Why do proteases mess up with antigen presentation by re-shuffling antigen sequences?

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Why do proteases mess up with antigen presentation by re-shuffling antigen sequences?

Juliane Liepe

¹

, Huib Ovaa

²

and Michele Mishto

³

ThesequenceofalargenumberofMHC-presentedepitopesis notpresentassuchintheoriginalantigenbecauseithasbeen re-shuffledbytheproteasomeorotherproteases.Whydo proteasesthrowaspannerintheworksofourmodelofantigen taggingandimmunerecognition?Wedescribeinthisreview whatweknowabouttheimmunologicalrelevanceofpost- translationallysplicedepitopesandwhyproteasesseemto haveasecond(dark)personality,whichiskeentocreatenew peptidebonds.

Addresses

1Max-Planck-InstituteforBiophysicalChemistry,37077Go¨ttingen, Germany

2DepartmentofChemicalImmunology,LeidenUniversityMedical Center,NL-2333ZALeiden,TheNetherlands

3CentreforInflammationBiologyandCancerImmunology(CIBCI)&

PeterGorerDepartmentofImmunobiology,King’sCollegeLondon, SE11ULLondon,UnitedKingdom

Correspondingauthor:Mishto,Michele(michele.mishto@kcl.ac.uk)

CurrentOpinioninImmunology2018,52:81–86

ThisreviewcomesfromathemedissueonAntigenprocessing EditedbyGennaroDeLibero

ForacompleteoverviewseetheIssueandtheEditorial https://doi.org/10.1016/j.coi.2018.04.016

0952-7915/ã2018TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creative- commons.org/licenses/by-nc-nd/4.0/).

Introduction

Epitopes can have their sequence re-shuffled by proteases, post-translationally modified, trimmed and bended onto MHC class I (MHC-I) molecules. Trans- formationscanbesodisguisingthatantigensmighthave trouble evenrecognizing themselves due to these non- canonical peptides. Nonetheless, the immune system seemstobeabletoselectivelyidentifythemnon-canoni- calepitopesandusethemforpatrollingthestatusofthe cell[1,2].

A growing number of studies about non-canonical epitopes has in part whipped out what we learned from textbooks about antigen presentation. For instance, intrinsiccharacteristics of non-canonical epitopes,espe- cially of those derived from peptide splicing,force the boundariesofourconceptualizationoftheimmunological self [3]. For example, a pre-requisite for streamlined

CD8⁺Tcellspatrollingbyrecognizingantigenicspliced peptides is that their generation is tightly regulated.

Indeed, ifanarbitrarypeptidefragment wereligatedto another fragment we would likely have dramatic pro- blems during thymocyte selection in the thymus due to animmensevarietyof splicedpeptidespresentedby corticalandmedullarythymicepithelialcells(cTECsand mTECs, respectively) and othermedullary professional antigenpresentingcells(APCs).Accordingtothethymic selectionmodels[4],onlyahandfulofthymocyteswould survive the negative selection with such an immense antigenic peptides’ variety presented by professional APCs. In agreement with the pre-requisite for stream- linedpatrollingbyCD8⁺Tcells,thereisagrowingbody of evidence that peptide splicing—and in particular proteasome-catalyzed peptide splicing (PCPS)—is not arandomprocess,andonlyaminorportionofthetheo- retical splicedpeptide isgenerated and presentedto T cells.Whatthesedrivingforcesare,andimplicationsthey can have on the immune response is still to be fully understood.

The MHC-I antigen presentation pathway is on the contrary welldescribed (Figure1).CD8⁺Tcellactivity is strongly regulated by which epitopes are presented onto MHC-I complexes, that is, the MHC-I immunopeptidome.AlterationsoftheMHC-Iimmunopeptidome affectthecytotoxicCD8⁺Tcellresponseagainstviruses andtheefficacyofanti-cancerimmunotherapies[5,6,7].

The immunopeptidome isinfluencedbyseveral factors (seeFigure1)includingantigenavailabilityand charac- teristics [8,9], proteasome processing [1], transport into theendoplasmicreticulum(ER)andloadingtothepep- tide loading complex (PLC) [10], trimming by ER amino peptidases (ERAPs) [7,11], as well as affinity to differentMHC-Iallotypes’clefts[12].

Part of theMHC-I immunopeptidome canderive from non-canonical reading frames [13], polymorphic or mutated sequences [2,14,15], non-coding sequences andDRiPs [16,17],orpost-translationallymodified peptides [18,19]. The predominant non-canonical peptide populationseemstobe,however,representedbyspliced peptides. Indeed,in theMHC-Iimmunopeptidomesof humanEBV-immortalizedBcellsandprimaryfibroblasts around20–30%ofpeptidesareproducedbyPCPS[20].

