359 Purification and Structural Analysis of the Novel Glycopro-tein Allergen Cyn d 24 From the Bermuda Grass Pollen

L. Chow1, L. Chiu1, K. Khoo2, S. Su3; 1Institute of Biochemistry andMolecular Biology, College of Medicine, National Taiwan Univ., Taipei,TAIWAN REPUBLIC OF CHINA, 2Institute of Biochemistry, AcademiaSinica, Taipei, Taiwan, Taipei, TAIWAN REPUBLIC OF CHINA,3Department of Medical Research and Education, Veterans General Hos-pital-Taipei, Taipei, Taiwan, Taipei, TAIWAN REPUBLIC OF CHINA.RATIONALE: Bermuda grass (Cynodon dactylon) pollen contains avery complexity of allergens. One of the allergens with molecular mass of21 kDa has been shown to bind serum IgE from 30% patients with BGPallergy. The aim of this study was to purify, characterize and clone the 21kDa allergen and investigate its glycan structure in IgE binding. METHODS: Bermuda grass pollen extract was fractionated by using ion-exchange chromatography and reverse phase HPLC. Immunoblotting andELISA were performed for investigating its allergenicity, and the endo-proteinase Lys-C digested peptides were determined by N-terminalsequencing. The cDNA sequence was analyzed by RACE PCR basedcloning. Putative glycan structures were elucidated using MALDI-TOFmass spectrometry. RESULTS: Cyn d 24allergen was purified from Bermuda grass pollenwith an observed molecular mass of 21 kDa and pI of 5.7. The cDNA ofCyn d 24 was predicted as a 153-aa mature protein with an N terminus atSer23. Sequence comparison revealed that Cyn d 24 possesses several fea-tures in common with the pathogen-related protein family. After removedthe N-glycan, Cyn d 24 reduced > 80% and >90% of binding activities tohuman IgE. Six oligosaccharides fractions were isolated and the mostabundant oligosaccharide (83.75%) was found to be Man3GlcNAc2Fuc. CONCLUSIONS: Cyn d 24, a novel glycoprotein allergen of Bermudagrass pollen has been isolated, characterized, and cloned. Its N-glycanstructure play an important role in the immune response, especially in theIgE binding.Funding: National Science Council

360 Cloning of the Major Cedar Pollen Allergens Jun a 1 and Cryj 1 and Expression of Recombinant Proteins

G. Schmidt, F. Ferreira; Department of Molecular Biology, University ofSalzburg, Salzburg, AUSTRIA.RATIONALE: Cedar/juniper pollen allergy is a very common worldwideallergy. Pollen of Mountain Cedar Juniperus ashei and the Japanese CedarCryptomeria japonica cause allergic disease in various areas includingNorthern America and Japan. Both major antigens Jun a 1 and Cry j 1encode for pectate lyases. Pectate Lyases resemble a large group ofenzymes expressed in ripening fruits and also in plant pathogenic bacte-ria. Due to lack of expressed soluble recombinant allergens of the pectatelyase group, testing of different prokaryotic protein expression protocolsfor production of soluble recombinant proteins is an important task.METHODS: After RNA purification out of homogenized pollen, codingcDNA sequences without signal sequence are amplified by one step RT-PCR using specific primers for Jun a 1 and Cry j 1 and ligated into expres-sion vectors containing various Tags for detection and purification. Dif-ferent prokaryotic expression systems including large-scale fermentationwere tested and the efficiency of soluble protein production was comparedby western blot technique.RESULTS: Recombinant allergens were expressed efficiently in all sys-tems. The amount of full length soluble protein produced varied dramati-cally depending on parameters like bacterial strain, expression vector,media and growth conditions.CONCLUSIONS: Our results demonstrate that tight expression controlis more important for reduction of product degradation than low temper-ature growth conditions.This work was supported by a grant from Biomay.Funding: Biomay

