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The Crystal Structure of Recombinant proDer p 1, a Major House Dust Mite Proteolytic Allergen This information is current as Kåre Meno, Peter B. Thorsted, Henrik Ipsen, Ole Kristensen, of September 26, 2021. Jørgen N. Larsen, Michael D. Spangfort, Michael Gajhede and Kaare Lund J Immunol 2005; 175:3835-3845; ; doi: 10.4049/jimmunol.175.6.3835 http://www.jimmunol.org/content/175/6/3835 Downloaded from References This article cites 49 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/175/6/3835.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Crystal Structure of Recombinant proDer p 1, a Major House Dust Mite Proteolytic Allergen1 Kåre Meno,2* Peter B. Thorsted,* Henrik Ipsen,* Ole Kristensen,† Jørgen N. Larsen,* Michael D. Spangfort,* Michael Gajhede,† and Kaare Lund* Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant molecule was produced. The sequence of the proDer p 1 allergen was modified to reduce glycosylation and to abolish enzymatic activity. The resulting rproDer p 1 preparation was homogenous and stable and yielded crystals diffracting to a resolution of 1.61 Å. The active site is located in a large cleft on the surface of the molecule. The 80-aa pro-peptide adopts a unique fold that interacts with the active site cleft and a substantial adjacent area on the mature region, excluding access to the cleft and the active site. Studies performed using crossed-line immunoelectrophoresis and IgE inhibition experiments indicated that several epitopes are Downloaded from covered by the pro-peptide and that the epitopes on the recombinant mature molecule are indistinguishable from those on the natural one. The structure confirms previous results suggesting a preference for aliphatic residues in the important P2 position in substrates. Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1. The Journal of Immunology, 2005, 175: 3835–3845. http://www.jimmunol.org/ nhalation allergy to house dust mite is among the most prev- (i.e., D. pteronyssinus and D. farinae). Because the house dust alent allergic diseases worldwide (1, 2). Allergy to house dust mites are taxonomically related, the different species contain ho- I mite causes symptoms typical for type 1 immediate hyper- mologous allergens with structural similarities, which causes IgE reactivity, such as rhinoconjunctivitis and asthma. The allergic cross-reactivity (5). Patients sensitized to one species therefore of- phenotype is characterized by a persistent inflammation in the air- ten also react to the other species (6, 7). way mucosa and the presence of allergen-specific IgE. Allergen- Several proteins derived from house dust mites have been specific IgE binds to high-affinity receptors on the surface of mast characterized as allergens. The most important allergens in cells and basophiles, thereby enabling these cells to become terms of prevalence of reactivity are the group 1, 2, 3, and 9 activated by allergen. After activation, mast cells and baso- allergens, to which Ͼ90% of mite allergic patients have IgE (8). by guest on September 26, 2021 philes release mediators, such as histamine, leukotrienes, en- Group 1 and group 2, however, account for most of the IgE on zymes, etc., that are the direct causes of the allergic symptoms. a quantitative basis. Whereas the biological function of the The binding of allergen molecules to receptor-bound IgE and group 2 mite allergen has not yet been identified, the other the cross-linking of these on the surface of mast cells and ba- mentioned allergens are proteases. Groups 3 and 9 are serine sophiles is therefore the key event in the triggering of a cascade proteases, and group 1 is a cysteine protease located in the of events leading directly to allergic symptoms. A thorough alimentary canal of the mite (5). understanding of the molecular events leading to activation of Derp1isamajor allergen from D. pteronyssinus (9). In one mast cells and basophiles is thus facilitated by the elucidation of study, the prevalence of IgE to Der p 1 was 97% in a