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. Determination of the crystal structure of the complex: monoclonal antibody-hyaluronidase will elucidate the allergenic determinant of this important allergen. (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 N-linked 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. Some of them were purified by nickel chelation chromatography. Both expressed and purified mutants retained IgG and IgE-binding activity as estimated by immunoblot with mouse monoclonal, rabbit polyclonal and human specific antibodies. This study suggests that the absence of N-linked carbohydrate residues does not affect significantly the antibody binding. Mutation of the glutamic acid residue in the position 113 significantly reduced enzymatic activity of recombinant hyaluronidase but not IgE - binding activity detected by RAST-inhibition assay. This finding confirms that glutamic acid-113 represents the part of the active site of this important enzyme and allergen. Several independent experiments were performed to remove the N-linked oligosaccharides from the recombinant hyaluronidase. They are (1) site-directed mutagenesis to modify all four potential N-linked glycosylation sites, (2) release of the oligosascharides using the enzyme Peptide N-Glycosidase F and (3) expression of the hyaluronidase in the presence of tunicamycin which blocks N-linked glycosylation. The carbohydrate residues were still detectable in all modified proteins, but IgG-binding activity was preserved as measured by immunoblot. All above data suggest the presence of additional to the N-linked glycosylation sites in recombinant hyaluronidase, probably O-linked glycosylation sites. We studied the carbohydrate composition of the recombinant hyaluronidase by Fluorophore Assisted Carbohydrate Electrophoresis. The monosaccharide composition includes the N-acetylglucosamine, mannose, fucose and glucose/galactose that is similar to the composition of the native counterpart. Fucose might represent the Crossreactive Carbohydrate Determinant (CCD) on the molecular surface of the hyaluronidase and, therefore, deserves a further study. N-linked oligosaccharide composition of the hyaluronidase is highly heterogeneous and includes a number of carbohydrate chains consisting of two to seven monosaccharides. This result confirms the well known feature of the baculovirus expression system. Extensive study of the oligosaccharide composition of the hyaluronidase and their mapping will contribute to the better understanding of the role of the glycosylation in the allergenicity of this important allergen 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 honeybee venom allergen, acid phosphatase. Dr. Soldatova has cloned the important allergen of honeybee venom, acid phosphatase, and determined 95% of cDNA sequence. Genomic DNA and mRNA from the venom glands were used as a template in the RT-PCR, classic PCR and 5'-RACE experiments. As a result, two overlapping nucleotide fragments that encoded the central region and N-terminal sequence of the acid phosphatase we generated. Directional Uni-ZAP XR cDNA library was constructed using mRNA isolated from honeybee venom glands. The 3'-end of the acid phosphatase gene was obtained by screening a cDNA library. Reconstitution of the 3'-end with the previously obtained nucleotide fragment resulted in full length cDNA sequence of acid phosphatase gene.The sequence has an open reading frame that encodes the putative leader sequence with the initiation codon followed by the mature enzyme.The deduced amino acid sequence is 323 amino acids in length, and appears to be identical to some of the known amino acid strands of bee venom acid phosphatase. A BLAST database search for nucleotide-protein sequence homologies confirmed approximately 30% homology of bee venom acid phosphatase with the Drosophila melanogaster lysosomal acid phosphatase precursor in both N-terminal and C-terminal regions of the protein. We found less homology (27%) to human prostatic acid phosphatase, mostly in C-terminal region of the protein. Therefore, we conclude that this allergen appears to be related both to lysosomal and to prostatic family of acid phosphatases with different degrees of homology. Successful expression of acid phosphatase gene in baculovirus-infected insect cells will provide an excellent tool to study this new important allergen-glycoprotein and, simultaneously, will contribute to a better characterization of the family of acid phosphatases.
