Glycosylation
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Basis of N- and O-linked glycobiology and glycosylation of therapeutic proteins in health and disease.
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Glycosylation
- 1. Antony, P., & Vijayan, R. (2021). Role of SARS-CoV-2 and ACE2 variations in COVID-19. Biomedical Journal, 44(3), 235–244. https://doi.org/10.1016/j.bj.2021.04.006 Protein Glycosylation 1 2 3 5 4 6
- 2. Glycosylation overview and linkage types Reily, C., Stewart, T. J., Renfrow, M. B., & Novak, J. (2019). Glycosylation in health and disease. Nature Reviews Nephrology, 15(6), 346–366. https://doi.org/10.1038/s41581-019-0129-4 1 2 3 5 4 6
- 3. Post-translational glycosidic modifications Overview of post-translational modifications: types of glycosylation (Thermo Fisher Scientific). Glycosidic linkage Glycan composition Glycan structure Glycan length 1 2 3 5 4 6
- 4. N-linked glycosylation Casas-Sanchez, A., Romero-Ramirez, A., Hargreaves, E., Ellis, C. C., Grajeda, B. I., Estevao, I. L., Patterson, E. I., Hughes, G. L., Almeida, I. C., Zech, T., & Acosta-Serrano, Á. (2022). Inhibition of Protein N-Glycosylation Blocks SARS-CoV-2 Infection. MBio, 13(1). https://doi.org/10.1128/mbio.03718-21 1 2 3 5 4 6
- 5. N-glycosylation: Precursor assembly and attachment Precursor glycan structure (Thermo Fisher Scientific) Precursor assembly and attachment (Thermo Fisher Scientific) 1 2 3 5 4 6
- 6. N-glycosylation: Glycan trimming Helenius, A., & Aebi, and M. (2001). Intracellular Functions of N-Linked Glycans. Science, 291(5512), 2364–2369. https://doi.org/10.1126/science.291.5512.2364 1 2 3 5 4 6
- 7. N-glycosylation: Glycan folding Słomińska-Wojewódzka, M., & Sandvig, K. (2015). The Role of Lectin-Carbohydrate Interactions in the Regulation of ER-Associated Protein Degradation. Molecules, 20(6), 9816–9846. https://doi.org/10.3390/molecules20069816 1 2 3 5 4 6
- 8. N-glycosylation: Golgi maturation Golgi maturation (Thermo Fisher Scientific) Higel, F., Seidl, A., Sörgel, F., & Friess, W. (2016). N-glycosylation heterogeneity and the influence on structure, function and pharmacokinetics of monoclonal antibodies and Fc fusion proteins. European Journal of Pharmaceutics and Biopharmaceutics, 100, 94– 100. https://doi.org/10.1016/j.ejpb.2016.01.005 1 2 3 5 4 6
- 9. N-glycosylation: Golgi maturation Golgi maturation (Thermo Fisher Scientific) 1 2 3 5 4 6
- 10. O-linked glycosylation 1 2 3 5 4 6
- 11. O-glycosidic linkage example: O-GalNac Wandall, H. H., Nielsen, M. A. I., King-Smith, S., Haan, N., & Bagdonaite, I. (2021). Global functions of O-glycosylation: promises and challenges in O‐glycobiology. The FEBS Journal, 288(24), 7183–7212. https://doi.org/10.1111/febs.16148 1 2 3 5 4 6
- 12. O-glycosylation functions Wandall, H. H., Nielsen, M. A. I., King-Smith, S., Haan, N., & Bagdonaite, I. (2021). Global functions of O-glycosylation: promises and challenges in O‐glycobiology. The FEBS Journal, 288(24), 7183–7212. https://doi.org/10.1111/febs.16148 1 2 3 5 4 6
- 13. Analysis and associations 1 2 3 5 4 6
- 14. Glycogenomic workflow analysis Schjoldager, K. T., Narimatsu, Y., Joshi, H. J., & Clausen, H. (2020). Global view of human protein glycosylation pathways and functions. Nature Reviews Molecular Cell Biology, 21(12), 729–749. https://doi.org/10.1038/s41580-020-00294-x 1 2 3 5 4 6
- 15. 173 gtfs predicted pathways (GWAS) https://doi.org/10.1038/s41580-020-00294-x 1 2 3 5 4 6
- 16. Transcriptionally active glycosylation genes (GWAS) https://doi.org/10.1038/s41580-020-00294-x 1 2 3 5 4 6
- 17. Glycosylation genes in human disease (GWAS) https://doi.org/10.1038/s41580-020-00294-x 1 2 3 5 4 6
- 18. Genome analysis in mice models (GWAS) https://doi.org/10.1038/s41580-020-00294-x 1 2 3 5 4 6
- 19. Glycosylation functional studies tool-box Wandall, H.H., Nielsen, M.A.I., King-Smith, S., de Haan, N. and Bagdonaite, I. (2021), Global functions of O-glycosylation: promises and challenges in O-glycobiology. FEBS J, 288: 7183-7212. https://doi.org/10.1111/febs.16148 1 2 3 5 4 6
