Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen
Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen
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DOI：
10.1034/j.1398-9995.2001.103125.x
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发表时间：
2001-10-01
期刊：
影响因子：
12.4
通讯作者：
Pini, C
中科院分区：
文献类型：
--
作者：
Alisi, C;Afferni, C;Pini, C
背景：建立了一种快速纯化柏木花粉的主要变应原--43 kDa变应原的方法。方法：用酸性缓冲液洗涤花粉，然后提取蛋白质，用层析进行纯化。免疫印迹、酶联免疫吸附试验和凝集素结合试验测定了粗提物和纯化后的Cup a1的等电点、部分氨基酸序列和糖链组成。结果：免疫化学分析证实，纯化后的Cup a1具有较高的致敏活性。部分氨基酸序列分析表明，Cupa1与柏科和杉科的致敏蛋白高度同源，显示出相似的分子质量。纯化后的蛋白质呈现一条等电点为5.2的条带。33份柏树过敏患者血清中有19份(57%)对纯化的CUA1有明显的反应性。MALDI-TOF质谱仪显示存在三种N-连接的寡糖结构：GnGnXF(3)(即辣根过氧化物酶类型的寡糖被两个非还原的N-乙酰氨基葡萄糖残基取代)，GGnXF(3)/GnGXF(3)(即GnGnXF带有一个非还原的半乳糖残基)，和(GF)GnXF-/Gn(GF)XF-(单臂上有Lewis(A)表位)的摩尔比为67：8：23。结论：快速纯化方法可对其性质和结构进行精细的研究，并可在自然条件下评价其I-E反应性。氨基酸序列的相似性和一些复杂的多糖结构可以解释柏科和杉科之间高度的交叉反应。
Background: A rapid method for the purification of the major 43-kDa allergen, of Cupressus arizonica pollen, Cup a 1, was developed.Methods: The salient feature was a wash of the pollen in acidic buffer, followed, by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino-acid sequence, and its glycan composition.Results: Immunochemical analysis of Cup a I confirmed that the allergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients allergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF(3) (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF(3)/GnGXF(3) (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF-/Gn(GF)XF- (with a Lewis(a) epitope on one arm) in the molar ratio 67:8:23.Conclusions: The rapid purification process of Cup a 1 allowed some fine studies on its properties and structure, as well as the evaluation of its I-E reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-reactivity among the Cupressaceae and Taxodiaceae families.