NADP-dependent mannitol dehydrogenase, a major allergen of Cladosporium herbarum.
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.
Simon-Nobbe
Birgit
B
Department of Cell Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria.
Denk
Ursula
U
Schneider
Peter Bernhard
PB
Radauer
Christian
C
Teige
Markus
M
Crameri
Reto
R
Hawranek
Thomas
T
Lang
Roland
R
Richter
Klaus
K
Schmid-Grendelmeier
Peter
P
Nobbe
Stephan
S
Hartl
Arnulf
A
Breitenbach
Michael
M
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
D000485:Allergens; D009841:Oligonucleotides; D007073:Immunoglobulin E; D000429:Alcohol Oxidoreductases; D008354:Mannitol Dehydrogenases; C116789:mannitol 2-dehydrogenase (NADP)