N-glycosylation is essential for protein folding in the endoplasmic reticulum (ER). Glycan attachment facilitates the binding of newly synthesized polypeptides to calnexin and calreticulin, two ER-resident lectins that act as chaperones and promote folding. The regulatory mechanism underlying this process is dictated by the glycan composition, and this study has elucidated the function of mannose trimming in the release of misfolded glycoprotein from ER quality control and subsequent transfer to ER-associated degradation (ERAD) in plants. Coimmunoprecipitation experiments were performed to investigate the interaction between the ERAD substrate SUBEX-C57Y and ER-resident lectins, providing evidence for a specific interaction with calnexin. Furthermore, the results show that the expression of calnexin and calreticulin can specifically inhibit the degradation of glycosylated ERAD substrates. However, overexpression of the α-mannosidase MNS4 was shown to overcome lectin-dependent accumulation and promote degradation via ERAD. MS-based analysis of released N-glycans and jack bean α-mannosidase-digested glycopeptides revealed the presence of monoglucosylated N-glycans even after mannose trimming. This finding is consistent with the observation that calnexin remained associated with SUBEX-C57Y after mannose removal from the C-branch of the N-glycan. In contrast, specific removal of the first α1,2-linked mannose from the A-branch or a block of glucose trimming of Glc3Man9GlcNAc2 prevented the interaction with calnexin. In conclusion, the study provides fundamental insights into the specific role of glycan residues for the interdependence between lectin-based ER quality control and glycan-dependent ERAD in plants.

The production of cannabinoid compounds such as Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) with potential pharmaceutical applications is growing sharply. However, challenges such as the low yield of minor cannabinoids, legal restrictions on cultivation, and the complexity and cost of purification from the Cannabis sativa plant necessitate a biotechnological approach. Since the biosynthetic pathway is disclosed, cannabinoids have been produced in yeast, insect cells and plants mainly by the heterologous expression of tetrahydrocannabinol acid synthase (THCAS). THCAS and cannabidiolic acid synthase (CBDAS) use cannabigerolic acid (CBGA) as a substrate. In this study, we transiently expressed recombinant forms of THCAS and CBDAS in leaves of Nicotiana benthamiana. Our results demonstrate that efficient expression in the secretory pathway relies on replacing the endogenous signal peptide with a heterologous one. Both proteins were successfully secreted to the apoplast. MS-based analysis of the purified proteins revealed that they are heavily glycosylated with mainly Golgi-processed complex type N-glycans. In planta enzymatic removal of N-glycans indicated that glycosylation plays a role for CBDAS protein folding or stability. Finally, in vitro assays with CBGA showed that the plant-made recombinant CBDAS and THCAS are enzymatically active.

Studying the interaction between the hemibiotrophic bacterium Pseudomonas syringae pv. tomato DC3000 and Arabidopsis thaliana has shed light onto the various forms of mechanisms plants use to defend themselves against pathogen attack. While a lot of emphasis has been put on investigating changes in protein expression in infected plants, only little information is available on the effect infection plays on the plants N-glycan composition. To close this gap in knowledge, total N-glycans were enriched from P. syringae DC3000-infected and mock treated Arabidopsis seedlings and analyzed via MALDI-TOF–MS. Additionally, fluorescently labelled N-glycans were quantified via HPLC-FLD. N-glycans from infected plants were overall less processed and displayed increased amounts of oligomannosidic N-glycans. As multiple peaks for certain oligomannosidic glycoforms were detected upon separation via liquid chromatography, a porous graphitic carbon (PGC)-analysis was conducted to separate individual N-glycan isomers. Indeed, multiple different N-glycan isomers with masses of two N-acetylhexosamine residues plus 8, 9 or 10 hexoses were detected in the infected plants which were absent in the mock controls. Treatment with jack bean α-mannosidase resulted in incomplete removal of hexoses from these N-glycans, indicating the presence of glucose residues. This hints at the accumulation of misfolded glycoproteins in the infected plants, likely because of endoplasmic reticulum (ER) stress. In addition, poly-hexose structures susceptible to α-amylase treatment were found in the DC3000-infected plants, indicating alterations in starch metabolism due to the infection process.

