title: NADP-dependent mannitol dehydrogenase, a major allergen of Cladosporium herbarum.
abstract: Cladosporium herbarum is an important allergenic fungal species that has been reported to cause allergic diseases in nearly all climatic zones. 5-30% of the allergic population displays IgE antibodies against molds. Sensitization to Cladosporium has often been associated with severe asthma and less frequently with chronic urticaria and atopic eczema. However, no dominant major allergen of this species has been found so far. We present cloning, production, and characterization of NADP-dependent mannitol dehydrogenase of C. herbarum (Cla h 8) and show that this protein is a major allergen that is recognized by IgE antibodies of approximately 57% of all Cladosporium allergic patients. This is the highest percentage of patients reacting with any Cladosporium allergen characterized so far. Cla h 8 was purified to homogeneity by standard chromatographic methods, and both N-terminal and internal amino acid sequences of protein fragments were determined. Enzymatic analysis of the purified natural protein revealed that this allergen represents a NADP-dependent mannitol dehydrogenase that interconverts mannitol and d-fructose. It is a soluble, non-glycosylated cytoplasmic protein. Two-dimensional protein analysis indicated that mannitol dehydrogenase is present as a single isoform. The cDNA encoding Cla h 8 was cloned from a lambda-ZAP library constructed from hyphae and spores. The recombinant non-fusion protein was expressed in Escherichia coli and purified to homogeneity. Its immunological and biochemical identity with the natural protein was shown by enzyme activity tests, CD spectroscopy, IgE immunoblots with sera of patients, and by skin prick testing of Cladosporium allergic patients. This protein therefore is a new major allergen of C. herbarum.
authors.affiliation: Department of Cell Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria.
authors.forename: Birgit
authors.initials: B
authors.lastname: Simon-Nobbe
authors.forename: Ursula
authors.initials: U
authors.lastname: Denk
authors.forename: Peter Bernhard
authors.initials: PB
authors.lastname: Schneider
authors.forename: Christian
authors.initials: C
authors.lastname: Radauer
authors.forename: Markus
authors.initials: M
authors.lastname: Teige
authors.forename: Reto
authors.initials: R
authors.lastname: Crameri
authors.forename: Thomas
authors.initials: T
authors.lastname: Hawranek
authors.forename: Roland
authors.initials: R
authors.lastname: Lang
authors.forename: Klaus
authors.initials: K
authors.lastname: Richter
authors.forename: Peter
authors.initials: P
authors.lastname: Schmid-Grendelmeier
authors.forename: Stephan
authors.initials: S
authors.lastname: Nobbe
authors.forename: Arnulf
authors.initials: A
authors.lastname: Hartl
authors.forename: Michael
authors.initials: M
authors.lastname: Breitenbach
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000485:Allergens / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D002963:Cladosporium / Q000737:chemistry / Q000276:immunology*; D003593:Cytoplasm / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry; D008354:Mannitol Dehydrogenases; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012882:Skin Tests
chemical_list: D000485:Allergens; D009841:Oligonucleotides; D007073:Immunoglobulin E; D000429:Alcohol Oxidoreductases; D008354:Mannitol Dehydrogenases; C116789:mannitol 2-dehydrogenase (NADP)