Breiteneder H, Ferreira F, Hoffmann-Sommergruber K, Ebner C, Breitenbach M, Rumpold H, Kraft D, Scheiner O
Institute of General and Experimental Pathology, University of Vienna, Austria.
Eur J Biochem. 1993 Mar 1;212(2):355-62. doi: 10.1111/j.1432-1033.1993.tb17669.x.
Previous studies showed that pollens from trees of the order Fagales (e.g. birch, alder, hazel and hornbeam) all contain one major allergen. These proteins are cross-reactive between these tree species, and approximately 95% of tree-pollen-allergic patients display IgE binding to these allergens. Using the reported N-terminal amino acid sequence of the hazel pollen allergen Cor a I, it was possible to amplify Cor-a-I cDNA by use of the polymerase chain reaction. Four clones with cDNA inserts were isolated. All four clones contained an open reading frame of 477 nucleotides (159 amino acids) but differed in length of their 3'-non-coding regions. Within the overlapping regions, the nucleotide sequence of the 3'-non-coding regions of the four clones were nearly identical. The open reading frames coded for different isoforms of the major hazel pollen allergen, Cor a I. The clones were designated Cor a I/5, 6, 11 and 16, respectively. Comparison of the deduced amino acid sequences of these Cor a I isoforms revealed identities of 96-99%. The sequence identities between the Cor a I isoforms and Bet v I, the major birch pollen allergen, were 71-73% (80.5-83% similarity). Comparing amino acid sequences of Cor a I isoforms with the published sequences of Aln g I, the major allergen from alder, and Car b I and isoforms, the major allergen from hornbeam, 75.5-76.7% identity (83.6-85% similarity) and 83.6-89.9% sequence identity (89.3-95% similarity), respectively, was found. The four Cor a I cDNAs were subcloned into plasmid pKK223-3 and expressed in Escherichia coli as non-fusion proteins; their capacity to bind serum IgE from tree-pollen-allergic patients was investigated. The four cloned isoforms showed an apparent molecular mass of 17 kDa in SDS/PAGE, identical to the natural, pollen-derived Cor a I. IgE antibodies from tree-pollen-allergic patients reacted with all four recombinant isoforms. However, we noted marked differences in the IgE-binding patterns of the distinct isoforms. Furthermore, Cor a I/11 was the only isoform recognized by the anti-(Bet v I) mAb, BIP 1. Our results demonstrate that Cor a I isoforms display different antigenic and allergenic properties, very likely due to few but significant changes in their amino acid sequences. These findings have implications for the development of reagents for diagnosis and immunotherapy of type I allergies.
先前的研究表明，壳斗目树木（如桦树、桤木、榛树和鹅耳枥）的花粉均含有一种主要过敏原。这些蛋白质在这些树种之间具有交叉反应性，约95%的对树花粉过敏的患者体内的免疫球蛋白E（IgE）会与这些过敏原结合。利用已报道的榛树花粉过敏原Cor a I的N端氨基酸序列，通过聚合酶链反应可以扩增出Cor-a-I互补DNA（cDNA）。分离出了四个带有cDNA插入片段的克隆。所有四个克隆都包含一个477个核苷酸（159个氨基酸）的开放阅读框，但它们3'非编码区的长度不同。在重叠区域内，四个克隆的3'非编码区的核苷酸序列几乎相同。这些开放阅读框编码了主要榛树花粉过敏原Cor a I的不同亚型。这些克隆分别被命名为Cor a I/5、6、11和16。对这些Cor a I亚型推导的氨基酸序列进行比较，发现其同一性为96 - 99%。Cor a I亚型与主要桦树花粉过敏原Bet v I之间的序列同一性为71 - 73%（相似性为80.5 - 83%）。将Cor a I亚型的氨基酸序列与已发表的桤木主要过敏原Aln g I以及鹅耳枥主要过敏原Car b I及其亚型的序列进行比较，分别发现同一性为75.5 - 76.7%（相似性为83.6 - 85%）和序列同一性为83.6 - 89.9%（相似性为89.3 - 95%）。将这四个Cor a I cDNA亚克隆到质粒pKK223 - 3中，并在大肠杆菌中作为非融合蛋白表达；研究了它们与树花粉过敏患者血清IgE结合的能力。这四个克隆的亚型在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）中显示出明显的分子量为17 kDa，与天然的、来自花粉的Cor a I相同。树花粉过敏患者的IgE抗体与所有四种重组亚型都发生了反应。然而，我们注意到不同亚型的IgE结合模式存在显著差异。此外，Cor a I/11是唯一被抗（Bet v I）单克隆抗体BIP 1识别的亚型。我们的结果表明，Cor a I亚型表现出不同的抗原性和过敏原性，很可能是由于其氨基酸序列中存在少量但显著的变化。这些发现对I型过敏的诊断和免疫治疗试剂的开发具有重要意义。
