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

Generation of Biologically Active Multi-Sialylated Recombinant Human EPOFc in Plants

<div><p>Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO). Recent progress in <em>N</em>-glycoengineering of non-mammalian expression hosts resulted in <em>in vivo</em> protein sialylation at great homogeneity. However the synthesis of multi-sialylated <em>N-</em>glycans is so far restricted to mammalian cells. Here we used a plant based expression system to accomplish multi-antennary protein sialylation. A human erythropoietin fusion protein (EPOFc) was transiently expressed in <em>Nicotiana benthamiana</em> ΔXTFT, a glycosylation mutant that lacks plant specific N-glycan residues. cDNA of the hormone was co-delivered into plants with the necessary genes for (i) branching (ii) β1,4-galactosylation as well as for the (iii) synthesis, transport and transfer of sialic acid. This resulted in the production of recombinant EPOFc carrying bi- tri- and tetra-sialylated complex <em>N-</em>glycans. The formation of this highly complex oligosaccharide structure required the coordinated expression of 11 human proteins acting in different subcellular compartments at different stages of the glycosylation pathway. <em>In vitro</em> receptor binding assays demonstrate the generation of biologically active molecules. We demonstrate the <em>in planta</em> synthesis of one of the most complex mammalian glycoforms pointing to an outstanding high degree of tolerance to changes in the glycosylation pathway in plants.</p> </div

Generation of tetra-sialylated structures in rhEPOFc.

<p>Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc co-expressed in <i>N. benthamiana</i> ΔXTFT with mammalian genes for synthesis of tetra-sialylated <i>N-</i>glycans (rhEPO_TetraSia). The analysis was performed on rhEPOFc_TetraSia present on fraction A of the 55kDa band (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone-0054836-g002" target="_blank">Figure 2B</a>, lane 4). Glycosylation patterns of rhEPO Gp1: E/A²²E<u>NIT</u>TGCAE³¹; Gp2: E/H³²CSLNE<u>NIT</u>VPDTK⁴⁵ and Gp3: R/G⁷⁷QALLV<u>NSS</u>QPWEPLQHLVDK⁹⁷ are shown. <i>N-</i>glycosylation profile of the Fc glycopeptide is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s001" target="_blank">Figure S1</a>. Glycosylation profile of rhEPOFc present on fraction B of the 55kDa band is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s003" target="_blank">Figure S3</a>. Peak labels were made according to the ProGlycAn system (<a href="http://www.proglycan.com" target="_blank">www.proglycan.com</a> Illustrations display <i>N</i>-glycans on assigned peaks, for interpretation of other assigned glycoforms see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s005" target="_blank">Figure S5</a>.</p

Generation of tri-sialylated structures in rhEPOFc.

<p>Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc co-expressed in <i>N. benthamiana</i> ΔXTFT with mammalian genes for synthesis of tri-antennary sialylated <i>N-</i>glycans (rhEPO_TriSia). The analysis was performed on rhEPOFc_TriSia present on fraction A of the 55kDa band (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone-0054836-g002" target="_blank">Figure 2B</a>, lane 3). Glycosylation patterns of rhEPO Gp1: E/A²²E<u>NIT</u>TGCAE³¹; Gp2: E/H³²CSLNE<u>NIT</u>VPDTK⁴⁵ and Gp3: R/G⁷⁷QALLV<u>NSS</u>QPWEPLQHLVDK⁹⁷ are shown. <i>N-</i>glycosylation profile of the Fc glycopeptide is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s001" target="_blank">Figure S1</a>. Glycosylation profile of rhEPOFc present on fraction B of the 55 kDa band is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s002" target="_blank">Figure S2</a>. Peak labels were made according to the ProGlycAn system (<a href="http://www.proglycan.com" target="_blank">www.proglycan.com</a>). Illustrations display <i>N</i>-glycans on assigned peaks, for interpretation of other assigned glycoforms see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s005" target="_blank">Figure S5</a>.</p

Nucleotide and Nucleotide Sugar Analysis by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry on Surface-Conditioned Porous Graphitic Carbon

We examined the analysis of nucleotides and nucleotide sugars by chromatography on porous graphitic carbon with mass spectrometric detection, a method that evades contamination of the MS instrument with ion pairing reagent. At first, adenosine triphosphate (ATP) and other triphosphate nucleotides exhibited very poor chromatographic behavior on new columns and could hardly be eluted from columns previously cleaned with trifluoroacetic acid. Satisfactory performance of both new and older columns could, however, be achieved by treatment with reducing agent and, unexpectedly, hydrochloric acid. Over 40 nucleotides could be detected in cell extracts including many isobaric compounds such as ATP, deoxyguanosine diphosphate (dGTP), and phospho-adenosine-5′-phosphosulfate or 3′,5′-cyclic adenosine 5'-monophosphate (AMP) and its much more abundant isomer 2′,3′-cylic AMP. A fast sample preparation procedure based on solid-phase extraction on carbon allowed detection of very short-lived analytes such as cytidine 5'-monophosphate (CMP)-2-keto-deoxy-octulosonic acid. In animal cells and plant tissues, about 35 nucleotide sugars were detected, among them rarely considered metabolites such as uridine 5'-diphosphate (UDP)-l-arabinopyranose, UDP-l-arabinofuranose, guanosine 5'-diphosphate (GDP)-l-galactofuranose, UDP-l-rhamnose, and adenosine diphosphate (ADP)-sugars. Surprisingly, UDP-arabinopyranose was also found in Chinese hamster ovary (CHO) cells. Due to the unique structural selectivity of graphitic carbon, the method described herein distinguishes more nucleotides and nucleotide sugars than previously reported approaches

Generation of GnGn structures in rhEPOFc.

