Accepted Article Research Triangle Park, NC of Health Department of Health, and Institutes Services, Human Sciences, National Health and Helmholtz Zentrum Augsburg,München, Germany Switzerland PhD BRANDSTETTER Hans LONDONE. PhD This by is protectedarticle reserved. Allrights copyright. doi: 10.1111/all.13948 lead to differences between this version and the throughbeen the copyediting, pagination typesetting, which process, may and proofreading hasThis article been accepted publicationfor andundergone peerfull but review not has # d c b a Wai Tuck SOH MSc Running Title: MULTIPLE ROLES OF BET V 1 LIGANDS AND MODULATION OFENDOSOMAL ACTIVITY Title:MULTIPLE LIGANDS ROLESINBET V1 ALLERGENSTABILIZATION OF :OriginalArticle: BasicTranslational and Article Allergy Immunology type PROF. FATIMA FERREIRA (Orcid ID: 0000-0003-0989-2335) PROF. CLAUDIA TRAIDL-HOFFMANN (Orcid ID: 0000-0001-5085-5179) 0000-0002-5159-2558) : ID (Orcid GILLES STEFANIE DR. DR. GEOFFREY MUELLER (Orcid ID : 0000-0001-8361-5323)

GILLES PhD PhD GILLES MSc MSc Chair and Institute of Medicine, UNIKA-T, Environmental Technical University Munich Christine-Kühne-Center for Allergy for Rese Christine-Kühne-Center Department of UniversityBiosciences, of Salzburg, Salzburg, Austria. These authors contributedequally to this work. Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Institute of BiologyGenome National Structural IntegrityLaboratory, and a , Tamara SCHEIDT MSc MSc SCHEIDT Tamara , c, d , Richard WEISSPhD b , Claudia TRAIDL-HOFFMANN PhD #a , LorenzAGLAS PhD a * a , Fatima, FERREIRA PhD , Peter M. THOMPSON PhD M. , Peter a , Sandra SCHEIBLHOFER PhD PhD SCHEIBLHOFER Sandra , arch andEducation(CK CARE),Davos, Version of Record. Please Version as this cite of Record. article #a , Geoffrey A.MUELLER PhD a *

c, d b , Peter BRIZA PhD PhD BRIZA , Peter , Chiara CABRELE PhD Chiara , CABRELE

a , Sara HUBER, b , Stefanie a , Robert , Robert a , Accepted Article This by is protectedarticle reserved. Allrights copyright. its allergenicity. molecules pollen-derived endolysosomalinfluence the processing Betvtherebyof 1, shaping incompletely extract,birch pollen unde remain sensitization, allergic of birch including roles of the Bet v 1andothercomponents of the birch major has been asthe However, pollen identified the molecular allergen. mechanisms Background ABSTRACT REL). (Z01-ES102906-01, Intramural Research Program of theNational by the inpart supported was study “Biomolecules” This Salzburg. of the of University Cancer-BioNano Research Centre”, byand theDoctorateSchool PlusProgram P23417FWF and Projects P27589), by theUniversity of Salzburgprogram “Allergy- priority Allergy”; and “Immunity inCancer W_01213, Program Funds(FWFPhD Science Austrian rat recombinant providing Vienna)for Sciences, providingand Lukas legumain, Dr. Mach (U We thank Dr. Elfriede (Department Dall of UniversityBiosciences, of Salzburg) for ACKNOWLEDGEMENTS Email: [email protected] +43-662-8044-5016,Tel.: Fax:+43-662-8044-745016 34, A-5020Austria Str. Hellbrunner Salzburg, ofDepartment Biosciences, Universityof Salzburg authors * Corresponding : Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 Betv pollen worldwide allergy. 1 people birch suffer from million 100 : Over

rstood. Here,rstood. we examined howknown birch Institute of Environmental Health Sciences Health Environmental of Institute B. Thiswork was supported by the niversity of Natural Resources and LifeResources and Natural niversity of Accepted Article Keywords: centered view to a more systemic view that includes the host endolysosomal . an allergen- from paradigma shift unveil . We by processingincluding cysteine its endolysosome, tothe When carried such modulate proteolytic compounds the can activity, Conclusion: aexhibited dualrole by stabilizing Betv 1 inhibitingand cathepsinprotease activity. This by is protectedarticle reserved. Allrights copyright. Results in BMDCs. processing by individualendolysosomal aswell as the T-cell epitope presentation v Bet kineticanalysis 1 of detailed bya followed ligands, and absence of the presence Bet v pr proteolytic theninvestigated We the 1. Methods BPE,pollen birch extract; BMDC, bone marrow-derived ; ANS, 8-anilinonapthalene-1-sulfonic acid; AMC, 7-amino-4-methylcoumarin; ABBREVIATIONS: lysosomal protease inhibition cleavage ofthesites endolysosomal proteases cathepsin S andlegumain. E preferential and kinetics degradation affecting v1, Bet of resistance proteolytic the enhanced : Weidentified E : We analyzed the biochemical and immunological interaction of ligands with ligands immunological interaction of biochemical Weanalyzedand the : allergenicity; birch pollen extract; E extract; pollen allergenicity; birch Bet v 1 can serve as a transporter of pollen-derived, bioactive compounds. pollen-derived, asatransporterof serve v1can Bet 1 phytoprostanes as novel Bet v 1 ligands. Pollen-derived ligands phytoprostanes Pollen-derived v novel as 1 ligands. Bet

ocessing of Bet v 1 by endosomal extracts in extracts ocessingBet v1 by endosomal of 1 phytoprostanes; ligandphytoprostanes; interaction; 1 phytoprostanes Accepted Article TLR, Toll-like receptor receptor Toll-like TLR, tris(2-carboxyethyl)phosphine; TCEP, SDS-PAGE, sodium dodecyl sulfate pol SAW, surface wave;acoustic Q3OS, quercetin3-O-sophoroside; Kdo This by is protectedarticle reserved. Allrights copyright. K spectroscopy; infrared Fourier-transform FTIR, Fc sorting; cell FACS, fluorescence-activated EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; DOC, sodium deoxycholate; DC, dendritic cell; CMK, chloromethyketones; CD, circulardichroism; PPA NMR, nuclear magnetic resonance; NLR, NOD-likereceptor; weight; MW, molecular dendritic cells; moDCs, monocyte-derived LTA, lipoteichoic acid; lipopolysaccharide;LPS, PPAR- d ε , equilibrium dissociationconstant; equilibrium , RI, high-affinityRI, IgE receptor; 2 1 , Kdo , PPB , γ , nuclear, peroxisome proliferator-activated receptor 2 1 -Lipid A; , PPE 1 , PPF , 1

