Glycan shield of the ebolavirus envelope glycoprotein GP

Abstract

The envelope glycoprotein GP of the ebolaviruses is essential for host cell entry and the primary target of the host antibody response. GP is heavily glycosylated with up to 17 N-linked sites, numerous O-linked glycans in its disordered mucin-like domain (MLD), and three predicted C-linked mannosylation sites. Glycosylation is important for host cell attachment, GP stability and fusion activity, and shielding from neutralization by serum antibodies. Here, we use glycoproteomics to profile the site-specific glycosylation patterns of ebolavirus GP. We detect up to 16 unique O-linked glycosylation sites in the MLD, and two O-linked sites in the receptor-binding GP1 subunit. Multiple O-linked glycans are observed within N-linked glycosylation sequons, suggesting crosstalk between the two types of modifications. We confirmed C-mannosylation of W288 in full-length trimeric GP. We find complex glycosylation at the majority of N-linked sites, while the conserved sites N257 and especially N563 are enriched in unprocessed glycans, suggesting a role in host-cell attachment via DC-SIGN/L-SIGN. Our findings illustrate how N-, O-, and C-linked glycans together build the heterogeneous glycan shield of GP, guiding future immunological studies and functional interpretation of ebolavirus GP-antibody interactions.

Fig. 1. Sequence and structure analysis of Filovirus GP glycosylation.

a Schematic of filovirus GP domain structure with annotated N-linked glycosylation and C-mannosylation. The cladogram on the left is based on the full GP sequences. Domain coloring as indicated below the diagram. Conserved N-linked glycans in ebolavirus species are annotated on top. b Pseudomodel of EBOV GP with core pentasaccharide of N-linked glycans shown as orange spheres. Model is built with GLYCAM based on PDB ID 5JQ3. The yellow lines indicate the approximate location of the MLD, connecting the respective termini of the glycan cap and GP2 subunits.

Fig. 2. N-linked glycosylation profiling of ebolavirus GP.

a Overview of site-specific N-linked glycan processing in ebolavirus GPΔTM from HEK293 and S2 cells as determined by LC-MS/MS. The glycans were classified by HexNAc content as truncated, paucimannose, oligomannose, hybrid or complex. Shown is the average of a duplicate experiment. b Pseudomodel of EBOV GP (as in Fig. 1), with glycans colored by main class (oligomannose, hybrid, complex) as observed in HEK293 cells.

Fig. 3. C-mannosylation in ebolavirus GP.

a schematic domain structure of EBOV GP with highlighted C-mannosylation sites. The modelled structure of C-mannosylated W288 was based on average F_o-F_c density (shown in green) of twelve isomorphic GP crystal structures. b LC-MS/MS spectrum of C-mannosylated W288 from BDBV GPΔTM from HEK293 cells. Note the prominent c11-c12 peaks provide direct evidence for the presence and localization of the Hex (+162 Da) modification of W288.

Fig. 4. O-linked glycosylation in the MLD of ebolavirus GP.

a GPΔTM from EBOV b GPΔTM from BDBV. Glycans drawn within a box represent multiple detected compositions per site. Glycans connected to multiple indicated sites could not be unambiguously localized from the LC-MS/MS data.

Fig. 5. O-linked glycosylation outside the MLD in ebolavirus GP.

a Pie charts represent the occupancy of the O-linked modification. b Sequence conservation of the detected O-linked glycosylation sites. c Distribution of glycan types at the indicated sites (bars indicate average of both replicate measurements shows as solid circles).

Fig. 6. Glycan-containing epitopes of monoclonal antibodies against ebolavirus GP.

a Overview of glycosylation sites associated with indicated epitopes. Direct contacts and clashes with CDRs are indicated in red, brushing interactions with framework regions in light orange. b Highlighted structure of 14G7 in complex with a linear epitope from the MLD (PDB ID:2Y6S), showing that the O-linked glycosylation site T485 is deeply buried in the cleft between heavy (dark red) and light chain (light pink).