The proline-rich motif of the proDer p 3 allergen propeptide is crucial for protease-protease interaction.

The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen.Peer reviewe

Etude du mécanisme d’activation du zymogène de l’allergène Der p 3 de l’acarien Dermatophagoides pteronyssinus

Der p 3, an allergen of the house dust mite Dermatophagoides pteronyssinus, is a serine protease of the trypsin-like family. In addition to allergenic properties, its protease activity might be involved in its adjuvant effect in the pathogenesis of allergy. The protease is synthesized as an inactive proDer p 3 zymogen formed by an 11 amino acid N-terminal propeptide and a protease domain of 232 amino acids. No recombinant source of Der p 3 has been described yet and the zymogen maturation mechanism remains to be elucidated. The presence of a Thr implicated in a potential N9AT N-glycosylation site at the C-terminal end of the Der p 3 propeptide suggests, however, a unique activation mechanism. The first part of this study was thus dedicated to express the zymogen in the yeast Pichia pastoris, to investigate its activation mechanism and to characterize the recombinant Der p 3 protease. With the use of both discontinuous and continuous enzymatic tests, we showed that the unusual intermolecular activation mechanism of proDer p 3 is mediated by the house dust mite cysteine protease Der p 1. Glycosylation of the propeptide was found to decrease the rate of maturation. We report for the first time a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. In the second part of this study, we analyzed the role of the propeptide and its particular polyproline rich motif (PRM) (NP2ILP5ASP8NAT) in the folding, thermal stability and maturation mechanism of the zymogen and also in the inhibition of Der p 3. Using non glycosylated zymogens, with one or all prolines replaced by alanine(s), we showed that, although the entire propeptide is not required to obtain a correctly folded protease, the PRM is important for resistance of proDer p 3 to undesired proteolysis. Amino-terminal deletions introduced in the propeptide did not influence the thermal stability of the protease. With the help of a quench flow system coupled to mass spectrometry, we determined the catalytic parameters (kcat, Km and kcat/Km) characteristic of the Der p 3 zymogen activation by Der p 1 and we demonstrated that prolines, mainly at positions 5 and 8, are crucial for activation. Finally, we showed that Der p 3 is inhibited by the free modified prosequence TP1R and that prolines affect the mode of inhibition of the protease.Two other allergens of the D. pteronyssinus species, termed Der p 6 and Der p 9, are serine proteases with chymotrypsin and elastase specificities, respectively. Similarly toDer p 1 and Der p 3, both are also expressed as inactive zymogen forms and matured into active proteases. These four proteases might be involved in the mite digestion. With the help of FRET synthetic peptides containing the activation sites of the four zymogen forms, we highlighted the proteolytic cascade occurring in the mite and, in particular, we elucidated the activation mechanism of proDer p 6 and proDer p 9./L’allergène de l’acarien Dermatophagoides pteronyssinus Der p 3 est une protéase à sérine active appartenant à la famille de la trypsine. Son activité protéolytique pourrait être un facteur adjuvant impliqué dans le développement et la chronicité de l’allergie. Cette protéase est synthétisée sous forme d’un précurseur inactif, proDer p 3, qui est constitué d’un propeptide 11 acides aminés suivi d’un domaine protéase de 232 acides aminés. A l’heure actuelle, le mécanisme d’activation de ce zymogène n’a pas encore été élucidé et il n’existe aucune forme recombinante de l’allergène Der p 3. La présence d’une thréonine à l’extrémité C-terminale du propeptide ainsi que celle d’un site potentiel de N-glycosylation N9AT suggèrent un mécanisme d’activation particulier. Durant la première partie de cette étude, nous nous sommes intéressés à l’expression recombinante de proDer p 3 en Pichia pastoris, à l’élucidation de son mécanisme d’activation et à la caractérisation de la forme mature Der p 3. Au moyen de tests enzymatiques continus et discontinus, nous avons mis en évidence que le mécanisme d’activation inter-moléculaire de proDer p 3 est médié par la protéase à cystéine active Der p 1. La cinétique d’activation était ralentie par la présence de la glycosylation au niveau du propeptide. Enfin, nous avons obtenu, pour la première fois, une forme recombinante de Der p 3 qui présente les mêmes propriétés enzymatiques que l’allergène naturel et la trypsine.Dans la deuxième partie de cette thèse, nous avons étudié le rôle du propeptide et en particulier celui des prolines du motif riche en prolines (PRM) (NP2ILP5ASP8NAT) dans le repliement, la stabilité thermique, le mécanisme d’activation ainsi que dans l’inhibition de la protéase Der p 3. Au moyen des zymogènes au sein desquels une ou les trois proline(s) a (ont) été remplacée(s) en alanine(s), nous avons pu mettre en évidence que bien que le propeptide en entier ne soit pas nécessaire à l’obtention de zymogènes correctement repliés, ce PRM est important pour la résistance de proDer p 3 à la protéolyse non spécifique. Les délétions amino-terminales introduites dans le propeptide de proDer p 3 n’ont pas modifié la stabilité thermique du zymogène. Au moyen d’un système couplant un système de quench-flow à un spectromètre de masse, nous avons pu déterminer les paramètres catalytiques (kcat, Km et kcat/Km) du mécanisme d’activation du zymogène proDer p 3 par Der p 1 et montrer ainsi que les prolines 5 et 8 du propeptide sont cruciales pour le mécanisme d’activation du pro-enzyme. Finalement, nous avons mis en évidence que Der p 3 est inhibée par la proséquence modifiée TP1R et que les prolines ont un rôle à jouer dans le modèle d’inhibition de la protéase.Deux autres allergènes appartenant à l’espèce D. pteronyssinus sont des protéases à sérine active de type chymotrypsine et élastase et sont nommées respectivement Der p 6 et Der p 9. Comme Der p 1 et Der p 3, ces allergènes sont synthétisés sous forme de précurseurs inactifs et doivent être ensuite activés en protéases matures. Ces quatre groupes d’allergènes seraient impliqués dans la digestion de l’acarien. Au moyen de peptides synthétiques basés sur la stratégie FRET et contenant les sites d’activation des quatre zymogènes, nous avons pu mettre en évidence la cascade protéolytique se déroulant chez l’acarien et plus particulièrement, nous avons élucidé le mécanisme d’activation des zymogènes proDer p 6 et proDer p 9

