International Journal of Molecular Sciences Article Profiling the Extended Cleavage Specificity of the House Dust Mite Protease Allergens Der p 1, Der p 3 and Der p 6 for the Prediction of New Cell Surface Protein Substrates Alain Jacquet 1,† , Vincenzo Campisi 2,3,†, Martyna Szpakowska 2,†, Marie-Eve Dumez 2,3, Moreno Galleni 3 and Andy Chevigné 2,* 1 Faculty of Medicine, Division of Research Affairs, Chulalongkorn University, 10330 Bangkok, Thailand; [email protected] 2 Department of Infection and Immunity, Luxembourg Institute of Health (LIH), 29, rue Henri Koch, L-4354 Esch-sur-Alzette, Luxembourg; [email protected] (V.C.); [email protected] (M.S.); [email protected] (M.-E.D.) 3 Laboratoire des Macromolécules Biologiques, Centre for Protein Engineering (CIP), University of Liège, 4000 Liège, Belgium; [email protected] * Correspondance: [email protected]; Tel.: +352-26-970-336; Fax: +352-26-970-390 † These authors contributed equally to this work. Received: 15 May 2017; Accepted: 21 June 2017; Published: 27 June 2017 Abstract: House dust mite (HDM) protease allergens, through cleavages of critical surface proteins, drastically influence the initiation of the Th2 type immune responses. However, few human protein substrates for HDM proteases have been identified so far, mainly by applying time-consuming target-specific individual studies. Therefore, the identification of substrate repertoires for HDM proteases would represent an unprecedented key step toward a better understanding of the mechanism of HDM allergic response. In this study, phage display screenings using totally or partially randomized nonameric peptide substrate libraries were performed to characterize the extended 0 substrate specificities (P5–P4 ) of the HDM proteases Der p 1, Der p 3 and Der p 6. The bioinformatics interface PoPS (Prediction of Protease Specificity) was then applied to define the proteolytic specificity profile of each protease and to predict new protein substrates within the human cell surface proteome, with a special focus on immune receptors. Specificity profiling showed that the nature of residues 0 in P1 but also downstream the cleavage sites (P positions) are important for effective cleavages by all three HDM proteases. Strikingly, Der p 1 and Der p 3 display partially overlapping specificities. Analysis with PoPS interface predicted 50 new targets for the HDM proteases, including 21 cell surface receptors whose extracellular domains are potentially cleaved by Der p 1, Der p 3 and/or Der p 6. Twelve protein substrate candidates were confirmed by phage ELISA (enzyme linked immunosorbent assay). This extensive study of the natural protein substrate specificities of the HDM protease allergens unveils new cell surface target receptors for a better understanding on the role of these proteases in the HDM allergic response and paves the way for the design of specific protease inhibitors for future anti-allergic treatments. Keywords: house dust mite; Dermatophagoides pteronyssinus; allergen; protease; phage display; cell surface proteome; phage substrate 1. Introduction House dust mites (HDMs) represent an important source of airborne allergens associated with various inflammatory diseases, such as allergic asthma, perennial rhinitis, conjunctivitis Int. J. Mol. Sci. 2017, 18, 1373; doi:10.3390/ijms18071373 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 1373 2 of 16 Int. J. Mol. Sci. 2017, 18, 1373 2 of 16 and atopic dermatitis [1]. Amongst the 20 allergens identified so far in the HDM species Dermatophagoidespteronyssinus (Available pteronyssinus online:(Available www.allergen.org), online: www.allergen.org Der p 1, Der p 3 ),and Der Der p 1,p 6 Der display p 3 andproteolytic Der p 6activities display proteolytic[2]. Der p activities1 is a papain-like [2]. Der p 1cysteine is a papain-like protease, cysteine whereas protease, Der p 3 whereas and Der Der p p6 3are and serine Der pproteases 6 are serine with proteases tryptic and with chymotryptic tryptic and chymotryptic activities, respectively activities, respectively[2]. Notably, [Der2]. Notably, p 1 is not Der only p 1 the is notmost only abundant the most HDM abundant allergen HDM in allergen house dust in house or in dust mite or cultures in mite culturesbut also buta potent also a allergenic potent allergenic protein proteinas more as than more 80% than of 80%the HDM of the allergic HDM allergicpopulation population develop develop high level high of levelIgE specific of IgE to specific this protease to this protease[3,4]. Der [3 p,4 ].1 has Der also p 1 hasbeen also demonstrated been demonstrated to act as tothe act activator as the activator of the precursors of the precursors of Der p of 3 Derand pDer 3 andp 6 Deraccording p 6 according to an uncommon to an uncommon