Although the average number of molecules of spliced peptidesboundtoMHC-Icomplexesissmallerthanthat of non-spliced peptides [20], spliced epitopes can be presented onto MHC-I complexes in the amount

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comparable to non-spliced epitopes [21]. For example, MHC-I-bound spliced epitopes have been found to prime a specific CD8⁺ T cell response during Listeria monocytogenesinfection[22].Furthermore,aspecificacti- vationof CD8⁺ Tcellstoward splicedepitopesderived from tumor-associated antigens is detectable in mela- noma patients [21] and has led to a regression of the tumormassinamelanomapatientandaleukemiamurine model [23,24]. As a consequence, spliced peptides are interestingnovelcandidatesfor anti-viralvaccinedevel- opment[25]andanti-cancerimmunotherapies[1,26].

Themolecular baseofthe doublelife of proteasome (andother proteases?) in permanentbalancebetween cleavagesand ligations

How does it come, however, that the proteasome and possiblyotherproteasesinhumancellsseemtobreakand buildagainpeptidessoefficiently?Wealreadyknowthat PCPSefficiencyispreservedalongevolution[27]andthat therearefactorsthatpromotePCPS.Forinstance,invitro assayssuggestthattheproteasome favorstheligationof somepeptidemotifs [27,28],and wheretheproteasome

preferstocleave,itdoesnotoftensplice[27].However,to understandwhytheproteasomecatalyzessooftenPCPS, weneed someinformationabout itsstructure. The proteasome core particle is a barrel-shaped multi-subunit complex. In its internalcavity, it can accumulateup to 200–300smallpeptides[29],or2–3proteins[30,31].Ithas three pairs of catalytic subunits (b1, b2, and b5). The activesite’s threoninenucleophilesfacetheproteasome internalchannel andare surrounded bythe non-primed andprimedsubstrate-bindingsites(Figure2a).Thesub- strate degradation rate is driven by the proteolytic-site activity as well as by the peptide transport along the internalchannel[29].The catalyticsubunit substitution, whichisthehallmarkdifferentiatingproteasomeisoforms, affects—atleast at aquantitative level—cleavage-site preferencesandthesubstratedegradationrate[29,32,33].

ItalsoimpactsPCPS,although,likely,onlyinasubstrate- specificmanner[21,27,34].In cells,PCPScan occur via eithertranspeptidationorcondensation[21,35],although the frequency of the latter mechanismstill needsto be assessed.Ininvitroassays,PCPScanefficientlyoccurby splicingfragmentsderivedfromthesamemolecule—cis PCPS—andfromdifferentmolecules—transPCPS—

Figure1

preferentially from which antigens?

proteasome

antigen fragments:

spliced and non-spliced

amino peptidases

TAP MHC-I- calreticulin

Tapasin ERp57

ERAP

PLC

Golgi

cytosol

antigen

preferentially from:

long, polar, acidic, disordered, short half-line, DRiPs antigens

CD8⁺ T cells

extracellular space secretory

vesicles

ER lumen

Current Opinion in Immunology

AntigenprocessingandpresentationbyMHC-IcomplexestoCD8⁺Tcells.Inthispathway,themajorityoftheantigensareprocessedby proteasome,whichproducessplicedandnon-splicedpeptidesinthecytosol.Peptidesarefurtherdegradedbyamino-peptidases,thereby regeneratingthecellularaminoacidpool.Fewpeptides,however,aretransportedintotheendoplasmicreticulum(ER)throughthetransporters associatedwithantigenprocessing(TAPs),whichispartofthepeptide-loadingcomplex(PLC).There,peptidescanbetrimmedbyER-resident aminopeptidases(ERAPs).MHC-I-peptidecomplexesundergomodifications,andaretransportedthroughtheGolgitothecellsurface.There,they canberecognizedbytheTcellreceptor(TCR),andinduceCD8⁺Tcellspriming/activation.

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(Figure2b)[27,28,36].Accordingtothetranspeptidation model[35],theproteasome’scatalyticN-terminalthreo- ninenucleophilebreaksthepeptidebondoftheresidue (PSP-P1) of the protein—thereby forming an acyl- enzymeintermediatewiththeN-terminalsplice-reactant, coupledtothereleaseoftheinterveningsequence—and, instead ofcatalyzingthe canonicalpeptide hydrolysis,it catalyzestheligationbetweenthePSP-P1residueofthe N-terminalsplice-reactantwiththeresiduePSP-P1⁰ofthe C-terminalsplice-reactant(Figure2b).Proteasome-mediated transpeptidation can alsoresult in isopeptide bond formation when a lysine side chain reacts with an acyl enzyme intermediate. Although it has also been shown thatsuchpeptidescaninduceanimmuneresponse,this processhowever remainstobeobservedinvivo[37].

