Colman P G, Nayak R C, Campbell I L, Eisenbarth G S
Joslin Diabetes Center, Brigham and Women's Hospital, Boston, Massachusetts 02215.
Diabetes. 1988 May;37(5):645-52. doi: 10.2337/diab.37.5.645.
With biochemical and enzymatic treatment of frozen sections of pancreas, we have previously shown that cytoplasmic islet cell antibodies (ICAs) react with carbohydrate determinants of islet cell glycoconjugates. As a first step toward purifying these glycoconjugates, human pancreas tissue was extracted in a mixture of chloroform and methanol, and the glycolipids were obtained by effecting a Folch partition. The protein pellet, lipid fraction, and glycolipid fraction so obtained were assessed for their ability to block the binding of ICAs to frozen sections of human pancreas, the effect being quantitated with a photometer. Only the glycolipid extract could block ICA binding, and blocking was dose dependent. Subfractionation of the glycolipid extract by hydrophobic interaction on C18 cartridges demonstrated that blocking activity resided in the fraction bound and eluted with methanol, consistent with the autoantigen being a glycolipid. Furthermore, the binding of an anti-islet cell ganglioside monoclonal antibody, 3G5, could be blocked with these extracts, whereas the binding of an anti-islet cell protein monoclonal antibody, 4F2, was unaffected. The major gangliosides of the pancreas were seen to be GM3 and GD3 by thin-layer chromatography (TLC). Fractions scraped and eluted from TLC plates were tested for their ability to block ICA binding to pancreatic sections. Neither GM3- nor GD3-containing fractions could block ICA binding; however, a fraction containing minor pancreatic gangliosides (including GM2) of monosialoganglioside mobility was a potent inhibitor of ICA binding to pancreas sections. TLC of a chloroform-methanol extract of human islets demonstrated that islets differentially express monosialogangliosides (especially GM2).
通过对胰腺冰冻切片进行生化和酶处理，我们之前已经表明，细胞质胰岛细胞抗体（ICAs）与胰岛细胞糖缀合物的碳水化合物决定簇发生反应。作为纯化这些糖缀合物的第一步，用人胰腺组织在氯仿和甲醇的混合物中进行提取，并通过Folch分配获得糖脂。对如此获得的蛋白质沉淀、脂质部分和糖脂部分进行评估，以确定它们阻断ICAs与人胰腺冰冻切片结合的能力，并用光度计对该效应进行定量。只有糖脂提取物能够阻断ICA结合，并且阻断呈剂量依赖性。通过在C18柱上进行疏水相互作用对糖脂提取物进行亚分级分离表明，阻断活性存在于用甲醇结合并洗脱的部分中，这与自身抗原为糖脂一致。此外，这些提取物可以阻断抗胰岛细胞神经节苷脂单克隆抗体3G5的结合，而抗胰岛细胞蛋白质单克隆抗体4F2的结合则不受影响。通过薄层色谱（TLC）观察到胰腺的主要神经节苷脂为GM3和GD3。对从TLC板上刮下并洗脱的部分进行测试，以确定它们阻断ICA与胰腺切片结合的能力。含GM3和含GD3的部分均不能阻断ICA结合；然而，一个含有单唾液酸神经节苷脂迁移率的少量胰腺神经节苷脂（包括GM2）的部分是ICA与胰腺切片结合的有效抑制剂。人胰岛氯仿 - 甲醇提取物的TLC表明，胰岛差异表达单唾液酸神经节苷脂（尤其是GM2）。
