Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus Journal Article
| Authors: | Colgrave, M. L.; Snelling, H. J.; Shiell, B. J.; Feng, Y. R.; Chan, Y. P.; Bossart, K. N.; Xu, K.; Nikolov, D. B.; Broder, C. C.; Michalski, W. P. |
| Article Title: | 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. © 2011 The Author. |
| Keywords: | controlled study; protein expression; human cell; nonhuman; binding affinity; mass spectrometry; mammalia; protein degradation; protein binding; hela cells; amino acid sequence; molecular sequence data; cell strain hek293; peptide fragments; recombinant proteins; recombinant protein; glycosylation; vaccinia virus; tandem mass spectrometry; crystal structure; models, molecular; crystallography, x-ray; x ray crystallography; polysaccharides; protein structure, quaternary; electrophoretic mobility shift assay; receptor, ephb2; carbohydrate analysis; carbohydrate sequence; glycopeptide; glycopeptides; electrophoretic mobility; amino acid motifs; polyacrylamide gel electrophoresis; sequence analysis, protein; equidae; carbohydrate conformation; hek293 cells; consensus sequence; ephrin b2; virus glycoprotein; protein glycosylation; glycan; viral envelope proteins; hendra virus; lectins; hendra virus (hev); n-and o-linked glycosylation; mannose; lectin binding |
| Journal Title: | Glycobiology |
| Volume: | 22 |
| Issue: | 4 |
| ISSN: | 0959-6658 |
| Publisher: | Oxford University Press |
| Date Published: | 2012-04-01 |
| Start Page: | 572 |
| End Page: | 584 |
| Language: | English |
| DOI: | 10.1093/glycob/cwr180 |
| PROVIDER: | scopus |
| PMCID: | PMC3287018 |
| PUBMED: | 22171062 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 1" - "Export Date: 2 April 2012" - "CODEN: GLYCE" - "Source: Scopus" |
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