, phytoprostane A phytoprostane , yacrylamide gel electrophoresis; 1 , B 1 , E 1 , and F , and 1 ; γ ; Accepted Article its major allergen Bet_v_1. allergen major its that, in the case of birch ( sensitizatio Bet_v_1, initial pollen allergen the primarylatterby triggered allergensuchasalone,the molecules the isolated canbe birch allergenicity. ofa determinant as properties concerninglipid-binding investigated allergens been have their reported as a physiological Bet_v_1 ligand. O previ Ina agonists. (TLR) receptor Toll-like (iii)microbe-derived and (ii)phytohormones, flavonoids, (i) which pollen-derived: are two of Three major groups of havecompounds beenpropos for a wide variety of natural hydrophobic ligands has been discussed. hasbeen hydrophobicligands natural of variety wide a for protein This by is protectedarticle reserved. Allrights copyright. by and byTh2 polarization a pronounced followed antibody an acute step. recognition Anresponse is allergic process, sensitizationtwo-step involvinga characterized step initial an INTRODUCTION even more intriguing.even more is structurally similar to brassinosteroids to similar structurally is deoxycholate(DOC), secondary generated acid microbiala as bile metabolicbyproduct a that Bet_v_1 have not beenyet reported for them of potential interest as a sensitization mechanism. sensitization as of potentialinteresta them to mammal functionally related ability of Bet_v_1 to bind brassinosteroids has demonstrated brassinosteroids been of Bet_v_1 bind ability to the While inpollen low-molecular-weight compoundsbrassinosteroids,extract. present are -sophoroside (Q3OS)was found to withBet_v_1 pollen thereforeco-purify from and 6

3,4 Bet_v_1’sIn thiscontext, abilityas orstorage tofunction a carrier Betula verrucosa) Betula 2 This observation This observation makes theroleBet_v_1 of amajor as allergen ian prostaglandins and possess prostaglandinsian Th2-skewing making and activity, phytoprostanes. Phytoprostanes like E Phytoprostanes like phytoprostanes. 10 and as serves anestablished ligand modelfor pollen allergy, Th2 polarization is not driven by is notdriven by polarization Th2 allergy, pollen ous study the glycosylated flavonoid quercetin 3- quercetin flavonoid glycosylated ous study the 7 Phytohormones, including and phytoprostanes n process is more complex. morecomplex. We recently is foundn process ed to interact or cooperate withBet_v_1, or cooperate interact ed to 9 Other ligands of interest includeinterest ligands of Other 8 , physical , interactions with 5 Indeed, several Indeed, 1 (PPE 1 While 1 ) are ) are Accepted Article Bet_v_1 ligands have been proposed either to exhibit direct immunomodulatory functions immunomodulatory to functions exhibit either direct Bet_v_1 proposed havebeen ligands legumain. Sand cathepsin particularly extracts, using purified and extracts reproduced microsomal detailedA description of methods theis providedin online the supplement. AND METHODS MATERIALS ourfor understanding of allergy. pollen-derived cathepsinscysteine inthe endolysosome. Wedi and allergenicity by influencing itsprocessing inthe endolysosome. change immunogenicity its could which indirectly, Bet_v_1conformation the or tostabilize been proposedbeen to interact with Bet_v_1. lipopolysaccharide, andthe LPS)have endotoxin acid, LTA, NLRP6agonistlipoteichoic and DOC. Remarkably, PPE Bet_v_1 present at approximately 50–70% recombinant Bet_v_1.0101 Bet_v_1(termed in themost the following), abundant isoform of In study,this webiochemically and immunologically dissected the ofinteractions This by is protectedarticle reserved. Allrights copyright. Bet_v_1 protease legumain. havebeenother proteases shown belonging to the papain belongingfamily, tothe endolysosomal proteases, thelarge family of cat 10,11 21

. In addition, immunomodulatory microbial compounds (such compounds as theTLR2 microbial and immunomodulatory In addition, . 20 As thedegradation endosomal such, of by Bet_v_1 modeled canbe 1 was notonly by retained Bet_v_1, but inhibited thealso

plays an important role in proteolytic inactivity. proteolytic plays role animportant to be relevant in antigen processing, including the cysteine including the antigen processing, relevant in to be

6,12-15 22

, with several ligands, including Q3OS, PPE includingQ3OS, ligands, with several , hepsins,which of most cysteineare proteases scuss the implications of new findingsscuss theimplications of these 19 17,18 Only a few few a Only Among 16 1 ,

Accepted Article phytoprostanes (PPB described elsewhere sophoroside (Q3OS) from Haihang Industry Co., Ltd. (Jinan PPE Ltd.(Jinan China). City, Co., Haihang(Q3OS) Industrysophoroside from USA);AL, 3- Inc. Lipids, andquercetin (Alabaster, Avanti Polar Korea) or South Incheon, (St. Louis, MO, USA);Kdo aureus DOC, 8-anilinonapthalene-1-sulfonic LTA (ANS), naringenin, acid from Investigated compounds and Bet_v_1 ligands This by is protectedarticle reserved. Allrights copyright. (K constantdissociation the of selected including determination of Bet_v_1compounds, the with interaction Surface wave acoustic technology (SAW) andNMR interaction Protein-ligand dissolved in DMSO. room at A incubated temperature. either 2h 4°Cratio and orfor overnight at 4C. Unless otherwise stated, molar Bet_v_1the ligandssix a 1:10 eachof wasmixed with in E performedng/ml) werepreviously as described. (<0.3 contamination endotoxin andmonitoring of Bet_v_1.0101 recombinant Production of Expression, andpurification, physicochemical characterization of recombinant Bet_v_1 (PPA JASCO Jasco, J-815 Tokyo, spectropolarimeter, Bet_v_1the thermal stability of elements and 1 -, and F 1 ) were purchased from Cayman Chemi , andLPS , from 1 -phytoprostanes (PP 23 1 ), F ), . Type I or/and type II phytoprostanes phytoprostanes type II I or/and Type . Escherichia coli Escherichia 1 d -phytoprostanes (PPF -phytoprostanes ). The of influence The ligand ). binding on secondarythe structure 2 -Lipid A (Kdo mix ) were produced by autoxidation of by) wereproduced autoxidation O111:B4 Inc. purchased were Sigma-Aldrich, from 2 ) from Adipogen, Inc. (Songdo-dong, Yeonsu-gu, (Songdo-dong, Inc. Adipogen, ) from was monitored using circular dichroism (CD, dichroism usingwas monitored circular 3,11 cals (Ann Arbor, MI, USA) and dried and and dried USA) MI, cals (AnnArbor, 1 ), and an isomeric mixture consisting of mixture of B isomeric andan consisting ),

Japan) and Fourier transform infrared Fourier transform and Japan) spectroscopy were used toobserve used spectroscopy the were were used, as inFigureindicated α -linolenic acid, as acid, -linolenic 1 -phytoprostanes Staphylococcus 1 , B O 1 1 -, - - Accepted Article vitro cell hybridomas were performe cell hybridomas were (moDCs) wasanalyzed aspreviously described. monocyte-derived ofcells maturation (BMDCs).The human dendritic cells dendritic described. excess)or andBet_v_1Q3OS in molar 10× wi This by is protectedarticle reserved. Allrights copyright. (Fc receptor IgE high-affinity human the with transfected cells, (RBL-2H3) basophil rat using assays by was mediator-release assessed degranulation ligand-loadedinduceand basophil Bet_v_1The ability of IgE-antigen-crosslinking to assaysImmunological detailed descriptionof these methods is USA). MA, A Billerica, Inc., Optics Bruker II system, FTIR (Tensor (FTIR) spectroscopy degradation assays.details Experimental described are the insupporting online information. PPE DOC, (either ligand-bound with was performed assay degradation The endolysosomal proteases endolysosomal In vitro vitro uptake of labeled Bet_v_1 was performed usingCD11c assaysis available (onlinesupporting information). simulation endolysosomal of using microsomesdegradation and individual 21 Recombinant human cathepsin S and human legumain were used in proteolytic used inproteolytic were legumain Sandhuman cathepsin human Recombinant d aspreviously described. available (online supporting information).supportingavailable (online 2 thout ligands (apo-Bet_v_1) as previously as previously (apo-Bet_v_1) ligands thout T-cell proliferation proliferation assays T-cell CD4 using ε 17 RI), as previously described. aspreviously RI), A detailed description of the A of description detailed the + murine bone marrow-derived bone murine 2,24 + T-

