Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus

Abstract

Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed.

Fig. 1.

Electrophoretic mobility, heterogeneity and lectin affinities of sG glycoproteins. (A) VV-sG HeV (i) and HEK293-sG HeV (ii) separated by SDS–PAGE on 4–12% gels and (B) transferred to PVDF membranes for detection with biotinylated lectins: NPL, SNA, UEAI, MAAII, GNA and DSA. Lectin affinities are shown for each of the sG glycoproteins. (C) VV-sG or HEK293-sG (15 μg) was separated by 2-DE using 7 cm (pH 3–11) non-linear immobilized pH gradient (IPG) strips and run on 4–20% SDS–PAGE gels. The protein spots were visualized by silver staining.

Fig. 2.

Glycopeptide identification and compositional analysis by LC-MS/MS. (A) Product ion spectra acquired on a linear ion trap mass spectrometer of the triply charged precursor ion at m/z 1305.21 eluting at 27.4 min from a tryptic digest of the VV-sG protein. Several b- and y-ions confirming the peptide as IHEC_(Cam)NISC_(Cam)PNPLPFR (HT2) are labeled. Carbohydrate diagnostic oxonium ions at m/z 204, 274, 366 and 528 were observed. The fragmentation observed from the spectra m/z 150–1600 revealed an N-glycan with the composition (HexNAc)₄(Hex)₅(NeuAc)₁(Fuc)₁. Note that not all y- and b-ions are labeled in this figure but are described in the text. (B) Product ion spectra acquired on a Q-TOF mass spectrometer of the triply charged precursor ion at m/z 1049.85 eluting at 49.6 min from a tryptic digest of the HEK293-sG protein. In this example, the peptide sequence was determined to be VSLIDTSSTITIPANIGLLGSK (HT12) and was O-glycosylated with a glycan of composition (HexNAc)₁(Hex)₁(NeuAc)₂.

Fig. 3.

Three-dimensional model of the HeV attachment glycoprotein and receptor complex. Three-dimensional models of a globular head domain of HeV sG (A) and in complex with ephrin-B2 (B). The glycans were modeled according to the electron density map of the crystal structure of sG/ephrin-B2 complex at 2.7 Å resolution. The glycan branches and some of the core structures were not modeled due to the weak signal that resulted from the flexibility of the glycan chains. N-linked glycosylation is shown at all five predicted sites on the head domain of the G protein. Glycan ⁵²⁵N forms close contacts with ephrin-B2.

Fig. 4.

Binding of different forms of the HeV-sG protein to ephrin-B2. The determination of interactions between different constructs of the sG (1 µM) and ephrin-B2 (EFN-B2) was performed using a tryptophan fluorescence quench assay on a SPEX FluoroMax-2 spectrofluorimeter (25°C, 10 mm path-length cuvette, excitation and emission wavelengths of 295 and 350 nm, respectively). Titrations were performed by a stepwise addition of small volumes of concentrated (0.5 mM) ephrin-B2 solutions to a 1 µM solution of sG constructs at pH 7.2. The difference between the fluorescence units of the complex and the sum of individual components was used to plot the results (filled square), insect cell-expressed sG head domain construct (low glycosylation level) and (filled diamond) HEK293 cell-expressed sG head domain construct.