BIOSYNTHESIS AND FUNCTION OF GLYCOSPHINGOLIPIDS AND OTHER GLYCOCONJUGATES

霍乱弧菌产生的霍乱毒素(CT)及其耐热性 由某些菌株产生的肠毒素(LT)是 霍乱和旅行者腹泻的病原体。这个 毒素在结构、免疫和功能上都是相似的：每一种 有一个五聚体B亚单位，它与细胞上的特定受体结合 肠粘膜细胞和参与激活的A亚基 腺苷环化酶。而CT使用神经节苷脂GM1作为其唯一的 受体，LT似乎识别G/M1和其他受体 肠道细胞。为了识别这些替代的LT受体，我们 使用人类肠道Caco-2细胞，它在培养中表现为 分化的肠道细胞是这两种毒素的天然靶点。 Caco-2细胞与LT的结合是CT的8倍，而LT只与CT结合 CT-B部分抑制，CT结合完全抑制 由LT-B执行。尽管G/M1是唯一被两者识别的Caco-2糖脂 CT和LT，一系列膜半乳蛋白(表观分子 体重在92 kDa到120 kDa之间)可被LT识别，但不能被CT识别。通过 以特异性糖苷酶、转铁蛋白和胎球蛋白为模型 半乳糖蛋白，我们确定了LT的结合决定因素 这些半乳糖蛋白是半乳糖(β1-4)N-乙酰氨基葡萄糖。 此外，我们能够证明这些半乳糖蛋白能够识别 用抗LT的特异性抗血清免疫沉淀 乳酸-N-新四糖类型的多乳糖化糖蛋白。最后， 其中一些LT的替代半乳糖蛋白受体似乎是 LT激活Caco-2细胞中腺苷酸环化酶的功能 CT-B仅部分抑制作用。因此，尽管高度的 LT和CT的同源性，LT可以识别与 与GM/1寡糖不同的碳水化合物结构 都能被两种毒素识别。此外，LT的表观能力 同时利用糖脂和糖蛋白作为受体增加了 LT可能使用一种以上的细胞途径 激活。在这方面，我们以前已经表明，反恐委员会必须 新神经节蛋白进入细胞的途径不同于 绑定到G/M1。

Cholera toxin (CT) produced by Vibrio cholerae and the heat-labile enterotoxin (LT) produced by certain strains of Escherichia coli are the causative agents of cholera and traveler's diarrhea, respectively. The toxins are structurally, immunologically and functionally similar: each has a pentameric B subunit which binds to specific receptors on the intestinal mucosal cell and an A subunit which is involved in activation of adenylylcyclase. Whereas CT uses the ganglioside GM1 as its only receptor, LT appears to recognize G/M1 as well as other receptors on intestinal cells. In order to identify these alternate LT receptors, we used human intestinal CaCo-2 cells, which behave in culture as differentiated enterocytes, the natural target for the two toxins. CaCo-2 cells bound 8-fold more LT than CT, and LT binding was only partially inhibited by CT-B whereas CT binding was completely inhibited by LT-B. Although G/M1 was the only CaCo-2 glycolipid recognized by both CT and LT, a series of membrane galactoproteins (apparent molecular weights between 92 and 120 kDa) were recognized by LT but not by CT. By using specific glycosidases and transferrin and fetuin as model galactoproteins, we established that the binding determinant for LT on these galactoproteins was galactose (beta1-4)N-acetylglucosamine. Furthermore, we were able to show that these galactoproteins recognized by LT were immunoprecipitated by specific antisera against polyactosylated glycoproteins of the lacto-N-neotetraosyl type. Finally, some of these alternate galactoprotein receptors for LT appear to be functional as activation of adenylylcyclase by LT in CaCo-2 cells was only partially inhibited by CT-B. Thus, despite the high degree of homology between LT and CT, LT can recognize glycoconjugates with carbohydrate structures distinct from the oligosaccharide of GM/1 which is recognized by both toxins. Furthermore, the apparent ability of LT to utilize both glycolipids and glycoproteins as receptors raises the possibility that LT may use more than one pathway for cellular activation. In this regard, we had shown previously that CT bound to neoganglioproteins enters the cell through a different pathway than when bound to G/M1.