The Fc RMSD is noticeably lower than the CMG2 RMSD for the Agly and GnGnXF glycoforms, indicating minor fold transitions in the CMG2 domains of these glycoforms.The hydrophobic solvent accessible surface area distributions are reported in Figure 11.The MAN8 glycoform has the highest amount of hydrophobic SASA in the simulation, while the aglycosylated has the lowest. Increased hydrophobic SASA likely yields a greater aggregation propensity . To elucidate which residues contribute most to the hydrophobic SASA difference between MAN8 and Agly, the average per-residue hydrophobic SASA was calculated, and the five residues with the greatest positive difference of MAN8 minus Agly hydrophobic SASA were obtained. These residues were Pro199, Pro201, Leu205, and Pro300 of one monomer, and Leu206 of the other monomer. All of these residues are located in close proximity to the N-terminal region of the Fc domain, shown in Figure 12. We see that these residues are highly spread out in the MAN8 structure, moderately spread out in the GnGnXF structure, and closely associating in the Agly structure. The increased SASA in the MAN8 glycoform is consistent with the existence of the high molecular weight band in the ER variant at ~250 kDa in the SDS-PAGE results .In this paper, experimental and computational techniques were employed to study the effects of N-glycosylation on the expression,what size pot for blueberries structure, function, and stability for anthrax decoy protein rCMG2-Fc.N-glycosylation was found to strongly stabilize rCMG2-Fc in planta as both APO and ER variants have over two-fold higher expression level than the Agly variant, shown in Figure 1.

The increase in expression level of glycosylated variants could be attributed to a decreased susceptibility of the APO and ER variants to proteases, where the steric hindrance of oligosaccharides inhibits proteolytic degradation of glycosylated rCMG2-Fc. From a manufacturing standpoint, producing glycosylated rCMG2-Fc would require less than half the production capacity of the aglycosylated form. Thus, when glycosylation is not detrimental, preserving natural N-glycosylation sites can enhance protein production. Alternatively, retaining the aglycosylated protein in the ER can also help improve yield, since the ER has fewer types of proteases than the apoplast . Furthermore, unlike the apoplast, the ER has chaperone proteins to provide folding support . It is also possible to enhance a glycoprotein’s function via modification of the N-glycosylation profile during expression. This can be achieved using subcellular targeting and/or the co-expression of glycan-processing enzymes or addition of enzyme inhibitors in the agroinfiltration buffer. For example, high mannose Fc N-glycosylation has been shown to enhance antibody-dependent cell-mediated cytotoxicity , which can be achieved by targeting the protein to the ER or addition of mannosidase I inhibitor to the agroinfiltration buffer or cell culture medium . It is worth noting that N-glycosylation of Fc is not strictly required when Fc is fused to the target protein for the purpose of increasing circulation half-life . SDS-PAGE and Western blot confirmed that intact rCMG2-Fc was produced, with bands near 50 kDa . There is also a band around 250 kDa in the non-reducing SDS-PAGE for the ER variant , which may correspond to a high molecular weight protein aggregate. The hypothesis of increased aggregation propensity of the ER variant is supported by the hydrophobic SASA predicted from our MD simulations. MAN8 was found to have significantly higher hydrophobic SASA, with the five strongest contributing residues in the N-terminal region of the Fc domain. Protein aggregation might reduce PA binding capacity, as it is evident in the functional ELISA , which is later discussed in the rCMG2-Fc function section.

The ability for rCMG2-Fc variants to sequester anthrax PA and prevent cell death was assessed using a cell-based TNA. Results are shown in Figure 5, where the ER variant has a statistically higher EC50 value than APO and Agly variants. The possibility of FcγR dependent toxin neutralization was ruled out as the EC50 values did not change upon FcγR blocking. This is likely because previous studies used antibodies or serum against PA that bind to PA but not necessarily blocks the its binding site to the anthrax cellular receptor CMG2. Thus, when incubated with cells, the antibody-bound PA can still bind to CMG2 and form prepore, resulting in LF and EF endocytosis. In this situation, Fc and FcγR could form an immune complex that is then sorted and degraded in the lysosome . In our experiments, rCMG2-Fc competitively inhibits binding between cellular receptor CMG2 and PA. This completely eliminates LF and EF internalization. The binding kinetics between rCMG2-Fc variants and PA were determined by BLI, results shown in Figure 6. The 37°C KD values were slightly lower than the 25°C KD values, but no appreciable difference in binding kinetics as a function of glycosylation was observed at 25 or 37°C. Considering the CMG2 domain is linked to Fc through a flexible linker, it is not surprising that the glycosylation of the Fc domain has minimal impact on the binding kinetics of the CMG2 domain with PA. Moreover, the sub-nanomolar affinity reported in this work is consistent with previous work on rCMG2 and PA binding kinetics , which is direct evidence that neither the fused Fc domain nor its glycosylation interferes with CMG2/PA binding kinetics. Even though the kinetics of all three variants were unaffected by glycosylation, it is possible that the fraction of functional protein changes over time. The BLI experiments only characterized the interaction kinetics, which are independent of the fraction of functional rCMG2-Fc on the sensor tip. The hypothesis that the fraction of functional protein at 37°C is glycosylation-dependent was confirmed with the functional rCMG2-Fc ELISA, where the ER variant lost the most activity overnight, consistent with the ER variant having the highest EC50. However, the MD simulation data exhibit high fold stability in all glycoforms, according to the RMSD of ordered domains as well as the secondary structure .

