Recombinant Envelope glycoprotein (GP)

Basic Research Questions

• What are the primary structural characteristics of viral envelope glycoproteins?

Viral envelope glycoproteins typically consist of distinct subunits with specialized functions. For HIV, the envelope glycoprotein complex comprises gp120 (surface) and gp41 (transmembrane) subunits forming functional trimers. The 3D structure of the gp120 core has been solved through X-ray crystallography at resolutions as fine as 2.2 Å, revealing important features including the core architecture and variable regions (V1-V5) .

Similarly, ebolavirus GP consists of GP1 and GP2 subunits, with GP1 containing the receptor-binding domain and a heavily glycosylated mucin-like domain (MLD), while GP2 mediates membrane fusion. The structure includes distinct domains such as the head, glycan cap, and membrane-proximal regions .

When designing experiments with recombinant GPs, researchers should consider:

• The impact of variable/flexible regions on protein stability and crystallization

• Strategic truncations (such as removing transmembrane domains) to improve solubility

• Whether the native oligomeric state (typically trimeric) is critical for the study objectives

• What types of glycosylation modifications occur in viral envelope glycoproteins?

Viral envelope glycoproteins exhibit multiple glycosylation types that collectively form a "glycan shield":

a) N-linked glycosylation: Occurs at asparagine residues within the Asn-X-Ser/Thr motif. Ebolavirus GP contains up to 17 N-linked glycosylation sites with heterogeneous glycan structures .

b) O-linked glycosylation: Attached to serine and threonine residues, particularly abundant in mucin-like domains. Ebolavirus GP features up to 16 unique O-linked glycosylation sites in its MLD, plus additional sites in the head and glycan cap domains .

c) C-linked mannosylation: A less common modification where mannose attaches to the C2 atom of tryptophan's indole group, typically in WXXW motifs. Ebolavirus GP contains confirmed C-mannosylation at W288 in the glycan cap .

Interestingly, glycosylation patterns show virus-specific conservation. For example, C-mannosylation motifs are conserved across ebolavirus species and Lloviu virus but absent in Marburg virus .

• How do different expression systems affect glycosylation patterns of recombinant GPs?

Expression systems significantly impact glycosylation profiles:

a) Mammalian cells (e.g., HEK293): Provide glycosylation patterns most similar to native viral proteins, including complex N-glycans, sialylated O-glycans, and C-mannosylation .

b) Insect cells (e.g., S2 cells): Produce simpler glycosylation patterns with paucimannose N-glycans, less sialylation, and typically lack C-mannosylation .

c) Plant expression systems (e.g., N. benthamiana): Can produce complex glycans but with plant-specific modifications like β1,2-xylose and α1,3-fucose .

A comparative analysis of ebolavirus GP expressed in different systems revealed:

• Both HEK293 and insect cells produced high-mannose glycans at conserved sites N257 and N563

• Only HEK293 cells supported C-mannosylation at W288

• O-glycans in HEK293-expressed GP showed more extended, sialylated structures compared to truncated forms in insect cells

This variability necessitates careful selection of expression systems based on research objectives.

• What functional roles do glycans play in viral envelope glycoproteins?

Glycans on viral envelope glycoproteins serve multiple critical functions:

a) Host cell attachment: Specific glycan structures mediate interactions with cell-surface lectins. The high-mannose glycans at sites N257 and N563 in ebolavirus GP likely facilitate attachment to DC-SIGN/L-SIGN receptors on target cells .

b) Protein folding and stability: Glycans contribute to proper folding, preventing aggregation and enhancing structural integrity. In HIV Env, glycosylation affects the stability of prefusion trimer conformations .

c) Immune evasion: The glycan shield masks potential antibody epitopes. In ebolavirus GP, the heavily glycosylated MLD functions primarily to shield critical receptor-binding sites from neutralizing antibodies .

d) Modulation of fusion activity: Glycosylation can influence the conformational changes required for membrane fusion during viral entry.

Understanding these functions is essential when designing glycan-modified immunogens or interpreting antibody neutralization data.

Advanced Research Questions

• What methodologies enable site-specific characterization of recombinant viral glycoprotein glycosylation?

Mass spectrometry-based glycoproteomics offers the most comprehensive approach for site-specific glycosylation analysis:

a) Sample preparation strategy:

• Multiple protease digestions (trypsin, chymotrypsin) to maximize sequence coverage

• Glycopeptide enrichment via hydrophilic interaction or lectin affinity chromatography

• Optional glycosidase treatments to confirm glycosylation types

b) LC-MS/MS analysis approach:

• Higher-energy collisional dissociation (HCD) for glycan fragmentation

• Electron transfer dissociation (ETD) for peptide sequence determination

• Specialized acquisition methods like stepped collision energy

c) Data analysis workflow:

• Glycopeptide identification software (e.g., Byonic, GPQuest)

• Manual validation of spectral assignments

• Quantitative analysis of glycoform distributions at each site

This approach has revealed important insights about ebolavirus GP, including heterogeneous complex N-glycans at most sites, enrichment of high-mannose glycans at N257 and N563, and multiple O-glycans within N-glycosylation sequons .