<p>Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc expressed in <i>N. benthamiana</i> ΔXTFT (rhEPOFc_ΔXTFT; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone-0054836-g002" target="_blank">Figure 2B</a>, lane 1). Glycosylation patterns of rhEPO glycopeptide 1 (Gp1): E/A²²E<u>NIT</u>TGCAE³¹; glycopeptide 2 (Gp2): E/H³²CSLNE<u>NIT</u>VPDTK⁴⁵ and glycopeptide 3 (Gp3): R/G⁷⁷QALLV<u>NSS</u>QPWEPLQHLVDK⁹⁷ are shown. The corresponding <i>N-</i>glycosylation profile of the Fc glycopeptide (R/EEQY<u>NST</u>YR) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s001" target="_blank">Figure S1</a>. Peak labels were made according to the ProGlycAn system (<a href="http://www.proglycan.com" target="_blank">www.proglycan.com</a>). Illustrations display <i>N</i>-glycans on assigned peaks, for interpretation of other assigned glycoforms see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s005" target="_blank">Figure S5</a>.</p

Expression of rhEPOFc in <i>N. benthamiana</i>.

<p>A. Western blot analysis of total soluble proteins extracted from <i>N. benthamiana</i> expressing rhEPOFc. 55 kDa protein reacts to both anti-EPO (α-EPO) and anti-Fc (α-Fc) antibodies while the ∼30 kDa band reacts only with α-Fc antibodies. B. Protein A purified rhEPOFc fractionated by SDS PAGE and stained with Coomassie-brilliant blue R-250. lane 1: rhEPOFc expressed in <i>N. benthamiana</i> mutants lacking plant specific β1,2-xylose and α1,3-fucose (rhEPOFc_ΔXTFT); lane 2: rhEPOFc co-expressed with mammalian genes for protein sialylation (GNE, NANS, CMAS, CST, ^STGalT and ST) (rhEPOFc_Sia,); lane 3: rhEPOFc co-expressed with mammalian genes necessary for sialylation and synthesis of tri-antennary <i>N-</i>glycans GnTIV or GnTV, (rhEPO_TriSia,); lane 4: rhEPOFc co-expressed with mammalian genes for sialylation and synthesis of tetra-antennary <i>N-</i>glycans, GnTIV and GnTV (rhEPO_TetraSia). A and B represent distinct protein fractions from the 55 kDa band of rhEpoFc_TriSia and rhEPO_TetraSia, used for N-glycan analysis; the ∼30 kDa band represent free Fc. C. Western blot analysis of total soluble proteins (5 µg TSP) extracted from <i>N. benthamiana</i> ΔXTFT mutants (control; lane 1) and of purified rhEPOFc_ΔXTFT (lane 2) using antibodies against Lewis-A epitopes (JIM 84). Several proteins in TSP and the 55 kDa protein band corresponding to intact rhEPOFc reacted to JIM 84 revealing the presence of <i>N-</i>glycans with Lewis-a epitopes. (M) protein marker.</p

<i>in vitro</i> activity of CHO- and plant-derived rhEPOFC.

<p><i>In vitro</i> activity assay of plant- and CHO- derived rhEPOFc. Half maximal effective doses (ED₅₀) are displayed. rhEPOFc was expressed in CHO cells (CHO); in <i>N. benthamiana</i> ΔXTFT mutants (ΔXTFT); co-expressed in ΔXTFT with mammalian genes for protein sialylation (Sia); co-expressed in ΔXTFT with mammalian genes for synthesis of tri-antennary sialylated <i>N-</i>glycans (TriaSia) and co-expressed in ΔXTFT with mammalian genes for synthesis of tetra-sialylated <i>N-</i>glycans (TetraSia).</p

Relative abundance of different complex glycoforms detected in rhEPOFc. (oligomannosidic structures that are present in all samples are not included).

<p>Relative abundance of complex <i>N</i>-glycans determined by LC-ESI-MS. <b>rhEPOFc_Sia</b>: rhEPOFc co-expressed with mammalian genes for protein sialylation; <b>rhEPOFc_TriaSia</b>: rhEPOFc co-expressed with mammalian genes for synthesis of tri-antennary sialylated <i>N-</i>glycans; <b>rhEPOFc_TetraSia:</b> rhEPOFc co-expressed with mammalian genes for synthesis of tetra-sialylated <i>N-</i>glycans. ΔXTFT was used as expression host. Values are in percentages. Quantifications were done for complex <i>N-</i>glycans (oligomannosidic structures were not included in calculations). Gp1: glycopeptide 1; Gp2: Glycopeptide 2; Gp3: Glycopeptide 3.</p

Generation of bi-sialylated structures in rhEPOFc.

<p>Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc co-expressed in <i>N. benthamiana</i> ΔXTFT with mammalian genes for protein sialylation (GNE, NANS, CMAS, CST, ^STGalT and ST) (rhEPOFc_Sia; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone-0054836-g002" target="_blank">Figure 2B</a>, lane 2). Glycosylation patterns of rhEPO Gp1: E/A²²E<u>NIT</u>TGCAE³¹; Gp2: E/H³²CSLNE<u>NIT</u>VPDTK⁴⁵ and Gp3: R/G⁷⁷QALLV<u>NSS</u>QPWEPLQHLVDK⁹⁷ are shown. <i>N-</i>glycosylation profile of the Fc glycopeptide is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s001" target="_blank">Figure S1</a>. Peak labels were made according to the ProGlycAn system (<a href="http://www.proglycan.com" target="_blank">www.proglycan.com</a>). Illustrations display <i>N</i>-glycans on assigned peaks, for interpretation of other assigned glycoforms see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s005" target="_blank">Figure S5</a>.</p