The glycosylation of proteins is an important modification of proteins and plays a vital role in the production of pharmaceuticals such as vaccines or anti-cancer medication. Through new advances in research, it becomes possible to characterize the influence glycoproteins have on the structure and binding properties of antibodies, vital knowledge that can be used to produce recombinant antibodies with desired characteristics.
By working with genetically modified versions like afucosylated antibodies especially, the production of more effective pharmaceuticals with less risk for cytotoxicity and cross-reactions was accomplished.
In this article, we will take a closer look at the process of glycosylation, its different types and why glycosylation is so important for afucosylated antibodies and biotechnology in general.
Glycosylation is defined as a co-translational or post-translational modification in newly synthesized proteins1, which takes place in the endoplasmic reticulum on amino acids with functional hydroxyl groups. The alterations caused by glycosylation are able to support cell structure, protein folding and quality control in cells.
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During this enzyme-catalysed reaction, carbohydrate molecules (sugar molecules) like N-glycans, glucose or galactose are linked to specific amino acid residues in the protein and transferred across the plasma membrane. This happens with the help of glycosidases or glycosyltransferases (processing enzymes). Other possible intermediates apart from enzymes are lectins. Typically, these amino acids are serine (Ser), threonine (Thr) or asparagine (Asn). The synthesis between the sugar molecules and amino acids is an essential process for physiological and pathological cellular functions and has many critical biochemical implications.
There are two major types of glycosylation which can be distinguished by the type of linkage of the oligosaccharide with the amino acid side chain: N-glyocsylation and O-glycosylation.
The glycosylation of proteins serves many different purposes in biology and biotechnology. The process protects proteins from degradation by proteases and helps to maintain their structural integrity. Further, it has an impact on protein folding and therefore conformational stability of proteins. The linkage formed by glycosylation on the cell surface also plays an important role in the adhesion and interaction of cells, affecting the activity of cell surface receptors and ligands. This improves cell-cell recognition and makes glycosylation an essential part of the immune response, as the interaction between immune cells is mediated by cell surface molecules.
The impact of glycosylation is also recognizable in biotechnological advances and is critical to the development and production of therapeutic proteins. By controlling glycosylation patterns, glycoengineering allows to optimize protein stability, efficacy, and safety. Glycobiology is therefore a significant aspect of biopharmaceutical research and manufacturing.2
There are various methods of glycosylation which differ in terms of their target amino acids, site of glycosylation and the generated glycan structures. The most common are N-glycosylation and O-glycosylation. Both are important for the proper function and regulation of glycoproteins in cells.
N-glycosylation is the most common type of co-translational modifications in eukaryotes and archae. The N-glycosylation site, where this process takes place, is the endoplasmic reticulum. Here, the sugar molecules are bound to asparagine residues or N-residues with the help of a lipid carrier molecule called dolichol phosphate. N-linked glycoproteins are also called N-acetylglucosamines.
After N-linked glycosylation occurred, the glycosylated protein undergoes extensive processing in the Golgi apparatus. Here, certain glucose and mannose residues are removed to generate a new range of glycan structures, a critical step, often referred to as “trimming”, for the functional diversity of glycoproteins.1
N-glycosylation serves several important functions. One of them is the assistance of N-glycans in proper protein folding and stability. They help to target degradation and maintain cellular homeostasis. Further, the n-glycans on the cell surface help cells recognize each other, bind more easily and shield the proteins from degradation through certain enzymatic reactions.
In O-glycosylation, the carbohydate molecules bind to serine (Ser) or threonine (Thr) side chains in protein. This glycosidic linkage takes place translationally in the Golgi apparatus, often on glycoproteins that were N-glycosylated in the endoplasmic reticulum.
While N-glycosylation is a rather stringent series of steps, O-glycosylation is more variable and shows a broader range of glycan structures. The glycoprotein, which is formed by binding nucleotide activated oligosaccharides with polypeptides, is called O-glcnac and interacts with the glycopeptide on the cell surface.
O-linked glycosylation is an important part of the biosynthesis of mucins (to form mucus or secretory liquids) or proteoglycan core proteins, which are integral to extracellular matrix components. Most importantly, a lot of antibodies rely on O-glycoproteins. The oligosaccharide structures of O-linked glycans are often less complex than N-linked glycans.3
Apart from N-glycosylation and O-glycasylation, there are several other examples for glycosylation with individual biological functions. Not all of them are researched, yet. As we cannot list them all, here is a selection of notable glycosylation types:
A lot of important advances in biotechnology rely on the use of glycans, such as the development of vaccines and therapeutics. They are an integral part of biotherapeutics like antibiotics and anti-cancer therapeutic agents.
The characterization of glycoproteome and glycome through mass spectrometry is of utter importance to better understand their different structures, functions, and biochemistry. As glycoproteins and polysaccharides have significant functions in the immune response, they can be used to design monoclonal antibodies.
The difficulty in research lies in the glycan’s structure, which does not only bind to certain site-specific antigens, e.g. in antibodies, but can interact with a number of peptides, enzymes etc. Once the glycan is linked to a protein, it has the ability to modulate the protein’s activity. The glycan’s structure is determined by cellular metabolism, cell type and other factors. On divergent substrate, proteome can form during different generations of glycosylated proteins, making the calculation of its effects even harder. The enormous diversity in the biological functions of glycans, such as glycoprotein modulation or localization, makes them hard to decode4 for genomics.
One solution to this problem is afucosylation, which makes it possible to regulate metabolic interactions of poly- and monosaccharides in monoclonal antibodies. The removal of oligosaccharides in the fc-region of the antibody has the potential to make antibody pharmaceutics more effective and enhances targeted cytotoxicity5 by removing the potential for heterogeneity.
Read more: Applications of afucoylated anitbodies
In afucosylated monoclonal antibodies, the oligosaccharides in the Fc region carry a low amount of fucose. Through afucosylation it becomes possible to increase the antibody-dependent cellular cytotoxity (ADCC).
Afucosylated monoclonal antibodies can be cultivated in different mammalian cell cultures. At evitiria, we use CHO cells, due to native glycoforms. With the help of the GlymaxX® platform, evitria is able to offer transiently expressed afucosylated variants using the same cells, the same DNA and without any modifications of the amino acid sequence, which provides a higher level of control in the production of highly effective therapeutic antibodies.
Learn more on how afucosylated antibodies are developed
Glycosylation is an important part of the production of genetically modified antibodies with excellent binding and targeting properties. The high demand for antibodies can only be met by high-standard technologies. One of those state-of-the-art systems that is used at evitria is the GlymaxX® platform, which allows us to transiently express both native and afucosylated antibodies.
This is made possible without having to apply any amino acid sequence modifications, reducing the risk of immunogenicity or cytokine release syndrome. evitria’s process of using the GlymaxX® platform enables us to offer a recombinant antibody expression service without having to rely on Fc mutations. This way of producing transient antibodies reduces the danger for potential undesired immune responses.
Most glycosylation processes occur in the endoplasmic reticulum and the Golgi apparatus.
Glycosylation is a co- and post-translational modification of proteins, which causes their alteration. It serves many purposes in the support of cell structure, protein folding and quality control.
Protein glycosylation ensures the stability of a cell through glycoproteins, which play a role in cell-cell adhesion and help detect cell degradation.