Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation

Metabolic labeling of glycans with a bioorthogonal chemical reporter such as the azide enables their visualization in cells and organisms as well as the enrichment of specific glycoprotein types for proteomic analysis. This process involves two steps. Azido sugars are fed to cells or organisms and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged with imaging probes or epitope tags, either ex vivo or in vivo, using an azide-specific reaction. This protocol details the syntheses of the azido sugars N-azidoacetylmannosamine (ManNAz), N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz) and 6-azidofucose (6AzFuc), and the detection reagents phosphine-FLAG and phosphine-FLAG-His6. Applications to the visualization of cellular glycans and enrichment of glycoproteins for proteomic analysis are described. The synthesis of the azido sugars (ManNAz, GalNAz, GlcNAz or 6AzFuc) or detection reagents (phosphine-FLAG or phosphine-FLAG-His6) can be completed in approximately 1 week. A cell metabolic labeling experiment can be completed in approximately 4 d.     

N-azidoacetylmannosamine-mediated chemical tagging of gangliosides

Peracetylated N-alpha-azidoacetylmannosamine (Ac(4)ManNAz) is metabolized by cells to CMP-azidosialic acid. It has been demonstrated previously that in this way azidosialic acid-containing glycoproteins are formed that can be labeled on the cell surface by a modified Staudinger ligation. Here, we first demonstrate that the same procedure also results in the formation of azidosialic acid-containing gangliosides. Deoxymannojirimycin, an inhibitor of N-glycan processing in proteins, decreases the total cell surface labeling in Jurkat cells by approximately 25%. Inhibition of ganglioside biosynthesis with N-[5-(adamantan-1-yl-methoxy)-pentyl]1-deoxynojirimycin reduces cell surface labeling by approximately 75%. In conclusion, exposure of cells to Ac(4)ManNAz allows in vivo chemical tagging of gangliosides.     

The humanization of N-glycosylation pathways in yeast

Yeast and other fungal protein-expression hosts have been extensively used to produce industrial enzymes, and are often the expression system of choice when manufacturing costs are of primary concern. However, for the production of therapeutic glycoproteins intended for use in humans, yeast have been less useful owing to their inability to modify proteins with human glycosylation structures. Yeast N-glycosylation is of the high-mannose type, which confers a short half-life in vivo and thereby compromises the efficacy of most therapeutic glycoproteins. Several approaches to humanizing yeast N-glycosylation pathways have been attempted over the past decade with limited success. Recently however, advances in the glycoengineering of yeast and the expression of therapeutic glycoproteins with humanized N-glycosylation structures have shown significant promise - this review summarizes the most important developments in the field.     

Glycoengineering the N-acyl side chain of sialic acid of human erythropoietin affects its resistance to sialidase

Abstract During the last years, the use of therapeutic glycoproteins has increased strikingly. Glycosylation of recombinant glycoproteins is of major importance in biotechnology, as the glycan composition of recombinant glycoproteins impacts their pharmacological properties. The terminal position of N-linked complex glycans in mammals is typically occupied by sialic acid. The presence of sialic acid is crucial for functionality and affects the half-life of glycoproteins. However, glycoproteins in the bloodstream become desialylated over time and are recognized by the asialoglycoprotein receptors via the exposed galactose and targeted for degradation. Non-natural sialic acid precursors can be used to engineer the glycosylation side chains by biochemically introducing new non-natural terminal sialic acids. Previously, we demonstrated that the physiological precursor of sialic acid (i.e., N-acetylmannosamine) can be substituted by the non-natural precursors N-propanoylmannosamine (ManNProp) or N-pentanoylmannosamine (ManNPent) by their simple application to the cell culture medium. Here, we analyzed the glycosylation of erythropoietin (EPO). By feeding cells with ManNProp or ManNPent, we were able to incorporate N-propanoyl or N-pentanoyl sialic acid in significant amounts into EPO. Using a degradation assay with sialidase, we observed a higher resistance of EPO to sialidase after incorporation of N-propanoyl or N-pentanoyl sialic acid.     

Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins

Nipah virus (NiV) and Hendra virus (HeV) are novel paramyxoviruses from pigs and horses, respectively, that are responsible for fatal zoonotic infections of humans. The unique genetic and biological characteristics of these emerging agents has led to their classification as the prototypic members of a new genus within the Paramyxovirinae subfamily called HENIPAVIRUS: These viruses are most closely related to members of the genus Morbillivirus and infect cells through a pH-independent membrane fusion event mediated by the actions of their attachment (G) and fusion (F) glycoproteins. Understanding their cell biological features and exploring the functional characteristics of the NiV and HeV glycoproteins will help define important properties of these emerging viruses and may provide new insights into paramyxovirus membrane fusion mechanisms. Using a recombinant vaccinia virus system and a quantitative assay for fusion, we demonstrate NiV glycoprotein function and the same pattern of cellular tropism recently reported for HeV-mediated fusion, suggesting that NiV likely uses the same cellular receptor for infection. Fusion specificity was verified by inhibition with a specific antiserum or peptides derived from the alpha-helical heptads of NiV or HeV F. Like that of HeV, NiV-mediated fusion also requires both F and G. Finally, interactions between the glycoproteins of the paramyxoviruses have not been well defined, but here we show that the NiV and HeV glycoproteins are capable of highly efficient heterotypic functional activity with each other. However, no heterotypic activity was observed with envelope glycoproteins of the morbilliviruses Measles virus and Canine distemper virus.     

A high-throughput method for quantification of glycoprotein sialylation

Sialic acid can improve qualities of therapeutic glycoproteins such as circulatory half-life, biological activity, and solubility. In production of therapeutic glycoproteins, a high-throughput method is required for process monitoring and optimization to ensure consistent and optimal sialic acid content. Current methods for quantifying sialic acid, however, require chromatographic separation that is time-consuming and cannot rapidly analyze many samples in parallel. Here we present a novel high-throughput method for quantifying glycoprotein sialylation. Using chemical reduction, enzymatic release of sialic acid, and chemical derivatization of the sialic acid, the method can accurately, rapidly (15 min), and specifically analyze many samples in parallel. It requires only 45 μl of sample and has a quantitation limit of 2 μM sialic acid. It has also been validated for monitoring sialylation of recombinant interferon gamma (IFN-γ) produced in Chinese hamster ovary (CHO) cell culture. This method is useful for various applications in upstream and downstream bioprocesses.     

Production of therapeutic glycoproteins through the engineering of glycosylation pathway in yeast

The application of recombinant DNA technology to restructure metabolic networks can change metabolite and protein products by altering the biosynthetic pathways in an organism. Although some success has been achieved, a more detailed and thorough investigation of this approach is certainly warranted since it is clear that such methods hold great potential based on the encouraging results obtained so far. In last decade, there have been tremendous advances in the field of glycobiology and the stage has been set for the biotechnological production of glycoproteins for therapeutic use. Today glycoproteins are one of the most important groups of pharmaceutical products. In this study the attempt was made to focus on identifying technologies that may have general application for modifying glycosylation pathway of the yeast cells in order to produce glycoproteins of therapeutic use. The carbohydrates of therapeutic recombinant glycoproteins play very important roles in determining their pharmacokinetic properties. A number of biological interactions and biological functions mediated by glycans are also being targeted for therapeutic manipulationin vivo. For a commercially viable production of therapeutic glycoproteins a metabolic engineering of a host cell is yet to be established.     

Measurement of lysosomal glucocerebrosidase activity in mouse liver using a fluorescence-activated cell sorter assay

Lysosomal acid beta-glucocerebrosidase hydrolyzes glucocerebroside to glucose ceramide. Patients diagnosed with Gaucher disease, however, lack this enzyme, leading to the accumulation of glucocerebroside in tissue macrophages within multiple organs. Such patients can receive enzyme replacement therapy during which a human placental-derived or recombinant form of acid beta-glucocerebrosidase is targeted to the macrophages. As part of evaluating the effectiveness of such therapies, currently available methodologies for measuring acid beta-glucocerebrosidase activity are primarily conducted in cultured cell lines or tissue culture. However, these in vitro assays are limited by their ability to evaluate the efficacy of in vivo acid beta-glucocerebrosidase replacement therapy in animal models. In particular, there is an unmet need to simultaneously define cellular localization and evaluate enzyme activity following treatment in vivo. In addition, results of commonly used fluorescent-based assays for enzyme activity are difficult to compare day to day and/or across laboratories due to the variability inherent in flow cytometric measurement. In this article, we describe a reproducible and consistent quantitative method for the combined measurement of fluorescein intensity from enzyme-substrate conversion and cell localization by phenotype-specific phycoerythrin-antibody staining. Following infusion of recombinant human acid beta-glucocerebrosidase in mice, nonparenchymal cells are prepared from the livers of treated and control animals. Acid beta-glucocerebrosidase activity is measured in molecules of equivalent soluble fluorophore units within Kupffer cell populations as defined by phenotype-specific monoclonal antibodies. This assay should be applicable to investigations of other Gaucher disease treatments in both human and animal models.     

