Hakkaart G A, Aalberse R C, van Ree R
Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, University of Amsterdam, The Netherlands.
Allergy. 1998 Feb;53(2):165-72. doi: 10.1111/j.1398-9995.1998.tb03865.x.
Recombinant Der p 2, expressed in the baker's yeast Saccharomyces cerevisiae, was used as a tool to determine IgE- and monoclonal antibody (mAb)-binding sites on this allergen. For this purpose, mutant molecules were produced by application of site-directed mutagenesis. The amino-acid residues spanning cys21-cys27 and cys73-cys78 were deleted, thus preventing loop formation through disulfide bonds. Charged residues in three predicted antigenic sites (residues 45-48, 67 + 69, and 88-90) were replaced by alanine residues, IgE- and mAb reactivity to these mutants was compared to that to "wild type" Der p 2. Residues spanning cys73-cys78 were involved in the antigenic binding site for mAb alpha DpX. Mutations in the areas adjacent to this loop (i.e., 67 + 69 and 88-90) had similar effects on this mAb (10- to 20-fold decreases in reactivity were observed), supporting the suggestion that these areas are involved in this antigenic structure. The area of residues 45-48 was shown to be involved in an epitope for mAb 2B12. The reactivity of mAb 7A1 was influenced by substitutions of residues 45-48 as well as 88-90. Deletion of the residues spanning cys21-cys27 resulted in decreased reactivity to three mAbs (10E11, alpha DpX, and 7A1). From these observations, it may be concluded that binding of different mAbs is influenced by the same mutations and that the binding of single mAbs is influenced by two or more mutations scattered over the allergen molecule. These findings can point in two directions: minor amino-acid changes result in disruption of the overall conformation of the allergen, or distant sites are close together in the three-dimensional structure of the allergen. Decreased IgE reactivity was observed with all mutant molecules, varying between patients. The observed effects ranged from 5- to 1000-fold. Deletion of the amino-acid residues spanning cys21-cys27 and cys73-cys78 had the strongest effect on IgE reactivity, where decreases up to 1000-fold were observed. Such mutants might be useful tools to improve the safety of allergen-specific immunotherapy.
在面包酵母酿酒酵母中表达的重组变应原Der p 2被用作一种工具，以确定该变应原上的IgE和单克隆抗体（mAb）结合位点。为此，通过定点诱变产生突变分子。删除了跨越cys21 - cys27和cys73 - cys78的氨基酸残基，从而阻止了通过二硫键形成环。三个预测抗原位点（残基45 - 48、67 + 69和88 - 90）中的带电荷残基被丙氨酸残基取代，将这些突变体与“野生型”Der p 2的IgE和mAb反应性进行了比较。跨越cys73 - cys78的残基参与了mAbαDpX的抗原结合位点。该环相邻区域（即67 + 69和88 - 90）的突变对该mAb有类似影响（观察到反应性降低10至20倍），支持了这些区域参与该抗原结构的观点。残基45 - 48区域被证明参与了mAb 2B12的一个表位。mAb 7A1的反应性受到残基45 - 48以及88 - 90取代的影响。删除跨越cys21 - cys27的残基导致对三种mAb（10E11、αDpX和7A1）的反应性降低。从这些观察结果可以得出结论，不同mAb的结合受到相同突变的影响，并且单个mAb的结合受到分散在变应原分子上的两个或更多突变的影响。这些发现可以指向两个方向：微小的氨基酸变化导致变应原整体构象的破坏，或者在变应原的三维结构中远距离位点彼此靠近。在所有突变分子中均观察到IgE反应性降低，不同患者之间存在差异。观察到的效应范围为5至1000倍。删除跨越cys21 - cys27和cys73 - cys78的氨基酸残基对IgE反应性的影响最强，观察到降低幅度高达1000倍。此类突变体可能是提高变应原特异性免疫疗法安全性的有用工具。
