Small Molecules Targeting Human UDP-GlcNAc 2-Epimerase.

Uridine diphosphate N-acetylglucosamine 2-epimerase (GNE) is a key enzyme in the sialic acid biosynthesis pathway. Sialic acids are primarily terminal carbohydrates on glycans and play fundamental roles in health and disease. In search of effective GNE inhibitors not based on a carbohydrate scaffold, we performed a high-throughput screening campaign of 68,640 drug-like small molecules against recombinant GNE using a UDP detection assay. We validated nine of the primary actives with an orthogonal real-time NMR assay and verified their IC50 values in the low micromolar to nanomolar range manually. Stability and solubility studies revealed three compounds for further evaluation. Thermal shift assays, analytical size exclusion, and interferometric scattering microscopy demonstrated that the GNE inhibitors acted on the oligomeric state of the protein. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed which sections of GNE were shifted upon the addition of the inhibitors. In summary, we have identified three small molecules as GNE inhibitors with high potency in vitro, which serve as promising candidates to modulate sialic acid biosynthesis in more complex systems.

Seamless Coupling of Chemical Glycan Release and Labeling for an Accelerated Protein N-Glycan Sample Preparation Workflow.

Analytical methods fr direct quantitative N-glycan analysis require a sequence of sample preparation and clean-up steps that result in reduced glycan recovery. Therefore, we aimed to combine glycan release and labeling steps. Based on the hypothesis that the reaction mechanism for oxidative chemical glycan release comprises a stable glycan isocyanate intermediate, we investigated whether this could be exploited for the in-situ preparation of fluorescent glycan conjugates. ANTS-labeled N-glycans were derived from chicken ovalbumin via an in-situ chemical release/coupling approach and by standard Peptide-N-Glycosidase F (PNGase F) digestion/reductive amination. Synoptic fluorescence-assisted carbohydrate electrophoresis with UV detection (FACE-UV) analysis yielded matching patterns of fluorescent N-glycan bands in the expected electrophoretic mobility range between hexose units GU-5 and GU-11 of the standard. Anthranilamide (2-AB)-glycan conjugates prepared from a test glycoprotein carrying a predominant Core-F glycan gave single predominant peaks in hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) and electrospray ionization mass spectrometry (ESI-MS) spectra in agreement with sodiated triply charged Core-F-AB conjugates for both the standard and the in-situ coupling methods. The Core-F-AB conjugate prepared by the in-situ coupling approach had a slightly elevated retention time on HILIC-FLD and an ESI-MS m/z peak in line with a urea-bonded glycan-AB conjugate, with closed pyran ring structures on the glycan moiety. Glycan isocyanates intermittently formed during chemical glycan release, which could be utilized to prepare labeled glycan samples directly from glycoproteins and fluorescent dyes bearing a primary amine functional group.

Real-time monitoring of the sialic acid biosynthesis pathway by NMR.

Sialic acids are part of the outermost component of the glycocalyx of all vertebrates; as such, they are fundamental markers in physiological and pathological processes. In this study, we introduce a real-time assay to monitor individual enzymatic steps of sialic acid biosynthesis, either with recombinant enzymes, in particular using UDP-N-acetylglucosamine 2-epimerase (GNE) or N-acetylmannosamine kinase (MNK), or in cytosolic rat liver extract. Using state-of-the-art NMR techniques, we are able to follow the characteristic signal of the N-acetyl methyl group, which displays different chemical shifts for the biosynthesis intermediates UDP-N-acetylglucosamine, N-acetylmannosamine (and its 6-phosphate) and N-acetylneuraminic acid (and its 9-phosphate). Pseudo 2- and 3-D NMR demonstrated that in rat liver cytosolic extract, the phosphorylation reaction of MNK is exclusive for N-acetylmannosamine generated by GNE. Thus, we speculate that phosphorylation of this sugar from other sources (e.g. external application to cells) or N-acetylmannosamine derivatives often applied in metabolic glycoengineering is not conducted by MNK but by a yet unknown sugar kinase. Competition experiments with the most prevalent neutral carbohydrates demonstrated that of these, only N-acetylglucosamine slowed N-acetylmannosamine phosphorylation kinetics, suggesting an N-acetylglucosamine-preferring kinase as the acting enzyme.

