Exploring Site-Specific N‑Glycosylation of HEK293 and Plant-Produced Human IgA Isotypes

The full potential of recombinant Immunoglobulin A as therapeutic antibody is not fully explored, owing to the fact that structure–function relationships of these extensively glycosylated proteins are not well understood. Here monomeric IgA1, IgA2m(1), and IgA2m(2) variants of the anti-HER2 antibody (IgG1) trastuzumab were expressed in glyco-engineered <i>Nicotiana benthamiana</i> plants and in human HEK293-6E cells. All three IgA isotypes were purified and subjected to biophysical and biochemical characterization. While no differences in assembly, antigen binding, and glycosylation occupancy were observed, both systems vary tremendously in terms of glycan structures and heterogeneity of glycosylation. Mass-spectrometric analysis of site-specific glycosylation revealed that plant-produced IgAs carry mainly complex-type biantennary N-glycans. HEK293-6E-produced IgAs, on the contrary, showed very heterogeneous N-glycans with high levels of sialylation, core-fucose, and the presence of branched structures. The site-specific analysis revealed major differences between the individual N-glycosylation sites of each IgA subtype. Moreover, the proline-rich hinge region from HEK293-6E cell-derived IgA1 was occupied with mucin-type O-glycans, whereas IgA1 from <i>N. benthamiana</i> displayed numerous plant-specific modifications. Interestingly, a shift in unfolding of the CH2 domain of plant-produced IgA toward lower temperatures can be observed with differential scanning calorimetry, suggesting that distinct glycoforms affect the thermal stability of IgAs

DataSheet_1_Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBDs.pdf

Subunit vaccines based on recombinant viral antigens are valuable interventions to fight existing and evolving viruses and can be produced at large-scale in plant-based expression systems. The recombinant viral antigens are often derived from glycosylated envelope proteins of the virus and glycosylation plays an important role for the immunogenicity by shielding protein epitopes. The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a principal target for vaccine development and has been produced in plants, but the yields of recombinant RBD variants were low and the role of the N-glycosylation in RBD from different SARS-CoV-2 variants of concern is less studied. Here, we investigated the expression and glycosylation of six different RBD variants transiently expressed in leaves of Nicotiana benthamiana. All of the purified RBD variants were functional in terms of receptor binding and displayed almost full N-glycan occupancy at both glycosylation sites with predominately complex N-glycans. Despite the high structural sequence conservation of the RBD variants, we detected a variation in yield which can be attributed to lower expression and differences in unintentional proteolytic processing of the C-terminal polyhistidine tag used for purification. Glycoengineering towards a human-type complex N-glycan profile with core α1,6-fucose, showed that the reactivity of the neutralizing antibody S309 differs depending on the N-glycan profile and the RBD variant.</p

Expression cassettes for hemagglutinin (HA) in plants.

<p>HAs were stably expressed in both leaves and seeds under the control of the CaMV 35S and the seed-specific promoters as the naked form (H5), hydrophobin I fusion protein (H5-HFBI) and ELPylated H5 (H5-ELP). All recombinant HAs contained His and c-myc tags for affinity chromatography purification and Western blotting, respectively. The LeB4 signal peptide and KDEL motif were used to ensure ER retention.</p

Glycosylation profile of recombinant HAs from leaves and seeds.

<p>Man_{6, 7, 8}: Mannose _{6, 7, 8}; Gn: <i>N</i>-acetylglucosamine; X: Xylose; F: Fucose.</p

Western blot analysis of purified HAs treated/untreated with PNGase F.

<p>Purified HAs from leaves were deglycosylated using the commercial PNGase F enzyme described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099347#s2" target="_blank">Materials and Methods</a> section. PNGase F-treated and untreated proteins were then separated in 10% SDS-PAGE. Recombinant proteins were detected using an anti-c-myc monoclonal antibody. “−” and “+” indicate PNGase F-untreated and treated samples, respectively.</p

Localization of hemagglutinin-hydrophobin I fusions in tobacco seeds.

<p>A. Fluorescence microscopy. B, C. Electron microscopy. B. Endosperm. C. Embryo. Scarce hydrophobin bodies in the endosperm (arrowheads, A, B). Abundant hydrophobin bodies in the embryo cells (arrowheads, A, C). Hydrophobin bodies show non-uniform electron density (*, C). Endosperm (end), embryo (emb), protein storage vacuole (PSV), ribosomes (arrow). Bars 25 µm (A), 0.5 µm (B, C).</p

Localization of hemagglutinin-ELP fusions in tobacco seeds.

<p>A, B. Fluorescence microscopy.C, D. Electron microscopy.A, C. Endosperm. B, D. Embryo. Note the ELP bodies (arrowheads, A, B) and those that are loosely packed (arrowheads, C, D). Cell wall (cw), oil bodies (ob), protein storage vacuole (arrows), nucleus (n). Bars 50 µm (A, B), 1 µm (C, D).</p

Influence of hydrophobin and elastin-like polypeptide on hemagglutinin accumulation in transgenic plants under the control of either the CaMV 35S promoter (A) or the seed-specific USP promoter (B).

<p>Each column shows the mean value of expression level of the transgenic plants, and the error bars indicate the standard deviation. TSP: total soluble protein.</p

Purification of ELPylated hemagglutinin (H5-ELP) from transgenic leaves and seeds by membrane-based ITC.

<p>ELPylated hemagglutinins from leaves (A) and seeds (B) were purified by the standard or improved mITC methods (C) described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099347#s2" target="_blank">Materials and Methods</a> section. Proteins in the raw plant extract (RE), in the supernatant after passage through a 0.2 µm cellulose acetate membrane (Sm) and in the eluent (Pm) were collected during the mITC purification process and separated by 10% SDS-PAGE. Recombinant proteins were then detected using Coomassie Brilliant Blue staining (left) or an anti-c-myc monoclonal antibody (right).</p

Immunofluorescence analysis of recombinant HAs in plant leaves.

<p>Leaves were fixed, embedded in PEG and sectioned. Recombinant HAs were immunodecorated with an anti-c-myc monoclonal antibody followed by incubation with secondary antibody (anti-mouse-IgG conjugated with AlexaFluor488) and counterstaining with DAPI. A. H5; B. H5-HFBI; C. H5-ELP; D. wild type. Bars represent 50 µm.</p