Agusti R, Couto A S, Campetella O, Frasch A C, de Lederkremer R M
CIHIDECAR (CONICET), Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Mol Biochem Parasitol. 1998 Nov 30;97(1-2):123-31. doi: 10.1016/s0166-6851(98)00137-6.
Both, culture-derived and metacyclic trypomastigotes of Trypanosoma cruzi shed a glycoprotein, the shed acute phase antigen, that is responsible for the trans-sialidase activity. In the present work the structure of the glycosylphosphatidylinositol membrane anchor of the trans-sialidase isolated from metacyclic forms was determined. Parasites were metabolically labelled with [9, 10(n)3H]-palmitic acid and the glycoprotein was purified by immunoprecipitation with a monoclonal antibody directed against the repetitive aminoacid sequence. Treatment of the glycoprotein with phosphatidylinositol phospholipase C (PI-PLC) from Bacillus thuringiensis rendered a lipid that comigrated in TLC with a standard of ceramide. No alkylglycerol was detected in contrast with the results previously obtained for the trans-sialidase isolated from culture-derived trypomastigotes where both lipids were found. Chemical and chromatographic analysis showed that the lipid moiety is palmitoyldihydrosphingosine with a minor amount of stearoyldihydrosphingosine. The glycan constituent of the glycosylphosphatidylinositol-anchor was analysed by nitrous acid deamination of the aqueous phase of the PI-PLC treatment, followed by reduction with NaBH4 and hydrolysis of the phosphodiester with aqueous hydrofluoric acid. A major oligosaccharide was obtained and enzymatic treatment with exoglycosidases and further chromatography in a high pH anion exchange system showed that the trimannosyl core backbone is substituted by an alpha-galactose. A comparison between the lipid constituent of the glycosylphosphatidylinositol anchor of several proteins and their spontaneous shedding by the action of an endogenous PI-PLC was made.
克氏锥虫的培养来源型和循环后期锥鞭毛体均分泌一种糖蛋白，即分泌型急性期抗原，它负责转唾液酸酶活性。在本研究中，确定了从循环后期形式分离的转唾液酸酶的糖基磷脂酰肌醇膜锚定结构。用[9,10(n)3H]-棕榈酸对寄生虫进行代谢标记，并用针对重复氨基酸序列的单克隆抗体通过免疫沉淀纯化糖蛋白。用来自苏云金芽孢杆菌的磷脂酰肌醇磷脂酶C（PI-PLC）处理该糖蛋白，得到一种脂质，其在薄层层析（TLC）中与神经酰胺标准品共迁移。与先前从培养来源的锥鞭毛体分离的转唾液酸酶的结果相反，未检测到烷基甘油，在后者中发现了两种脂质。化学和色谱分析表明，脂质部分是棕榈酰二氢鞘氨醇，还有少量硬脂酰二氢鞘氨醇。通过对PI-PLC处理水相进行亚硝酸脱氨，然后用NaBH4还原并用水相氢氟酸水解磷酸二酯，分析了糖基磷脂酰肌醇锚定的聚糖成分。得到了一种主要的寡糖，用外切糖苷酶进行酶处理并在高pH阴离子交换系统中进一步色谱分析表明，三甘露糖核心骨架被一个α-半乳糖取代。对几种蛋白质的糖基磷脂酰肌醇锚定的脂质成分及其通过内源性PI-PLC作用的自发脱落进行了比较。