In in 1 , Accepted Article is similar to prev to similar is Bet_v_1 interacts with high affinity with pollen-derived PPE with pollen-derived Bet_v_1 interacts withhighaffinity RESULTS (Bachem) as a fluorogenic substrate. andpapain Vienna, Austria) (Merck, Mach) recomb of Activities TCEP. mM DTT with0.5 As a reference ligand, the binding of ANS to Bet_v_1 was determined (K determined wasBet_v_1 to ANSof the binding ligand, reference As a similarbuthomogenous immune structure activity stimulatory whencompared LPS.to native LPS-substructure Kdo LPS-substructure more quantitative previously qualitativeapproach than assays. described determined the dissociation constants (K components, Q3OS andPPE To assess the interactions between Bet_v_1 PPE DOC, andQ3OS, DOC, compound but LTA ornot LPS.with brassinosteroid-like This by is protectedarticle reserved. Allrights copyright. of PPE The in inhibitory was effect parallel. assessed activities S andlegumaincathepsin onthe The effect (BPE)(20–200µg/mL) of pollen birch extract mM and 2 5 mMEDTA, chloride, 0.1 acetateM Msodium pH5.0, (0.1 sodium substrate buffer in digestion fluorogenic of ligand (unless stated) of 50 µM and otherwise was of protease incubated with 100 µM Sandlegumainon cathepsin of 10nM influence Bet_v_1 ligands the To evaluate activities, Enzymatic activity assays respectively. The bacterial TLR agonists, LTA (199.8 µM) and LPS (185.0 µM), and and the and LPSµM), (185.0 Therespectively. bacterial TLR agonists, µM) LTA (199.8 iouslyK published 2 -Lipid A (Kdo

1 , exhibitedhigh withK , binding affinities DTT), as described in the online supporting information . supporting online information the asdescribed in DTT), 2 ) was used for bindingstudies, due itsmore used to for ) was d d µM) (18.5 values ) using binding SAW assays S1), 1, (Table Fig. a at 10 nM were assayed at10nM were Z-FR-AMCusing inant rat (provided by Dr. Lukas cathepsin B(providedby Dr. inant rat 27 1 . The two pollen-derived The two. pollen-derived 1 wasassessed byreplacing and Q3OS and with the and withthe Q3OS and 1 , LTA, or LPS, we LPS, or LTA, , d d 11,26 of 32.7 µM) which µM) 32.7 of = 1.5 and 0.5 µM, = 1.5 and 0.5 µM, In addition, the Inaddition, Accepted Article differences between the for LTA, LPS, or Kdo or LPS, LTA, for phytoprostanes (PP structuresecondary S4). (Fig. content indicating lower binding affinities. For the phytoprostane derivatives, PPB phytoprostane Forthe affinities. binding indicating lower PPE This by is protectedarticle reserved. Allrights copyright. and Kdo model substances, DOC µM) (58.8 specific binding PPE of To thevalidate interactions determined by SAW, we used NMR respectively. as for a physiologically relevant isomeric mixture aphysiologicallyas consisting of for mixture isomeric B relevant approximately 4°Cto DOCPPE nearly 7°C Bet_v_1 boundand for and 2). Bet_v_1Binding of DOC, Q3OS or PPE Q3OSor DOC, Bet_v_1Bindingof 2). prevented direct measurement. The commercially available PPA but affinity, intermediate exchange and with a a poor signal-to-noiselow tosub-µM ratio Moreover, using CD and FTIR spectroscopy we observed an increased melting point (T meltingpoint increased observedwe an spectroscopy CDandusing FTIR Moreover, onSepharose Strep-Tactinimmobilized beads (Fig. S3). Bet_v_1, consistent withLPSpull-down assays biotinylated using LPS and Bet_v_1 presence of PPE 1 to identify the phytoprostane (s). No significant interactionsobserved Nosignificant were binding site(s). phytoprostane the identify to 1 confirmed that confirmed the allergen specifically binds PPE mix 2 ), we observed dissociation constants of 1.0, 2.4, and 1.2 µM, 1.21.0,), µM, and weobserveddissociationconstants 2.4, of , indicating that these indicating bacterialdo notspecifically to, that compounds bind 1 1 , LPS,LTA, andKdo, H- 15 N HSQC spectra of 2 (379.8 µM), demonstrated higherK (379.8 µM), 1 2 toBet_v_1 significantlydid not alterits to Bet_v_1 Fig.(Table 1, S2). Substantial 15 N-labelled Bet_v_1 intheabsence and 1 was used as a substitute foras a wasused

1 spectroscopy theto test . The K 1 , respectively(Table 1 and PPF and 1 -, E -, d wasconsistent 1 -, and -,F and 1 d , as well values, values, m ) of ) of 1 - Accepted Article the resulting PPE diversitythe upon Bet_v_1bindingof peptide wasreduced peptideswhereasthe withinof peptide different ofBet_v_1, thanwiththeform clusters apo 2-fold diversity higher ina Q3OS binding resulted of The 1C). proteases (Fig. endolysosomal the presenceof PPE Bet_v_1in proteolytic stability of anenhanced B) revealed SDS-PAGE 1A, analysis (Fig. of modelligands towardsendolyscomplex withthe 53.9% and respectively).53.9% 69.7%, and In approach, semi-quantitative a thegenerated peptides were presentation Given the relevance of stabilityconformational and resistance proteolytic MHCIIfor influence Bet_v_1 itsLigandinteractions lysosomal with processing. This by is protectedarticle reserved. Allrights copyright. presencethe of plant-derived(Q3OS, PPE Bet_v_1 ligands assessed by None the readouts above RBLof wasS6). by(Fig. influenced described assay in cell supernatants culture S5B,C). (Fig. IgE cross-linking by was Bet_v_1-complexes andby Thpolarization-associated determinationcytokinesmaturation markerexpression of potential was assessed of byon thelevel dendritic cells flow cytometric analysis of S5A) (Fig. with ligands or without Bet_v_1, pHrodo™was Red-labeled response. assessedAntigen byuptake of uptake immune testWe nextoutto for set effects onBet_v_1-complexes ondifferent stages allergicof the dendritic cells. basophil ortheactivation of degranulation Ligandto Bet_v_1doesnot binding affect ensuing response immune the of quantity allergens of with the quality correlates resistance and lysosomal As the the first 12h.observation This correlate 28 ,

