ABSTRACT
Der p 5 is one of the important house dust mite allergens in Algeria; this allergen is frequently recognized by patients with allergic asthma. However, there is no information on its IgG-binding epitopes. In the present study, rabbits were immunized with recombinant Der p 5 allergen, and serum samples were obtained. Recognition of linear IgG epitopes of Der p 5 was determined using synthesized peptides derived from the allergen sequence. The results showed that serum from immunized rabbits recognized three linear epitopes from Der p 5 (28EDKKHDYQNEFDFLLMERIHEQIK43), (37IHEQIKKGELALFYLQEQ55) and (92LMQRKDLDIFEQYNLEMAKKS112). More interestingly, we observed that the 92L-S112 amino acid sequence is well recognized by both IgE and IgG antibodies. Der p 5 stimulates the synthesis of specific IgG antibodies which recognize common but also novel epitopes compared to IgE antibody binding. Indeed, the potential to induce IgG antibodies can be used to inhibit human IgE binding to allergens which may be part of the mechanism of action of specific immunotherapy.
ABSTRACT
BACKGROUND: The allergen Der p 3 is underrepresented in house dust mite (HDM) extracts probably due to autolysis. Recombinant stable molecule of the allergen is thus needed to improve the diagnosis of allergy and the safety and efficacy of immunotherapy. OBJECTIVE: The current study reports the immunological characterization of two recombinant molecules of the HDM allergen Der p 3 as useful tools for diagnosis and immunotherapy. METHODS: Recombinant mature (rDer p 3) and immature (proDer p 3) Der p 3 and their corresponding S196A mutants were produced in Pichia pastoris and purified. The stability, IgE-binding capacity and allergenicity of the different proteins were analysed and compared with those of the major mite allergen Der p 1 used as a reference. Additionally, the immunogenicity of the different allergens was evaluated in a murine model of Der p 3 sensitization. RESULTS: Compared to the IgE reactivity to recombinant and natural Der p 3 (nDer p 3), the mean IgE binding of patient's sera to rDer p 3-S196A (50%) was higher. The poorly binding to nDer p 3 or rDer p 3 was due to autolysis of the allergen. Contrary to Der p 3, proDer p 3 displayed very weak IgE reactivity, as measured by sandwich ELISA and competitive inhibition, rat basophil leukaemia degranulation and human basophil activation assays. Moreover, proDer p 3 induced a TH 1-biased immune response that prevented allergic response in mice but retained Der p 3-specific T-cell reactivity. CONCLUSION: rDer p 3-S196A should be used for the diagnosis of HDM allergy elicited by Der p 3, and proDer p 3 may represent a hypoallergen of Der p 3.
Subject(s)
Antigens, Dermatophagoides/immunology , Arthropod Proteins/immunology , Dermatophagoides pteronyssinus/immunology , Hypersensitivity/diagnosis , Hypersensitivity/therapy , Immunotherapy , Recombinant Proteins/immunology , Serine Endopeptidases/immunology , Allergens/immunology , Animals , Antigens, Dermatophagoides/metabolism , Arthropod Proteins/metabolism , Basophils/immunology , Basophils/metabolism , Cytokines/metabolism , Female , Humans , Immunization , Immunoglobulin E/blood , Immunoglobulin E/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Protein Binding , Proteolysis , Rats , Recombinant Proteins/metabolism , Serine Endopeptidases/metabolismABSTRACT
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, K(I) and [Formula: see text] 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.