- 20. Glycomics limitations 1 2 3 5 4
- 21. Glycosylation of mAbs 1 2 3 5 4 6
- 22. Glycosylation of IgG antibodies Jennewein, M. F., & Alter, G. (2017). The Immunoregulatory Roles of Antibody Glycosylation. Trends in Immunology, 38(5), 358–372. https://doi.org/10.1016/j.it.2017.02.004 1 2 3 5 4 6
- 23. IgG glycoprotein diversity Jennewein, M. F., & Alter, G. (2017). The Immunoregulatory Roles of Antibody Glycosylation. Trends in Immunology, 38(5), 358–372. https://doi.org/10.1016/j.it.2017.02.004 1 2 3 5 4 6
- 24. Role of Fc región and receptors Jennewein, M. F., & Alter, G. (2017). The Immunoregulatory Roles of Antibody Glycosylation. Trends in Immunology, 38(5), 358–372. https://doi.org/10.1016/j.it.2017.02.004 1 2 3 5 4 6
- 25. IgG glycoprotein diversity Jennewein, M. F., & Alter, G. (2017). The Immunoregulatory Roles of Antibody Glycosylation. Trends in Immunology, 38(5), 358–372. https://doi.org/10.1016/j.it.2017.02.004 1 2 3 5 4 6
- 26. Antibody glycosylation in nature and disease 1 2 3 5 4 6
- 27. Natural glycovariation 1 2 3 5 4 6
- 28. Antibodies glycosylation profiles 1 2 3 5 4 6 Jennewein, M. F., & Alter, G. (2017). The Immunoregulatory Roles of Antibody Glycosylation. Trends in Immunology, 38(5), 358–372. https://doi.org/10.1016/j.it.2017.02.004
- 29. Possibilities 1 2 3 5 4 6 Shade, K.-T., & Anthony, R. (2013). Antibody Glycosylation and Inflammation. Antibodies, 2(4), 392–414. https://doi.org/10.3390/antib2030392
- 30. Possibilities 1 2 3 5 4 6 Gerdes, C. A., Nicolini, V. G., Herter, S., van Puijenbroek, E., Lang, S., Roemmele, M., Moessner, E., Freytag, O., Friess, T., Ries, C. H., Bossenmaier, B., Mueller, H. J., & Umaña, P. (2013). GA201 (RG7160): A Novel, Humanized, Glycoengineered Anti-EGFR Antibody with Enhanced ADCC and Superior In Vivo Efficacy Compared with Cetuximab. Clinical Cancer Research, 19(5), 1126–1138. https://doi.org/10.1158/1078-0432.CCR-12-0989
- 31. Possibilities 1 2 3 5 4 6 Gerdes, C. A., Nicolini, V. G., Herter, S., van Puijenbroek, E., Lang, S., Roemmele, M., Moessner, E., Freytag, O., Friess, T., Ries, C. H., Bossenmaier, B., Mueller, H. J., & Umaña, P. (2013). GA201 (RG7160): A Novel, Humanized, Glycoengineered Anti-EGFR Antibody with Enhanced ADCC and Superior In Vivo Efficacy Compared with Cetuximab. Clinical Cancer Research, 19(5), 1126–1138. https://doi.org/10.1158/1078-0432.CCR-12-0989
- 32. Possibilities 1 2 3 5 4 6 Gerdes, C. A., Nicolini, V. G., Herter, S., van Puijenbroek, E., Lang, S., Roemmele, M., Moessner, E., Freytag, O., Friess, T., Ries, C. H., Bossenmaier, B., Mueller, H. J., & Umaña, P. (2013). GA201 (RG7160): A Novel, Humanized, Glycoengineered Anti-EGFR Antibody with Enhanced ADCC and Superior In Vivo Efficacy Compared with Cetuximab. Clinical Cancer Research, 19(5), 1126–1138. https://doi.org/10.1158/1078-0432.CCR-12-0989
- 34. Bibliography ● Wandall, H. H., Nielsen, M. A. I., King‐Smith, S., Haan, N., & Bagdonaite, I. (2021). Global functions of O‐glycosylation: promises and challenges in O‐glycobiology. The FEBS Journal, 288(24), 7183–7212. https://doi.org/10.1111/febs.16148 ● Schjoldager, K. T., Narimatsu, Y., Joshi, H. J., & Clausen, H. (2020). Global view of human protein glycosylation pathways and functions. Nature Reviews Molecular Cell Biology, 21(12), 729–749. https://doi.org/10.1038/s41580-020-00294-x ● Casas-Sanchez, A., Romero-Ramirez, A., Hargreaves, E., Ellis, C. C., Grajeda, B. I., Estevao, I. L., Patterson, E. I., Hughes, G. L., Almeida, I. C., Zech, T., & Acosta-Serrano, Á. (2022). Inhibition of Protein N-Glycosylation Blocks SARS-CoV-2 Infection. MBio, 13(1). https://doi.org/10.1128/mbio.03718-21 ● Helenius, A., & Aebi, and M. (2001). Intracellular Functions of N-Linked Glycans. Science, 291(5512), 2364–2369. https://doi.org/10.1126/science.291.5512.2364 ● Higel, F., Seidl, A., Sörgel, F., & Friess, W. (2016). N-glycosylation