Webb H, Carnall N, Carrington M
Department of Biochemistry, Cambridge University, England.
Braz J Med Biol Res. 1994 Feb;27(2):349-56.
The glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei exhibits exquisite specificity for the GPI-anchor of the variant specific glycoprotein (VSG). However the evidence that it is involved in VSG metabolism in the living trypanosome is circumstantial; it shows the same life cycle stage regulated expression as the VSG, no feasible alternative substrate has been identified, and it metabolises the VSG efficiently in vitro and in vivo on hypotonic lysis. Against these considerations are the observations that the GPI-PLC is found on the cytoplasmic face of vesicles so it could not gain access to the VSG through normal vesicle fusion and that the accelerated loss of VSG from bloodstream forms on differentiation to procyclic forms occurs through the action of a protease. To try to determine the role of the GPI-PLC, a homozygous null mutant T. brucei has been constructed. The null mutant was created by replacement of the entire gene at both alleles with selectable antibiotic resistance markers in procyclic form trypanosomes. The GPI-PLC gene is not usually expressed in procyclic forms and so, as would be expected, the null procyclics display no obvious phenotype. The null procyclics have been used to infect tsetse flies and it remains to be seen whether it is possible for them to differentiate to bloodstream forms and, if so, what the antigenic variation phenotype of the null bloodstream forms would be.
来自布氏锥虫的糖基磷脂酰肌醇特异性磷脂酶C（GPI-PLC）对变异特异性糖蛋白（VSG）的GPI锚具有极高的特异性。然而，关于它在活锥虫中参与VSG代谢的证据是间接的；它与VSG表现出相同的生命周期阶段调控表达，尚未鉴定出可行的替代底物，并且在体外和体内低渗裂解时能有效代谢VSG。与这些考虑因素相悖的是，观察到GPI-PLC存在于囊泡的细胞质面，因此它无法通过正常的囊泡融合接触到VSG，而且从血流形式分化为前循环形式时VSG的加速丢失是通过蛋白酶的作用发生的。为了试图确定GPI-PLC的作用，构建了纯合缺失突变体布氏锥虫。该缺失突变体是通过用可选择的抗生素抗性标记物替换前循环形式锥虫两个等位基因上的整个基因而产生的。GPI-PLC基因通常在前循环形式中不表达，因此，正如预期的那样，缺失前循环体没有明显的表型。这些缺失前循环体已被用于感染采采蝇，它们是否能够分化为血流形式以及如果可以的话，缺失血流形式的抗原变异表型会是什么，还有待观察。