we preparedendosomal extracts to theassess resistance of Bet_v_1 in 1 andDOC. ByQ3OS contrast, only weakly stabilizing had a over effect 17 , we, analyzed the peptides generated after with 12 h incubation d with ourwithd data. thermalstability , and subsequent FACS andsubsequent analysis.Sensitizing, osomal proteases over 48 h. Densitometrich. proteases over 48 osomal 1 , and DOC)., and 1 and DOC (to DOC and Accepted Article orthogonal catalytic mechanisms. w families protease endolysosomal cysteine enzymatic activity towards substrates of andcathepsin twoprominent legumain, Based on previously described enzymatic data further analyzing of ligand theinfluence binding to processingBet_v_1 ontheir capability. down thecomplexity theassay of by identifying is various aimedtobreakSincewe an acomplexmixtureof , extract endosomal of basis degradation. mechanistic attenuatedthe microsomalModeling the Bet_v_1 of processing by S legumaincathepsin reveals and peptide poolavailable MHCII presentation isfor the byaffected ligands. the andqualityof quantity both the show that data these Bet_v_1Together, 1C). in Fig. box identified (grey of peptides number by asindicated the epitope, T-cell immunodominant of the generation moreefficient in a processing, whichresulted of proteolytic patternaltered an showed withQ3OS orDOC complex peptides. Bet_v_1 in cluster terminal This by is protectedarticle reserved. Allrights copyright. the presence of PPE In ligands. of presence bythe elimination wasaffected and/or production corepeptide of rate grouped main intoseven with th clusters core by PPE proteases cathepsinalsoby inthecase of S, the individual and, DOC, was strongly retarded degradationendolysosomal apo kinetics of Consequently, we tested cathepsin whether activities were specifically by cathep inhibited cathepsin-like andlegumain-like enzymatic activities in extracts,microsomal theseand 1 . Other reported ligands Bet_v_1 . 1 orDOC, Bet_v_1 processing preferentially theaccumulated two N- 29 Consistent with the literature the with Consistent 11 had (Naringenin) a minor ornodetectableeither and ligand-bound Bet_v_1. Indeed, processing ligand-bound Indeed, by and Bet_v_1. sin S/B and legumain inhibitors (Fig. S7). (Fig. inhibitorslegumain S/Bsin and ith subs complementary eir abundanceseir relative shown The 1D). (Fig. S or legumain qualitatively reproduced qualitativelyS orlegumain the reproduced key of proteases microsomalthe extracts and 21,25 , we tested microsomalthe for fraction we , 30 trate preferences and , detected bothwe , Accepted Article cleavage sites. Although ligands cleavageaffected patternother as the PPE well, protease an exhibits asparaginyl-peptidase activity cleavage sitesusingasparagine as after knownwere legumain, noother only reproduced ge were clusters peptide the of majority The vulnerablesites to the protease. can be reconciledexposes byassuming thebinding additional toBet_v_1 SDS that of thereduced substrates cleavage fluorogenic of bycathepsinThese observations S(Fig. 3A). significantly accelerated degradation its by This by is protectedarticle reserved. Allrights copyright. (PPB exclusively by the interaction with Bet_v_1. Therefore, whether we tested the withBet_v_1. Therefore, ligandsexclusively by theinteraction We wondered whether observedthe of the(de)stabilizing ligandseffects were caused Birchpollen extract reducescathepsin activity in adose-dependent manner. of peptides available for MHCpresentation is strongly affected bythe presenceof ligands. Asp157analysis this (Fig. strongly Overall, 2C). relativeshows increasedabundance that the terminal region; upon incubation with legumain, cleavage Asn120 frequency after and with cathepsin S,preferential cleavage was observed after Phe20,Lys21, and inthe C- illustrate the effect of ligand binding onthegeneration of cleavage sites: upon incubation PPE apo form. in the not presentcleavage new sitesrarelycleavages Bet_v_1, generated at certain but sites within frequency of the affected mostly ligands of presence The resultingthe of peptides. abundance Bet_v_1of processing were byaffected the presenceofrelativewe ligands, analyzedthe peptideC-terminal clusters1C, S8). (Fig.of 1 , ANS) effect on its proteolytic resistance. SDS, whichalso bindsBet_v_1 SDS, resistance. proteolytic onits ANS)effect , 1 induced prominent changes prominent induced relative preference of in the Bet_v_1 nerated Susing cathepsin alone;however, several To To understand how patternthe kinetics the and both proteases (Fig. 3A, B).SDS proteases Bycontrast, (Fig. both 32 , particularly relevant for the production particularly the relevant , for 1

wasusedto 31 , Accepted Article PPE To further investigate the mechanism of PPE of mechanism investigate the To further can expect about 150 pmol PPE proteases and PPEfound activity was completely abolished bycompletelyactivity abolished PPE was (TCEP) tris(2-carboxyethyl)phosphine and (ii) class.Therefore, we compared the effect of reducingtwo (i) agents, the thiol-containingDTT protease for this in characteristic a activethe thiol nucleophilic site, cysteine the with reacting of DTT (Fig. 4D, S9). This differential Thisdifferential effect 4D, S9). of DTT(Fig. This by is protectedarticle reserved. Allrights copyright. onlyof PPElosses, come withsignificant typeand II affected protease activity peptidictowards small substrates. Surprisingly, PPE Since approximately 0.5 µg of PPE of µg 0.5 Since approximately cathepsininhibited butnotlegumain 3A). S, (Fig. cathepsin S activity (Fig. 4B, C). We wanted to examine whether PPE not inhibited (Fig. 4A). structurally the (Fig. PPB not inhibited 4A). similar Importantly, cysteine cathepsins. By legumain, contrast, whic cathepsin Smay inhibition partially be caused by PPE effects.exclude possible substrate competition wasused to µg/ml) observed. (200 theBet_v_1 highest concentration wascathepsin at S 3B). marginalto activity, adose-dependent onlegumain contrast effects the inhibition In of (Fig. activity legumain and S cathepsin on BPE of influence the investigated we Therefore, that BPEatcorresponding concentrations PPE 1 1 , in agreement , concentrationwith thereported range inhibits lysosomal cathepsins by blocking their catalytic cysteine. catalytic their byblocking lysosomal cathepsins inhibits 1 -mediated inhibition of the papain-like protease family, such such as protease family, of the papain-like inhibition -mediated 1 in 100 µg of of protein pollen-extracted per mL, i.e. 150nM in100 µg 1 is present in 1 mg of birch pollen-extractedprotein ispresentof in1mg 1 in the presence of TCEP, but inthepresenceof TCEP, thenot presence in can be understood by DTT thiols competing for competing for thiols by understood DTT be can , which lacks any which , Cathepsin thiol groups. S should alsoattenuate activity. proteolytic These data data These suggest BPE-mediated that the h belongs to h belongs a different protease class, was 1 -mediated inhibition, we inhibition, we -mediated analyzed other 1 1 is an active inhibitor, we hypothesized we is active inhibitor, an . 33 . Although the extraction will extraction Although the . 1 andPPF 1 exerts its effect by itseffect exerts

