Identification of cholera toxin B subunit binding protein and its bioactive analysis

霍乱毒素(CT)由两个亚基组成，一个是毒素A亚基(CT-A)，负责激活腺苷环化系统；另一个是五元B亚单位(CT-B)，负责CT与细胞膜GM1神经节苷脂的结合。CT是最有效和被广泛研究的黏膜佐剂之一。虽然ADP核糖化的CT-A具有增强免疫应答的作用，但受体结合的CT-B具有更强的免疫原性，可能是一种无潜在毒性的佐剂活性储存库。CT-B分子改变免疫反应的能力可能需要GM1。然而，新的未知的CT-B受体对免疫调节具有重要意义，可能是这种信号转导的原因。为了阐明CT-B体外免疫调节的机制，我们用生物素CT-B和亲和素-琼脂糖凝胶对细胞表面CT-B结合受体p32(32 kDa蛋白)进行了纯化。经p32凝胶内切和MALDI-MS指纹图谱分析，确定p32为哺乳动物细胞中已知的蛋白质。CT-B受体p32在CT-B免疫调节中的作用将继续被研究。

The Cholera toxin (CT)is composed of two subunits, a toxigenic A subunit (CT-A)which activates the adenylyl cyclase system and a pentamaric B subunit (CT-B)which is responsible for CT binding to the cell membrane GM1 gangliosides. The CT is one of the most effective and widely studied mucosal adjuvants. Although the ADP-ribosylating CT-A has been implicated in augmenting immune responses, the receptor-binding CT-B has a greater immunogenicity and may be a repository of adjuvant activity without potential toxicity In general knowledge, the only CT-B receptor is the cell membrane GM1 ganglioside, which expresses in all mammalian cells. The GM1 may be required for the ability of CT-B molecules to alter immunoresponse. However, novel unknown CT-B receptor, which is significant for immune modulation, may account for this signal transduction. In order to elucidate mechanisms of immune modulation by CT-B in vitro, we purified cell surface CT-B binding receptor p32 (32 kDa protein)on SDS-PAGE gel by pull-down method with biotinyl CT-B and avidin-Sepharose. By in-gel digestion of p32 and MALDI-MS fingerprint analysis, p32 was determined as a well-known protein on mammalian cells. The role of CT-B receptor p32 for immune modulation by CT-B will continue to be examined.