{"paper_id":"496bab88-7a59-4f69-8ddc-2d65f151a215","body_text":"Abstract\nImmune evasion in many pathogens relies on sequence variation to generate antigenic diversity. African trypanosomes use an additional strategy where O-glucosylation of variant surface glycoproteins (VSGs) generates heterogeneous glycans that alter antibody epitope recognition, influencing infection outcome. However, the VSG O-glycosylation enzyme has remained unknown. Here, we identify ESAG3 as the glycosyltransferase required for generating these O-glycan-dependent epitopes. ESAG3 depletion in vivo abolishes O-glycosylation-specific monoclonal antibody recognition, while complementation restores binding. ESAG3 has strict UDP-glucose specificity, manganese dependence, and modifies serine/threonine residues within cysteine-flanked VSG peptides. Single-particle cryo-electron microscopy reveals that ESAG3 forms an unprecedented octadecameric architecture with C3 symmetry at 3.4 angstrom resolution, a novel quaternary organisation for a glycosyltransferase. Structure-guided mutagenesis demonstrates essential active-site residues for catalysis, while interface mutations disrupt octadecamer assembly and enhance substrate turnover, indicating that oligomeric architecture regulates catalytic output. This work establishes ESAG3 as a kinetoplastid-specific glucosyltransferase and reveals the molecular basis whereby VSG O-glycosylation generates epitope diversity alongside sequence-based antigenic variation.\nSignificance statement African trypanosomes evade host immunity by switching variant surface glycoproteins (VSGs) from a vast gene archive, generating extreme antigenic diversity through sequence polymorphism. O-glucosylation adds a second layer where heterogeneous O-linked sugars at the very top of surface-exposed VSG loops diversify the epitope space accessible to host antibodies. We identify ESAG3 as the O-glucosyltransferase responsible for this modification and reveal it forms an unprecedented 18-subunit architecture. Critically, ESAG3 is encoded within VSG expression sites, coupling O-glycosylation to monoallelic transcriptional control. When parasites switch expression sites, VSG sequence changes alongside co-transcribed O-glycosylation machinery, integrating genetic and chemical variation into a unified immune evasion strategy. This work provides the molecular and structural basis for O-glycan-mediated epitope control on the trypanosome surface.\nCompeting Interest Statement\nThe authors have declared no competing interest.","source_license":"CC-BY-4.0","license_restricted":false}