Molecular cloning and functional expression of beta1, 2-xylosyltransferase cDNA from Arabidopsis thaliana

The transfer of xylose from UDP-xylose to the core beta-linked mannose of N-linked oligosaccharides by beta1,2-xylosyltransferase (XylT) is a widespread feature of plant glycoproteins which renders them immunogenic and allergenic in man. Here, we report the isolation of the Arabidopsis thaliana XylT gene, which contains two introns and encodes a 60.2 kDa protein with a predicted type II transmembrane protein topology typical for Golgi glycosyltransferases. Upon expression of A. thaliana XylT cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited XylT activity in vitro. Furthermore, the recombinant enzyme displayed XylT activity in vivo in the insect cells, as judged by the acquired cross-reaction of cellular glycoproteins with antibodies against the beta1,2-xylose epitope. The cloned XylT cDNA as well as the recombinant enzyme are essential tools to study the role of beta1,2-xylose in the immunogenicity and allergenicity of plant glycoproteins at the molecular level.     

Establishment of N-acetylmannosamine (ManNAc) analogue-resistant cell lines as improved hosts for sialic acid engineering applications

Metabolic substrate-based sialic acid engineering techniques, where exogenously supplied N-acetylmannosamine (ManNAc) analogues are utilized by the sialic acid biosynthetic pathway, allow the cell surface to be endowed with novel physical and chemical properties and show promise for increasing the quality of recombinant glycoproteins. The in vitro toxicity of many ManNAc analogues, however, hinders the large-scale adoption of this technology. In this study, we used a selection strategy where cells were subjected to progressively higher levels of ManNAc analogues to establish novel cell lines that showed decreased sensitivity to analogue-induced in vitro toxicity. The decreased sensitivity to sugar analogue-induced apoptosis, demonstrated by the Annexin V-FITC detection method and DNA fragmentation assays, corresponded to increased sialic acid production in the resistant cell lines. The ManNAc analogue-resistant cell lines exhibited cross-resistance to apoptosis induced by staurosporine and an apoptosis-activating Fas antibody. We propose that the selection strategy employed to develop these novel cell lines, which serve as superior hosts for substrate-based sialic acid engineering applications, will generally apply to the development of host cell lines for biotechnology applications.     

Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells

Recombinant glycoproteins produced by mammalian cells represent an important category of therapeutic pharmaceuticals used in human health care. Of the numerous sugars moieties found in glycoproteins, the terminal sialic acid is considered particularly important. Sialic acid has been found to influence the solubility, thermal stability, resistance to protease attack, antigenicity, and specific activity of various glycoproteins. In mammalian cells, it is often desirable to maximize the final sialic acid content of a glycoprotein to ensure its quality and consistency as an effective pharmaceutical. In this study, CHO cells overexpressing recombinant human interferon gamma (hIFNγ) were treated using short interfering RNA (siRNA) and short‐hairpin RNA (shRNA) to reduce expression of two newly identified sialidase genes, Neu1 and Neu3. By knocking down expression of Neu3 we achieved a 98% reduction in sialidase function in CHO cells. The recombinant hIFNγ was examined for sialic acid content that was found to be increased 33% and 26% respectively with samples from cell stationary phase and death phase as compared to control. Here, we demonstrate an effective targeted gene silencing strategy to enhance protein sialylation using RNA interference (RNAi) technology. Biotechnol. Bioeng. 2010;105: 1094–1105. © 2009 Wiley Periodicals, Inc.     

Modified GM3 gangliosides produced by metabolic oligosaccharide engineering

Metabolic oligosaccharide engineering is powerful approach to altering the structure of cellular sialosides. This method relies on culturing cells with N-acetylmannosamine (ManNAc) analogs that are metabolized to their sialic acid counterparts and added to glycoproteins and glycolipids. Here we employed two cell lines that are deficient in ManNAc biosynthesis and examined their relative abilities to metabolize a panel of ManNAc analogs to sialosides. In addition to measuring global sialoside production, we also examined biosynthesis of the sialic acid-containing glycolipid, GM3. We discovered that the two cell lines differ in their ability to discriminate among the variant forms of ManNAc. Further, our data suggest that modified forms of sialic acid may be preferentially incorporated into certain sialosides and excluded from others. Taken together, our results demonstrate that global analysis of sialoside production can obscure sialoside-specific differences. These findings have implications for downstream applications of metabolic oligosaccharide engineering, including imaging and proteomics.     

