sign in sign up
<<
Home , Deamidation

An antigenic produced by reverse splicing PNAS PLUS and double deamidation

Alexandre Daleta, Paul F. Robbinsb, Vincent Stroobanta, Nathalie Vignerona, Yong F. Lib, Mona El-Gamilb, Ken-ichi Hanadab, James C. Yangb, Steven A. Rosenbergb, and Benoît J. Van den Eyndea,1 aLudwig Institute for Cancer Research, Brussels Branch and de Duve Institute, Université Catholique de Louvain, B-1200 Brussels, Belgium; and bSurgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 AUTHOR SUMMARY

Cytolytic T lymphocytes (CTL) ments can be assembled either in are a class of immune cells that CTL the same or reverse order to that kill diseased cells such as can- in which they appear in the pa- cerous or virus-infected cells. To rental (3). do so, they must recognize pep- Three antigenic have tide fragments produced when V. cytosol been described where an as-

viral or cancer-related N-linked paragine residue was converted located inside the cell are bro- into an . This con- N version was analyzed in detail ken down by the proteasome, NH2 N COOH for one of the peptides, and it a catalytic complex responsible D D tyrosinase MHC for the bulk of protein degrada- molecule ERAD was shown to occur through the IV. tion in the cytosol of animal endoplasmic reticulum I. removal of the group of cells. The peptide fragments are TAP the of the asparagine N II. NH2 (5). As a result, the peptide presented at the surface of dis- N III. COOH eased cells by a different group D contains an aspartic acid (D in NH NH2 2 IYMDGTADFSF COOH D peptide N-glycanase of immune proteins called MHC COOH the one-letter code) at position 3 of its sequence, class I molecules. Peptides rec- proteasome ognized by CTL usually consist which reads YMDGTMSQV, whereas the sequence of the of fragments of 8 to 11 amino Fig. P1. Production of the deamidated reverse-spliced antigenic acids that are transported into peptide derived from tyrosinase. Misfolded N-glycosylated tyrosinase parental protein has an aspara- the endoplasmic reticulum proteins are retrotranslocated from the ER into the cytosol (I), where gine at this position (ER), an organelle surrounding peptide N-glycanase converts two into aspartates upon (YMNGTMSQV). This aspara- the nucleus, by a protein dubbed removal of the asparagine-associated sugars (II). This process is gine is a site where sugar moie- “ followed by cleavage and reverse splicing of the deamidated protein ties are normally added to some transporter associated with an- fi tigen processing” (TAP). Once by the proteasome (III), leading to production of the nal antigenic cellular proteins, a process peptide IYMDGTADFSF, which is then transported back into the ER by N inside the ER, the peptides are known as -. Dea- the TAP transporter (IV). The peptide/MHC complex then migrates to midation of the asparagine of loaded onto MHC class I mole- the cell surface where it can be recognized by the CTL (V). ERAD, cules. Stable peptide/MHC ER-associated degradation. the peptide was found to result from the removal of the at-

complexes can then exit the ER IMMUNOLOGY and reach the cell surface, where tached sugar moiety before the they can be recognized by surrounding lymphocytes. Most anti- breakdown of the protein by the proteasome. This deglycosyla- N genic peptides consist of a fragment of the parental protein. tion is performed by an enzyme called peptide- -glycanase However, some antigenic peptides undergo modifications (PNGase), which removes not only the sugar moiety but also the resulting in a change of their sequence. The extent of such nitrogen of the side chain of the asparagine where the sugar was modifications and their significance remain unclear. Here, we attached, thereby converting the amide into a carboxylate group. describe a highly unexpected peptide, which combines two Hence, the asparagine becomes an aspartic acid (5). complex modifications. One is the splicing of two peptide In the present study, we describe an antigenic peptide that combines these two posttranslational modifications. It is recog- fragments that are not contiguous in the parental protein. The + other is a conversion of asparagine amino acid residues into nized by CD8 cytotoxic T lymphocytes isolated from a human aspartic acid residues. melanoma, a potentially fatal skin cancer. The parental protein, Three antigenic peptides produced by peptide splicing in the the enzyme tyrosinase, is responsible for the production of proteasome have been described so far, and the biochemical mechanism of splicing has been detailed (1–4). In the course of Author contributions: A.D., P.F.R., V.S., N.V., K.-i.H., J.C.Y., S.A.R., and B.J.V.d.E. designed protein degradation by the proteasome, each peptide fragment research; A.D., P.F.R., V.S., N.V., Y.F.L., and M.E.-G. performed research; P.F.R. contributed resulting from cleavage transiently forms an in- new reagents/analytic tools; A.D., P.F.R., V.S., and B.J.V.d.E. analyzed data; and A.D., P.F.R., termediate in which the peptide fragment remains attached to the and B.J.V.d.E. wrote the paper. proteasome catalytic subunit by an ester bond. Usually, this in- The authors declare no conflict of interest. termediate is rapidly hydrolyzed by surrounding water molecules. This article is a Direct Submission. However, during peptide splicing, the amino terminus of other Freely available online through the PNAS open access option. peptide fragments present in the catalytic chamber competes with 1To whom correspondence should be addressed. E-mail: [email protected]. water molecules and occasionally leads to the formation of a new org. peptide bond that links the two fragments. This process is called See full research article on page E323 of www.pnas.org. transpeptidation. Interestingly, the noncontiguous peptide frag- Cite this Author Summary as: PNAS 10.1073/pnas.1101892108.

