Mw. Devouge et al., ISOLATION AND EXPRESSION OF A CDNA CLONE ENCODING AN ALTERNARIA-ALTERNATA ALT-A-1 SUBUNIT, International archives of allergy and immunology, 111(4), 1996, pp. 385-395
Alternaria alternata is recognized as an important source of fungal ae
roallergens. Alt a 1, the major allergen of this mold, is a dimer of d
isulfide-linked sub-units that migrate in SDS-PAGE under reducing cond
itions 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 ind
ividuals. Previous studies from this laboratory showed that the N-term
ini 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 gt11, using rabbit IgG antiser
um against partially purified Alt a 1. One of nineteen clones selected
from screens totalling 305,000 pfu (rb51) was sequenced, and determin
ed to harbor an insert of 660 bp. An in-frame open reading frame withi
n the cloned insert encodes a peptide of M(r) 16,960 that bears no sig
nificant homology to known allergens or proteins. The size of the rb51
transcript was determined to be approximate to 0.7 kb by Northern ana
lysis of A. alternata total RNA. The largely hydrophobic N-terminal re
gion of the peptide contains an alpha-helical domain and other feature
s characteristic of membrane targeting or secretory signals. The pepti
de 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 Pic
hia pastoris exists as a dimer in conditioned medium, as shown by immu
noblotting under nonreducing conditions. Recombinant Alt a 1, like the
natural allergen in A. alternata extracts, is also reactive with seru
m IgE from A. alternata-sensitive individuals.