Receptor specificity and functional comparison of recombinant sea bass (Dicentrarchus labrax) gonadotropins (FSH and LH) produced in different host systems

Different yields, biopotency, and in vivo pharmacokinetics are obtained for recombinant sea bass gonadoltropins depending on the production system and DNA construct, but they show specific activation of their corresponding receptors. Gonadotropins (GTHs) are glycoprotein hormones that play a major role in the regulation of gonadal functions. Recently, we succeeded in isolating the native sea bass Fsh from sea bass pituitaries, but to ensure the availability of bioactive GTHs and no cross-contamination with other related glycoproteins, recombinant sea bass GTHs were produced using two expression systems-insect and mammalian cells-and different constructs that yielded tethered or noncovalently bound dimers. Their production levels, binding specificity to their homologous cognate receptors, and bioactivity were investigated and compared. Both expression systems were successful in the generation of bioactive recombinant GTHs, but insect Sf9 cells yielded higher amounts of recombinant proteins than mammalian Chinese Hamster Ovary (CHO) stable clones. All recombinant GTHs activated their cognate receptors without cross-ligand binding and were able to stimulate sea bass gonadal steroidogenesis in vitro, although with different biopotencies. To assess their use for in vivo applications, their half-life in sea bass plasma was evaluated. Sf9-GTHs had a lower in vivo stability compared with CHO-GTHs due to their rapid clearance from the blood circulation. Cell-dependent glycosylation could be contributing to the final in vivo stability and biopotency of these recombinant glycoproteins. In conclusion, both insect and mammalian expression systems produced bioactive sea bass recombinant gonadotropins, although with particular features useful for different proposes (e.g., antibody production or in vivo studies, respectively).     

Exploiting bacterial glycosylation machineries for the synthesis of a Lewis antigen-containing glycoprotein

Glycoproteins constitute a class of compounds of increasing importance for pharmaceutical applications. The manipulation of bacterial protein glycosylation systems from Gram-negative bacteria for the synthesis of recombinant glycoproteins is a promising alternative to the current production methods. Proteins carrying Lewis antigens have been shown to have potential applications for the treatment of diverse autoimmune diseases. In this work, we developed a mixed approach consisting of in vivo and in vitro steps for the synthesis of glycoproteins containing the Lewis x antigen. Using glycosyltransferases from Haemophilus influenzae, we engineered Escherichia coli to assemble a tetrasaccharide on the lipid carrier undecaprenylphosphate. This glycan was transferred in vivo from the lipid to a carrier protein by the Campylobacter jejuni oligosaccharyltransferase PglB. The glycoprotein was then fucosylated in vitro by a truncated fucosyltransferase from Helicobacter pylori. Diverse mass spectrometry techniques were used to confirm the structure of the glycan. The strategy presented here could be adapted in the future for the synthesis of diverse glycoproteins. Our experiments demonstrate that bacterial enzymes can be exploited for the production of glycoproteins carrying glycans present in human cells for potential therapeutic applications.     

Enhanced sialylation of EPO by overexpression of UDP-GlcNAc 2-epimerase/ManAc kinase containing a sialuria mutation in CHO cells

Sialylation (e.g. expression of sialic acid) plays a crucial role for function and stability of most glycoproteins. The key enzyme for the biosynthesis of sialic acid is the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE). Mutations in the binding site of the feedback inhibitor CMP-sialic acid of the GNE leads to sialuria, a disease in which patients produce sialic acid in gram scale. Here, we report on the use in biotechnology of sialuria-mutated GNE. Expression of the sialuria-mutated GNE in CHO-cells leads to increased sialylation of recombinant expressed erythropoietin (EPO). Our data show that sialuria-mutated-GNE over-expressing cells are the perfect platform to express highly sialylated therapeutic proteins, such as EPO.     

Knockout of sialidase and pro-apoptotic genes in Chinese hamster ovary cells enables the production of recombinant human erythropoietin in fed-batch cultures

Sialic acid, a terminal monosaccharide present in N-glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode. This is mainly due to the accumulation of extracellular sialidases released from the cells. To overcome this hurdle, three sialidase genes (Neu1, 2, and 3) were initially knocked-out using the CRISPR/Cas9-mediated large deletion method in the rhEPO-producing rCHO cell line. Unlike wild type cells, sialidase knockout (KO) clones maintained the sialic acid content and proportion of tetra-sialylated rhEPO throughout fed-batch cultures without exhibiting a detrimental effect with respect to cell growth and rhEPO production. Additional KO of two pro-apoptotic genes, BAK and BAX, in sialidase KO clones (5X KO clones) further improved rhEPO production without any detrimental effect on sialylation. On day 10 in fed-batch cultures, the 5X KO clones had 1.4-times higher rhEPO concentration and 3.0-times higher sialic acid content than wild type cells. Furthermore, the proportion of tetra-sialylated rhEPO on day 10 in fed-batch cultures was 42.2–44.3% for 5X KO clones while it was only 2.2% for wild type cells. Taken together, KO of sialidase and pro-apoptotic genes in rCHO cells is a useful tool for producing heavily sialylated glycoproteins such as rhEPO in fed-batch mode.     