heterogeneity and the influence on structure, function and pharmacokinetics of monoclonal antibodies and Fc fusion proteins. European Journal of Pharmaceutics and Biopharmaceutics, 100, 94–100. https://doi.org/10.1016/j.ejpb.2016.01.005 ● Słomińska-Wojewódzka, M., & Sandvig, K. (2015). The Role of Lectin-Carbohydrate Interactions in the Regulation of ER-Associated Protein Degradation. Molecules, 20(6), 9816–9846. https://doi.org/10.3390/molecules20069816 ● Reily, C., Stewart, T. J., Renfrow, M. B., & Novak, J. (2019). Glycosylation in health and disease. Nature Reviews Nephrology, 15(6), 346–366. https://doi.org/10.1038/s41581-019-0129-4
- 35. ● Oliveira, T., Thaysen-Andersen, M., Packer, N. H., & Kolarich, D. (2021). The Hitchhiker’s guide to glycoproteomics. Biochemical Society Transactions, 49(4), 1643–1662. https://doi.org/10.1042/BST20200879 ● Singh, A. (2021). Glycoproteomics. Nature Methods, 18(1), 28–28. https://doi.org/10.1038/s41592-020-01028-9 ● Illiano, A., Pinto, G., Melchiorre, C., Carpentieri, A., Faraco, V., & Amoresano, A. (2020). Protein Glycosylation Investigated by Mass Spectrometry: An Overview. Cells, 9(9), 1986. https://doi.org/10.3390/cells9091986 ● Kattla, J. J., Struwe, W. B., Doherty, M., Adamczyk, B., Saldova, R., Rudd, P. M., & Campbell, M. P. (2011). Protein Glycosylation. In Comprehensive Biotechnology (pp. 467–486). Elsevier. https://doi.org/10.1016/B978-0-08-088504-9.00230-0 ● Eichler, J. (2019). Protein glycosylation. Current Biology, 29(7), R229–R231. https://doi.org/10.1016/j.cub.2019.01.003 ● Eichler, J., & Koomey, M. (2017). Sweet New Roles for Protein Glycosylation in Prokaryotes. Trends in Microbiology, 25(8), 662–672. https://doi.org/10.1016/j.tim.2017.03.001 ● Pinho, S. S., & Reis, C. A. (2015). Glycosylation in cancer: mechanisms and clinical implications. Nature Reviews Cancer, 15(9), 540– 555. https://doi.org/10.1038/nrc3982 ● Magalhães, A., Duarte, H. O., & Reis, C. A. (2021). The role of O-glycosylation in human disease. Molecular Aspects of Medicine, 79, 100964. https://doi.org/10.1016/j.mam.2021.100964 ● Marth, J. D., & Grewal, P. K. (2008). Mammalian glycosylation in immunity. Nature Reviews Immunology, 8(11), 874–887. https://doi.org/10.1038/nri2417 Bibliography
- 36. ● Gerdes, C. A., Nicolini, V. G., Herter, S., van Puijenbroek, E., Lang, S., Roemmele, M., Moessner, E., Freytag, O., Friess, T., Ries, C. H., Bossenmaier, B., Mueller, H. J., & Umaña, P. (2013). GA201 (RG7160): A Novel, Humanized, Glycoengineered Anti-EGFR Antibody with Enhanced ADCC and Superior In Vivo Efficacy Compared with Cetuximab. Clinical Cancer Research, 19(5), 1126– 1138. https://doi.org/10.1158/1078-0432.CCR-12-0989 ● Shade, K.-T., & Anthony, R. (2013). Antibody Glycosylation and Inflammation. Antibodies, 2(4), 392–414. https://doi.org/10.3390/antib2030392 ● Jennewein, M. F., & Alter, G. (2017). The Immunoregulatory Roles of Antibody Glycosylation. Trends in Immunology, 38(5), 358–372. https://doi.org/10.1016/j.it.2017.02.004 ● Gasdaska, J. R., Sherwood, S., Regan, J. T., & Dickey, L. F. (2012). An afucosylated anti-CD20 monoclonal antibody with greater antibody-dependent cellular cytotoxicity and B-cell depletion and lower complement-dependent cytotoxicity than rituximab. Molecular Immunology, 50(3), 134–141. https://doi.org/10.1016/j.molimm.2012.01.001 Bibliography
Editor's Notes