www.pnas.org/cgi/doi/10.1073/pnas.1101892108 PNAS | July 19, 2011 | vol. 108 | no. 29 | 11741–11742 Downloaded by guest on September 26, 2021 melanin in pigmented cells. The peptide, with the sequence neling the antigenic peptides back to the ER for assembly with IYMDGTADFSF, is made up of the fragments ADFSF and MHC molecules. This result further confirmed the production IYMDGT, which are separated by 27 amino acids in the parental of this peptide in the cytosol by PNGase and the proteasome, protein. In the peptide, these fragments are spliced in the reverse followed by its transport into the ER by the TAP transporter. order to that in which they appear in the tyrosinase protein. In Together, our findings indicate that the processing of this addition, the peptide IYMDGTADFSF contains, in positions 4 unusual antigenic peptide requires the translation and the gly- and 8, two aspartate residues resulting from deamidation of the cosylation of the parental protein in the ER followed by its asparagines encoded by the parent gene. CTL assays and tandem retrotranslocation into the cytosol, where PNGase converts the mass spectrometry analyses on peptides eluted from the surface two glycosylated asparagine residues into aspartates (Fig. P1). of melanoma cells confirmed that the peptide IYMDGTADFSF This process is followed by cleavage and reverse splicing of the was naturally presented by antigen-presenting MHC molecules appropriate fragments by the proteasome, and additional trans- on melanoma cells. port of the resulting peptide into the ER through the Using a proteasome inhibitor, we showed that the proteasome TAP transporter. was required for the production of the peptide IYMDG- The description of this antigenic peptide and the character- TADFSF. Purified proteasomes incubated with an extended ization of its mode of production expand our knowledge on precursor peptide containing the two aspartates were able to modified antigenic peptides. In particular, this fourth example of produce the spliced peptide in vitro. The splicing reaction pro- spliced peptide suggests that spliced peptides are not exceptional ducing the reordered peptide IYMDGTADFSF seemed to occur and may represent a significant part of the repertoire of antigenic by transpeptidation, as in the previous examples of spliced peptides presented to T lymphocytes by antigen-presenting peptides (2–4). molecules. Moreover, the relevance of such modified peptides is Chemical inhibition of PNGase impaired the presentation of strengthened by the fact that the peptide described here is the the spliced peptide in both melanoma cells and transfected cells target of T lymphocytes whose infusion into a melanoma patient expressing the wild-type tyrosinase but not in cells expressing was followed by tumor regression, indicating that this spliced fi a modi ed tyrosinase containing aspartic residues instead of peptide is a relevant target of antitumor immune responses and fi asparagines at the relevant positions. This result con rmed that might be useful for cancer immunotherapy. PNGase was responsible for the conversion of the encoded as-

paragine residues into aspartates, presumably through removal 1. Hanada K, Yewdell JW, Yang JC (2004) Immune recognition of a human renal cancer of the sugar moieties attached to these N-glycosylation sites. antigen through post-translational protein splicing. Nature 427:252–256. Because PNGase is located in the cytosol and protein N-glyco- 2. Vigneron N, et al. (2004) An antigenic peptide produced by peptide splicing in the sylation occurs in the ER, the parental protein seems to be proteasome. Science 304:587–590. 3. Warren EH, et al. (2006) An antigen produced by splicing of noncontiguous peptides in retrotranslocated from the ER to the cytosol before removal of the reverse order. Science 313:1444–1447. the sugar moieties. Such retrotranslocation is known as a critical 4. Dalet A, Vigneron N, Stroobant V, Hanada K, Van den Eynde BJ (2010) Splicing of step of ER-associated degradation (ERAD), a process allowing distant peptide fragments occurs in the proteasome by transpeptidation and produces proteasomal degradation in the cytosol of misfolded proteins the spliced antigenic peptide derived from fibroblast growth factor-5. J Immunol 184: 3016–3024. located in the ER. 5. Altrich-VanLith ML, et al. (2006) Processing of a class I-restricted epitope from Presentation of the spliced antigenic peptide was also im- tyrosinase requires peptide N-glycanase and the cooperative action of endoplasmic paired by blocking the activity of TAP, the transporter chan- reticulum aminopeptidase 1 and cytosolic proteases. J Immunol 177:5440–5450.

11742 | www.pnas.org/cgi/doi/10.1073/pnas.1101892108 Dalet et al. Downloaded by guest on September 26, 2021