Effects of N-Glycosylation on the Structure, Function, and Stability of a Plant-Made Fc-Fusion Anthrax Decoy Protein

Abstract

Protein N-glycosylation is an important post-translational modification and has influences on a variety of biological processes at the cellular and molecular level, making glycosylation a major study aspect for glycoprotein-based therapeutics. To achieve a comprehensive understanding on how N-glycosylation impacts protein properties, an Fc-fusion anthrax decoy protein, viz rCMG2-Fc, was expressed in Nicotiana benthamiana plant with three types of N-glycosylation profiles. Three variants were produced by targeting protein to plant apoplast (APO), endoplasmic reticulum (ER) or removing the N-glycosylation site by a point mutation (Agly). Both the APO and ER variants had a complex-type N-glycan (GnGnXF) as their predominant glycans. In addition, ER variant had a higher concentration of mannose-type N-glycans (50%). The decoy protein binds to the protective antigen (PA) of anthrax through its CMG2 domain and inhibits toxin endocytosis. The protein expression, sequence, N-glycosylation profile, binding kinetics to PA, toxin neutralization efficiency, and thermostability were determined experimentally. In parallel, we performed molecular dynamics (MD) simulations of the predominant full-length rCMG2-Fc glycoform for each of the three N-glycosylation profiles to understand the effects of glycosylation at the molecular level. The MAN8 glycoform from the ER variant was additionally simulated to resolve differences between the APO and ER variants. Glycosylation showed strong stabilizing effects on rCMG2-Fc during in planta accumulation, evidenced by the over 2-fold higher expression and less protein degradation observed for glycosylated variants compared to the Agly variant. Protein function was confirmed by toxin neutralization assay (TNA), with effective concentration (EC 50 ) rankings from low to high of 67.6 ng/ml (APO), 83.15 ng/ml (Agly), and 128.9 ng/ml (ER). The binding kinetics between rCMG2-Fc and PA were measured with bio-layer interferometry (BLI), giving sub-nanomolar affinities regardless of protein glycosylation and temperatures (25 and 37°C). The protein thermostability was examined utilizing the PA binding ELISA to provide information on EC 50 differences. The fraction of functional ER variant decayed after overnight incubation at 37°C, and no significant change was observed for APO or Agly variants. In MD simulations, the MAN8 glycoform exhibits quantitatively higher distance between the CMG2 and Fc domains, as well as higher hydrophobic solvent accessible surface areas (SASA), indicating a possibly higher aggregation tendency of the ER variant. This study highlights the impacts of N-glycosylation on protein properties and provides insight into the effects of glycosylation on protein molecular dynamics.

Development and simulation of fully glycosylated molecular models of ACE2-Fc fusion proteins and their interaction with the SARS-CoV-2 spike protein binding domain

“…Steps 4 and 7 exhibit new, publicly available software under GNU General Public Licenses. GlyRot has previously been used to model glycosylated butyrylcholinesterase and CMG2-Fc [ 13 , 14 ]. Forcefield topologies were generated using the AmberFF14SB [ 48 ]forcefield for protein atoms, the Glycam06-j [ 49 ] forcefield for glycan atoms, and the SPC/E water model [ 50 ].…”

“…Very recently a short simulation of the Spike protein with glycosylation has been published which is enabling longer studies [28]. Our group has made significant progress in the field of glycan modeling in recent years [13,14,29].…”

“…While the availability of recombinant sources for S and ACE2 glycoproteins have greatly contributed to our understanding of the structure and interactions between these proteins, it is important to recognize that glycosylation of recombinant S and ACE proteins will depend on the host cell [9], the recombinant protein, as well as production [10,11] and purification methods [12]. As molecular models and molecular dynamics simulations can describe the interactions of proteins with glycans and the modulation of protein structure by glycans [13,14] they are powerful tools to assess the significance of glycosylation on 3D structure and binding site interactions between the SARS-CoV-2 spike protein and the human ACE2 receptor, and to design novel biotherapeutics including optimizing glycosylation.…”

Development and simulation of fully glycosylated molecular models of ACE2-Fc fusion proteins and their interaction with the SARS-CoV-2 spike protein binding domain

“…Consequently, only a few simulations of related fully glycosylated proteins available (19)(20)(21)(22) among them very recently a proposed glycosylated model of the Spike protein (23) . Our group has made significant progress in the field of glycan modeling in recent years (9,10,24). N-glycan structure is highly heterogeneous, and the relative abundance of glycans depends on the expression system for glycoprotein production.…”

Effects of Kifunensine on Production and <em>N</em>-glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor

“…Protein expression in plant systems has the potential to provide a safe, cost-effective, and scalable method to meet the increasing need for therapeutic protein production. Plantbased expression offers several advantages to the biopharmaceutical industry, including decreased cost of production, scalability, a lack of susceptibility to mammalian pathogens, elimination of animal or human sourced raw materials, and the ability to produce complex proteins with post-translational modifications such as N-glycosylation [1][2][3][4][5]. For many therapeutic proteins, N-glycosylation is essential for protein folding, oligomerization, quality control, enzyme activity, ligand interactions, localization, and trafficking [6,7].…”