1 notinhibitdid 1 specifically specifically 9 , we Accepted Article enhancing of TCEPversusDTTeffect is due its to strongerreducing capacity acidic at pH. slightly activity- The stronger S9). DTT (Fig. 4D, and TCEP both of in the presence substrate of the pollen process tothe of the of extract significantly sensitization.allergic contribute by explained cannot allergenbe Th2 polarization DISCUSSION ligandsanddetectedthe different by manner,DCs inatime-dependent weincubated BMDCs Bet_v_1with complexwith in of theidentified to test relevance the In order was found. wasPPE found. absence Inthe of published values for Q3OS and DOC. and Q3OS for values published PPE with incomplex Interestingly, Bet_v_1 5). (Fig. secretion byIL-2 indirectly was monitored T-cell immune-dominant the for specific cells This by is protectedarticle reserved. Allrights copyright. PPE on the site the reactive of up Da. to 1,400 hydrophobic byBet_v_1’s propertyis encoded the search for additional factors complementing allergenicity. Bet_v_1 factors additional search for the context, in compoundsable bind pollen-derived promisingto allergens represent candidates and PPE but TLR notthe phytoprostanes, agonists LTA PPE contrast, Q3OS andPPB In 5F). only48h (Fig. presentation after wasaffected whereas incomplexwith DOC epitope 1 1 consistently affected the MHCII presentation of Bet_v_1 on affected MHCII 5B of epitope the DCs presentation (Fig. consistently and DOC affect Bet_v_1 processing and presentation in DCs 1 , , well as DOC, haveas micromolar to affinities Bet_v_1, comparable to previously

40,41 1 did not theaffect ofBet_v_1. presentation Here, for the first time, we observed that Bet_v_1 binds Bet_v_1that observed we first time, the Here, for 1 inhibitor. By contrast, no inhibitory effect on legumain by PPE by on legumain effect noinhibitory By contrast, inhibitor. presentation byusingCD4 ofBet_v_1 1 we found high cathepsin Sactivity highwe cathepsin fluorogenictowards afound 22,27

Bet_v_1 processing ligandsin and presentation epitope (Thr142-Ala153). T-cell proliferation T-cell (Thr142-Ala153). epitope binding pocket, which can compounds harbor can which binding pocket, and LPS. The pollen-derived pollen-derived Q3OSligands The LPS. and ic exclusively; instead, components 11,35-39 + T-cell hybridoma T-cell Structurally, this 2 Inthis - 34 F),

1

Accepted Article κ immunogenicity/allergenicity. stability tocorrelatewi thermal Increased tended revealedOur results that ligand binding resultedin overall protein-stabilizing an effect. endolysosomalsubstantially extracts was TLR4can induce expression ofmaturation markers of whichviaactivation withLPS, treatment additional discrepancies by explained be the can expression, neither did Bet_v_1 alone norBet_v_1 complex with Q3OSin orDOC. These didnotupregulate cytokinemarkers nor maturation additional LPS-co-stimulation alter

loading and density of class IIMHC-peptide complexes, thus favoring Th2 polarization. beenIt has proteolytic suggested that diminished processing of inlowantigens results This by is protectedarticle reserved. Allrights copyright. PPE Consequently, legumainConsequently, was inthe included component of the endolysosomal fraction, withlowerfluorescencealbeit signal. an allowingS, by largely pattern ustoestablish cathepsin degradation bemimicked but its can thereaction becomplexity, of endolysosomal conditions the fraction controlled, cannot easily contrast, we found that of stimulation bymoDCs PPE different from cathepsins. properties, substrate profiles, mechanistica legumain is not a member of papain-like the protease clan andtherefore possesses B and activation of PPAR- B andactivation of in vitro 1 inhibits the production of IL-12p70 in LPS-stimulated human DCs via blocking of NF- of blocking humanDCsvia LPS-stimulated in IL-12p70 of production the inhibits degradation system. degradation 29

17 γ , thus favoring a Th2-dominated thus favoring, immune Th2-dominated response. a Indeed, Indeed, the susceptibility Bet_v_1to degradation byof 21 Here, Here, we revealed significant legumain activity a as reduced by the ligands DOC and PPE ligands DOC bythe and reduced nd profiles inhibition that fundamentally are th proteolytic stability, which in turn affects in which turn stability, proteolytic th in vitro in 42 . 1 in complex withBet_v_1 complex without in degradation system. Importantly, system. degradation 1 . Due to its 23,33 By 18

Accepted Article such an effectwasnot observed for PPB when Bet_v_1 was complexed with PPEwith Bet_v_1when wascomplexed reduction showingcell proliferation of T-cellepitopesunique presentation assay, the a the in The immunological relevance of these unexpected findings was demonstrated even in T-a and, in particular, was identified in birch pollen. wasin particular, identified and,

PPE possesses bethis inhibitory can explained structure function PPE bythe chemical of This by is protectedarticle reserved. Allrights copyright. PPE available Bet_v_1peptides ligand for Secondly,MHC presentation. the newly identified affected theallergenprocessing primarily respect relative with to the ofabundance generated Bet_v_1 endolysosomal processing intwo mech usingInvestigation the differs from PPB from differs spontaneously undergo dehydration concentrations ranging 4.5 from 61 toper ng gramof weight dry by inhibited PPE explanation the for S-inhibitory effectcathepsin underacidic is conditions, PPEthat, active of site papain-like proteases the than controlled stringently more sterically is legumain of the to access The 4E). of the thiolate cysteine, therebycatalytic covalently (Fig. active site protease blocking the nucleophilic the of addition the favors cyclopentenone The acceptor. Michael electrophilic legumain. WhyPPElegumain. 1 1 ’s cysteine cathepsin-inhibition function, andfunction, cathepsin-inhibition ’s cysteine selectively inhibited cathepsin S and othe 1 . The . The reactive 3-hydroxy-cyclopent 1 and PPF 1 , butnot two , phytoprostanesthe structurally PPBrelated in vitro in 1 at the five-membered ring the five-membered at degradation system revealed that Bet_v_1 ligands can tune 32 , explaining why legumain neither reacts with nor is with reacts neither explaining why , legumain 43 1 , converting the five-membered ring into an ring into five-membered the converting , . 1 . This drastic effect can mostlyby can effect explained drastic This be . anistically different ways. Firstly, ligands Firstly, ways. different anistically r papain-like cysteine butproteases, not hardly to its stabilizingproperties since 9, 45 anone is anone commonly in found plants PPE 43 (Fig. 4C). The mechanistic 44 1 . was found in plants in plants at wasfound 1 andPPF 1 , which 1 can 44 1 ,

Accepted Article immunogenicity. proteases. Increased proteolytic resistanceof Bet_v_1 drastically affects its allergenicityand of an endosomal inhibitor, which interferes with the main class of antigen-processing carrier unexpected mechanism servesas a by identifiedwhichwe To an Bet_v_1 summarize, This by is protectedarticle reserved. Allrights copyright. Bet_v_1homologues, suchas 1Cor a are required relevant clinically of binding ligand investigatingstudies syndrome, future pollen-food the of in light Especially, orIgE-binding. the proliferations, T-cell processing, stability, proteolytic able tobind whichligands, enables themtofurt or other it isunknown sources from Sopollen Bet_v_1 food whether homologues are far, interaction interaction of Bet_v_1 with ligands receptors. these receptorsNLRs. TLRs like directthereimmune and a intracellular be may Additionally, presentedand immunopeptidome of butalsothe endosomalthe activation proteolytic interest. interest. of conflicts authors declare norelevant remaining theythat have The USA).AllergenOnline, CH; SIAF, Davos, NL; Advisory (HAL Allergy, Scientific Boards a memberof is Ferreira F. CONFLICT OF INTEREST interpreted data. W.T.S., L.A., H.B., F.F wrote the manuscript. and experiments the devised F.F. H.B., C.C., C.T-H., R.L, S.G., G.M., L.A., W.T.S, W.T.S., L.A., S.H.,S.G., T.S., P.T., P.B.,G.M. performed the experiments. CONTRIBUTIONS AUTHOR finding thatfinding the sensitizationprocess birch by pollen extractsindependent is Bet_v_1.from to reconcilethe intriguing help molecules can non-proteinogenic activation bypollen-derived 17 Furthermore, such broad-spectrumlikely inhibition only is tochange not her increase their alle their her increase 46 47 . The relevance of such direct or indirect orindirect suchof direct relevance The rgenicity in terms of interms rgenicity 2