CD19 is an essential protein in personalized CD19-targeting chimeric antigen receptor (CAR)-T cell-based cancer immunotherapies and CAR-T cell functionality evaluation. However, the recombinant expression of this “difficult to-express” (DTE) protein is challenging, and therefore, commercial access to the protein is limited. We have previously described the successful stable expression of our soluble CD19-AD2 fusion protein of the CD19 extracellular part fused with human serum albumin domain 2 (AD2) in CHO-K1 cells. The function, stability, and secretion rate of DTE proteins can be improved by culture conditions, such as reduced temperature and a shorter residence time. Moreover, glycosylation, as one of the most important post-translational modifications, represents a critical quality attribute potentially affecting CAR-T cell effector function and thus impacting therapy’s success. In this study, we increased the production rate of CD19-AD2 by 3.5-fold through applying hypothermic culture conditions. We efficiently improved the purification of our his-tagged CD19-AD2 fusion protein via a Ni-NTA-based affinity column using a stepwise increase in the imidazole concentration. The binding affinity to commercially available anti-CD19 antibodies was evaluated via Bio-Layer Interferometry (BLI). Furthermore, we revealed glycosylation patterns via Electrospray Ionization Mass Spectrometry (ESI–MS), and five highly sialylated and multi-antennary N-glycosylation sites were identified. In summary, we optimized the CD19-AD2 production and purification process and were the first to characterize five highly complex N-glycosylation sites.

