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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.


MALDI-TOF/TOF-MS/MS analysis of N-glycan precursor ions in MS spectra.Precursor ions were subjected to MS/MS analysis to obtain cleavages, including glycosidic cleavages and cross-ring cleavages. Structures of cleavage ions and m/z values are shown in tandem mass spectra. Three major N-glycan peaks are indicated: (A) m/z 1419.476, (B) m/z 1663.581, and (C)m/z 2028.714.
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pone.0132122.g008: MALDI-TOF/TOF-MS/MS analysis of N-glycan precursor ions in MS spectra.Precursor ions were subjected to MS/MS analysis to obtain cleavages, including glycosidic cleavages and cross-ring cleavages. Structures of cleavage ions and m/z values are shown in tandem mass spectra. Three major N-glycan peaks are indicated: (A) m/z 1419.476, (B) m/z 1663.581, and (C)m/z 2028.714.

Mentions: MALDI-TOF/TOF-MS/MS was performed to obtain detailed information regarding substitutions and branching patterns of the studied monosaccharide constituents. To investigate the exact pattern of N-linked oligosaccharides on mature DENV-2 virions, MALDI-TOF-MS/MS analysis was employed. MALDI-TOF-MS spectra of N-linked oligosaccharides on DENV-2 with signal-to-noise ratios >4 were annotated using Glyco Workbench software (Fig 6). DENV showed 19 distinct m/z N-glycans, and hybridtype-N-glycans made up an important class of glycans isolated from DENV, such as the N-glycans at m/z1622.4, 1825.6, 1987.6 and 2133.6. Detailed information on these predicted N-glycans are shown in Fig 7. More remarkably,the hybrid type-N-glycan at 2133 m/z (Fuc1Hex7HexNAc4) is the most abundant N-glycan in DENV. In addition, several high-mannose type-glycans were identified in DENV, such as the species at m/z 1257.3, 1419.4, 1581.4, 1743.5 and 1905.5, which are shown in Fig 6. When we determined the N-glycan composition of DENV by MALDI-TOF-MS, we observed that galactosylated N-glycans dominated the N-linked oligosaccharides (15 of 19 N-glycans were galactosylated). MALDI-TOF-MS analysis also exhibited that some N-glycans were further modified with fucose and sialic acid; N-glycans at m/z 2133.6, 2174.6, 2289.8, 2341.7, 2654.8 and 2983.9 were modified with fucose moieties linked to either internal or external HexNAc residues (shownin Fig 6). Moreover, N-glycans at m/z 2289.8, 2341.7, 2654.8 and 2983.9 had terminal sialic acids (shown in Fig 6). To obtain detailed information on the N-linked oligosaccharides of DENV, the peaks that were detected in the N-glycan spectra were subjected to MALDI MS/MS analysis. MS/MS spectra of precursor ions at m/z 1419.380, 1663.468 and 2028.648 are shown in Fig 8A-8D. An oligosaccharide with mannose branches was revealed by fragment ions B3Y3β (671.201) and B4αY4β(1095.370)at m/z 1419.476(shown in Fig 8A). The existence of GalNAc residues in the N-glycan profile of DENV was identified byB3Y5α(388.121) at m/z 1663.581 and by B3Y4α(550.174) and Y4α(1663.581) at m/z 2028.714(shown in Fig 8B and 8C). In conclusion, MS profiling in combination with tandem mass spectrometry provided detailed information on the N-glycan profile of DENV.


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)

MALDI-TOF/TOF-MS/MS analysis of N-glycan precursor ions in MS spectra.Precursor ions were subjected to MS/MS analysis to obtain cleavages, including glycosidic cleavages and cross-ring cleavages. Structures of cleavage ions and m/z values are shown in tandem mass spectra. Three major N-glycan peaks are indicated: (A) m/z 1419.476, (B) m/z 1663.581, and (C)m/z 2028.714.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132122.g008: MALDI-TOF/TOF-MS/MS analysis of N-glycan precursor ions in MS spectra.Precursor ions were subjected to MS/MS analysis to obtain cleavages, including glycosidic cleavages and cross-ring cleavages. Structures of cleavage ions and m/z values are shown in tandem mass spectra. Three major N-glycan peaks are indicated: (A) m/z 1419.476, (B) m/z 1663.581, and (C)m/z 2028.714.
Mentions: MALDI-TOF/TOF-MS/MS was performed to obtain detailed information regarding substitutions and branching patterns of the studied monosaccharide constituents. To investigate the exact pattern of N-linked oligosaccharides on mature DENV-2 virions, MALDI-TOF-MS/MS analysis was employed. MALDI-TOF-MS spectra of N-linked oligosaccharides on DENV-2 with signal-to-noise ratios >4 were annotated using Glyco Workbench software (Fig 6). DENV showed 19 distinct m/z N-glycans, and hybridtype-N-glycans made up an important class of glycans isolated from DENV, such as the N-glycans at m/z1622.4, 1825.6, 1987.6 and 2133.6. Detailed information on these predicted N-glycans are shown in Fig 7. More remarkably,the hybrid type-N-glycan at 2133 m/z (Fuc1Hex7HexNAc4) is the most abundant N-glycan in DENV. In addition, several high-mannose type-glycans were identified in DENV, such as the species at m/z 1257.3, 1419.4, 1581.4, 1743.5 and 1905.5, which are shown in Fig 6. When we determined the N-glycan composition of DENV by MALDI-TOF-MS, we observed that galactosylated N-glycans dominated the N-linked oligosaccharides (15 of 19 N-glycans were galactosylated). MALDI-TOF-MS analysis also exhibited that some N-glycans were further modified with fucose and sialic acid; N-glycans at m/z 2133.6, 2174.6, 2289.8, 2341.7, 2654.8 and 2983.9 were modified with fucose moieties linked to either internal or external HexNAc residues (shownin Fig 6). Moreover, N-glycans at m/z 2289.8, 2341.7, 2654.8 and 2983.9 had terminal sialic acids (shown in Fig 6). To obtain detailed information on the N-linked oligosaccharides of DENV, the peaks that were detected in the N-glycan spectra were subjected to MALDI MS/MS analysis. MS/MS spectra of precursor ions at m/z 1419.380, 1663.468 and 2028.648 are shown in Fig 8A-8D. An oligosaccharide with mannose branches was revealed by fragment ions B3Y3β (671.201) and B4αY4β(1095.370)at m/z 1419.476(shown in Fig 8A). The existence of GalNAc residues in the N-glycan profile of DENV was identified byB3Y5α(388.121) at m/z 1663.581 and by B3Y4α(550.174) and Y4α(1663.581) at m/z 2028.714(shown in Fig 8B and 8C). In conclusion, MS profiling in combination with tandem mass spectrometry provided detailed information on the N-glycan profile of DENV.

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.