Selective cloning of allergens from the skin colonizing yeast Malassezia furfur by phage surface display technology.
The yeast Malassezia furfur, also known as Pityrosporum orbiculare (ovale), is part of the normal microflora of the human skin but has also been associated with different skin diseases including atopic dermatitis. More than 50% of atopic dermatitis patients have positive skin test and specific IgE to M. furfur extracts; however, the pathophysiologic role of these IgE-mediated reactions in the development of the disease remains unknown. The yeast is able to produce a wide panel of IgE-binding proteins, variably recognized by sera of individual patients. In order to assess the contribution of individual components to the disease, highly pure allergen preparations are required. We have cloned M. furfur allergens from a cDNA library displayed on the phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. Phage displaying IgE-binding proteins were selectively enriched from the library using IgE from a M. furfur-sensitized atopic dermatitis patient as a ligand. We were able to identify five different inserts coding for IgE-binding polypeptides. Three of the sequenced cDNA encode incomplete gene products with molecular masses of 21.3 kDa (MF 7), 14.4 kDa (MF 8), and 9.7 kDa (MF 9), respectively, having no sequence similarity to known proteins. The other two cDNA encode allergens of 18.2 kDa (Mal f 5) and 17.2 kDa (Mal f 6). Mal f 5 shows significant homology to M. furfur allergens Mal f 2, Mal f 3 and an Aspergillus fumigatus allergen Asp f 3. Mal f 6 has significant homology with cyclophilin. All of the recombinant polypeptides were capable of binding serum IgE from atopic dermatitis patients in immunoblotting experiments. The availability of pure recombinant M. furfur allergens will allow the careful investigation of the role of IgE-binding proteins in atopic dermatitis.
Lindborg
M
M
Department of Laboratory Medicine, Karolinska Institute and Hospital, Stockholm, Sweden.
Magnusson
C G
CG
Zargari
A
A
Schmidt
M
M
Scheynius
A
A
Crameri
R
R
Whitley
P
P
D000485:Allergens; D000946:Antigens, Fungal / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry; D003876:Dermatitis, Atopic / Q000276:immunology / Q000382:microbiology; D015723:Gene Library; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000378:metabolism; D008290:Malassezia / Q000235:genetics / Q000276:immunology*; D011485:Protein Binding; D011852:Radioallergosorbent Test; D011994:Recombinant Proteins / Q000096:biosynthesis; D012867:Skin / Q000276:immunology*
D000485:Allergens; D000946:Antigens, Fungal; D018076:DNA, Complementary; D004271:DNA, Fungal; D011994:Recombinant Proteins; D007073:Immunoglobulin E