Analysis of tertial structure and function of non-toxic components associating with Clostridium botulinum neurotoxins.

A型至D型肉毒杆菌血凝素阳性祖毒素（PTX）由三种不同的成分组成：神经毒素（NTX）、血凝素（HA）和无毒的非HA （NTNH）。HA由HA1 （~ 33kDa）、2 （~ 17kDa）、3a （~ 22kDa）和3b (~ 53kDa) 4个子组分组成。我们建立了一种简单的纯化B型ha阳性PTX和NTX的方法，采用亲和凝胶柱连接乳糖。通过使用纯化的A- D型毒素及其每个HA亚组分制备成gst融合蛋白，我们已经证明HA，特别是HA1和HA3b，在HA阳性ptx附着在小肠上部微绒毛和红细胞上发挥粘附素的作用。A型（和B型）毒素主要通过HA1与半乳糖结合，而C型（和D型）毒素主要通过HA3b与唾液酸结合。通过对HA1和HA3b的一级和三级结构分析，发现HA1具有两个类似蓖麻毒素凝集素b链的β-三叶结构域，而HA3b具有唾液粘附素家族（Siglec家族）的碳水化合物识别结构域（CRD）。在与C ha阳性毒素和人结肠癌HT-29细胞的结合和内化实验中，毒素与高分子量的糖蛋白结合，在4 min内内化，但经神经氨酸酶预处理后，毒素未在4 min内内化，说明糖蛋白唾液酸是C型毒素的受体，毒素能快速内化到细胞内。还发现HA1和HA3b均具有佐剂活性。

Clostridium botulinum type A to D hemagglutinin positive progenitor toxins (PTX) consist of three distinct components : neurotoxin (NTX), hemagglutinin (HA), and non-toxic non-HA (NTNH). The HA consists of four subcomponents designated HA1 (〜33kDa), 2 (〜17kDa), 3a (〜22kDa), and 3b (〜53kDa).We have established an easy procedure for purifying type B HA-positive PTX and NTX by employing an affinity gel column-linked lactose. By employing the purified type A to D toxins and their each HA subcomponents prepared as GST-fusion proteins, we have shown that HA, especially HA1 and HA3b, works as an adhesin in the attachment of HA-positive PTXs to the-microvilli of the upper small intestine as well as erythrocytes. Type A (and B) toxin bound to the galactose mainly via HA1, whereas type C (and D) bound to the sialic acid mainly via HA3b. By analyses of primary and tertiary structure of HA1 and HA3b, It became clear that HA1 has two β-trefoil domains like as lectin B-chain of the ricin, whereas HA3b has carbohydrate recognition domain (CRD) of sialo-adhesin family (or Siglec family). In the binding and internalization tests with C HA-positive toxin and human colon carcinoma HT-29 cells, the toxin bound to glycoproteins with high molecular mass, and internalized within 4 min, but not if the cells were pretreated with neuraminidase, indicating that sialic acid of glycoprotein is the receptor for type C toxin and the toxin can internalize quickly into the cells. It was also found that both HA1 and HA3b have adjuvant activity.