353 Analysis of the Crystal Structure of rproDer p 1 With Empha-sis on Differences in Antibody Binding of Pro/Mature Der p 1

K. Meno1, P. B. Thorsted1, H. Ipsen1, O. Kristensen2, M. Gajhede2, K.Lund1; 1Research Department, ALK-Abello A/S, Horsholm, DENMARK,2Department of Medicinal Chemistry, The Danish University of Pharma-ceutical Sciences, Copenhagen, DENMARK.RATIONALE: Recombinant Der p 1 as a tool for allergy diagnosisand/or a vaccine candidate is an attractive prospect, but the proteolyticactions of an active cysteine protease or presence of the pro peptide on theimmature protein have been of concern for some time.METHODS: An enzymatically inactive rproDer p 1 variant has beencrystallised and the structure was solved by X-ray crystallography. Theantibody binding properties of nDer p 1, rDer p 1 and rproDer p 1 wereassessed by in situ crossed line immune electrophoresis (CLIE).RESULTS: Here we present the production and crystallisation of astable recombinant proDer p 1 variant. The mature region adopts aconformation similar to the mature form of other cysteine proteasessuggesting that no major structural changes are induced by maturation.The pro region adopts a unique fold, which interacts with the activesite cleft and a substantial area adjacent to this on the mature region.From the fermentation culture we have furthermore succeeded in puri-fying a fraction of the recombinant molecules which have sponta-neously matured. This enzymatically inactive mature rDer p 1 variantshowed antibody binding properties indistinguishable from nDer p 1 inCLIE.CONCLUSIONS: We show here for the first time the crystal structure ofrproDer p 1. The pro peptide is placed in a position where it shields cer-tain B-cell epitopes, compared to rDer p 1 or nDer p 1. Furthermore, itwas proven possible to purify a mature but inactive variant of rDer p 1from the culture medium.

The Major Mite Allergen From Dermatophagoides

S88 Abstracts J ALLERGY CLIN IMMUNOL

FEBRUARY 2005

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351 Catalogue of the Major Transcripts of the Storage Mite, Aleu-roglyphus ovatus: Revealing the Putative Allergenic Reper-toire

S. F. R. Ramjan1, A. H. B. Loo1, Y. P. Lim2, F. T. Chew1; 1Department ofBiological Sciences, National University of Singapore, Singapore, SIN-GAPORE, 2Bioinformatics Institute, Singapore, SINGAPORE.RATIONALE: Aleuroglyphus ovatus (Acaridae) is a storage mite that has aworldwide distribution and is found mainly in stored bran and wheat. Sensi-tization to this contaminating mite has been reported in several populations.METHODS: An Expressed Sequence Tag (EST) catalogue of this mitewas created in an attempt to identify the major expressed proteins and itsallergenic components. Homologues to allergenic components were iden-tified and isolated for further studies.RESULTS: The initial 2063 reliable sequences with a read-length ofmore than 500 nucleotides were compared to the public sequence data-bases using the BLASTX algorithm. A total of 47% showed significantmatches to known proteins in the public databases. The ESTs were cate-gorized into 11 functional groups of which allergen homologues (5%)were of special interest. Homologues to 28 different putative allergens,including the mites group 1, 2, 3, 5, 6, 7, 8, 10, 13, 14, 18 allergens as wellas a number of well documented non-mite allergens including enolase,arginine kinase, phospholipase A1, thioredoxin and thaumatin-like pro-tein, were identified. The mite group 8 allergen was most highly repre-sented (17.9% of all the allergen transcripts identified), and had as manyas six different putative isoforms. The mite group 13 allergen was the sec-ond most abundant (16.84%) with four distinct isoforms. Paralogousforms of the group 2, 5, and 13 allergens were also observed.CONCLUSIONS: Systematic analysis of the major transcriptome hasproven to be useful in identifying the putative allergen repertoire, includ-ing possible isoforms, in a particular source material.Funding: Biomedical Research Council, Singapore

352 Solution Structure and Epitope Mapping of a Major Group 13Allergen From the House Dust Mites, Dermatophagoidesfarinae

S. L. Chan, S. T. Ong, T. C. Ong, F. T. Chew, Y. K. Mok; Department ofBiological Science, National University of Singapore, Singapore, SIN-GAPORE.RATIONALE: We recently identified two paralogous Group 13 allergensfrom dust mite, Dermatophagoides farinae (Der f), one which bound spe-cific-IgE strongly in approximately 40% of Der f sensitized individualswhile the other bound less than 10%. We determined the structure of thisallergen by NMR spectroscopy and observed that it is structurally similarto the human fatty acid binding protein. The human counterpart did notbind IgE and was thus cross-compared to the dust mite allergen to high-light structural differences that may point to potential IgE binding epitopes.METHODS: We carried out site-directed mutagenesis on the dust miteallergen and screened the mutants by ELISA to identify residues that maybe critical for IgE binding.RESULTS: Sequence alignment of the allergen with human fatty acidsbinding proteins revealed 18 mismatched residues. These were mainlycharged and polar residues and reside on the surface of the allergen.Four of these residues were identified to be critical for IgE binding;Glu-41, Lys-63, Lys-91 and Lys-103. These residues were separatedinto 2 groups (Glu-41 and Lys-63; Lys-91 and Lys-103) and distributedat 2 distinct regions on opposite surfaces of the protein. Two doublemutants (E41A+K63A and K91A+K103A) were generated and shownto have further reduced IgE binding and in vivo skin prick reactivity.CONCLUSIONS: Two major IgE binding epitope regions were identi-fied on the Der f 13 allergens and mutants of these were found to haveboth reduced IgE binding and in vivo skin prick reactivity.Funding: Biomedical Reseach Council, Singapore

354 pteronyssinus, Der p 2, Is a Sterol Binding ProteinK. Reginald1, M. R. Wenk2, F. T. Chew1; 1Department of Biological Sci-ence, National University of Singapore, Singapore, SINGAPORE,2Department of Biochemistry, National University of Singapore, Singa-pore, SINGAPORE.RATIONALE: Crystal structure of Der p 2, a major allergen from Der-matophagoides pteronyssinus, has previously been reported to have anunidentified hydrophobic ligand. Another molecule, NPC2 (Niemann-Pick disease type C2 protein), which is structurally similar to Der p 2, hasbeen shown to bind cholesterols. Due to their high structural similarities,we hypothesize that Der p 2 may be a lipid binding molecule.METHODS: To evaluate this, we performed pull down assays by incu-bating recombinant Der p 2 with liposomes prepared from total lipidbovine brain extracts with and without increasing concentrations of cho-lesterol. In a separate experiment, we attempted to narrow down the iden-tity of the ligand by delipidating Der p 2 and separating the lipid fractionon thin layer chromatography together with several standards from differ-ent classes of lipids.RESULTS: Der p 2 bound to liposomes with cholesterol, in a dosedependent fashion. Nevertheless, Der p 2 was found to bind the lipo-somes alone at lower levels, suggesting that some components of theliposome were able to interact with Der p 2. The GST-fusion tag, act-ing as a non-specific control, did not bind to liposomes with or withoutcholesterol. The results suggest that cholesterol may be a possible bind-ing partner of Der p 2. Separation on thin layer chromatographyshowed that Der p 2 had a bound ligand that migrated at the same rateas cholesterol.CONCLUSIONS: Der p 2 is able to bind sterol molecules, in particularcholesterol, and may function as a lipid binding protein.Funding: Biomedical Research Council, Singapore