides (39).…”

“…Standard proteasomes were able to produce the antigenic peptide IYMDGTADFSF, whereas immunoproteasomes were not. Such differences between the two proteasome types to produce spliced peptides were already observed for the three known spliced peptides and resulted from a different capacity to produce the peptidic partners of the splicing reaction (39).…”

The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing

“…These antigenic peptides can be produced by the proteasome by normal peptide-bond hydrolysis or by proteasomecatalyzed peptide splicing. The latter reaction generates peptides that have a different sequence than the original antigen, and it can occur by binding fragments of a single molecule (cis proteasome-catalyzed peptide splicing) or of two distinct molecules (trans proteasome-catalyzed peptide splicing) (1-4).In vitro experiments performed with purified 20S proteasomes were shown to closely reflect the in vivo situation, making it an ideal platform to study the generation of non-spliced and spliced antigenic peptides in vitro (1,(5)(6)(7)(8)(9)(10)(11). Under ideal conditions the 20S proteasome exists in two isoforms, i.e.…”

“…In vitro experiments performed with purified 20S proteasomes were shown to closely reflect the in vivo situation, making it an ideal platform to study the generation of non-spliced and spliced antigenic peptides in vitro (1,(5)(6)(7)(8)(9)(10)(11). Under ideal conditions the 20S proteasome exists in two isoforms, i.e.…”

Immunoproteasomes Shape the Transcriptome and Regulate the Function of Dendritic Cells

“…Vertebrates also express immunoproteasomes (IPs), in which the catalytic b-subunits are replaced by IFN-g-inducible homologs: LMP2 (aka b1i, Psmb9) for b1 (Psmb6), MECL1 (aka b2i, Psmb10) for b2 (Psmb7), and LMP7 (aka b5i, Psmb8) for b5 (Psmb5). The best-described nonredundant role ascribed to IPs is their enhanced ability to generate MHC class I-associated peptides (MIPs) (2)(3)(4)(5).…”