Cloning, characterization and expression of complete coding sequences of three IgE binding Malassezia furfur allergens, Mal f 7, Mal f 8 and Mal f 9.
Malassezia furfur, formerly known as Pityrosporum orbiculare or P. ovale, is a yeast that colonizes human skin. Normally, this yeast is nonpathogenic but under the influence of predisposing factors it may induce IgE reactivity in patients with atopic dermatitis. Approximately 40-65% of atopic dermatitis patients have IgE antibodies and/or skin reactivity against M. furfur and a higher T-cell response against this yeast is found in atopic dermatitis patients than in healthy individuals. By making a cDNA library displayed on a phage surface, we previously cloned five different IgE-binding proteins, Mal f 5, Mal f 6, MF 7, MF 8 and MF 9, from this yeast. The cDNAs encoding these allergens were sequenced and expressed in Escherichia coli. The sequences of MF 7, MF 8 and MF 9 were not full length (missing their 5'-ends) giving only partial gene products. To obtain complete cDNA sequences, we performed RACE-PCR to amplify the 5'-ends of each cDNA. These PCR products were sequenced and analyzed. The coding sequences of Mal f 7, Mal f 8 and Mal f 9 encode proteins with ORFs of 141 (16.2 kDa), 179 (19.2 kDa) and 126 (14.0 kDa) amino-acid residues, respectively. None of the putative proteins showed significant sequence homology with other known proteins in the searched database. The proteins encoded by the complete cDNA sequences were expressed in E. coli as recombinant proteins. Immunoblotting and radioallergosorbant test data showed that all of the expressed recombinant proteins have the ability to bind serum IgE from atopic dermatitis patients and furthermore, the M. furfur extract could specifically inhibit this IgE binding.
Rasool
O
O
Department of Medicine,Unit of Clinical Allergy Research, Karolinska Institutet and Hospital, Stockholm, Sweden. omid.rasool@cck.ki.se
Zargari
A
A
Almqvist
J
J
Eshaghi
H
H
Whitley
P
P
Scheynius
A
A
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000485:Allergens / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000946:Antigens, Fungal / Q000096:biosynthesis / Q000235:genetics*; D052179:Antigens, Plant; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D003876:Dermatitis, Atopic / Q000097:blood / Q000276:immunology*; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D015723:Gene Library; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology* / Q000378:metabolism; D008290:Malassezia / Q000235:genetics / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA
D000485:Allergens; D000946:Antigens, Fungal; D052179:Antigens, Plant; D018076:DNA, Complementary; C412786:Mal f 7 allergen; C412787:Mal f 8 allergen; C412788:Mal f 9 allergen; D011994:Recombinant Proteins; D007073:Immunoglobulin E