title: Isolation and expression of a cDNA clone encoding an Alternaria alternata Alt a 1 subunit.
abstract: Alternaria alternata is recognized as an important source of fungal aeroallergens. Alt a 1, the major allergen of this mold, is a dimer of disulfide-linked subunits that migrate in SDS-PAGE under reducing conditions at apparent M(r)s of 14,500 and 16,000. IgE antibodies to this protein are present in the sera of >90% of A. alternata-sensitive individuals. Previous studies from this laboratory showed that the N-termini twenty amino acids of the purified subunits are nearly identical. We now report the isolation of clones from an A. alternata (strain 34-016) cDNA library constructed in lambda(gt)11, using rabbit IgG antiserum against partially purified Alt a 1. One of nineteen clones selected from screens totalling 305,000 pfu (rb51) was sequenced, and determined to harbor an insert of 660 bp. An in-frame open reading frame within the cloned insert encodes a peptide of M(r) 16,960 that bears no significant homology to known allergens or proteins. The size of the rb51 transcript was determined to be approximately 0.7 kb by Northern analysis of A. alternata total RNA. The largely hydrophobic N-terminal region of the peptide contains an alpha-helical domain and other features characteristic of membrane targeting or secretory signals. The peptide sequence downstream of this region matches previously sequenced Alt a 1 N-terminal from two independent sources at 17 of 20, and 24 of 26 positions. Recombinant Alt a 1 expressed as a secreted protein in Pichia pastoris exists as a dimer in conditioned medium, as shown by immunoblotting under nonreducing conditions. Recombinant Alt a 1, like the natural allergen in A. alternata extracts, is also reactive with serum IgE from A. alternata-sensitive individuals.
authors.affiliation: Life Sciences Division, Bureau of Drug Research, Health Canada, Ottawa.
authors.forename: M W
authors.initials: MW
authors.lastname: De Vouge
authors.forename: A J
authors.initials: AJ
authors.lastname: Thaker
authors.forename: I H
authors.initials: IH
authors.lastname: Curran
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.forename: G
authors.initials: G
authors.lastname: Muradia
authors.forename: H
authors.initials: H
authors.lastname: Rode
authors.forename: H M
authors.initials: HM
authors.lastname: Vijay
mesh_terms: D000485:Allergens / Q000096:biosynthesis / Q000235:genetics; D000528:Alternaria / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D052179:Antigens, Plant; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D000939:Epitopes / Q000096:biosynthesis / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: C096258:ALTA1 protein, Alternaria alternata; D000485:Allergens; D052179:Antigens, Plant; D018076:DNA, Complementary; D000939:Epitopes; D005656:Fungal Proteins; D011994:Recombinant Proteins