Accepted Article This by is protectedarticle reserved. Allrights copyright. HoneyRudensky K, AY. Lysosomal cysteine proteasesregulate antigenpresentation. 19. FreierE, R,Dall Brandstetter H. recognition Protease sites in Bet_v_1a are cryptic, 18. Y, Machado Freier S, R,Scheiblhofer Foldet during stability endolysosomalal. 17. Ligacheva AA, Danilets MG, Trofimova ES, et al.Signaling Events during 16. Hara Seregin H, SS, Yanget D,al. TheNLRP6 Inflammasome Recognizes 15. EC, Trevethick HulmeLigand MA. binding assaysvalidation atequilibrium: and 14. WyerJR, Willcox BE,Gao GF,et al. T cell receptor and coreceptor CD8 alphaalpha 13. McKenna Posselt OE, G, P, Briza etal. Multi-Approach Analysis the for 12. Asam Kofler S, C,M, EckhardU, WallnerBrandstetter FerreiraF, H. 11. Markovic-Housley DeganoZ, M,Lamba D, et al. structure Crystal of a 10. C,Mariani Traidl-Hoffmann V, Hochrein H, et al. phytoprostanes Pollen-associated 9. Chwastyk M, Jaskolski M, Cieplak M. Structure-based analysis of thermodynamic 8. Seutter vonC,Hoffmann Loetzen Hartl T, MJ, et al. Secretof the birchmajor pollen 7. Bublin M, Eiwegger T, Breiteneder H. Do 6. Radauer H. C,The LacknerBet_v_1P, Breiteneder scaffold versatile ancient,fold: an 5. M, Smith S, Jager Berger U,etal. Geographic temporalpollenandin variations 4. Asam C, BatistaAL,Moraes AH,et al. Bet_v_1--a Trojan horse smallfor ligands 3. Aglas L, Gilles Bauer S, R, al.et Contextmatters: TH2 polarization resultingfrom 2. O,AkdisM, Palomares M, Akdis Martin-Fontecha CA. Mechanisms ofimmune 1. REFERENCES NatImmunol. Rev itsexplainingprocessing slow to relevant its allergenicity. pollen allergen. acidification is a key factorforof immunogenicity and allergenicity the birch major Med+. Activation Betulapendulawith RothPectic Polysaccharides. LipoteichoicAcid and Gram-Positive Regulates Infection.Pathogen interpretation. 1999;10(2):219-225. bind peptide-MHC independentlydistinctwith and kinetics. of within Identification Proteases Pollen.Birch of Bet_v_1. Birch Allergen Pollen Isoforms MappedCrystallographically Ligand High in Binding Differs IgE Binding Lowand biological function as planta steroid carrier. hypoallergenic ofthe isoform pollenmajor birch allergen Bet_v_1 itslikelyand polarization. cellinhibit dendritic interleukin-12production and Taugment helper type 2 cell pathogenesis-related proteins of class 10. and mechanical of properties cavity-containing proteins--case studyplant of 2014;457(3):379-390. allergenBet_v_1: identification of the physiological ligand. process? bindingfor oflarge,ligands.hydrophobic Europe. exposure across sensitization? boosting allergic Immunol. pollen composition and not from protein-intrinsic allergenicity. 2017;278(1):219-236. in the diseases: regulation allergic 2014;156(4):465-469. 2014;156(4):465-469. J Allergy Clin Immunol.

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Po 23. of Recognition Ligand O, al. et Hartl-Spiegelhauer T, Jacob C, Loetzen von Seutter 22. Egger M, Jurets A, Wallner M, et al. Assessing Protein Immunogenicity with a 21. E, Dall Brandstetter H.Structure of function and legumain in health anddisease. 20. Biochimie. 2013;191(4):1529-1535. 2013;191(4):1529-1535. cat dander proteinFel d 1 enhances TLRactivationby lipid ligands. 477. native recombinanthouse and mite dust Der allergen 1. p deliverydisruption oftight by junctions. Than Dithiothreitol. Carboxyethyl)Phosphine, anOdorlessReducing AgentStable More Effectiveand vivo. of aqueousbirch pollen extracts phytoprostanesand primary on immuneresponses in 2018;46(D1):D624-D632. withcomparison peptidases in the PANTHER database. databaseproteolytic of substratesenzymes, their 2017 ininhibitors and and a 2014;107(12):2972-2981. ofdynamics andbirch compactness the pollen major allergen. Peptidases. 2013;110(27):10940-10945. zymogenicity, distinct activation pathways, and regulation. protein structure bylimitedproteolysis. Chem. allergen, Bet_v_1,shows affinity for a broad spectrumof physiological ligands. Major BirchAllergen Pollen Bet_v_1Isoform is Dependent. Peptides.Epitope-Containing Ole e1-LikeProteins: Analysis ofProteolytic Cleavage Sites Revealing T Cell Proteolytic Resistance to Cathepsin S. 2009;182(11):6653-6658. PPAR-gamma NF-kappaB-dependentand mechanisms. 2015;10(6):e0128677. the Major Birch Pollen Allergen Bet_v_1 is Isoform Dependent. Dendritic Line-DerivedCell Endolysosomal Degradome. J Allergy Clin Immunol. 2002;277(26):23684-23692. 2002;277(26):23684-23692. 2016;122:126-150. Academic Press; 2013. Anal Biochem. Anal Biochem. Introduction:Families of Clans and The Cysteine Int J Mol Sci. Mol Int J 2007;120(2):293-299. 2007;120(2):293-299. al. Ligandal. bindingmodulates the structural 1994;220(1):5-10. . Allergens. as immunomodulatorythe proteins: Int J Mol Sci. Int MolSci. J llen-derivedsignal E1-phytoprostanes via Acta BiochimActa Pol. ffmann C, ffmann et al.Immunomodulatory effects J Clin Invest. Invest. J Clin 2017;18(8). 2017;18(10). 1999;104(1):123-133. 1999;104(1):123-133. J Immunol. Nucleic Acids Res. Allergy. Allergy. Plos One. 2004;51(2):299-321. P Natl SciUSA. Acad Plos One. Biophys J.Biophys PLoS One.PLoS 2009;64(3):469- 2011;6(2). 2011;6(2). J Immunol. 2015;10(6). J Biol Accepted Article This by is protectedarticle reserved. Allrights copyright. J. March MB, Smith 48. BonhamKS, Kagan JC. EndosomesNOD-like as platforms receptor for signaling. 47. a area: birch-free a in sensitization pollen Fagales F. Ferreira M, Wallner A, Mari 46. Oeder Alessandrini S, F, Wirz OF, et al. Pollen-derivedsubstancesnonallergenic 45. S, Parchmann Mueller MJ. Evidence ofdinor for the isoprostanesformation E-1 from 44. Mueller MJ. Isoprostane nomenclature: Inherentproblems may cause setbacksfor the 43. Shen H,Tesar BM, Walker WE, Goldstein DR.Dual signaling ofMyD88 and TRIF 42. HP. Size Erickson and shapeof molecules protein at the nanometer determined level 41. Chwastyk M,Jaskolski CieplakM,M. The volumeofin cavities proteins and virus 40. A, Trompette Divanovic Visintin S, A, et 39. RoyerEmara PJ, M, YangC, et The al. mannosereceptor mediates theuptakeof 38. diverse nativediverse allergens bydendritic cells andallergen-induced determines Tcell Cell HostMicrobe. v 2,rBet v 4). rBet (rBet_v_1, allergens respiratory cohortsurveyusing Fagales pollenextracts andbirchrecombinant enhance Th2-induced IgE productionin B cells. 32655. plants.in acidalpha-linolenic ofdevelopment isoprostanoid the field. 2008;181(3):1849-1858. is forcritical maximalTLR4-induced dendritic cell maturation. 2009;11:32-51. and microscopy. by gelelectron filtration, sedimentation, capsids. ofreceptor mimicry aToll-like protein. complex polarization ofIDOthrough modulation activity. Proteins. 2016;84(9):1275-1286. 2016;84(9):1275-1286. 2014;15(5):523-525. 2014;15(5):523-525. March's Advanced Organic Chemistry. Journal of Biological Chemistry. Prostag Leukotr Ess. Clin Exp Allergy. Allergy. Exp Clin al. Allergenicity resulting from functionalAllergenicity from al. resulting Allergy. Allergy. Nature. J Immunol. 2015;70(11):1450-1460. 2015;70(11):1450-1460. 2009;457(7229):585-588. 2009;457(7229):585-588. Biol Proced Online. Online. Biol Proced 2003;33(10):1419-1428. 2003;33(10):1419-1428. 6ed: Wiley; 2007. 2010;185(3):1522-1531. 2010;82(2-3):71-81. 2010;82(2-3):71-81. 1998;273(49):32650- J Immunol. Accepted Article CD, circular dichroism; FTIR, Fourier transform infrared spectroscopy; Tm, melting point; SD, Standard deviation compounds Bacteria-derived binding groups essential mimicking Model compounds compounds Pollen-derived