For comprehensive characterization, researchers should:

• Analyze multiple batches to assess reproducibility

• Compare glycosylation across different expression systems

• Validate key findings using orthogonal methods (e.g., lectin binding)

• How can researchers effectively study glycan shields of viral envelope glycoproteins?

Studying glycan shields requires integrated structural and functional approaches:

a) Structural methods:

b) Glycan accessibility mapping:

• Lectin binding assays to probe surface-exposed glycan structures

• Limited proteolysis with LC-MS/MS analysis

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

c) Immunological approaches:

• Neutralization assays with glycosylation site mutants

• Binding studies using glycan-specific antibodies

• Structural analysis of antibody-GP complexes

Studies on ebolavirus GP have employed these approaches to understand how glycans contribute to and restrict neutralization epitopes, providing a framework to guide immunogen design .

An effective experimental design should:

• Compare wild-type and glycan-modified variants

• Analyze GP in multiple contexts (soluble, membrane-anchored, virus-like particles)

• Include cross-strain comparisons to identify conserved glycan functions

• How can researchers optimize recombinant GP expression for structural studies?

Optimization strategies include:

a) Construct design considerations:

• Remove disordered regions (e.g., mucin-like domains) that hinder crystallization

• Include stabilizing mutations (e.g., I559P in HIV Env)

• Replace transmembrane domains with trimerization motifs

• Optimize leader sequences (e.g., LPH for plants, TPA for mammalian cells)

b) Expression system selection based on research goals:

• HEK293 cells for native-like glycosylation

• GnTI-deficient HEK293S cells for homogeneous high-mannose glycans

• Insect cells for reduced glycan heterogeneity

• Plant expression systems for cost-effective scale-up

c) Purification and quality control:

• Size exclusion chromatography to isolate properly folded trimers

• Blue native PAGE to assess oligomeric state

• Negative-stain EM to verify protein morphology

One successful approach for HIV Env gp140 and MARV GPΔTM combined:

• Replacement of natural signal peptides with LPH or TPA leader peptides

• Insertion of a flexible linker (GGGGS)2 in place of the furin cleavage site

• Removal of transmembrane and cytoplasmic domains

• Addition of stabilizing mutations where necessary

• What is the significance of C-mannosylation in viral envelope glycoproteins?

C-mannosylation represents an underexplored but potentially important modification:

a) Biochemical characteristics:

• Covalent attachment of α-mannose to the C2 atom of tryptophan's indole group

• Occurs at the first W in WXXW sequences

• Present in ebolavirus GP at W288 in the glycan cap

b) Functional implications:

• Likely roles in protein folding and stability

• May influence trafficking of secreted glycoproteins

• Evolutionarily conserved across ebolavirus species and Lloviu virus, but absent in Marburg virus

c) Detection considerations:

• Requires specialized mass spectrometry methods

• Only occurs in mammalian expression systems, not in insect cells

• Previously confirmed in the secreted GP (sGP) and now in full-length trimeric GP

The biological significance of C-mannosylation in viral infection remains unclear, making it an important area for future research. Its conservation suggests it may play a crucial role in GP function or immune evasion.

• How do high-mannose and complex glycans differentially affect GP function?

The distribution of high-mannose versus complex glycans on GPs has important functional implications:

a) Host cell attachment mechanisms:

• High-mannose glycans at N257 and N563 in ebolavirus GP likely facilitate binding to DC-SIGN/L-SIGN lectins on target cells

• Complex glycans may interact with different receptors or modulate access to protein epitopes

b) Differential immune recognition:

• High-mannose glycans can be targets for broadly neutralizing antibodies

• Complex glycans generally provide better shielding from antibody recognition

c) Strategic glycan processing:

• Sites N257 and N563 in ebolavirus GP consistently display high-mannose and hybrid glycans despite expression system differences

• This conservation suggests evolutionary pressure to maintain these structures for functional reasons

Research approaches to investigate these differences include:

• Glycosidase treatments (EndoH vs PNGaseF sensitivity)

• Expression in cells with modified glycosylation pathways

• Site-directed mutagenesis combined with functional assays

• What techniques are most effective for analyzing interactions between antibodies and glycosylated epitopes?

Modern approaches include:

a) Structural analysis methods:

• X-ray crystallography of antibody-GP complexes

• Cryo-EM of full-length GP-antibody complexes

• Computational modeling of glycan contributions to epitopes

b) Biophysical characterization:

• Surface plasmon resonance with wild-type and glycan-modified variants

• Bio-layer interferometry for kinetic analysis

• Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

c) Functional and immunological assays:

• Neutralization testing with glycosidase-treated virions

• Glycan "knockout" panels through site-directed mutagenesis

• Competition binding studies with glycan-specific lectins

Integration of these approaches has revealed how glycans on ebolavirus GP contribute to and restrict neutralization epitopes . For example, glycoproteomics data combined with antibody-GP complex structures can explain why certain epitopes remain resistant to neutralization.

When designing such studies, researchers should:

• Consider glycan heterogeneity when interpreting binding data

• Use complementary techniques to build a comprehensive picture

• Compare results across antibody classes from different sources (e.g., vaccination vs infection)