Comparative study of mature and zymogen mite cysteine protease stability and pH unfolding.

BACKGROUND: Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes. METHODS: To have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1. RESULTS: We showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen. GENERAL SIGNIFICANCE: Finally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis

Orchestration of an uncommon maturation cascade of the house dust mite protease allergen quartet.

In more than 20% of the world population, sensitization to house dust mite allergens triggers typical allergic diseases such as allergic rhinitis and asthma. Amongst the 23 mite allergen groups hitherto identified, group 1 is cysteine proteases belonging to the papain-like family whereas groups 3, 6, and 9 are serine proteases displaying trypsin, chymotrypsin, and collagenolytic activities, respectively. While these proteases are more likely to be involved in the mite digestive system, they also play critical roles in the initiation and in the chronicity of the allergic response notably through the activation of innate immune pathways. All these allergenic proteases are expressed in mite as inactive precursor form. Until recently, the exact mechanisms of their maturation into active proteases remained to be fully elucidated. Recent breakthroughs in the understanding of the activation mechanisms of mite allergenic protease precursors have highlighted an uncommon and unique maturation pathway orchestrated by group 1 proteases that tightly regulates the proteolytic activities of groups 1, 3, 6, and 9 through complex intra- or inter-molecular mechanisms. This review presents and discusses the currently available knowledge of the activation mechanisms of group 1, 3, 6, and 9 allergens of Dermatophagoides pteronyssinus laying special emphasis on their localization, regulation, and interconnection

Cross-Reactivity between Major IgE Epitopes of Family 5 Allergens from Dermatophagoides pteronyssinus and Blomia tropicalis