SAW interaction studies and binding confirmation by NMR spectroscopy spectroscopy NMR by and bindingconfirmation studies SAW interaction This by is protectedarticle reserved. Allrights copyright. PPE1 DOC Q3OS - T Ligand Table 2:Influenceon of ligand th interaction Table 1:Binding (K affinity TABLES

06 ±01 6.1 ± 0.05 ± 4.36 ± 1.77 69.31 66.6 ± 2.24 65.26 ± 0.15 ± 0.58 63.38 70.62 ± 0.10 67.44 ± 0.06 64.04 63.68 m

C DC T SDCD CD Kdo LPS LTA ANS DOC PP Q3OS PPB opudM D] K MW Compound [Da] PPA PPF PPE mix 2 1 1 1 1

d ) of ) of Bet_v_1 tothe compounds determined selected as by 2,306.8 2,306.8 10,000-20,000 4,000-8,000 308.4 356.5 328.4 308.4 626.5 299.34 414.6 m FTIR SD FTIR FTIR SD FTIR ermal ofBet_v_1in stability (values °C) 379.8 379.8 185.0 199.8 n.d. n.d. 0.5 2.4 1.0 1.2 1.5 32.7 32.7 58.8 d [µM] SD [µM] NMR [µM] [µM] NMR SD [µM] [µM] ±62.8 ±123.1 ±55.7 n.d. n.d. ±0.1 ±0.5 ±0.4 ±0.1 ±0.1 ±0.3 ±0.3 ±24.3 Δ + 6.94 + 3.81 + 0.36 CD No significant interactions No significant interactions No significant interactions 0.1-1 0.1-1 n.d. n.d. n.d. [7] [11] [11] [11] Δ + 5.93 + 3.22 + 1.88 FTIR FTIR Accepted Article This by is protectedarticle reserved. Allrights copyright. BPE A,activities. ligandsbirch Effect polle of 3. and Fig. peptidepresented profiles are inFig. S8. one The individualsites ligand. for given of emphasizes varyingthe kinetic accessibility representationof Thisis availableligand. adirect rather not the cleavage but sites, therespective peptidefor abundant themost found to peptide intensity wasnormalized basedwere on relative by peptidesthe measured of abundance spectrometry mass the and C, was analyzedprofile Blue-stained byCoomassie and SDS-PAGE degradation The ligands. various of presence or absence the in legumain and S cathepsin degradation of ligands Effect 2. on Bet_v_1 Fig. The ofunique number intensities. peptide grouped their as clusters abundance, intoseven with MS from degradation derived relative and 48h 0to points from time at different analysis of Ligandthe 1. Fig. interaction prot alters LEGENDSFIGURE fluorogenic fluorogenic activity 15min. was measured after Bet_v_1 wasRecombinant used as control proteolytic degradation proteolytic degradation analyzed by massspectrometry. (Bio-Rad). Software 4.0.1 Lab Image Bet_v_1 cleavage site frequency analyses of on lysosomal protease activity. protease on lysosomal in vitro Effect of ligands on fluorogenic activity of ligands lysosomal proteases. Effect of onfluorogenic endolysosomal degradation of Bet_v_1 with and without ligand recorded ligandrecorded without ofBet_v_1 withand endolysosomal degradation

BPE C,

Generated peptide clusters obtained after 12 h of12 h obtained after clusters peptide Generated sequences is shownsequences in is brackets. was incubated with the with respectivewas incubated protease and the B, eolytic susceptibility of Bet_v_1. A, n extract (BPE) on cathepsin S and legumain legumain Sand oncathepsin (BPE) n extract densitometric analysis thereof, interpreted with the degradation assay in(A). The analyses in vitro.