Plant-produced glycoproteins carry α1,3-linked core fucosylated N-glycans, and the reduction/elimination thereof often confers beneficial features (Strasser et al., 2008; Zeitlin et al., 2011). Here, we evaluated the possibility of altering fucosylation in planta by overexpressing a bacterial GDP-6-deoxy-d-lyxo-4-hexulose reductase (RMD), an enzyme negatively interfering with the GDP-l-fucose biosynthesis pathway (von Horsten et al., 2010). RMD gene from Pseudomonas aeruginosa (Figure S1) was transiently expressed in Nicotiana benthamiana wild-type (WT) plants (Figure S2). MS-based N-glycosylation profiles from total soluble proteins (TSP) showed that overall fucosylation decreased from 64% to 36% compared to TSP lacking RMD (Figure 1a) while leaving the overall N-glycan composition largely unchanged. MALDI-TOF MS peaks of fucosylated glycans were accompanied by peaks which, at a cursory glance, could have been interpreted as potassium adduct ions. A more consistent interpretation, however, identifies these peaks as consisting mainly of glycans that contain hexose (possibly l-galactose) instead of fucose. Next, different monoclonal antibodies (Abs; Rx, rituximab, Cx, cetuximab, 2G12 and an Fc-fragment, Fcab) were transiently expressed (Figure S3; Castilho et al., 2015; Li et al., 2016; Strasser et al., 2008) and subjected to glycopeptide-analyses. The Fc-N-glycan composition of all mAbs expressed without RMD carried mainly complex N-glycans decorated with core xylose and fucose (Figure 1a), ranging from 70% to 90%. Upon RMD expression, the amounts of fucosylated N-glycans were significantly reduced with otherwise only minor changes in the N-glycan profile (Figure 1a,b). The most prominent fucose reduction was observed for Fcab (from 70% to <2%). Cx, which carries two GSs, was also expressed in N. benthamiana X1, a xylosyltransferase RNAi line synthesizing complex N-glycans largely devoid of β1,2-xylose (Strasser et al., 2008). Both Cx GSs exhibited significantly reduced fucosylated N-glycans upon RMD expression, from 40% to 10% and from 97% to 50%, at Fc and Fab, respectively (Figure 1a,b). Next, IgA2 an Ab isotype that carries four GSs (GS 1–4 from N- to C- terminus) was expressed in WT N. benthamiana plants (Figure S3). GS1 and GS3 carried 85%–90% fucosylated N-glycans, whereas GS4 exhibited only about 40% thereof and GS2 N-glycans lack fucose (Figure 1c). Upon RMD co-expression, a significant reduction of fucose-carrying N-glycans was observed at GS1, 3 and 4, while at the same time, overall glycosylation was largely unchanged. Interestingly, upon RMD expression MS spectra revealed extra peaks that were not present in the absence of RMD, especially at GS1, GS3 and GS4 (Figure 1c) and on the Fab N-glycan from cetuximab (Figure 1b). Some peaks were accompanied by masses of additional ~162 Da, suggesting the attachment of a hexose (e.g. galactose) instead of fucose (146 Da). To investigate if the attachment of this additional hexose is dependent on the α1,3-fucosyltransferase, IgA2 was expressed in N. benthamiana ΔXT/FT background (RNAi downregulated fucosyl/xylosyltransferase), that synthesize complex N-glycans largely devoid of xylose and fucose (Strasser et al., 2008). ΔXT/FT-derived IgA2 exhibits complex N-glycans largely devoid of plant-specific residues and co-expression of RMD further pronounced fucose reduction (Figure 1c). In contrast, by co-expressing a plant α1,3 fucosyltransferase (Castilho et al., 2015) and RMD in ΔXT/FT, glycans with the additional 162 Da peak appeared (up to 59%), indicating a fucosyltransferase-mediated attachment. PNGase F treatment was performed to analyse these RMD-associated peaks (Figure 1d). The enzyme digested structures without core-GlcNAc modification (i.e., oligomannosidic glycans, MGnX, GnGnX and MMX), whereas glycan peaks (assigned as MMX + Hex, MGnX+Hex, GnGnX+Hex) remained unmodified. These results indicate the attachment of a hexose at the core GlcNAc thereby blocking PNGase F activity (Figure 1d). The peaks with the highest intensity (i.e. MMXF and MMX + Hex) were subjected to collision-induced fragmentation (MS/MS, Figure 1d). The respective MMXF profile exhibited a peak representing a peptide carrying GlcNAc and fucose (+146.1 Da), as expected for core fucosylated N-glycans. However, upon RMD co-expression, the MMX + Hex peak was fragmented in a peptide with a GlcNAc carrying an additional hexose (+162.1 Da, Figure 1d), suggesting the addition of a core hexose (most probably galactose) to the core GlcNAc residue. Notably, the additional hexose was not detected at all glycosites. Similar differential behaviour was observed for ΔXTFT RNAi knock-down plants, where the remaining low levels of FucT sufficed to add some fucose to Fab glycans, while Fc glycans remained essentially unfucosylated (Castilho et al., 2015). Finally, functional activities were determined for Rx expressed in WT plants with and without RMD. Similar antigen binding of both Ab variants was observed (Figure 1e). In contrast, compared to Rx, Rx + RMD exhibited increased binding to cellular receptor FcγRIIIa (Figure 1f), as expected for IgG1 Abs with reduced core fucosylation. The experiments show no functional impairments upon RMD expression. Collectively, we demonstrate the expression of a functionally active bacterial RMD in plants. Notably, our data suggest, that suppressed availability of GDP-l-fucose allows the core α1,3-fucosyltransferase to use the structurally related UDP-l-galactose as donor substrate, in line with other results that report the transfer of l-galactose to N-glycans in case of GDP-l-fucose shortage (Ohashi et al., 2017; Rayon et al., 1999). This should be considered by RMD-based glycan engineering for commercial purposes (Puklowski et al., 2013). In summary, transient co-delivery of bacterial RMD into WT plants provides a straightforward alternative for the removal of core fucose compared to laborious genome editing approaches (Jansing et al., 2019). See Supporting information. The authors declare no conflicts of interest. HS, BK, SK, RS, FA, JG, and LS designed research; BK, RP, DM, JG, and LS performed experiments; all contributed to data interpretation and manuscript writing. Appendix S1 Material and methods. Figure S1 Schematics of RMD expression construct. Figure S2 Heterologous expression of RMD in N. benthamiana. Figure S3 Schematic presentation of reporter glycoproteins. Table S1 Glycopeptides after enzymatic digest. Table S2 Quantified glyco-profile of TSP and purified antibodies. Table S3 Quantified glyco-profile of IgA2m1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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.