Development of a successful antitumor therapeutic model combining in vivo dendritic cell vaccination with tumor irradiation and intratumoral GM-CSF delivery

Vaccination of dendritic cells (DC) combined with GM-CSF secreting tumor cells has shown good therapeutic efficacy in several tumor models. Nevertheless, the engineering of GM-CSF secreting tumor cell line could represent a tedious step limiting its application for treatment in patients. We therefore developed in rats, an “all in vivo” strategy of combined vaccination using an in vivo local irradiation of the tumor as a source of tumor antigens for DC vaccines and an exogenous source of GM-CSF. We report here that supplying recombinant mGM-CSF by local injections or surgical implantation of osmotic pumps did not allow reproducing the therapeutic efficacy observed with in vitro prepared combined vaccines. To bypass this limitation possibly due to the short half-life of recombinant GM-CSF, we have generated adeno-associated virus coding for mGM-CSF and tested their efficacy to transduce tumor cells in vitro and in vivo. The in vivo vaccines combining local irradiation and AAV2/1-mGM-CSF vectors showed high therapeutic efficacy allowing to cure 60% of the rats with pre-implanted tumors, as previously observed with in vitro prepared vaccines. Same efficacy has been observed with a second generation of vaccines combining DC, local tumor irradiation, and the controlled supply of recombinant mGM-CSF in poloxamer 407, a biocompatible thermoreversible hydrogel. By generating a successful “all in vivo” vaccination protocol combining tumor radiotherapy with DC vaccines and a straightforward supply of GM-CSF, we have developed a therapeutic strategy easily translatable to clinic that could become accessible to a much bigger number of cancer patients.     

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Mammalian brains contain relatively high amounts of common and uncommon sialylated N-glycan structures. Sialic acid linkages were identified for voltage-gated potassium channels, Kv3.1, 3.3, 3.4, 1.1, 1.2 and 1.4, by evaluating their electrophoretic migration patterns in adult rat brain membranes digested with various glycosidases. Additionally, their electrophoretic migration patterns were compared with those of NCAM (neural cell adhesion molecule), transferrin and the Kv3.1 protein heterologously expressed in B35 neuroblastoma cells. Metabolic labelling of the carbohydrates combined with glycosidase digestion reactions were utilized to show that the N-glycan of recombinant Kv3.1 protein was capped with an oligo/poly-sialyl unit. All three brain Kv3 glycoproteins, like NCAM, were terminated with alpha2,3-linked sialyl residues, as well as atypical alpha2,8-linked sialyl residues. Additionally, at least one of their antennae was terminated with an oligo/poly-sialyl unit, similar to recombinant Kv3.1 and NCAM. In contrast, brain Kv1 glycoproteins consisted of sialyl residues with alpha2,8-linkage, as well as sialyl residues linked to internal carbohydrate residues of the carbohydrate chains of the N-glycans. This type of linkage was also supported for Kv3 glycoproteins. To date, such a sialyl linkage has only been identified in gangliosides, not N-linked glycoproteins. We conclude that all six Kv channels (voltage-gated K+ channels) contribute to the alpha2,8-linked sialylated N-glycan pool in mammalian brain and furthermore that their N-glycan structures contain branched sialyl residues. Identification of these novel and unique sialylated N-glycan structures implicate a connection between potassium channel activity and atypical sialylated N-glycans in modulating and fine-tuning the excitable properties of neurons in the nervous system.     

Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli

Background  

The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically in vitro but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity.     

Estimating glycoprotein carbohydrate chain structures by lectin reactivities in polyacrylamide gels

Complex mixtures of cellular glycoproteins contain a myriad of different carbohydrate chains that cannot be easily analyzed without rigorous purification of each individual glycoprotein. We have analyzed the carbohydrate chains in complex mixtures of cellular glycoproteins by separation using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and interacting the gels with several 125I-labeled lectins. By use of in situ chemical modifications of the glycoproteins after their electrophoretic separation together with the known carbohydrate-binding specifities of several lectins, it has been possible to estimate glycoprotein carbohydrate chain structures. As an example we have examined the cellular glycoproteins of a ovary-colonizing metastatic variant of B16 melanoma and report the types of carbohydrate chains that are found on various melanoma glycoproteins.