D, A, The peptide The sequences were Bet_v_1 degradation assay by B, densitometric analysis. densitometric B, SDS-PAGE SDS-PAGE Effect of Effect Accepted Article elimination, resulting in PPA resulting in elimination, (Thr142-Ala153) upon presentation by presentation BMDCs. (Thr142-Ala153) upon pg/ml) thelogarithmic peptideconcentration ofto thecorresponding immune-dominant A, T-cells. phytohormones used in (B). phytohormones usedin(B). activity. activity Fluorogenic was 15 after recorded min. Fig. 5. Effect of ligand binding on the Be the on of ligand binding 5. Effect Fig. adduct donor) the at S(Michael ofcathepsin nucleophilic cysteine This by is protectedarticle reserved. Allrights copyright. inPPF andisdisfavored ring, hydroxylgroup inthe min. wereincubatedlegumain PPE with (ra proteases Papain-like cysteine not legumain. PPE of mechanism 4. Inhibition Fig. over bars Error indicate buffer control. standard deviations. for apossible substrate competitioneffect. The percent fluorogenicactivitywas calculated PPE and legumain. Theto ability proteases cleave of resulting PPA resulting mechanism of cathepsin S inhibition by PPE by cathepsin Sinhibition of mechanism p <0.05. with statisticalsignificance indicates Asterisk deviations. standard were Z-VVR-AMCand legumain and Z-AAN-AM presentation by BMDCs 16 from to 48h. 1 (5 µM) in the presence ofDTTand TCEP. the in µM) (5 B, 48 Effect of phytohormones (0.1 mM) structurally related toPPE mM)structurally of phytohormones (0.1 Effect related , thus inhibiting cathepsin S inhibiting cathepsin activity. , thus IL-2 the relating curve Dose response 1 is an electrophile (Michael acceptor) and can be by readily attacked the and can acceptor) (Michael isanelectrophile D, 1 . Effect of reducing of onPPE Effect agents 43 This reaction does not occur with PPB with occur reaction This does not 1 (5 µM) and fluorogenic activities were recorded after 15 after were recorded activities fluorogenic and (5µM) 1 . A, . A, C-F t_v_1-specific presentation BMDCsto CD4 of t_v_1-specific 1 PPE . PPE , comparison comparison , of the presented Bet_v_1 T-cell andthe fluorogenic substrates with without 1 t cathepsin B, cathepsin B,t cathepsin cathepsin andpapain) andS, inhibits papain-like papain-like but proteases,cysteine inhibits Fluorogenic substrates used for cathepsinsubstrates S used for Fluorogenic 1 undergoes spontaneous dehydration by B secretion T-cellsecretion of (in hybridoma cells 1 , kinetics of Bet_v_1 T-cell epitope Bet_v_1 T-cell of kinetics , due to the missing ketone group. themissing The ketone dueto C, respectively. Error bars indicate indicate Error bars respectively. C,

β carbon to form a covalent toform carbon C, 1 inhibition of of cathepsin inhibition S Chemical structure Chemical of 1 1 on cathepsin S , whichlacks a E, Proposed β + -

Accepted Article Fig. S2. Fig. PPA Therefore high lower affinity ligands concentrations. ligand commercially availablerequire limited ratio poor signal-to-noise a exchange and (green), LTA (red), LPS (light blue), orPPA blue), LPS (light LTA (red), (green), A, S4. Fig. beads Sepharose Strep-Tactin on immobilized S3. LPSpull-down and LPS assay usingFig. biotinylated Bet_v_1 binding pocket of Bet_v_1. the line residues that sheet other various in and helix C-terminal of the interior the residues in This by is protectedarticle reserved. Allrights copyright. P-values were calculated withone-way ANOVA and a dependency 24, 32 epitope in timepoint (16, ateach ligand ofinvolvedindividual and 48h). spectra of

Fig. S1.Fig. *P using statisticalAll performed calculations Ns, P> Prism GraphPad 7software; were 0.05; the binding As the affinities. thea reference, K SAW phase changes were recorded over time at different ligand concentrations to calculate Secondary structureelements were analyzed byCD and ≤ 1 0.05; **P was used to identify the phytoprostane binding site(s). The signals The affected to binding identifyphytoprostane site(s). belonged wasusedto the

Ligand interaction does not affect the se NMR NMR Surface acoustic (SAW) interact wave 15 N-labelled Bet_v_1 theabsence andpresenceN-labelled ofin (black) PPE 1 ≤ H- 0.01; ***P 15 N HSQCN ofBet_v_1 spectra ligands. andthepossible ≤ 0.001;****P d of ANS was determined to be 32.72 µM. µM. be32.72 to determined was ANS of ≤ 0.0001. direct measurement. NMR measurements of NMRmeasurement. measurements direct 1 (dark blue). Intermediate conformational Intermediate (darkblue). ion studies immobilizedof Bet_v_1. condary structure elementsBet_v_1. of

Tukey´s multiple comparisons test. test. Tukey´s multiple comparisons B, FTIR measurements. 1 Overlay of two (violet), Kdo (violet), The 2

Accepted Article s. 900 for monitored were assays The enzymatic respectively. Ac-YVAD-CMK, and Z-FL-COCHO were and Z-AAN-AMC, for S inhibitors used respectively.cathepsin The peptidic andlegumain substrates Thefluorogenic DC). (murine used substrates. fluorogenic S7.Fig. Detection of cathepsin and legumain inactivities DC microsomalextract using shown in A. half representation mediatormaximal releaseAggregated on of dependent Bet_v_1ligands as A, S6.was Mediator-releaseFig. significantly changedbyBet_v_1 ligands. not *P >0.05; P Ns, software; statisticalcalcul All independent experiments. were P-values calculated SEM. with ANOVA. Thedata at twoderived are least from representeddata asthe mean and are The ratio. 1:10 molar in a pre-incubation ligand without presented Bet_v_1 as medianof with or fluorescence. MoDCs were with1 treated µg/ml Maturation results and wereligands. experiments cytometry, the performedareflow by with donors either Bet_v_1 upon alone stimulation or incombination with pollen-derived IL-1(CCL17, DR, CD83, CD80, and CD86)and This by is protectedarticle reserved. Allrights copyright. CD11cPercentage of A, DCs. human and murine by Bet_v_1 ligand-bound uptake of and Activation S5. Fig. Q3OS, or PPE Q3OS, or releaseprofilesdependentPatient mediator β 1 , IL-6, IL-10, and IL-6, TNF , ) monitored over 24 h. h. over) monitored 24 + BMDCs taking up labeled Bet_v_1 in the presence of a ligand (DOC, ligand presence a of the BMDCs in Bet_v_1 uplabeled taking The microsomal The extract line was the JAWSII cell fromisolated ≤ 0.05; **P C, ≤ 0.01; ***P B, α secretion Thpolarization-associated of cytokines ) by moDCs derived from atopic non-atopicatopic ) by moDCsderivedfromand Expression of maturation markers (CD40, HLA- markers (CD40, maturation of Expression for cathepsin and legumain were Z-VVR-AMC were legumain and cathepsin for ations were performed using were ations performed GraphPad Prism 5 on thepresence ofBet_v_1 the ligands. ≤ 0.001. B,

Accepted Article Fig. S9. Effect of PPE of reducing S9. agents on Effect Fig. ( This by is protectedarticle reserved. Allrights copyright. of ligands. bytw ofBet_v_1 S8.profile Degradation Fig. cathepsin S and legumain were Z-VVR-AMCandZ-AAN-AMC, respectively. presence of DTT and TCEP was monitored fo ability of proteases to cleave the fluor to cleave the proteases ability of B ) in the) in presence ofvarious ligands. Thepeptides were identifiedbyspectrometry. mass Time-dependent degradation profiles Bet_v_1 of byS ( cathepsin ogenic substrates with and without PPEand without substratesogenic with 1 inhibition of cathepsin S and legumain. S and cathepsin of inhibition o endolysosomalproteases inthepresence r 30 min. Fluorogenic substrates used for substratesused Fluorogenic rmin. 30 A 1 ) and legumain (5 µM) in the inthe µM) (5 The Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.