Physicochemical Characterization, Glycosylation Pattern and Biosimilarity Assessment of the Fusion Protein Etanercept.

Etanercept is a soluble fusion protein of the tumor necrosis factor receptor (TNFR) extracellular domain, linked to an Fc part of IgG1. It possesses three N- and 13 O-glycosylation sites. Due to its complex structure, an analytical challenge is facing the development and approval of biosimilars. In the current study, physicochemical characterization using state-of-the-art analytics was performed to analyze intact and subunit masses, post-translational modifications (PTMs), higher order structure and potency of Etanercept originator Enbrel® and its biosimilar Altebrel™ (AryoGen Pharmed) in accordance to critical quality attributes of biopharmaceuticals. Intact mass and subunit analysis revealed a size of about 126 kDa for both biologicals. Similar glycoprotein species for the complete monomer and the Fc domain of originator and follow-on product were observed, however, small differences in lysine variants and oxidation were found. N-Glycopeptide analysis with UHPLC-QTOF-MSE confirmed the N-glycosylation sites (N149, N171 and N317) as well as Fc-specific glycosylation on N317, and TNFR-specific highly sialylated glycans on N149 and N171 on both investigated products. Small quantitative variations in the N-glycan profile were detected, although the N-glycans were qualitatively similar. Four different O-glycopeptides bearing core 1-type glycans were detected. For both, N- and O-glycopeptide analysis, determination was achieved without prior cleavage of the sialic acid residues for the first time. In addition, ion mobility spectrometry data confirmed close similarity of higher-order structure of both biologics. Furthermore, a neutralization assay, investigating the impact of altered PTMs on potency, indicated that the differences within all batches are still in the acceptable range for biosimilarity.

Bioengineering of rFVIIa Biopharmaceutical and Structure Characterization for Biosimilarity Assessment.

Eptacog alfa (NovoSeven®) is a vitamin K-dependent recombinant Factor VIIa produced by genetic engineering from baby hamster kidney (BHK) cells as a single peptide chain of 406 residues. After activation, it consists of a light chain (LC) of 152 amino and a heavy chain (HC) of 254 amino acids. Recombinant FVIIa undergoes many post-translational modifications (PTMs). The first ten glutamic acids of the N-terminal moiety are γ-carboxylated, Asn145 and Asn322 are N-glycosylated, and Ser52 and Ser60 are O-glycosylated. A head-to-head biosimilarity study was conducted for the originator and the first biosimilar AryoSeven™ to evaluate comparable bioengineering. Physicochemical properties were analyzed based on mass spectrometry, including intact mass, PTMs and higher-order structure. Both biotherapeutics exhibit a batch-to-batch variability in their N-glycan profiles. N-Glycopeptide analysis with UHPLC-QTOF-MSE confirmed N-glycosylation sites as well as two different O-glycopeptide sites. Ser60 was found to be O-fucosylated and Ser52 had O-glucose or O-glucose-(xylose)1,2 motifs as glycan variants. Ion mobility spectrometry (TWIMS) and NMR spectroscopy data affirm close similarity of the higher-order structure of both biologicals. Potency of the biodrugs was analyzed by a coagulation assay demonstrating comparable bioactivity. Consequently, careful process optimization led to a stable production process of the biopharmaceuticals.

Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans.

Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one of the main causes of respiratory tract infection in infants and elderly, as a model protein for vaccination. The RSV-F protein was expressed in CHO-DG44 cells, which were further modified by co-expression of ß1,2-xylosyltransferase from Nicotiana tabacum. Xylosylation of RSV-F N-glycans was shown by monosaccharide analysis and MALDI-TOF mass spectrometry. In immunogenic studies with a human artificial lymph node model, the engineered RSV-F protein revealed improved vaccination efficacy.

In Vitro Evaluation of Glycoengineered RSV-F in the Human Artificial Lymph Node Reactor.

Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN®). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences.

Werner Reutter: A Visionary Pioneer in Molecular Glycobiology.

A creative pioneer: Werner Reutter (1937-2016) was a scientist who both made fundamental discoveries in glycobiology and reached out to disciplines beyond his core field. Many of his former colleagues and students will remember his desire to exchange research ideas, which ultimately contributed to the birth of new research fields.

