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Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells.

Lei Y, Yu H, Dong Y, Yang J, Ye W, Wang Y, Chen W, Jia Z, Xu Z, Li Z, Zhang F - PLoS ONE (2015)

Bottom Line: By combining these methods, a high heterogeneity of DENV N-glycans was found.Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found.For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, School of Preclinical Medicine, The Fourth Military Medical University, 169 Changle Xi Road, Xian, Shaanxi 710032 China.

ABSTRACT
DENV envelope glycoprotein (E) is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies. It is well known that DENV E glycoprotein has two potential N-linked glycosylation sites at Asn67 and Asn153. The N-glycans of E glycoprotein have been shown to influence the proper folding of the protein, its cellular localization, its interactions with receptors and its immunogenicity. However, the precise structures of the N-glycans that are attached to E glycoprotein remain elusive, although the crystal structure of DENV E has been determined. This study characterized the structures of envelope protein N-linked glycans on mature DENV-2 particles derived from insect cells via an integrated method that used both lectin microarray and MALDI-TOF-MS. By combining these methods, a high heterogeneity of DENV N-glycans was found. Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found. Furthermore, a complex between a glycan on DENV and the carbohydrate recognition domain (CRD) of DC-SIGN was mimicked with computational docking experiments. For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

No MeSH data available.


Related in: MedlinePlus

N-Linked glycosylation status of purified DENV-2 was analyzed by SDS-PAGE and WB after PNGase F treatment.(A) Purified virus was observed by SDS-PAGE. (B) Purified virus was digested with PNGaseF (+) or mock digested (-) and evaluated by SDS-PAGE. (C) Western blotting was performed to show the deglycosylation patterns of envelope proteins of DENV-2 virions using anti-DENV-2 hyperimmune serum.
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pone.0132122.g003: N-Linked glycosylation status of purified DENV-2 was analyzed by SDS-PAGE and WB after PNGase F treatment.(A) Purified virus was observed by SDS-PAGE. (B) Purified virus was digested with PNGaseF (+) or mock digested (-) and evaluated by SDS-PAGE. (C) Western blotting was performed to show the deglycosylation patterns of envelope proteins of DENV-2 virions using anti-DENV-2 hyperimmune serum.

Mentions: To evaluate the glycosylation patterns of envelope proteins on purified DENV-2 produced by insect cells, a deglycosylation assay using N-endoglycosidase PNGase F digestion was performed. PNGase F removes all types of N-linked oligosaccharides from glycoproteins. Fig 3A & 3B show that without PNGase F treatment a 70kDa band was detected by SDS-PAGE, whereas with PNGase F treatment DENV-2 envelope bands were reduced to a 60kDa band. A similar deglycosylation pattern was observed in the envelope proteins of DENV-2 virions with western-blot (Fig 3C). Thus, these results revealed that the envelope proteins of DENV-2 virions, when produced by insect cells, posses a high degree of N-linked glycosylation.


Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells.

Lei Y, Yu H, Dong Y, Yang J, Ye W, Wang Y, Chen W, Jia Z, Xu Z, Li Z, Zhang F - PLoS ONE (2015)

N-Linked glycosylation status of purified DENV-2 was analyzed by SDS-PAGE and WB after PNGase F treatment.(A) Purified virus was observed by SDS-PAGE. (B) Purified virus was digested with PNGaseF (+) or mock digested (-) and evaluated by SDS-PAGE. (C) Western blotting was performed to show the deglycosylation patterns of envelope proteins of DENV-2 virions using anti-DENV-2 hyperimmune serum.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4514477&req=5

pone.0132122.g003: N-Linked glycosylation status of purified DENV-2 was analyzed by SDS-PAGE and WB after PNGase F treatment.(A) Purified virus was observed by SDS-PAGE. (B) Purified virus was digested with PNGaseF (+) or mock digested (-) and evaluated by SDS-PAGE. (C) Western blotting was performed to show the deglycosylation patterns of envelope proteins of DENV-2 virions using anti-DENV-2 hyperimmune serum.
Mentions: To evaluate the glycosylation patterns of envelope proteins on purified DENV-2 produced by insect cells, a deglycosylation assay using N-endoglycosidase PNGase F digestion was performed. PNGase F removes all types of N-linked oligosaccharides from glycoproteins. Fig 3A & 3B show that without PNGase F treatment a 70kDa band was detected by SDS-PAGE, whereas with PNGase F treatment DENV-2 envelope bands were reduced to a 60kDa band. A similar deglycosylation pattern was observed in the envelope proteins of DENV-2 virions with western-blot (Fig 3C). Thus, these results revealed that the envelope proteins of DENV-2 virions, when produced by insect cells, posses a high degree of N-linked glycosylation.

Bottom Line: By combining these methods, a high heterogeneity of DENV N-glycans was found.Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found.For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, School of Preclinical Medicine, The Fourth Military Medical University, 169 Changle Xi Road, Xian, Shaanxi 710032 China.

ABSTRACT
DENV envelope glycoprotein (E) is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies. It is well known that DENV E glycoprotein has two potential N-linked glycosylation sites at Asn67 and Asn153. The N-glycans of E glycoprotein have been shown to influence the proper folding of the protein, its cellular localization, its interactions with receptors and its immunogenicity. However, the precise structures of the N-glycans that are attached to E glycoprotein remain elusive, although the crystal structure of DENV E has been determined. This study characterized the structures of envelope protein N-linked glycans on mature DENV-2 particles derived from insect cells via an integrated method that used both lectin microarray and MALDI-TOF-MS. By combining these methods, a high heterogeneity of DENV N-glycans was found. Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found. Furthermore, a complex between a glycan on DENV and the carbohydrate recognition domain (CRD) of DC-SIGN was mimicked with computational docking experiments. For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

No MeSH data available.


Related in: MedlinePlus