To support its ongoing monitoring program of therapeutic and diagnostic allergen preparations, LIB has initiated a multifaceted study of the relationship between the structure and function of allergens. These include: 1. Physico-chemical identification of allergens. We have initiated studies on the use of HPLC, MALDI-TOF mass-spectroscopy and microarray analyses in the characterization of target allergens. In general, the MALDI-TOF spectra correlated with the SDS-PAGE patterns of standardized allergen vaccine references. Major allergens - Amb a 1, Fel d 1, cat albumin, phopholipase A2, hyaluronidase and antigen 5 - were readily discernable, and the molecular weights of isoforms could be identified. Protein profiles were more difficult to ascertain for the more complex protein mixtures. In one study, the MALDI- TOF spectrum of honeybee venom was substantially improved by the chromatographic removal of mellitin prior to analysis. MALDI-TOF mass spectrometry may be a valuable adjunct to currently available techniques for allergen identification and characterization. Surface enhanced laser desorption/ionization (SELDI) permits the use of protein chip arrays to do rapid protein profiling of extracts from cells, tissues and biofluids. We adapted this technology for the study of the epitopes of the latex allergen Hev b 5. We have developed a practical method for the study of allergen epitopes. Although the binding surface of the activated chip has not yet been standardized, the highly accurate determination of the molecular mass of desorbed proteins makes this technique attractive for the study of allergens and their epitopes. 2. The study of the allergenic epitopes of honeybee hyaluronidase. This allergen is being studied for allergenic structural determinants in order to modify them, thus producing allergens with decreased IgE binding activity but preserved ability to stimulate a T-cell immunity. (1) The search of protein surface IgE binding epitopes will be conducted using monoclonal antibodies against hyaluronidase, which compete with IgE antibodies. 31 monoclonal antibodies have been produced and characterized by competitive binding to hyaluronidase in ELISA and by immunoblot analysis. Selected monoclonal antibodies will be used for the screening of synthetic fragments and/or tryptic peptides of hyaluronidase in order to detect the binding epitopes. An alternative approach will be to produce Fab fragments of selected monoclonal antibodies and co-crystallize them with hyaluronidase. (2) Role of glycosylation in allergen recognition and processing. Recent advances in the bioengineering and cloning of allergens have raised new questions about the immunogenicity of non-glycosylated proteins. We are studying the role of glycosylation in the immunogenicity of recombinant bee venom hyaluronidase by searching for potential carbohydrate epitopes. Preliminary data on the importance of carbohydrate chains of allergens were obtained when Dr. Soldatova expressed hyaluronidase in bacteria and in baculovirus-infected insect cells. The biological activity of two differently produced proteins was compared and revealed the superior enzymatic and IgE binding activity of baculovirus-expressed hyaluronidase in comparison to E.coli-expressed allergen. Based on these data, the role of the glycosylation for the biological activity of this allergen seems to be important. The expression of the biologically active hyaluronidase in baculovirus system made it possible to determine the crystal structure of this important allergen (in collaboration with Dr. Housley Basel Biocenter, Switzerland). A series of active site and glycosylation site mutants for hyaluronidase have been obtained in collaboration with Dr. Housley (Basel Biocenter, Switzerland). In our laboratory we expressed four potential active site and glycosylation site mutants in baculovirus-infected insect cells. All expressed mutants preserved IgG-binding activity according to immunoblot analysis. Mutation of the glutamic acid residue in the position 113 significantly reduced not only enzymatic activity of recombinant hyaluronidase but also IgE - binding activity detected by RAST-inhibition assay. Preliminary data on the glycosylation site mutants suggest that the absence of carbohydrate residues does not affect significantly their biological activity. 3. Enzymatic activity as a measure of allergenicity. Current standards require the presence of enzymatically active hyaluronidase and phospholipase in venom extracts. We have initiated developing of the ELISA-based assay for quantification of hyaluronidase using a set of monoclonal antibodies raised against recombinant hyaluronidase. For this purpose the pairs of monoclonal antibodies were identified that recognize different epitopes of recombinant hyaluronidase. These pairs will be used in the trapping ELISA for quantification of hyaluronidase to pg/ml level. 4. Molecular cloning of a new bee venom allergen, acid phosphatase. Dr. Soldatova has cloned the important allergen of honeybee venom, acid phosphatase, and determined 70% of cDNA sequence. A 30% of nucleotide sequence homology was found with Drosophila and human lysosomal acid phosphatases suggesting that honeybee acid phosphatase is potentially membrane protein. Original data from direct protein sequencing of acid phosphatase correlate only in part with the partial sequence of cloned isoform of the allergen suggesting the presence of other isoforms in the honeybee venom. Since other cloned genes of honeybee allergens represent rare copy genes, we will search for other acid phosphatase isoforms using a cDNA library of honeybee and not by RT-PCR method. Directional Uni-ZAP XR cDNA library was constructed using mRNA isolated from honeybee venom glands. Screening of honeybee cDNA library is currently performed in order to accomplish a sequencing of the found isoform, and to detect and to clone the other important isoforms of acid phosphatase.