Comparability study of Rituximab originator and follow-on biopharmaceutical.

Immunglobolin G (IgG)-based biopharmaceuticals are emerging on the pharmaceuticals market due to their high target selectivity in different diseases. In parallel, a growing interest by other companies to produce similar or highly similar follow-on biologics exits, once the patent of blockbuster biotherapeutics is about to expire. In correlation to their complex structure, an analytical challenge is facing the approval of these biosimilars. Health authorities (e.g. FDA and EMA) have issued several guidelines to define critical quality attributes during manufacturing process changes. In the current study, physicochemical characterization using state-of-the-art analytics was applied to analyse intact mass, post-translational modifications (PTMs) and higher order structure of Rituximab and one of its biosimilars. Intact mass analysis, middle-up approach as well as subunit analysis revealed similar glycoforms but additional lysine variants in the biosimilar. The N-glycosylation site was confirmed for both, the originator and the biosimilar. PTMs and higher order structure were confirmed to be similar. A special focus was given to N-glycosylation due to its potential to monitor the batch-to-batch consistency and alteration during the production bioprocess. Comparison of the N-glycosylation profiles obtained from three batches of the biosimilar and the reference product showed quantitative variations, although the N-glycans were qualitatively similar. Furthermore, a head-to-head comparability of functional properties was performed to investigate the impact of glycosylation alteration and PTMs on potency within the biosimilar batches and between originator and follow-on biodrug. The data affirm that the difference is still in the acceptable range for biosimilarity.

Small Molecules Targeting Human N-Acetylmannosamine Kinase.

N-Acetylmannosamine kinase (MNK) plays a key role in the biosynthesis of sialic acids and glycosylation of proteins. Sialylated glycoconjugates affect a large number of biological processes, including immune modulation and cancer transformation. In search of effective inhibitors of MNK we applied high-throughput screening of drug-like small molecules. By applying different orthogonal assays for their validation we identified four potential MNK-specific inhibitors with IC50 values in the low-micromolar range. Molecular modelling of the inhibitors into the active site of MNK supports their binding to the sugar or the ATP-binding pocket of the enzyme or both. These compounds are promising for downregulation of the sialic acid content of glycoconjugates and for studying the functional contribution of sialic acids to disease development.

The Interaction of UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) and Alpha-Actinin 2 Is Altered in GNE Myopathy M743T Mutant.

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the gene mutated in GNE myopathy. In an attempt to elucidate GNE functions that could account for the muscle pathophysiology of this disorder, the interaction of GNE with α-actinins has been investigated. Surface plasmon resonance and microscale thermophoresis analysis revealed, that in vitro, GNE interacts with α-actinin 2, and that this interaction has a 10-fold higher affinity compared to the GNE-α-actinin 1 interaction. Further, GNE carrying the M743T mutation, the most frequent mutation in GNE myopathy, has a 10-fold lower binding affinity to α-actinin 2 than intact GNE. It is possible that this decrease eventually affects the interaction, thus causing functional imbalance of this complex in skeletal muscle that could contribute to the myopathy phenotype. In vivo, using bi-molecular fluorescent complementation, we show the specific binding of the two proteins inside the intact cell, in a unique interaction pattern between the two partners. This interaction is disrupted in the absence of the C-terminal calmodulin-like domain of α-actinin 2, which is altered in α-actinin 1. Moreover, the binding of GNE to α-actinin 2 prevents additional binding of α-actinin 1 but not vice versa. These results suggest that the interaction between GNE and α-actinin 1 and α-actinin 2 occur at different sites in the α-actinin molecules and that for α-actinin 2 the interaction site is located at the C-terminus of the protein.

Recombinant glycoproteins: The impact of cell lines and culture conditions on the generation of protein species.

