Glycoengineering Strategies Research Articles

Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases.

Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy. [BMB Reports 2015; 48(8): 438-444]

Strategies for Engineering Protein N-Glycosylation Pathways in Mammalian Cells.

Complexity and heterogeneity of oligosaccharides present a considerable challenge to the biopharmaceutical industry to manufacture biotherapeutics with reproducible and consistent glycoform profiles. Mammalian cells, especially Chinese hamster ovary cells, are the most widely used platform for the production of biotherapeutics. The glycans produced are predominantly of the complex type, with some differences between human and nonhuman mammalian glycosylation existing. This review briefly summarizes metabolic glyco-engineering strategies used in mammalian cells in order to alter the glycosylation patterns attached to proteins applied for diverse biotechnology applications.

Differences in the production of hyperglycosylated IFN alpha in CHO and HEK 293 cells

Background IFN alpha is an important cytokine of the immune system. It has the ability to interfere with virus replication exerting antiviral activity. Moreover, it displays antiproliferative activity and can profoundly modulate the immune response. IFN4N (or hyperglycosylated IFN alpha) is an IFN-alpha2b mutein developed in our laboratory using glycoengineering strategies. This molecule contains 4 potential N-glycosylation sites together with the natural O-glycosylation site in Thr106 [1]. The resulting Nand O-glycosylated protein shows higher apparent molecular mass and longer plasmatic half-life compared to the nonglycosylated IFN-alpha produced in bacterial systems and used for clinical applications. As a consequence, the correct glycosylation of our modified cytokine is very important for its in vivo activity. For this reason, it is of great relevance the evaluation of different mammalian host cells for its production. While hamster-derived CHO cells are widely used for large scale production of recombinant therapeutic glycoproteins, human HEK cells are a promising system because they are easy to grow and transfect [2]. In this work, we performed a comparison between both production systems in terms of cell growth, culture parameters and specific productivity of hyperglycosylated IFN alpha.

Integrating Bioinformatics Tools to Handle Glycosylation

This tutorial is planned for biologists and computational biologists interested in bioinformatics applications to study protein glycosylation. Glycosylation is a co- and post-translational modification that involves the selective attachment of carbohydrates to proteins. The enhancement of glycosylation by applying glycoengineering strategies has become widely used to improve properties for protein therapeutics. In this tutorial, the use of bioinformatics to assist the rational design and insertion of N-glycosylation sites in proteins is described.

Neisseria gonorrhoeae O‐linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure

Neisseria gonorrhoeae expresses an O-linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a ‘top-down’ mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O-acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N-linked glycosylation system that adds N-acetylgalactosamine onto undecaprenylpyrophosphate-linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O-linked di- and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid-linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O- and N-linked pathways can be combined in novel glycoengineering strategies.