361 Recombinant Weed Pollen Allergens for Diagnosis of Rag-weed and Mugwort Allergies

N. Wopfner1, R. Asero2, G. Gadermaier1, G. Hubinger1, P. Gruber1, F.Ferreira1; 1Department of Molecular Biology, University of Salzburg,Salzburg, AUSTRIA, 2Ambulatorio di Allergologia, Clinica San Carlo,Paderno Dugnano, ITALY.RATIONALE: Ragweed pollen is one of the most important sources ofallergenic proteins in different parts of North America but can also befound in Canada, Japan, Australia and Europe. Mugwort is native toEurope and Asia, but is now also found throughout the eastern US. In bothweeds several allergens have been identified and the natural and recom-binant proteins have been isolated and characterized.METHODS: In this study we tested five recombinant mugwort allergens(Art v 1, Art v 4, 2 and 3 EF-hand calcium binding proteins and the Amba 1 homologue in mugwort) and six recombinant ragweed allergens (Amba 1, Amb a 5, Amb a 6, 2 and 3 EF-hand calcium binding proteins and pro-filin) with patients from the area of Milan mainly sensitized to ragweedpollen. All recombinant proteins were produced as his-tagged fusion pro-teins. To test the IgE-reactivity, the purified proteins were dotted onto anitrocellulose membrane and exposed to patients`sera. Bound IgE wasdetected using 125 I-rabbit anti-human IgE.RESULTS: As expected, most of the patients reacted with the major rag-weed pollen allergen Amb a 1, but also the minor allergens Amb a 5,Amb a 6 and profilin were recognized. In the case of the mugwort aller-gens, Art v 1 was the most frequently recognized protein followed by Artv 4.CONCLUSIONS: In summary, our results demonstrated that ragweedand mugwort share common allergens. However, there seems to be limit-ed cross-reactivity due to differences in the sensitization patterns ofpatients exposed to mugwort and ragweed pollen.Funding: Fonds zur Förderung der Wissenschaftlichen Forschung (FWF)

S90 Abstracts J ALLERGY CLIN IMMUNOL

FEBRUARY 2005

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362 Molecular Cloning and Characterization of a Group 5 Par-alogue From Blomia tropicalis

Y. F. Gao1, X. Z. Bi1, H. S. Shang1, D. Y. Wang2, F. T. Chew1; 1Depart-ment of Biological Science, National University of Singapore, Singapore,SINGAPORE, 2Department of Otolaryngology, National University ofSingapore, Singapore, SINGAPORE.RATIONALE: In the tropics, the dust mite Blomia tropicalis is an impor-tant source of indoor allergens and Blo t 5 is the major allergen. Here, wereport a new group 5 paralogue from this dust mite and cross comparedthe two paralogues.METHODS: Our in-house Blomia tropicalis ESTs database showed thepresence of two clusters of cDNA sharing some sequence identity to Blot 5 (U59102). The coding regions of these antigens were cloned andexpressed in Escherichia coli. Allergenicity of these paralogues was eval-uated via immunoblotting.RESULTS: The Blo t 5 paralogue cDNA is 609bp, containing an openreading frame of 390bp encoding 129 amino acid residues. The twoparalogues share only 55% identity at the nucleic acid level and 36%identity and 46% similarity at the protein level. Genomic studiesrevealed that the genes encoding these products have a small intron atthe 5’ terminal region, with an intron size of 55 and 54 nt, respective-ly. The studies suggested that these group 5 paralogues are translatedfrom different genes, which possibly originated from the same evolu-tionary ancestor. Recombinant Blo t 5 was found to bind specific IgEin 70 of the 133 Singapore atopic sera tested, while its paralogue onlyreacts to 50 individuals in the same sera set with lower intensity. Cross-reaction studies showed that these two paralogues could only partiallyinhibit each other.CONCLUSIONS: We have identified a new Blo t 5 paralogue, which haslower IgE binding capacity but share partial cross reactivity only.Funding: Biomedical Research Council, Singapore