Background: The aim of this work was to understand the molecular features that trigger the cross-reactivity observed between Der p 5 from Dermatophagoides pteronyssinus, Blo t 5 from Blomia tropicalis, and Der f 5 from D. farinae. Methods: We collected serum from 60 house dust mite (HDM)-allergic patients residing in the Dellys area of Boumerdès province in northern Algeria. The presence of specific IgE to Der p 5, Der f 5, and Blo t 5 was analyzed. We performed in silico analysis of the structure of the different allergens in order to identify epitopes that can elicit the cross-reactivity of the sera. Synthetic peptides corresponding to the linear epitope sequence of Der p 5, Der f 5, and Blo t 5 were used to evaluate its implication in the cross-reactivity between the allergens. We also modified the sequence of the conformational epitope of Der p 5 by site-directed mutagenesis to mimic Blo t 5. Results: Several sera of patients allergic to HDM contained specific IgE antibodies to Der p 5 and Blo t 5. We demonstrated that the linear epitope of Der p 5 and Blo t 5 is not involved in the cross-reactivity of the sera. Furthermore, mutations introduced in the sequence of Der p 5 to mimic Blo t 5 could not modulate the cross-reactivity between them. Conclusions: The major linear IgE epitopes of Der p 5 and Blo t 5 are involved in species-specific recognition. Our results may be useful for the development of a hypoallergenic vaccine against HDM group 5 allergens.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Der p 1 is the primary activator of Der p 3, Der p 6 and Der p 9 the proteolytic allergens produced by the house dust mite Dermatophagoides pteronyssinus

Background: The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade. Methods: The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Förster Resonance Energy Transfermethod. The proDer p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanismbymite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus. Results: All peptides were specifically cleaved by Der p 1, such as proDer p 6. The propeptide of proDer p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite. Conclusions: Der p 1 in either its recombinant formor in the natural context of house dustmite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases. General significance: This finding suggests that Der p 1 may be valuable target against mites

The Lys-Asp-Tyr Triad within the Mite Allergen Der p 1 Propeptide Is a Critical Structural Element for the pH-Dependent Initiation of the Protease Maturation.

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element

Immunodominant IgE Epitopes of Der p 5 Allergen.

Der p 5 is an important allergen of Dermatophagoides pteronyssinus that plays a key role in allergic airway diseases. Its three dimensional structure (PDB 3MQ1) consists of three anti-parallel α-helices arranged in a helical bundle. Aggregation of Der p5 can modulate its allergenicity. This study aimed to identify the key residues of IgE binding epitopes of Der p 5.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Inhibition of the keratinolytic subtilisin protease Sub3 from Microsporum canis by its propeptide (proSub3) and evaluation of the capacity of proSub3 to inhibit fungal adherence to feline epidermis

Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis, mainly in cats, dogs and humans. Proteolytic enzymes have been postulated to be key factors involved in the invasion of the stratum corneum and keratinized epidermal structures. Among these proteases, the secreted subtilisin protease Sub3 was found to be required for adherence of M. canis arthroconidia to feline epidermis. This protease is synthetized as a preproenzyme consisting of a signal peptide followed by the propeptide and the protease domain. In order to assess whether the enzymatic activity of Sub3 could be responsible for the role of the protease in the adherence process, we expressed and characterized the propeptide of Sub3 and demonstrated that this propeptide is a strong inhibitor of its mature enzyme. This propeptide acts as a noncompetitive inhibitor with dissociation constants, Ki and K?i of 170 and 130 nM respectively. When tested for its capacity to inhibit adherence of M. canis to feline epidermis using an ex vivo adherence model made of feline epidermis, the propeptide does not prevent adherence of M. canis arthroconidia because it loses its capacity to inhibit rSub3 following a direct contact with living arthroconidia, presumably through inactivation by fungal membrane-bound proteases.Fil: Baldo, A.. Université de Liège; BélgicaFil: Chevigné, A.. Université de Liège; BélgicaFil: Dumez, M. E.. Université de Liège; BélgicaFil: Mathy, A.. Université de Liège; BélgicaFil: Power, Pablo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Université de Liège; Bélgica. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tabart, J.. Université de Liège; BélgicaFil: Cambier, L.. Université de Liège; BélgicaFil: Galleni, Lorena Alejandra. Université de Liège; BélgicaFil: Mignon, María Belen. Université de Liège; Bélgic