Elevated plasma and tissues histamine concentrations can cause severe symptoms in mast cell activation syndrome, mastocytosis or anaphylaxis. Endogenous and recombinant human diamine oxidase (rhDAO) can rapidly and completely degrade histamine, and administration of rhDAO represents a promising new treatment approach for diseases with excess histamine release from activated mast cells. We recently generated heparin-binding motif mutants of rhDAO with considerably increased in vivo half-lives in rodents compared with the rapidly cleared wildtype protein. Herein, we characterize the role of an evolutionary recently added glycosylation site asparagine 168 in the in vivo clearance and the influence of an unusually solvent accessible free cysteine 123 on the oligomerization of diamine oxidase (DAO). Mutation of the unpaired cysteine 123 strongly reduced oligomerization without influence on enzymatic DAO activity and in vivo clearance. Recombinant hDAO produced in ExpiCHO-S™ cells showed a 15-fold reduction in the percentage of glycans with terminal sialic acid at Asn168 compared with Chinese hamster ovary (CHO)-K1 cells. Capping with sialic acid was also strongly reduced at the other glycosylation sites. The high abundance of terminal mannose and N-acetylglucosamine residues in the four glycans expressed in ExpiCHO-S™ cells compared with CHO-K1 cells resulted in rapid in vivo clearance. Mutation of Asn168 or sialidase treatment also significantly increased clearance. Intact N-glycans at Asn168 seem to protect DAO from rapid clearance in rodents. Full processing of all glycoforms is critical for preserving the improved in vivo half-life characteristics of the rhDAO heparin-binding motif mutants.

Plant glycoproteins display a characteristic type of O-glycosylation where short arabinans or larger arabinogalactans are linked to hydroxyproline. The conversion of proline to 4-hydroxyproline is accomplished by prolyl-hydroxylases (P4Hs). Eleven putative Nicotiana benthamiana P4Hs, which fall in four homology groups, have been identified by homology searches using known Arabidopsis thaliana P4H sequences. One member of each of these groups has been expressed in insect cells using the baculovirus expression system and applied to synthetic peptides representing the O-glycosylated region of erythropoietin (EPO), IgA1, Art v 1 and the Arabidopsis thaliana glycoprotein STRUBBELIG. Unlike the situation in the moss Physcomitrella patens, where one particular P4H was mainly responsible for the oxidation of erythropoietin, the tobacco P4Hs exhibited rather similar activities, albeit with biased substrate preferences and preferred sites of oxidation. From a biotechnological viewpoint, this result means that silencing/knockout of a single P4H in N. benthamiana cannot be expected to result in the abolishment of the plant-specific oxidation of prolyl residues in a recombinant protein.

Background: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Methods: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests’ broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. Findings: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. Interpretation: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. Funding: WWTF, Project No. COV20–016; BOKU, LBI/LBG

Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection. While first-generation antibody tests have primarily provided qualitative results with low specificity, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Here, we describe two quantitative ELISA antibody tests based on the SARS-CoV-2 spike receptor-binding domain and the nucleocapsid protein. Comparative expression in bacterial, insect, mammalian and plant-based platforms enabled the identification of new antigen designs with superior quality and high suitability as diagnostic reagents. Both tests scored excellently in clinical validations with multi-centric specificity and sensitivity cohorts and showed unprecedented correlation with SARS-CoV-2 neutralization titers. Orthogonal testing increased assay specificity to 99.8%, thereby enabling robust serodiagnosis in low-prevalence settings. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19.

The tobacco variant Nicotiana benthamiana has recently emerged as a versatile host for the manufacturing of protein therapeutics, but the fidelity of many recombinant proteins generated in this system is compromised by inadvertent proteolysis. Previous studies have revealed that the anti‐HIV‐1 antibodies 2F5 and PG9 as well as the protease inhibitor α 1 ‐antitrypsin (A1AT) are particularly susceptible to N . benthamiana proteases. Here, we identify two subtilisin‐like serine proteases (NbSBT1 and NbSBT2) whose combined action is sufficient to account for all major cleavage events observed upon expression of 2F5, PG9 and A1AT in N. benthamiana . We propose that downregulation of NbSBT1 and NbSBT2 activities could constitute a powerful means to optimize the performance of this promising platform for the production of biopharmaceuticals. Databases NbSBT sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers MN534996 to MN535005.

