Isoform identification and characterization of Art v 3, the lipid-transfer protein of mugwort pollen.
Art v 3, the lipid-transfer protein (LTP) of Artemisia vulgaris pollen is a relevant allergen showing frequent cross-reactivity with homologues in other plants. Here we report the identification of four full-length Art v 3 sequences obtained by cDNA cloning using mass spectrometry-based sequencing. Two isoforms, Art v 3.0201 and Art v 3.0301 were expressed as soluble proteins in Escherichia coli Rosetta-gami B(DE3) pLysS using different expression systems. Purified natural and recombinant Art v 3 demonstrated similar secondary structures in circular dichroism analysis. All preparations showed high thermal stability but low resistance to gastric digestion with pepsin. Patient-specific IgE reactivity patterns to natural or recombinant isoallergens were observed among Art v 3-sensitized subjects. Using Immuno Solid-phase Allergen Chip (ISAC) assays, frequent cross-reactivity of Art v 3 with LTPs from peach and hazelnut was shown. The biological activity of both isoforms was comparable to the natural allergen in basophil release assays. The newly identified sequences provide the basis for recombinant mugwort LTP production enabling batch-to-batch reproducibility and thus ensuring high-quality products for diagnosis and therapy.
Gadermaier
Gabriele
G
Christian Doppler Laboratory for Allergy Diagnosis and Therapy, Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria. Gabriele.Gadermaier@sbg.ac.at
Harrer
Andrea
A
Girbl
Tamara
T
Palazzo
Paola
P
Himly
Martin
M
Vogel
Lothar
L
Briza
Peter
P
Mari
Adriano
A
Ferreira
Fatima
F
D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D052179:Antigens, Plant; D019306:Artemisia / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology*; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology*; D011058:Pollen / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment
D000485:Allergens; D052179:Antigens, Plant; C495881:Art v 3 allergen, Artemisia vulgaris; D002352:Carrier Proteins; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C030363:lipid transfer protein; D007073:Immunoglobulin E