Fig 3.

Mapping isoform-specific O-glycosites on EBOV GP. (A) Identified O-glycosites and most-complex unambiguously assigned site-specific structures are shown in the context of EBOV GP primary sequence, where VLP-derived sites are shown above the sequence and recombinant GP-derived sites—below the sequence. Glycosites with median quantification ratios of singly glycosylated peptides below 0.5 were considered as isoform regulated and are outlined in cyan, maroon, and red for GalNAc-T1, GalNAc-T2, and GalNAc-T3, respectively. A simplified cartoon above summarizes the data, where orange bars represent all identified O-glycosites. Color-coded arrows indicate isoform-regulated glycosites. Epitopes of protective antibodies derived from convalescent or vaccinated individuals, as well as epitopes for several protective mAbs, are annotated in the sequence (52–55). Amino acids for which combined substitutions to Ala abolish cytopathic effect are also highlighted (15). (B) TMT quantification ratios of single-site peptides, where each dot represents a separate PSM, and horizontal bars indicate median values. (C) Molecular modeling of EBOV GP with the identified O-glycans attached. The MLD was built de novo based on available cryo-EM/ET density maps (56, 57) of virion- and VLP-derived GP and combined with an atomic resolution structure of the GP lacking the MLD (PDB: 6HS4). The MLD is shaded in iceblue and the GCD is shaded in lime. GalNAc-T1, -T2, and -T3 regulated O-glycosites are highlighted in cyan, purple, and red, respectively. The remaining O-glycans are shown in white. Chain A contains VLP-derived glycosites, chain B contains recombinant GP-derived glycosites, and chain C contains combined maximum capacity. (D) Identified O-glycans are colored based on the longest site-specific structure identified, as indicated in the legend. Putative N-glycans were included in the model and are shown in blue.