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

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.

Global aspects of viral glycosylation

“…Applying mass spectrometry-based analysis to viral glycans released from mature viral particles illustrated the heterogeneity of N-glycan structures present on a single protein. For example, detailed studies on Dengue virions revealed enormous heterogeneity of N-glycan structures modifying two putative sites of Dengue virus glycoprotein, with 19 distinct structures identified (Lei et al 2015). In addition, MS-based glycoprofiling demonstrated distinct glycosylation patterns in different host species for Chikungunya virus (Lancaster et al 2016), confirming earlier observations on togaviruses (Hsieh et al 1983).…”

“…Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is one of the most commonly used tools in modern day glycan analysis. Combining MALDI-MS with lectin microarray enables more reliable characterization of glycan structures, where lectin binding profiles can provide additional information on the glycosidic linkage (Mechref et al 2003;Lei et al 2015). Moreover, tandem MALDI-MS or tandem electrospray ionization MS allows fragmentation of selected precursor ions, enabling more detailed characterization of structures of a given composition (Mechref et al 2003;Ritchie et al 2010).…”

Glycosylation of viral surface proteins probed by mass spectrometry

“…utilized; however, it is less efficient [55,56]. Machupo GP 1 [57], HIV-1 gp120 [45,58], Dengue virus GP E [59], chikungunya virus E1 and E2 proteins [60], and the LASV GP complex (GPC) [6 ] are a few examples of Examples of site-specific glycan profile outputs. All data obtained from publicly available HIV-1 recombinant gp120 data sets [24 ,94 ].…”

Viral glycoproteomes: technologies for characterization and outlook for vaccine design

“…Predicted numbers of N‐ and O‐glycosylation sites were used to generate the pie charts where no glycoproteomic data were available ( EBOV N‐ and O‐ glycosites; DENV O‐glycosites; HSV ‐1 N‐glycosites). Data presented is based on the following references: HIV ‐1 , HCV , FLUAV , EBOV , DENV , HSV ‐1 , HENV .…”

“…Developments in MS‐based proteomics have enabled sensitive detection and comprehensive analysis of proteins and their post‐translational modifications. It has been widely used for discovery and analysis of glycosylation on viral envelope glycoproteins . Several types of information are desired when investigating glycosylation of proteins.…”