Proteasome-generated sliced epitopes are however not the only examples of spliced epitopes.Indeed, Delong andcolleagues[38]identifiedsomehybridinsulinpep- tides(HIPs), derivedfromtheligation of afragmentof proinsulinwithpeptidesoriginatingfromotherantigens present inthegranules ofthepancreaticb cells.These trans spliced epitopes are presented by MHC-II complexes,therebytriggeringaspecificresponseinCD4⁺T cells in type 1 diabetic patients [38,39]. In general, extracellular antigenscan beinternalized, processed by proteolysis in the lysosome, bind the MHC class II molecules,andthencirculatetothecellsurfaceandback

to the lysosome [40].Although thelysosomal proteases also rely on(thio)ester intermediates, theenzyme cata- lyzing theproductionof theHIPs isstillunknown.

Whydoproteases(frequently)behaveasligases,too?In principle, any proteasethatusesa nucleophiletopro- motehydrolysisthroughanesterintermediatecancat- alyzetranspeptidation.Henceanyproteasecouldplaya roleintheformationofsplicedpeptides.Althoughitis notunderstoodwhytheproteasomeinparticularseems tocatalyzethisprocesssoefficiently,wecanspeculate on the reasons. Transpeptidation efficiency depends highly on three factors: firstly, high concentration of theaminenucleophilemustbepresentinordertofavor theformation ofanovelpeptide-bondoverhydrolysis;

secondly,theesterneedstohaveasufficientlifetimein ordertoallowthereactionandpeptidebondformation over hydrolysis; thirdly, the active site in which this esterintermediateisformedmustbesufficientlyacces- sible for nucleophilesto react.The proteasome struc- turecanfavorallthesethreeconditions,asit’saclosed barrel that could have a high local concentration of peptide products, and use substrate-binding sites in proximitytotheproteasome’scatalyticN-terminalthre- oninenucleophile.Anotherresultofpeptides confine- ment in the proteasome barrel could be the fact that transPCPSseemstooccurlessfrequentlythancisPCPS [27,28,36].

16nm gates

10nm

N

N N

N

C C

antechamber (59nm³)

antechamber (59nm³) chamber

(84nm³)

PSP-P1

cleavages by Thr1

spliced peptides

trans PCPS

PSP-P1 PSP-P1′

PSP-P1′

normal cis PCPS reverse cis PCPS

intervening sequences

(a) (b)

Molecularbasefortheunexpectedhighfrequencyofpeptidesplicing.(a)Thehuman20Scoreparticleoftheproteasomeisshownbasedonthe structuregeneratedby[45].ThechainsB,C,H,I,J,Q,R,S,YandZarehiddenfromthestructureinordertoseetheinnerproteasomecavities withthecentralchamberanditstwoantechambers.Theaandbsubunitsarecoloredingreyandblue,respectively.Asanexampleofacatalytic subunit,heb2subunitisshowninpinkwithitsactivesitethreonineinred.(b)Proteasome-generatedsplicedpeptidescanbeformedby:firstly, cisPCPS,whenthetwosplice-reactantsderivefromthesamepolypeptidemolecule;theligationcanoccurinnormalorder,thatis,followingthe orientationfromN-terminustoC-terminusoftheparentalprotein(normalcisPCPS),orinthereverseorder(reversecisPCPS);secondly,trans PCPS,whenthetwosplice-reactantsoriginatefromtwodistinctproteinmoleculesortwodistinctproteins.

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TheoreticalimpactofPCPSinrecognizingthe immunological self

One majorfeatureof peptidesplicing isthe theoretical increaseofthenumberofsequencesthatcanbederived fromtheantigenpoolandbeallocatedintheMHC-Iand MHC-II clefts. This enlargement could have implica- tionsin therecognitionof theimmunological selfbyT cells.Indeed,itcouldincreasetheriskofmimicry,which isthephenomenonwherebytwoepitopeshavesequence similaritiesandarerecognizedbythesameTcellclone [1].Inparticular,wenameas‘zwitterpeptide’apeptide thatcanbederivedfromthehumanself-proteomeaswell as fromapathogenproteome (Figure 3).In2012,Calis et al. [41] investigated the sequence overlap between humanself-peptidesandalargesetofnon-self-peptides derivedfromvirusesandbacteriainthecontextofCD8⁺ Tcellrecognition. Theyfound thatless than 1%of all theoretical possible 9-mernon-spliced peptidesderived frompathogenshaveasequenceidenticaltothetheoret- icalhumannon-splicedpeptides,thatis,arezwitterpep- tides. If the zwitter antigenic peptides were presented similarlybymTECsandotherprofessionalAPCsinthe medullary thymus and by dendritic cells (DCs) in the periphery,wewouldexpecttheabsence,attheperiphery, ofCD8⁺T-cellclonesrecognizing,withhighaffinity,the zwitter peptides presented by DCs, because they have beeneliminatedduringthethymicnegativeselection[4].

