Major Histocompatibility Complex Class I-presented Antigenic Peptides Are Degraded in Cytosolic Extracts Primarily by Thimet Oligopeptidase

Abstract

Nearly all peptides generated by proteasomes during protein degradation are digested rapidly to amino acids, but a few proteasomal products escape this fate and are presented to the immune system on cell surface major histocompatibility complex class I molecules. To test whether these antigenic peptides may be inherently resistant to cytosolic peptidases, six different antigenic peptides were incubated with HeLa cell extracts. All six were degraded rapidly by a process involving o-phenanthroline-sensitive metallopeptidases. One antigenic peptide, FAPGNYPAL, was rapidly destroyed in the extracts by a bestatin-sensitive exopeptidase, apparently by the puromycin-sensitive aminopeptidase. The disappearance of the other five was reduced 30 -90% by a specific inhibitor of the cytosolic endopeptidase, thimet oligopeptidase (TOP) (EC 3.4.24.15), whose physiological function(s) have been unclear and controversial. All these peptides were sensitive to pure recombinant TOP. Furthermore, upon fractionation of the extracts, the major peptidase peak that degraded the ovalbumin-derived epitope, SIINFEKL, co-purified with TOP. In the extracts, TOP also catalyzed rapid degradation of Nextended variants of SIINFEKL and of other antigenic peptides, which in vivo can serve as precursors of these major histocompatibility complex-presented epitopes. This enzyme (unlike cell proteins that promote production of antigenic peptides) is not regulated by interferon-␥. TOP seems to be primarily responsible for the rapid breakdown of antigenic peptides in cytosolic extracts, and our related studies

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes

“…Purified TOP, A CTL epitope from M. Tuberculosis Hsp65 with undefined aa sequence has previously been shown to rely on TOP 36 . A possible role for TOP in the protection of some cytosolic peptides from further destruction has been documented 37 , however, this could not be confirmed 38 . To the contrary, biochemical and class I expression results 5,6,38,39 led to the current notion that TOP has solely a destructive role in class I antigen processing 1,2 .…”

“…A possible role for TOP in the protection of some cytosolic peptides from further destruction has been documented 37 , however, this could not be confirmed 38 . To the contrary, biochemical and class I expression results 5,6,38,39 led to the current notion that TOP has solely a destructive role in class I antigen processing 1,2 . However, destruction of defined epitopes by TOP as studied by CTL recognition of target cells with suppressed expression of TOP, has thus far only been shown for the mouse H2-K b -presented SIINFEKL epitope from ovalbumin 5 .…”

“…Thus the role of TOP in antigen processing appears to be twofold. On the one hand, TOP has a destructive role 5,38 -limiting presentation of epitopes whose C-terminus has already been made by e.g. the proteasome, like SIINFEKL 5,8 .…”

Indoleamine 2,3-dioxygenase–expressing dendritic cells form suppurative granulomas following Listeria monocytogenes infection

“…Immunoblot analysis was performed as previously described (33,78). The following antibodies were used: monoclonal (Chemicon International) or polyclonal anti-IDO antibody (AbD Serotec); polyclonal anti-COX-2 antibody (IBL); and monoclonal β-actin antibody (Sigma-Aldrich).…”

Generation of a functional, soluble tapasin protein from an alternatively spliced mRNA

“…[4][5][6][7][8][9][10] The first five of these associate to form the peptide-loading complex (PLC), which is believed to promote the assembly of peptide-receptive complexes with peptide within the ER. Unlike calreticulin, calnexin and ERp57, which have general chaperone functions, tapasin has a specialised role in MHC class I assembly.…”