349 Characterization of the Enzymatic Activities Present inExtracts of Various Mites

M. S. Morgan, L. G. Arlian; Wright State University, Dayton, OH.RATIONALE: Many of the previously-characterized allergens of housedust mites are known to be proteases and this enzymatic activity isthought to contribute to their allergenicity. Other astigmatid mites are alsoknown to be allergenic but little or nothing is known about their enzy-matic activities. The purpose of this study was to characterize the enzy-matic activities present in extracts from 9 species of astigmatid mites, sev-eral of which have not previously been investigated.METHODS: Extracts were prepared from 5 stored product mites (Chor-toglyphus arcuatus, Lepidoglyphus destructor, Blomia tropicalis, Tyroph-agus putrescentiae, Acarus siro), 3 house dust mites (Dermatophagoidesfarinae, D. pteronyssinus, Euroglyphus maynei) and one parasitic mite(Sarcoptes scabiei). ApiZym strips were used to screen for the presence of19 individual enzyme activities. Digestion of 9 other substrates was alsoevaluated by spectrophotometric or electrophoretic methods.RESULTS: All mites exhibited some form of phosphatase, esterase,aminopeptidase and glycosidase activity although their substrate speci-ficities varied considerably. Scabies mites did not possess detectable ser-ine peptidase activity nor were they able to hydrolyze gelatin or caseinwhile all other mites exhibited these activities.CONCLUSIONS: Storage mites possessed enzymes capable of degrad-ing the widest range of substrates while scabies mites had the most limit-ed proteolytic capacity.Funding: NIH-NIAID

Functional Study of the Cockroach Allergen Bla g 2

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347 Allergome—a Database of Allergenic Molecules: Structureand Data Implementations of a Web-Based Resource

A. Mari, V. Mari, A. Ronconi; Allergy Data Laboratories, Latina, ITALY.RATIONALE: In 2002 the Allergome database (http://www.aller-gome.org/) has been created to arrange in the most rationale way the cur-rent knowledge on allergenic molecules. Dedicated software has beendeveloped using world wide web tools, and a second version includingnew functions and data has been released in 2003. The current data entryis up-to-date and processing of the past data is almost completed. TheAllergome database host also monographs on allergenic sources lackingidentified allergens.METHODS: To define the current status of the Allergome database the over-all amount of data and links available within the database have been evaluat-ed, as well as the number of molecules and sources hosted by it. Specific eval-uation has been done on how to improve data display and database content.RESULTS: Monographs dedicated to allergenic molecules in the Aller-gome database are 1231, of which 300 dedicated to isoallergens. Theallergenic molecules being denominated by the IUIS subcommittee are443. Allergenic sources without identified allergenic molecules are 404.The overall number of links to external web sites is 62263, and that ofinternal ones is 46981. Data feeding the database have been obtained byprocessing about 5400 papers published in the last 35 years.CONCLUSIONS: Version 3 of the Allergome software and database willprovide 1) more data for the identification and description of the aller-genic molecules and sources; 2) an easier access to the database introduc-ing the search in different languages; 3) more graphical representation ofdata cross-linking different allergen monographs.Funding: Pharmacia, InBio, Leti

348 Predicting and Cataloging Putative Fungal Allergens From theFungal Protein Sequences in the GenBank

S. S. Joshi, C. Y. Kong, F. T. Chew; Department of Biological Science,National University of Singapore, Singapore, SINGAPORE.RATIONALE: Fungi are associated with a number of allergic reactionscharacterized by increased specific IgE antibodies. Sequence analysisplays an important role in assessing potential allergens. Our previousstudies show that sequences which are similar to known allergen wouldhave high probability to be coding for allergenic components themselves.METHODS: We evaluated the number of putative allergens that could beidentified from 198,909 fungal protein sequences in National Center forBiotechnology Information (NCBI) GenBank (as on May 2004) using theBLASTP global alignment algorithm. Sequences following significantsimilarity criteria: bit score > 100, E-value 40% and has > 8 contiguousamino acids matching any known allergens, were analyzed.RESULTS: 1319 (0.66%) of the total fungal protein sequences analyzedhad significant identities to known allergens based on the selection crite-ria. A total of 66 groups of these putative allergens can be classified into5 major protein catagories: metabolism (23%), stress response (34%),structural (7%), pathogenesis-related (17%) or unknowns (19%). Heatshock proteins and cyclophilins made up the largest groups of putativeallergens. Sequences showing similarity to fungal as well as non fungalallergens (dust mites, food, plant pollen and venom allergens) wereobtained. Most of these allergens were stress response and pathogenesis-related proteins (~51%).CONCLUSIONS: To-date, only 80 fungal allergen sequences are avail-able (at www.allergen.org). Integration of structural and immunologicinformation to these putative allergen sequences will help in our under-standing of the different classes of proteins that may be allergenic.Funding: Biomedical Research Council, Singapore

350S. Wünschmann1, M. D. Chapman1, A. Gustchina2, A. Wlodawer2, M.Li2, A. Pomés1; 1INDOOR Biotechnologies Inc., Charlottesville, VA,2Macromolecular Crystallography Laboratory NCI, Frederick, MD.RATIONALE: Allergen proteolytic activity has been associated with IgEproduction and asthma. However, the major cockroach allergen Blag2,although homologous to aspartic proteinases, was inactive in standardaspartic proteinase assays. The absence of catalytic activity was attributedto critical amino acid changes in the catalytic site of the molecule.Aim: To study the function and the potential for proteolytic activity ofBlag2 by reverse engineering of the catalytic site.METHODS: Site-directed mutagenesis was used to introduce mutationsin both catalytic triads (S33T, T34G, S217G) and the “flap” region (F75Y,F75a/deletion, or F75aA). Recombinant Blag2-wildtype (Wt) and mutants(Mut) were expressed in Pichia pastoris and affinity purified using 7C11Mab. Blag2-Wt and Mut were tested for catalytic activity using a hemo-globin assay.RESULTS: Recombinant Blag2 (Wt and Mut) was inactive at concen-trations of up to 80�g/ml when assayed in standard conditions, wherepepsin is active at concentrations below 10�g/ml. However, using amodified assay with an extended incubation time of 16h, we detectedresidual activity not only for the mutant GTGK1 at 40�g/ml, but alsofor the Wt at 160�g/ml, whereas pepsin was already active at a 1000fold lower concentration. Inhibition by pepstatin, but not PMSF (a ser-ine protease inhibitor) indicated that the activity was aspartic pro-teinase.CONCLUSIONS: Residual proteolytic activity was found for Blag2 atconcentrations of ~4mM. However, this very low activity suggests thatproteolysis is not the primary function of this allergen and that it is unlike-ly to contribute to the allergenicity of Blag2.Funding: INDOOR Biotechnologies Inc., Philip Morris Inc.

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