N-glycosylation is a highly abundant protein modification present in all domains of life. Terminal sugar residues on complex-type N-glycans mediate various crucial biological processes in mammals such as cell-cell recognition or protein-ligand interactions. In plants, the Lewis A trisaccharide constitutes the only known outer-chain elongation of complex N-glycans. Lewis A containing complex N-glycans appear evolutionary conserved, having been identified in all plant species analyzed so far. Despite their ubiquitous occurrence, the biological function of this complex N-glycan modification is currently unknown. Here, we report the identification of Lewis A bearing glycoproteins from three different plant species: Arabidopsis thaliana, Nicotiana benthamiana, and Oryza sativa. Affinity purification via the JIM84 antibody, directed against Lewis A structures on complex plant N-glycans, was used to enrich Lewis A bearing glycoproteins, which were subsequently identified via nano-LC-MS. Selected identified proteins were recombinantly expressed and the presence of Lewis A confirmed via immunoblotting and site-specific N-glycan analysis. While the proteins identified in O. sativa are associated with diverse functions, proteins from A. thaliana and N. benthamiana are mainly involved in cell wall biosynthesis. However, a Lewis A-deficient mutant line of A. thaliana showed no change in abundance of cell wall constituents such as cellulose or lignin. Furthermore, we investigated the presence of Lewis A structures in selected accessions from the 1001 genome database containing amino acid variations in the enzymes required for Lewis A biosynthesis. Besides one relict line showing no detectable levels of Lewis A, the modification was present in all other tested accessions. The data provided here comprises the so far first attempt at identifying Lewis A bearing glycoproteins across different species and will help to shed more light on the role of Lewis A structures in plants.

Here, we expressed two neutralizing monoclonal antibodies (Abs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; H4 and B38) in three formats: IgG1, IgA1 monomers (m), and IgA1 dimers (d) in glycoengineered Nicotiana benthamiana plants. All six Ab variants assembled properly and exhibited a largely homogeneous glycosylation profile. Despite modest variation in antigen binding between Ab formats, SARS-CoV-2 neutralization (NT) potency significantly increased in the following manner: IgG1 < IgA1-m < IgA1-d, with an up to 240-fold NT increase of dimers compared to corresponding monomers. Our results underscore that both IgA's structural features and multivalency positively impact NT potency. In addition, they emphasize the versatile use of plants for the rapid expression of complex human proteins.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein plays a key role in the virus-host cell interaction, and viral infection. The RBD is a major target for neutralizing antibodies, whilst recombinant RBD is commonly used as an antigen in serological assays. Such assays are essential tools to gain control over the pandemic and detect the extent and durability of an immune response in infected or vaccinated populations. Transient expression in plants can contribute to the fast production of viral antigens, which are required by industry in high amounts. Whilst plant-produced RBDs are glycosylated, N-glycan modifications in plants differ from humans. This can give rise to the formation of carbohydrate epitopes that can be recognized by anti-carbohydrate antibodies present in human sera. For the performance of serological tests using plant-produced recombinant viral antigens, such cross-reactive carbohydrate determinants (CCDs) could result in false positives. Here, we transiently expressed an RBD variant in wild-type and glycoengineered Nicotiana benthamiana leaves and characterized the impact of different plant-specific N-glycans on RBD reactivity in serological assays. While the overall performance of the different RBD glycoforms was comparable to each other and to a human cell line produced RBD, there was a higher tendency toward false positive results with sera containing allergy-related CCD-antibodies when an RBD carrying β1,2-xylose and core α1,3-fucose was used. These rare events could be further minimized by pre-incubating sera from allergic individuals with a CCD-inhibitor. Thereby, false positive signals obtained from anti-CCD antibodies, could be reduced by 90%, on average.

Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.

