Fu Zhuji, Chen Chen, Barbieri Joseph T, Kim Jung-Ja P, Baldwin Michael R
Department of Biochemistry, Medical Collegeof Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
Biochemistry. 2009 Jun 23;48(24):5631-41. doi: 10.1021/bi9002138.
Botulinum neurotoxin causes rapid flaccid paralysis through the inhibition of acetylcholine release at the neuromuscular junction. The seven BoNT serotypes (A-G) have been proposed to bind motor neurons via ganglioside-protein dual receptors. To date, the structure-function properties of BoNT/F host receptor interactions have not been resolved. Here, we report the crystal structures of the receptor binding domains (HCR) of BoNT/A and BoNT/F and the characterization of the dual receptors for BoNT/F. The overall polypeptide fold of HCR/A is essentially identical to the receptor binding domain of the BoNT/A holotoxin, and the structure of HCR/F is very similar to that of HCR/A, except for two regions implicated in neuronal binding. Solid phase array analysis identified two HCR/F binding glycans: ganglioside GD1a and oligosaccharides containing an N-acetyllactosamine core. Using affinity chromatography, HCR/F bound native synaptic vesicle glycoproteins as part of a protein complex. Deglycosylation of glycoproteins using alpha(1-3,4)-fucosidase, endo-beta-galactosidase, and PNGase F disrupted the interaction with HCR/F, while the binding of HCR/B to its cognate receptor, synaptotagmin I, was unaffected. These data indicate that the HCR/F binds synaptic vesicle glycoproteins through the keratan sulfate moiety of SV2. The interaction of HCR/F with gangliosides was also investigated. HCR/F bound specifically to gangliosides that contain alpha2,3-linked sialic acid on the terminal galactose of a neutral saccharide core (binding order GT1b = GD1a >> GM3; no binding to GD1b and GM1a). Mutations within the putative ganglioside binding pocket of HCR/F decreased binding to gangliosides, synaptic vesicle protein complexes, and primary rat hippocampal neurons. Thus, BoNT/F neuronal discrimination involves the recognition of ganglioside and protein (glycosylated SV2) carbohydrate moieties, providing a structural basis for the high affinity and specificity of BoNT/F for neurons.
肉毒杆菌神经毒素通过抑制神经肌肉接头处乙酰胆碱的释放导致快速弛缓性麻痹。已提出七种肉毒杆菌神经毒素血清型（A - G）通过神经节苷脂 - 蛋白质双受体与运动神经元结合。迄今为止，肉毒杆菌神经毒素F型（BoNT/F）宿主受体相互作用的结构 - 功能特性尚未明确。在此，我们报告了肉毒杆菌神经毒素A型（BoNT/A）和F型（BoNT/F）受体结合域（HCR）的晶体结构以及BoNT/F双受体的特性。HCR/A的整体多肽折叠与BoNT/A全毒素的受体结合域基本相同，除了与神经元结合相关的两个区域外，HCR/F的结构与HCR/A非常相似。固相阵列分析确定了两种HCR/F结合聚糖：神经节苷脂GD1a和含有N - 乙酰乳糖胺核心的寡糖。使用亲和色谱法，HCR/F作为蛋白质复合物的一部分与天然突触小泡糖蛋白结合。使用α(1 - 3,4)-岩藻糖苷酶、内切β - 半乳糖苷酶和PNGase F对糖蛋白进行去糖基化破坏了与HCR/F的相互作用，而HCR/B与其同源受体突触结合蛋白I的结合不受影响。这些数据表明HCR/F通过突触小泡蛋白2（SV2）的硫酸角质素部分与突触小泡糖蛋白结合。我们还研究了HCR/F与神经节苷脂的相互作用。HCR/F特异性结合在中性糖核心的末端半乳糖上含有α2,3 - 连接唾液酸的神经节苷脂（结合顺序为GT1b = GD1a >> GM3；不与GD1b和GM1a结合）。HCR/F假定的神经节苷脂结合口袋内的突变降低了与神经节苷脂、突触小泡蛋白复合物和原代大鼠海马神经元的结合。因此，BoNT/F对神经元的识别涉及神经节苷脂和蛋白质（糖基化的SV2）碳水化合物部分的识别，为BoNT/F对神经元的高亲和力和特异性提供了结构基础。