population the three-dimensional structure of the allergen molecule. Fur- of 35 house dust mite allergic individuals, as measured by radioal- thermore, allergen structures facilitate the rational design of lergosorbent tests using purified nDer p 1 (8). Derp1isa25-kDa allergen variants with reduced IgE binding, which has possible cysteine protease that is present in mite feces in high concentra- uses in specific allergy vaccination (3). tions. The proteolytic activity of Der p 1 has been proposed to Several species of house dust mites have been identified con- enhance the capacity of the molecule to sensitize human beings tributing to the environmental exposure of human beings (4). The (10). The gene encoding Der p 1 has been cloned and sequenced most prevalent species belong to the genus Dermatophagoides (11) and shown to display isoallergenic variation (12). The open reading frame encodes an 18-aa signal peptide, an 80-aa pro-pep- tide, and the mature region that comprises an additional 222 aa. *ALK-Abello´A/S, Research Department, Hørsholm, Denmark; and †Department of The sequence includes four potential N-linked glycosylation sites, Medicinal Chemistry, Danish University of Pharmaceutical Sciences, Copenhagen, three in the mature sequence and one in the pro-peptide. Der p 1 Denmark is produced in the mite as an enzymatically inactive pro-enzyme Received for publication March 16, 2005. Accepted for publication June 27, 2005. that becomes enzymatically active after cleavage and detachment The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance of the pro-peptide. Apart from inhibiting the activity of the pro- with 18 U.S.C. Section 1734 solely to indicate this fact. enzyme, the pro-peptide may also act as a folding scaffold for 1 The coordinates and structure factors of rproDer p 1-CN have been deposited with mature Der p 1, as suggested for other proteases (13, 14). When the Protein Data Bank coordinate file 1XKG and related structure factor file Derp1isextracted from mite feces, it is present in the mature R1XKGSF. form (nDer p 1). Recombinant Der p 1 has been produced in Esch- 2 Address correspondence and reprint requests to Dr. Kåre Meno, ALK-Abello´A/S, Research Department, Bøge Alle´ 6–8, DK-2970 Hørsholm, Denmark. E-mail ad- erichia coli, but the resulting protein had much reduced IgE bind- dress: [email protected] ing indicating improper folding (15). In Pichia pastoris, rproDer p Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 3836 CRYSTAL STRUCTURE OF proDer p 1 Table I. Primer sequences Primer No. Primer Sequence 5Ј-3Ј Primer 1, forward GTCGCTCGAGAAAAGAAGACCATCTTCTATTAAGACTTTCGAAGAATAC Primer 2, reverse GCCTCTAGATCAGTGATGGTGGTGATGGTGACCAGTTTGACCCAAAATGACAACGTATGGATATTCTTC Primer 3, reverse GTTCAGCAAGATCCAATGATTGGTCACGGTAAGCCAAATAAGC Primer 4, forward GCTTATTTGGCTTACCGTGACCAATCATTGGATCTTGCTGAAC Primer 5, reverse ACCAGAGAAAGCCCAAGCTGAACCACAGCC Primer 6, forward GGCTGTGGTTCAGCTTGGGCTTTCTCTGGT Primer 7, reverse GTTCAGCAAGATCCAATGATTGTTCACGGTAAGCCAAATAAGC Primer 8, forward GCTTATTTGGCTTACCGTGAACAATCATTGGATCTTGCTGAAC 1 can be produced as a hyperglycosylated pro-enzyme with re- Construction of site-specific mutations C114A and N132D was done by the duced enzymatic activity and IgE binding (16). After in vitro mat- overlap extension method (21). Briefly, primer 1 (Table I) was used together uration, however, enzymatic activity and IgE binding can be re- with primer 5 in one PCR, primer 3 was used together with primer 6 in another PCR, and primer 2 was used together with primer 4 in a third PCR, all with stored independently of glycosylation (17). proderp1 as a template. The full fragment containing the mutations was then The cysteine proteases are divided into five clans, each containing created by using the three resulting PCR products together with primers 1 and a number of families. Der p 1 belongs to clan CA, family C1, which 2 in a final PCR, followed by directional cloning of the 957-bp XhoI-XbaI Downloaded from also includes papain and its
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