In vitro translation in a cell-free system from Trypanosoma brucei yields glycosylated and glycosylphosphatidylinositol-anchored proteins

African trypanosomes escape many cellular and unspecific immune reactions b y the expression of a protective barrier formed from a repertoire of severa l hundred genes encoding immunologically distinct variant surface glycoprot eins (VSGs). All mature VSGs are glycosylphosphatidylionositol-anchored and N-glycosylated. To study trypanosome-specific post-translational modifications of VSG, a ce ll-free system capable of in vitro translation, translocation into the roug h endoplasmic reticulum, N-glycosylation and glycosylphosphatidylinositol-a nchor addition was established using lysates of the bloodstream form of Try panosoma brucei. Monitoring protein synthesis by [S-35]methionine incorpora tion, labeled protein bands were readily detected by fluorography following SDS/PAGE. Appearance of these bands increased during a time-course of 45 m in and was sensitive to cycloheximide but not chloramphenicol treatment. Ef ficiency of this system, in terms of incorporation of radiolabeled amino ac ids into newly formed proteins, is similar to reticulocyte lysates. The sys tem does not, however, allow initiation of protein synthesis. Depending on the clone used, immunoprecipitation revealed one or two newly formed VSG bands. Upon digestion with N-glycosidase F these bands resulted in a single band of a lower apparent molecular mass, indicating that newly synthesized VSG underwent translocation and glycosylation in the cell-free system. Biotinylation of VSG and a combination of precipitation with immobi lized avidin and detection of VSG using antibodies specific for clones and cross-reacting determinants revealed that newly formed VSG contained the gl ycosylphosphatidylinositol anchor.