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

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 (EC₅₀) 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 EC₅₀ 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.