Mogensen Jesper E, Wimmer Reinhard, Larsen Jørgen N, Spangfort Michael D, Otzen Daniel E
Department of Biotechnology, Institute of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, Aalborg DK-9000, Denmark.
J Biol Chem. 2002 Jun 28;277(26):23684-92. doi: 10.1074/jbc.M202065200. Epub 2002 Apr 12.
Bet v 1 is a 17-kDa protein abundantly present in the pollen of the White birch tree and is the primary cause of birch pollen allergy in humans. Its three-dimensional structure is remarkable in that a solvent-accessible cavity traverses the core of the molecule. The biological function of Bet v 1 is unknown, although it is homologous to a family of pathogenesis-related proteins in plants. In this study we first show that Bet v 1 in the native state is able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS). ANS binds to Bet v 1 with 1:1 stoichiometry, and NMR data indicate that binding takes place in the cavity. Using an ANS displacement assay, we then identify a range of physiologically relevant ligands, including fatty acids, flavonoids, and cytokinins, which generally bind with low micromolar affinity. The ability of these ligands to displace ANS suggests that they also bind in the cavity, although the exact binding sites seem to vary among different ligands. The cytokinins, for example, seem to bind at a separate site close to ANS, because they increase the fluorescence of the ANS. Bet v 1 complex. Also, the fluorescent sterol dehydroergosterol binds to Bet v 1 as demonstrated by direct titrations. This study provides the first qualitative and quantitative data on the ligand binding properties of this important pollen allergen. Our findings indicate that ligand binding is important for the biological function of Bet v 1.
Bet v 1是一种17千道尔顿的蛋白质，大量存在于白桦树的花粉中，是人类桦树花粉过敏的主要原因。其三维结构很显著，因为一个溶剂可及腔贯穿分子核心。尽管Bet v 1与植物中一类病程相关蛋白同源，但其生物学功能尚不清楚。在本研究中，我们首先表明天然状态的Bet v 1能够结合荧光探针8-苯胺基-1-萘磺酸（ANS）。ANS以1:1的化学计量比与Bet v 1结合，核磁共振数据表明结合发生在腔内。然后，我们使用ANS置换试验鉴定了一系列生理相关配体，包括脂肪酸、黄酮类化合物和细胞分裂素，它们通常以低微摩尔亲和力结合。这些配体置换ANS的能力表明它们也在腔内结合，尽管不同配体的确切结合位点似乎有所不同。例如，细胞分裂素似乎在靠近ANS的一个单独位点结合，因为它们增加了ANS - Bet v 1复合物的荧光。此外，通过直接滴定证明荧光固醇脱氢麦角固醇与Bet v 1结合。本研究提供了关于这种重要花粉过敏原配体结合特性的首批定性和定量数据。我们的研究结果表明配体结合对Bet v 1的生物学功能很重要。
