Steric constraints control processing of glycosylphosphatidylinositol anchors in .

The transferrin receptor (TfR) of the bloodstream form (BSF) of is a heterodimer comprising glycosylphosphatidylinositol (GPI)-anchored expression site-associated gene 6 (ESAG6 or E6) and soluble ESAG7. Mature E6 has five -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 -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--glycosidase F treatment, lectin pulldowns, and exoglycosidase treatment, we have now investigated TfR -glycan and GPI processing. E6 increased ∼5 kDa during maturation, becoming reactive with both TL and lectin (ECL, terminal LacNAc), indicating synthesis of poly-LacNAc on paucimannose -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--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.