Steric constraints control processing of glycosylphosphatidylinositol anchors in Trypanosoma brucei

The transferrin receptor (TfR) of the bloodstream form (BSF) of Trypanosoma brucei is a heterodimer comprising glycosylphosphatidylinositol (GPI)-anchored expression site–associated gene 6 (ESAG6 or E6) and soluble ESAG7. Mature E6 has five N-glycans, consisting of three oligomannose and two unprocessed paucimannose structures. Its GPI anchor is modified by the addition of 4 – 6 α-galactose residues. TfR binds tomato lectin (TL), specific for N-acetyllactosamine (LacNAc) repeats, and previous studies have shown transport-dependent increases in E6 size consistent with post-glycan processing in the endoplasmic reticulum. Using pulse-chase radiolabeling, peptide-N-glycosidase F treatment, lectin pulldowns, and exoglycosidase treatment, we have now investigated TfR N-glycan and GPI processing. E6 increased ∼5 kDa during maturation, becoming reactive with both TL and Erythrina cristagalli lectin (ECL, terminal LacNAc), indicating synthesis of poly-LacNAc on paucimannose N-glycans. This processing was lost after exoglycosidase treatment and after RNAi-based silencing of TbSTT3A, the oligosaccharyltransferase that transfers paucimannose structures to nascent secretory polypeptides. These results contradict previous structural studies. Minor GPI processing was also observed, consistent with α-galactose addition. However, increasing the spacing between E6 protein and the GPI ω-site (aa 4 –7) resulted in extensive post-translational processing of the GPI anchor to a form that was TL/ECL-reactive, suggesting the addition of LacNAc structures, confirmed by identical assays with BiPNHP, a non-N-glycosylated GPI-anchored reporter. We conclude that BSF trypanosomes can modify GPIs by generating structures reminiscent of those present in insect-stage trypanosomes and that steric constraints, not stage-specific expression of glycosyltransferases, regulate GPI processing.