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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.


Relative expression levels of DENV-2 glycan binders by lectin microarray.The glycans binders were categorized into five types. (A) The GlcNAc binder DSA showed a stronger binding signal than the others. (B) (GlcNAc)n binders. (C) Bisecting and biantennary GlcNAc binders. (D) Mannose binders. (E) Gal binders. (F) Fucose binders. (G) Sialic acid binders.
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pone.0132122.g005: Relative expression levels of DENV-2 glycan binders by lectin microarray.The glycans binders were categorized into five types. (A) The GlcNAc binder DSA showed a stronger binding signal than the others. (B) (GlcNAc)n binders. (C) Bisecting and biantennary GlcNAc binders. (D) Mannose binders. (E) Gal binders. (F) Fucose binders. (G) Sialic acid binders.

Mentions: To identify patterns of glycosylation on DENV, we performed an analysis using a lectin microarray with Cy3 labeled-DENV-2. Aresultant image of the incubationis shown in Fig 4A, and the fluorescent intensities of the assessed lectins are shown in Fig 4B. According to the results, 21 out of 37 lectins showed positive binding signals. Among these, the GlcNAc binder DSA showed a stronger binding signal than the others (see Fig 5A), which indicated that GlcNAc was a dominant glycopattern in DENV-2. Meanwhile, the binding signal produced by PHA-E was stronger than those produced by other lectins, except DSA, which indicated that glycopatterns inclusive of bisected GlcNAc and biantennary N-glycans are in high abundance on DENV-2 (shown in Fig 5C). The values of the binding signals of HHL and GNA, which were identified as high mannose, were also greater than 500, demonstrating that high mannose is an N-glycan component in DENV-2. According to the specificity of lectins, the resultant glycan profile of DENV provided the following information: (i) gal, α-gal structures exist in the glycan profile of DENV and are associated with signals from ACA, RCA120, EEL, BS-I, BPL and PTL-II (Fig 5D);(ii) a glycoform of high-mannose was detected in DENV, which was associated with signals from GNA, HHL NPA and ConA (Fig 5E);(iii) fucose content (especiallyfucoseα-1,6GlcNAc) was evident in the glycan profile of DENV in AAL, PSA and LCA (Fig 5F);(iv) sialic acid content (Sia2-3/6Gal, Multivalent Sia) also emerged in the glycan profile of DENV-2 and was evident by signals corresponding to MAL-II, SNA and WGA (Fig 5G).


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)

Relative expression levels of DENV-2 glycan binders by lectin microarray.The glycans binders were categorized into five types. (A) The GlcNAc binder DSA showed a stronger binding signal than the others. (B) (GlcNAc)n binders. (C) Bisecting and biantennary GlcNAc binders. (D) Mannose binders. (E) Gal binders. (F) Fucose binders. (G) Sialic acid binders.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132122.g005: Relative expression levels of DENV-2 glycan binders by lectin microarray.The glycans binders were categorized into five types. (A) The GlcNAc binder DSA showed a stronger binding signal than the others. (B) (GlcNAc)n binders. (C) Bisecting and biantennary GlcNAc binders. (D) Mannose binders. (E) Gal binders. (F) Fucose binders. (G) Sialic acid binders.
Mentions: To identify patterns of glycosylation on DENV, we performed an analysis using a lectin microarray with Cy3 labeled-DENV-2. Aresultant image of the incubationis shown in Fig 4A, and the fluorescent intensities of the assessed lectins are shown in Fig 4B. According to the results, 21 out of 37 lectins showed positive binding signals. Among these, the GlcNAc binder DSA showed a stronger binding signal than the others (see Fig 5A), which indicated that GlcNAc was a dominant glycopattern in DENV-2. Meanwhile, the binding signal produced by PHA-E was stronger than those produced by other lectins, except DSA, which indicated that glycopatterns inclusive of bisected GlcNAc and biantennary N-glycans are in high abundance on DENV-2 (shown in Fig 5C). The values of the binding signals of HHL and GNA, which were identified as high mannose, were also greater than 500, demonstrating that high mannose is an N-glycan component in DENV-2. According to the specificity of lectins, the resultant glycan profile of DENV provided the following information: (i) gal, α-gal structures exist in the glycan profile of DENV and are associated with signals from ACA, RCA120, EEL, BS-I, BPL and PTL-II (Fig 5D);(ii) a glycoform of high-mannose was detected in DENV, which was associated with signals from GNA, HHL NPA and ConA (Fig 5E);(iii) fucose content (especiallyfucoseα-1,6GlcNAc) was evident in the glycan profile of DENV in AAL, PSA and LCA (Fig 5F);(iv) sialic acid content (Sia2-3/6Gal, Multivalent Sia) also emerged in the glycan profile of DENV-2 and was evident by signals corresponding to MAL-II, SNA and WGA (Fig 5G).

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.