Glycosylation is the most complex post-translational modification. Thus, it contributes to versatile chemical compositions of proteins, leading to high amounts of protein species. The structural heterogeneity of glycoproteins was also described by the definition of glycoforms. We therefore introduced a new term called "glycoprotein species" to join the two concepts from different fields of biology. In this study, we further determined the theoretical numbers of glycoprotein species of two recombinant glycoproteins - a therapeutical antibody and the human protease inhibitor alpha-1-antitrypsin (A1AT) - based on structural analysis of their N-glycans. Moreover, we showed that variations in the used cell lines and their cultivation conditions strongly influence the number of glycoprotein species in case of recombinant A1AT production. BIOLOGICAL SIGNIFICANCE: Protein glycosylation is a major source for the huge amount of protein species. This study extends the sight of protein species by the following contributions: 1) The new term "glycoprotein species" was defined to introduce the concept of glycoforms into the field. 2) An estimation of the number of potential glycoprotein species of two particular glycoproteins was given. 3) The influence of production conditions for recombinant glycoproteins on glycoprotein species generation was displayed.

Inhibition of the key enzyme of sialic acid biosynthesis by C6-Se modified N-acetylmannosamine analogs.

Synthetically accessible C6-analogs of N-acetylmannosamine (ManNAc) were tested as potential inhibitors of the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE/MNK), the key enzyme of sialic acid biosynthesis. Enzymatic experiments revealed that the modification introduced at the C6 saccharide position strongly influences the inhibitory potency. A C6-ManNAc diselenide dimer showed the strongest kinase inhibition in the low µM range among all the substrates tested and successfully reduced cell surface sialylation in Jurkat cells.

The human rhabdomyosarcoma cell line TE671--Towards an innovative production platform for glycosylated biopharmaceuticals.

The market of therapeutic glycoproteins (including coagulation factors, antibodies, cytokines and hormones) is one of the profitable, fast-growing and challenging sectors of the biopharmaceutical industry. Although mammalian cell culture is still expensive and technically complex, the ability to produce desired post-translational modifications, in particular glycosylation, is a major issue. Glycans can influence ligand binding, serum half-life as well as biological activity or product immunogenicity. Aiming to establish a novel production platform for recombinant glycoproteins, the human TE671 cell line was investigated. Since the initial analysis of cell membrane proteins showed a promising glycosylation of TE671 cells for biotechnological purposes, we focused on the recombinant expression of two model glycoproteins of therapeutical relevance. The optimization of the cell transfection procedure and serum-free expression succeeded for the human serine protease inhibitor alpha-1-antitrypsin (A1AT) and the hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). N-glycan analyses of both purified proteins by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provided first fundamental insights into the TE671 glycosylation potential. Besides protein specific pattern, strong distinctions - in particular for N-glycan fucosylation and sialylation - were observed depending on the medium conditions of the respective TE671 cell cultivations. The cell line's ability to synthesize complex and highly sialylated N-glycan structures has been shown. Our results demonstrate the TE671 cell line as a serious alternative to other existing human expression systems.

Applying Acylated Fucose Analogues to Metabolic Glycoengineering.

Manipulations of cell surface glycosylation or glycan decoration of selected proteins hold immense potential for exploring structure-activity relations or increasing glycoprotein quality. Metabolic glycoengineering describes the strategy where exogenously supplied sugar analogues intercept biosynthetic pathways and are incorporated into glycoconjugates. Low membrane permeability, which so far limited the large-scale adaption of this technology, can be addressed by the introduction of acylated monosaccharides. In this work, we investigated tetra-O-acetylated, -propanoylated and -polyethylene glycol (PEG)ylated fucoses. Concentrations of up to 500 µM had no substantial effects on viability and recombinant glycoprotein production of human embryonic kidney (HEK)-293T cells. Analogues applied to an engineered Chinese hamster ovary (CHO) cell line with blocked fucose de novo synthesis revealed an increase in cell surface and recombinant antibody fucosylation as proved by lectin blotting, mass spectrometry and monosaccharide analysis. Significant fucose incorporation was achieved for tetra-O-acetylated and -propanoylated fucoses already at 20 µM. Sequential fucosylation of the recombinant glycoprotein, achieved by the application of increasing concentrations of PEGylated fucose up to 70 µM, correlated with a reduced antibody's binding activity in a Fcγ receptor IIIa (FcγRIIIa) binding assay. Our results provide further insights to modulate fucosylation by exploiting the salvage pathway via metabolic glycoengineering.

UDP-GlcNAc 2-Epimerase/ManNAc Kinase (GNE): A Master Regulator of Sialic Acid Synthesis.