Thisphenomenoncouldinpartexplaintheoccurrenceof holesintheTcellrepertoireandintheirabilitytotackle

viralinfections [41].Onthecontrary,if thezwitteranti- genic peptides were efficiently presented by DCs and otherAPCsintheperipherybutnotbymTECsandother professional APCs in the thymic medulla, we would expectattheperipherythepresenceofpotentiallyauto- reactiveCD8⁺T-cellclones,whichcouldbeprimedand activatedbyDCsandotherAPCsinlymphnodesduring thepathogeninfectionandafterwardsattackhumancells andparticipateto inanautoimmuneresponse.

Inmultiplesclerosis,forinstance,myelin-reactiveCD8⁺ T cell are theorized to mediate the cytotoxic activity againsttheoligodendrocytesleadingtothecharacteristic de-myelinationandplaqueformation.Furthermore,asso- ciationsbetweenmultiplesclerosis,someMHC-Ivariants (e.g.HLA-B*07)andEpstein-Barrvirus(EBV)infection havebeenreported,andithasbeenhypothesizedthatan EBVinfection couldtriggertheprimingofautoreactive CD8⁺Tcellclonesthroughmimicry[42].Usingasimilar approachasCalisetal.[41],wecancomparetheoverlapof theoretical9merpeptides(eithersplicedornon-spliced) derived from 24 human myelin proteins (MBP, MAG, MOG,PLPandisoforms)andfrom9EBVantigens(i.e.

LMP1, LMP2, BMLF1, BMRF1, BZLF1, BRLF1, BNRF1,BLLF1,EBNA3).All27peptidestheoretically common to myelin and EBVantigens are spliced peptides, since there are no identical non-spliced peptide sequencesbetweenthesetwosetsofantigens.Amongthe 27theoreticalzwitterpeptides,13peptidesarepredicted

Figure3

Pathogen: EBV Host: Human

EBV latent membrane protein 1 (LMP1) Myelin oligodendrocyte glycoprotein (MOG)

zwitter spliced peptide

IC₅₀(HLA-B*07:02) = 58 nM

ExampleofzwitterpeptidepotentiallygeneratedfrombothEBVandmyelinantigens.Thetheoreticalzwitterpeptide[GPR][LLLLLL]canbe generatedfromboth,theEBVantigenLMP1andthehumanmyelinproteinMOG,throughcispeptidesplicing.Thiszwitterpeptideispredictedto bindtheHLA-B*07:02variantwithanIC₅₀of58nM,anditisoneofthe13peptidesthatarepredictedtostronglybindtothemostcommon MHC-Imolecules.InthisanalysisthebindingaffinityispredictedapplyingtheSMMpredictionmethod[46],filteredforpeptideswithrank1.The 13theoreticalzwitterpeptidesarepredictedtobindoneofthefollowingvariants:HLA-A*01:01,HLA-A*02:01,HLA-B*07:02,HLA-B*08:01,orHLA- B*40:01(datanotshown).

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accountsfor onlythetheoretical presenceor absenceof apeptidein theimmunopeptidomeof APCs.To better estimatetherealprevalenceofzwitterantigenicpeptides andtheirrecognitionbyCD8⁺Tcells,weshouldconsider the TCR degeneracy, theaffinity/avidity of TCRs and MHC-I-peptides,andthedynamicsofthedifferentsteps of theMHC-Iantigenpresentation(Figure1)including the,onlypartiallydescribed,drivingforcesofPCPS.This preliminaryinsilicoresult,however,confirmsthatPCPS could play a particularly relevant role in the central tolerance, the occurrence of large holes in the T cell repertoire, and the autoimmune response mediated by CD8⁺Tcells.

Concludingremarks

Thesurprisingevidencesreportedinthelastyears,which suggestthatMHC-I(andinpartMHC-II)immunopep- tidomes are populated by spliced peptides, need to be confirmedbyapplyingdifferentapproachesbeforeunder- standing the magnitude of their immunological rele- vance. However, the implications of peptide splicing couldexceedtheedgesof antigenpresentation.Ifpep- tidesplicingwereacommonreactionforotherproteases rather than proteasome, we could speculate that post- translationally spliced peptides (and why not spliced proteins?) could be involved in other aspects of the immune response and cell metabolism, as it has been proved for proteasome-processed non-spliced peptides and proteins [43,44]. If this hypothesis were correct, peptide splicing could be a further regulatory layer in thelifeofacellandanorganism.

Conflictof intereststatement Nothing declared.

Acknowledgements

WethankDarijaMuharemagicforproofreadingthemanuscript.Workin theOvaalabissupportedbytheInstituteforChemicalImmunology,a gravitationprogrammefinancedtheNetherlandsFoundationforScientific Research(N.W.O.).

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