activation activation cascade cascade [5–7]. In [5 –contrast7]. In contrast to Der top Der1, little p 1, is little known is known about aboutthe concentration the concentration of Der of p Der3 and p 3Der and p Der6 in pmite 6 in cultures mite cultures and their and IgE their binding IgE binding frequencies frequencies remain remainpoorly poorlycharacterized characterized with IgE with prevalence IgE prevalence ranging ranging from 10% from to 10%50% for to 50% Der forp 3 Derand paround 3 and around40% for 40%Der forp 6 Der[8–10]. p 6 [8–10]. ItIt is is now now well well established established that that the the proteolytic proteolytic activities activities of HDMof HDM allergens allergens drastically drastically influence influence the developmentthe development of the of allergic the allergic response response through throug differenth different mechanisms mechanisms [11], including: [11], including: (I) the disruption (I) the ofdisruption the epithelial of the barrier epithelial integrity barrier through integrity cleavages through of the cleavages lung epithelium of the lung surfactant epithelium proteins surfactant SP-A, SP-Dproteins [12], SP-A, the tight SP-D junction [12], the proteins tight junction occludin, protei zonans occludin, occludens-1 zona (ZO-1) occludens-1 and cadherins (ZO-1) and [13 cadherins]; (II) the activation[13]; (II) the of activation damage-associated of damage-associated molecular patterns molecular (DAMPS) patterns such(DAMPS) as uric such acid as [uric14,15 acid]; (III) [14,15]; the direct(III) the activation direct activation of protease-activated of protease-activated receptors receptors (PARs) (PARs) expressed expressed on airway on airway epithelial epithelial cells andcells keratinocytesand keratinocytes [16– 18[16–18];]; (IV) (IV) the the cleavage cleavage of immuneof immune receptors receptors expressed expressed by by dendritic dendritic (CD40 (CD40 and and DC-SIGN)DC-SIGN) [ 19[19],], B B (CD23) (CD23) [ 20[20]] or or T T (CD25) (CD25) [ 21[21,22],22] cells; cells; and and (V) (V) thethe inactivationinactivation of of proteaseprotease inhibitors inhibitors suchsuch as as the theα α1-antitrypsin1-antitrypsin [[23],23], thethe elastase-specificelastase-specific inhibitorinhibitor (elafin)(elafin) andand secretorysecretory leukocyteleukocyte proteaseprotease inhibitorinhibitor and and homeostasis homeostasis proteins proteins [ 24[24,25].,25]. Altogether,Altogether, the cleavages cleavages of ofthese these cellular cellular recept receptorsors and secreted and secreted proteins proteins by the HDM by the proteases HDM proteasesinfluence influencethe initiation the initiationof allergic of sensitization allergic sensitization and may and lead may to leadthe exacerbation to the exacerbation of allergic of allergicinflammation inflammation by promoting by promoting a pro-Th2 a pro-Th2 environment environment and/or and/or by by downregulating downregulating thethe Th1/TregTh1/Treg differentiationdifferentiation [[1,11,26].1,11,26]. ItIt shouldshould bebe pointedpointed outout thatthat sosofar far the the different different human human protein protein substrates substrates identifiedidentified forfor DerDer pp 1,1, DerDer pp 33 or or Der Der p p 6 6 were were discovered discovered essentially essentially byby individualindividual andand targetedtargeted studiesstudies [ 16[16,17,22,27],17,22,27] and and that that the the identification identification of theirof their complete complete repertoire repertoire of cellular of cellular substrates substrates is still is unachievedstill unachieved [19]. [19]. Notably, Notabl withy, with the exception the exception of the of tight the tight junction junction proteins, proteins, the airway the airway epithelial epithelial cell surfacecell surface receptor(s) receptor(s) targeted targeted by Der by pDe 1r remain p 1 remain curiously curiously unknown unknown [1,2]. [1,2]. TheThe interplay interplay between between a a defined defined protease protease and and its it correspondings corresponding substrates substrates is mainly is mainly mediated mediated by theby structurethe structure of the of active the active site cleft, site whichcleft, which determines determines the type the of residuestype of residues compatible compatible with the differentwith the substratedifferent bindingsubstrate sites/pockets binding sites/pockets (subsites S1–5 (subsites and S1 S1–50–40). Determiningand S1′–4′). Determining the substrate specificitythe substrate of aspecificity protease consistsof a protease in identifying consists consensusin identifying residues consensus located residues upstream located (P5–P upstream1) and/or (P downstream5–P1) and/or 0 0 (Pdownstream1 –P4 ) of the (P peptide1′–P4′) cleavageof the peptide site, which cleavage can be bestsite,