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme of sialic acid biosynthesis in vertebrates. It catalyzes the first two steps of the cytosolic formation of CMP-N-acetylneuraminic acid from UDP-N-acetylglucosamine. In this review we give an overview of structure, biochemistry, and genetics of the bifunctional enzyme and its complex regulation. Furthermore, we will focus on diseases related to UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.

The analysis of N-glycans of cell membrane proteins from human hematopoietic cell lines reveals distinctions in their pattern.

Human cell lines are often different in their features and present variations in the glycosylation patterns of cell membrane proteins. Protein glycosylation is the most common posttranslational modification and plays a particular role in functionality and bioactivity. The key approach of this study is the comparative analysis of five hematopoietic cell lines for their N-glycosylation pattern. The N-glycans of membrane proteins were elucidated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MALDI-TOF/TOF-MS analyses. Furthermore, the expression of a set of glycosyltransferases was determined via RT-PCR. The B-lymphoma BJA-B and promyelocytic HL-60 cell lines distinguish in levels and linkages of glycan-bound sialic acids. Furthermore, subclones of BJA-B and HL-60 cells, which completely lack UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis, contained almost no sialylated N-glycans. Compared to wild-type cells, the GNE-deficient cells presented a similar cell surface N-glycosylation pattern in terms of antennarity and fucosylation. The Jurkat T-cell line revealed only partially sialylated N-glycans. Additionally, the different hematopoietic cell lines vary in their level of bisecting GlcNAcylation and antennary fucosylation with the quantities of bisecting N-acetylglucosamine (GlcNAc) and core fucose coinciding with the expression of GnT-III and FucT-VIII. Of note is the occurrence of N-acetyllactosamine (LacNAc) extensions on tetraantennary structures in GNE-deficient cell lines.

Crystal structures of N-acetylmannosamine kinase provide insights into enzyme activity and inhibition.

Sialic acids are essential components of membrane glycoconjugates. They are responsible for the interaction, structure, and functionality of all deuterostome cells and have major functions in cellular processes in health and diseases. The key enzyme of the biosynthesis of sialic acid is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase that transforms UDP-N-acetylglucosamine to N-acetylmannosamine (ManNAc) followed by its phosphorylation to ManNAc 6-phosphate and has a direct impact on the sialylation of cell surface components. Here, we present the crystal structures of the human N-acetylmannosamine kinase (MNK) domain of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase in complexes with ManNAc at 1.64 Å resolution, MNK·ManNAc·ADP (1.82 Å) and MNK·ManNAc 6-phosphate · ADP (2.10 Å). Our findings offer detailed insights in the active center of MNK and serve as a structural basis to design inhibitors. We synthesized a novel inhibitor, 6-O-acetyl-ManNAc, which is more potent than those previously tested. Specific inhibitors of sialic acid biosynthesis may serve to further study biological functions of sialic acid.

Loss of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) induces apoptotic processes in pancreatic carcinoma cells.

Early invasive growth and metastasis are features of pancreatic cancer that rely on its resistance to anoikis, an apoptosis program activated on loss of matrix anchorage. How anoikis is regulated is unclear. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine-kinase (GNE) was silenced, or p16 was overexpressed, in human pancreatic carcinoma cells. Gene expression profiling, enzymatic assays, Western blotting, and cell cycle analysis were conducted. Silencing of GNE, the key enzyme of sialic acid biosynthesis, sensitizes pancreatic cancer cells to anoikis. Accordingly, we observed a loss of GNE enzyme activity in cells, which became anoikis susceptible after transfection with the tumor suppressor p16. Similarly, studies of another cell line with low GNE activity revealed strong anoikis susceptibility, confirming the association of low GNE activity and anoikis susceptibility. Gene expression profiling demonstrated that the loss of GNE triggered the transcriptional activation of the ATF4-ATF3-CHOP pathway, leading to apoptosis in the framework of the unfolded protein response. In silico analysis showed that GNE up-regulation occurred predominantly in pancreatic cancer but also in other malignancies. Delineation of GNE-dependent signaling pathways may provide targets that control anchorage dependence and/or restore drug efficacy, which is of utmost relevance for the treatment of pancreatic cancer.