Thus, we hypothesize that the reduction in activity is not due reduced fold stability. Moreover, the MAN8 had the highest hydrophobic SASA among the three simulated glycoforms,flood and drain tray indicating a higher aggregation propensity. The residues that contributed the most to the decreased hydrophobic SASA in the simulated Agly from the MAN8 were located in the N-terminal region of the Fc domain, just after the C-terminal region of the linker . This region is also more extended in the MAN8 glycoform, as shown in the final conformation and the COM distance distribution . This exposure of hydrophobic residues in the thinly extended N-terminal region of the Fc domain could facilitate the aggregation with other rCMG2-Fc or protein fragments. This could explain the reduced activity for ER variant in the functional ELISA and the 250 kDa band observed in the nonreducing SDS-PAGE gel for the ER variant . Fusion protein aggregation has been observed in the literature for another Fc fusion protein ALK1-Fc, where a high abundance of MAN5 glycoform was found as high molecular weight aggregates . It is worth noting that a recent study on high-mannose type IgG showed a decrease in protection factors of backbone amide nitrogen in the CH2 domain , which could also contribute to the reduced activity of the ER variant. Meanwhile, Lu et al., found that high mannose glycans have no detrimental effect on antibody stability and aggregate rate . The antibodies used in their study were IgG1 and IgG2, which has a molecular weight ~150 kDa. Since both the protein size and structure can affect protein stability, it is not surprising to see diverse protein stability results performed on different molecules. Within Fc-fusion proteins, structure can still vary depending on the fusion partner size and structure. However, we do expect Fc-fusion proteins with similar structure, molecular weight and conserved glycosylation site as rCMG2-Fc to likely exhibit similar behaviors. The APO and Agly variants had no significant difference in fraction of functional protein after being incubated overnight at 37°C. This result is in agreement with a previous study where human IgG1s stored at 37°C for 21 days had no difference in aggregation or fragmentation , suggesting the absence of glycans had no major impact on stability under physiological temperature. A previous study of rCMG2-Fc using a different linker exhibited variants with oligomannose glycan or no glycan and had similar TNA EC50 values for both variants , while our data indicate a significant increase in EC50 for the ER variant over the APO and Agly variants. The linker used in Wycoff et al.’s study and this study were two serine residues followed by the upper hinge of IgG1 and IgG2 , respectively. These linkers differ in both length and sequence. This study utilizes the IgG2 hinge to enhance protein dimerization due to the two additional cysteine residues for inter-hinge disulfide bond formation. Since both linker sequence and length can affect stability and function of fusion proteins , thus, it is not surprising to see a difference in toxin neutralization ability. In this study, the expressed N-glycosylation variants were not found to functionally affect rCMG2-Fc/PA binding. However, protein expression, integrity and thermostability were affected by glycosylation.

The APO variant showed the best overall performance with a high expression level, high protein integrity and thermostability. However, the debate on plant complex type N-glycosylation is ongoing. It has been shown that plant complex N-glycans are immunogenic by the detection of anti-plant glycoepitopes antibodies in human sera , despite that no adverse effects were observed when plant-made pharmaceuticals with complex N-glycans were applied to patients with IgE against plant glycoforms . In addition, the plant β1,2-xylose and α1,3-fucose moieties can potentially induce rapid clearance from circulation due to the presence of IgE against those epitopes. Similarly, variants containing mannose-type N-glycans can lead to a shorter circulation half-life compared to the Agly variant, due to the presence of mannose receptor in serum . However, the shorter half-life of rCMG2-Fc variants can turn into an advantage when using as a post exposure treatment , resulting in fast blood clearance of decoy-toxin complex. For prophylaxis, the Agly variant is likely the best option, considering its longer circulation half-life than other two variants.Glycosylation variants of an anthrax decoy protein rCMG2-Fc were successfully produced in Nicotiana benthamiana plants with distinct N-glycosylation patterns. The expression levels were in the range of 148–578 mg/kg LFW. The N-glycosylation profiles, characterized by mass spectrometry, were 50% high mannose type for the ER variant and 99% complex-type for the APO variant. The rCMG2-Fc variants were all functional with sub-nanomolar dissociation rate constants regardless of N-glycosylation pattern. The higher EC50 of the ER variant compared with the APO and Agly variants was likely due to the loss of activity during the 37°C incubation condition used in the TNA assay. The loss of activity could be explained by increased aggregation of the ER variant, consistent with the SDS-PAGE and MD simulation results. To better assess the effects of N-glycosylation on protein properties, in vitro enzymatic glycan modification can be employed to express more uniform glycoforms. This avoids glycan heterogeneity, allowing a more accurate comparison between experimental and MD simulation data. Moreover, other proteins, especially Fc-fusion proteins can be studied using the methodology provided in this work to assess the applicability of our findings and to optimize glycoprotein therapeutic design.Hyper accumulator plants, globally over 450 species , take up metal from soils, then translocate and store them in their above ground biomass . Considerable research has been done on hyper accumulators aiming at remediating soil and water contaminated with metals and determining mechanisms of metal accumulation in above ground biomass . Hyper accumulator rhizosphere chemistry affects metal plant availability and metal release from soil has been tied to nutrient scavenging . Manipulating metal solubility in soils to increase hyper accumulator uptake and therefore phytoextraction rates is attractive . However, in creases in metal solubility in soil could lead to metal leaching if the mobilized fraction is not completely taken up by the target plant, a potentially negative and poorly characterized outcome of phytoextraction. Extensive work has explored arsenic phytoextraction with the fern Pteris vittata as an in situ alternative to soil excavation-based arsenic remediation methods . The fern hyper accumulates arse nic under a range of soil physicochemical conditions . However, phytoextraction rates are slow even at moderate concentrations , leading to estimated remediation times on the order of decades or more to deplete soil arsenic to background levels .