An octadecameric O-glucosyltransferase generates diversity in antibody epitopes on variant surface antigens in African trypanosomes
Immune 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) alters antibody epitopes and influences infection outcome, but the responsible enzyme remained elusive. Here, we show that an expression-site-associated gene (ESAG3), which is co-transcribed with the active VSG, encodes an O-glucosyltransferase required for VSG3 O-glycosylation in vivo. ESAG3 depletion abolishes VSG3 O-glycosylation and recognition of live trypanosomes by an O-glycan-dependent monoclonal antibody, while complementation restores both. ESAG3 has strict UDP-glucose donor specificity and modifies serine/threonine residues within VSG3 peptides in vitro. Cryo-electron microscopy at 3.4 Angstrom resolution reveals that ESAG3 forms a homo-octadecameric ring with C3 symmetry, an architecture not previously observed among glycosyltransferases. Structure-guided mutagenesis shows that disruption of ESAG3 inter-subunit interfaces reduces higher-order assembly, enhances substrate conversion, and alters VSG glycoform composition. Furthermore, we show that VSG3 O-glycan occupancy correlates with susceptibility to complement-mediated trypanolysis in an antibody-dependent manner, establishing that ESAG3-mediated O-glucosylation shapes both antibody specificity and the functional outcome of the host response.