ABSTRACT Methylotrophic yeasts are considered to use alcohol oxidases to assimilate methanol, different to bacteria which employ alcohol dehydrogenases with better energy conservation. The yeast Komagataella phaffii carries two genes coding for alcohol oxidase, AOX1 and AOX2. The deletion of the AOX1 leads to the MutS phenotype and the deletion of AOX1 and AOX2 to the Mut– phenotype. The Mut– phenotype is commonly regarded as unable to utilize methanol. In contrast to the literature, we found that the Mut– strain can consume methanol. This ability was based on the promiscuous activity of alcohol dehydrogenase Adh2, an enzyme ubiquitously found in yeast and normally responsible for ethanol consumption and production. Using 13C labeled methanol as substrate we could show that to the largest part methanol is dissimilated to CO2 and a small part is incorporated into metabolites, the biomass, and the secreted recombinant protein. Overexpression of the ADH2 gene in K. phaffii Mut– increased both the specific methanol uptake rate and recombinant protein production, even though the strain was still unable to grow. These findings imply that thermodynamic and kinetic constraints of the dehydrogenase reaction facilitated the evolution towards alcohol oxidase-based methanol metabolism in yeast.

The importance of protein glycosylation in the biomedical field requires methods that not only quantitate structures by their monosaccharide composition, but also resolve and identify the many isomers expressed by mammalian cells. The art of unambiguous identification of isomeric structures in complex mixtures, however, did not yet catch up with the fast pace of advance of high-throughput glycomics. Here, we present a strategy for deducing structures with the help of a deci-minute accurate retention time library for porous graphitic carbon chromatography with mass spectrometric detection. We implemented the concept for the fundamental N-glycan type consisting of five hexoses, four N-acetylhexosamines and one fucose residue. Nearly all of the 40 biosynthetized isomers occupied unique elution positions. This result demonstrates the unique isomer selectivity of porous graphitic carbon. With the help of a rather tightly spaced grid of isotope-labeled internal N-glycan, standard retention times were transposed to a standard chromatogram. Application of this approach to animal and human brain N-glycans immediately identified the majority of structures as being of the bisected type. Most notably, it exposed hybrid-type glycans with galactosylated and even Lewis X containing bisected N-acetylglucosamine, which have not yet been discovered in a natural source. Thus, the time grid approach implemented herein facilitated discovery of the still missing pieces of the N-glycome in our most noble organ and suggests itself—in conjunction with collision induced dissociation—as a starting point for the overdue development of isomer-specific deep structural glycomics.

ABSTRACTThe importance of protein glycosylation in the biomedical field demands for methods capable of resolving and identifying isomeric structures of N-glycans. However, the unambiguous identification of isomeric structures from complex mixtures is currently not reasonably realized even by the most sophisticated approaches. Here we present a novel approach which uses stable isotope labelled reference N-glycans to establish a retention time grid (glyco-TiGr) on porous graphitized carbon. This furthermore enables retention as the primary criterion for the structural assignment of isomeric N-glycans.Moreover, we biosynthesized forty natural isomers of the fundamental N-glycan type consisting of five hexoses, four N-acetylhexosamines and one fucose residue. Nearly all of these isomers occupied unique positions on the retention time grid. Reference glycan assisted retention time determination with deci-minute accuracy narrowed the assignment space to very few, often only one possible glycan isomer.Application of the glyco-TiGr approach revealed yet undescribed isomers of Lewis x determinants in multimeric human IgA and hybrid type N-glycans in human brain with galactose and even fucose linked to the bisecting N-acetylglucosamine. Thus, the brain N-glycome displayed a degree of sophistication commensurate with this organ’s role.

N-glycosylation is defined as a key quality attribute for the majority of complex biological therapeutics. Despite many N-glycan engineering efforts, the demand to generate desired N-glycan profiles that may vary for different proteins in a reproducible manner is still difficult to fulfill in many cases. Stable production of homogenous structures with a more demanding level of processing, for instance high degrees of branching and terminal sialylation, is particularly challenging. Among many other influential factors, the level of productivity can steer N-glycosylation towards less mature N-glycan structures. Recently, we introduced an mRNA transfection system capable of elucidating bottlenecks in the secretory pathway by stepwise increase of intracellular model protein mRNA load. Here, this system was applied to evaluate engineering strategies for enhanced N-glycan processing. The tool proves to indeed be valuable for a quick assessment of engineering approaches on the cellular N-glycosylation capacity at high productivity. The gene editing approaches tested include overexpression of key Golgi-resident glycosyltransferases, partially coupled with multiple gene deletions. Changes in galactosylation, sialylation, and branching potential as well as N-acetyllactosamine formation were evaluated.