De Vouge M W, Thaker A J, Curran I H, Zhang L, Muradia G, Rode H, Vijay H M
Life Sciences Division, Bureau of Drug Research, Health Canada, Ottawa.
Int Arch Allergy Immunol. 1996 Dec;111(4):385-95. doi: 10.1159/000237397.
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.
链格孢被认为是真菌气传变应原的重要来源。该霉菌的主要变应原Alt a 1是由二硫键连接的亚基组成的二聚体，在还原条件下于SDS-PAGE中迁移，表观分子量分别为14,500和16,000。超过90%的对链格孢敏感个体的血清中存在针对该蛋白的IgE抗体。本实验室先前的研究表明，纯化亚基的N端二十个氨基酸几乎相同。我们现在报告，利用针对部分纯化的Alt a 1的兔IgG抗血清，从构建于λ(gt)11的链格孢（菌株34-016）cDNA文库中分离出克隆。从总计305,000个噬菌斑（rb51）的筛选中选出的19个克隆之一进行了测序，确定其插入片段为660 bp。克隆插入片段内的一个读码框编码一个分子量为16,960的肽段，该肽段与已知变应原或蛋白质无明显同源性。通过对链格孢总RNA的Northern分析，确定rb51转录本的大小约为0.7 kb。该肽段的大部分疏水N端区域包含一个α-螺旋结构域以及其他膜靶向或分泌信号的特征。该区域下游的肽段序列与先前从两个独立来源测序的Alt a 1 N端在20个位置中的17个以及26个位置中的24个相匹配。如在非还原条件下的免疫印迹所示，在毕赤酵母中作为分泌蛋白表达的重组Alt a 1在条件培养基中以二聚体形式存在。重组Alt a 1与链格孢敏感个体的血清IgE也有反应，就如同链格孢提取物中的天然变应原一样。
