Glycosylation | EPFL Graph Search

La glycosylation est une réaction enzymatique consistant à lier de façon covalente un glucide à une chaîne peptidique, une protéine, un lipide ou d'autres molécules. À ne pas confondre avec la glycation, réaction purement chimique et spontanée. Le processus de glycosylation débute dans le réticulum endoplasmique rugueux (RER) et s'achève dans l'appareil de Golgi. La glycosylation concerne essentiellement les protéines membranaires ainsi que les protéines sécrétées. Des remaniements de la glycosylation primaire, comme la suppression de certains résidus glucidiques se produisent dans l'appareil de Golgi distal (late Golgi). Il est néanmoins important de préciser que seules la N-glycosylation et la C-glycosylation débutent dans le réticulum endoplasmique (uniquement le RER en ce qui concerne les protéines) pour s'achever dans l'appareil de Golgi, car la O-glycosylation, qui se fait sur des résidus hydroxylés (hydroxylysine, hydroxytyrosine, hydroxyproline) est exclusivement réalisée par

Introducing tolerance to foreign and partly foreign proteins by erythrocyte binding and artificial glycosylations

Kym Lorenz Brünggel

Over the last few years, protein drugs have steadily gained importance in the clinic. Compared to small molecule drugs, protein drugs are taken up by antigen presenting cells, which degrade the proteins into peptides that are then presented on major histocompatability complex I and II to CD8+ respectively CD4+ T-cells. CD4+ T-cells are then able to activate B-cells, which upon activation will produce anti-drug antibodies. While anti-drug antibodies have mostly been viewed as a safety risk leading to drug injection related side effects, they have recently been proven to diminish the efficacy of the treatment. High antibody titers against Humira, a clinically approved monoclonal antibody used to treat rheumatoid arthritis, have been associated with low serum drug concentration and high disease activity score, ultimately leading to treatment failure. Two approaches to induce tolerance were developed in the Hubbell lab. One approach utilizes protein variants that are designed to bind to circulating erythrocytes. As 1 % of erythrocytes undergo apoptosis each day and are taken up by antigen presenting cells, along with the bound protein, they are presented to the immune system in a non-inflammatory tolerogenic fashion. Immunological tolerance is thereby induced against the apoptotic erythrocyte debris and the associated bound protein. The second approach uses synthetic glucose, galactose or mannose polymers conjugated to the target protein. The polymers target C-type lectins, which are known to clear apoptotic debris, leading to presentation of the protein by hepatic antigen presenting cells in a tolerogenic liver microenvironment. This thesis investigates the potential of these approaches to induce tolerance towards protein drugs. Arylsulfatase B was used as a model protein to investigate the potential of tolerance induction by targeting circulating erythrocytes, as 97% of patients receiving arylsulfatase B develop anti-drug antibodies. An erythrocyte binding variant of arylsufatase B was created by chemically conjugating the erythrocyte binding peptide ERY1 to arylsulfatse B. In mice receiving two doses of ERY1-arylsulfatase B one week apart followed by weekly doses of arylsulfatase B a significant delay of four week in the production of anti-drug Antibodies was found when compared to mice receiving weekly doses of only arylsulfatase B. To investigate the ability of synthetic glyco-polymers to induce tolerance towards protein drugs we used asparaginase as model protein. Between 37.5 % to 75% of patients treated with asparaginase develop anti-drug antibodies. Administering three intravenous injections of asparaginase-conjugated to glucose polymers before eight weeks of treatment with asparaginase significantly reduced anti-drug antibody titers by a factor of 125 when compared to animals only receiving eight weeks of treatment with asparaginase. Three pre-injections with asparaginase conjugated to the galactose or mannose polymer led to a reduction in anti-drug antibody titers by a factor of 12 respectively 400 after 5 weeks of treatment with asparaginase when compared to mice receiving only asparaginase for 5 weeks. These results indicates that synthetic glyco polymers, especially mannose and glucose based ones, are able to induce long term tolerance towards asparaginase. Synthetic glucose and mannose polymers therefore represent an excellent molecular approach to induce tolerance towards protein drugs.

EPFL2018

GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis

Laura Capolupo, Charlotte Julie Caroline Gehin

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.

WILEY2021

Transient Gene Expression in HEK-293E Cells for Recombinant Protein Production

Sophie Nallet

The growing demand of biopharmaceutical products is boosting the market for therapeutic recombinant proteins (r-proteins). More than half of the 140 r-proteins that have gained approval for human therapeutic use are manufactured in mammalian cells that make possible complex post-translational modifications, like glycosylation. In the industry, r-proteins are manufactured by stable gene expression (SGE) following Good Manufacturing Practice (GMP) standards. Transient gene expression (TGE) in mammalian cells is an alternative method for the expression of r-proteins whose main advantages are rapidity and flexibility. TGE involves the expression of the protein of interest from plasmids, which are transfected into the cells with a non-viral DNA transfecting agent, usually polyethylenimine (PEI). DNA is transcribed from the plasmids without the latter having been integrated into the host genome. Despite the recent improvements in r-protein titers by TGE and the scale up of this method to the 100-L scale, TGE remains so far a tool for protein production for research purposes. Therefore, to determine if TGE can be used for manufacturing therapeutic r-protein following GMP standards, different features related to reproducibility and product quality from TGE of a recombinant anti-rhesus D immunoglobulin G in HEK-293E cells using linear PEI were characterized. To test the effects of the size distribution and chemical structure of PEI on TGE, different commercially available PEIs were characterized and their impact on transfection and r-protein expression were assessed. The results showed that both the molecular weight distribution and the chemical structure of the PEI had an effect on TGE. However, the small variations in size distribution and structure between the batches of PEI from the same supplier did not affect significantly the transfection and r-protein production by TGE. Then, the fate of PEI and plasmid DNA in cell culture over time and their removal from the final r-protein during purification were determined and measured. Most of the PEI and pDNA remained in the cell culture medium after transfection. However, both PEI and plasmid DNA, which are additional components in TGE compared to processes based on recombinant cell lines, could be reduced to a concentration below the limit of detection of the assays after Protein A affinity purification of the antibody. To test the reproducibility of a TGE process, 10 batches of transfected cells were run in 500-mL orbitally shaken bottles. The cell specific productivity of the antibody was 20.2 ± 2.6 pg.cell-1.day-1 on average for the 10 batches. The purity and size consistency of the recombinant antibody produced in those 10 batches was demonstrated by gel electrophoresis and size exclusion chromatography. Then, the glycosylation profile of the antibody produced by TGE in HEK-293E cells was determined by mass spectrometry and was reproducible over the 10 batches. Moreover, it was similar to the glycosylation profile of the antibody produced by 3 stable HEK-293E cell lines. Finally, as a proof of concept, TGE was applied for the development of a vaccine against the respiratory syncytial virus. The fusion protein of the respiratory syncytial virus was produced by TGE and used as an antigen for the development of a recombinant vaccine. TGE enabled the rapid evaluation of the candidate vaccine in animal trials. Overall, the results of this study suggest that TGE is a reliable and reproducible method for manufacturing r-proteins of a consistent quality, provided that some particular features, such as reagents quality and process parameters, are well defined and controlled. Since TGE makes possible the rapid production of virtually any protein, even toxic, it could be used to provide GMP approved biopharmaceuticals for clinical trials in a short time, reducing development costs of biological therapeutic products.