- Definition Glycosylation is defined as the enzymatic process that produces glycosidic linkages of saccharides to other saccharides, proteins or lipids. Glycosylation is thought to be the most complex type of post-translational modification because of the large number of enzymatic processes it involves. The most common types of glycosylation are N-glycosylation and O-glycosylation (>90% of all glycosylations). Although glycosylation encompasses an important number of steps, the most common glycosidic linkages (Figure 1) include: N-linked glycans attached to a nitrogen of asparagine or arginine side-chains. N-linked glycosylation requires participation of a special lipid called dolichol phosphate. O-linked glycans attached to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains, or to oxygens on lipids such as ceramide C-linked glycans are a rare form of glycosylation where a sugar is added to a carbon on a tryptophan side-chain. Aloin is one of the few naturally occurring substances. Glypiation is the addition of a GPI anchor that links proteins to lipids through glycan linkages. Phosphoglycosylation includes a wide range of posttranslational modifications that further increase the diversity of glycoproteins in the proteome such as: Sulfation at Man and GlcNac residues in glycosaminoglycans (GAGs), acetylation of sialic acid and phosphorylation in the Golgi apparatus. Functions To monitor the status of protein folding, acting as a quality control mechanism for properly folded proteins to enter the Golgi trafficking. To mediate cell attachment or stimulate signal transduction pathways through acting as ligands for receptors on the cell surface. To facilitate or prevent proteins from binding to associated interaction domains by affecting protein-protein interactions. To alter the solubility of a protein through hydrophilic interactions.
- Glycosylation post-translational modifications provide as a result an unmatched increase of the proteome diversity which can be attained because almost every aspect of glycosylation can be modified by their enzymes. Glycosidic linkage (site of binding glycan). Glycan composition (types of sugars linked). Glycan structure (branched or unbranched). Glycan length. This diversity is mediated principally by glycosyltransferases (Gtfs) and glycosidases, which bind specifically to particular donor and substrate sugars and act independently of their enzymatic counter-parts. Enzyme availability is highly dependant on enzyme concentrations which is conciliated by cells compartmental divisions (e.g. Golgi cisternaes). As proteins begin to fold, specific amino acids become inaccessible for glycosidic binding, thus only allowing conformationally accesible glycosylation to occur
- Although most glycosylations patterns occur post-translationally, N-glycosylation takes place often co-translationally as the protein is being translated and transported in the endoplasmic reticulum (ER). It’s name denotes that this glycosylation type is covalently bound to carboxamido nitrogens on asparagine residues (Asn or N). While N-glycosylation initially occurs identically for all proteins, it’s diverse capacity occurs at the subsequent trimming and glycan maturation levels. Precursor glycan assembly in the ER Oligosaccharides attached by N-glycosylation linkages are derived from a 14-sugar precursor molecule made of N-acetylglucosamine (GlcNAc), mannose (Man) and glucose (Glc) which are consecutively added into dolichol (polyisoprenoid lipid carrier embedded in the E.R. membrane) in an orderly fashion. In the cytoplasm, UDP- and GDP (sugar nucleotides) bound to dolichol vía pyrophosphate linkage (-PP-) donate the first 7 sugars. When the Man5GlcNAc2-PP-dolichol intermediate is completed, the entire complex is flipped into the lumen of the ER. The final 7 sugars are donated from Man- and Glc-P-dolichol molecules to make the Gcl3Man9GlcNAc2-PP-dolichol precursor glycan. Glycan attachment N- terminal protein synthesis transports the increasingly growing polypeptide into the E.R. and protein folding occurs as the polypeptide enters. As protein folding continues, OSTase is less able to access the consensual sequence for glycan transfer. Interestingly, not every Asn with in-silico predicted consensual sequences are glycosylated.
- Glycan trimming Oligosaccharides are trimmed in the endoplasmic reticulum and the Golgi apparatus by glycosidases vía hydrolysis. In the endoplasmic reticulum Glucosidases I and II remove 2 terminal Glc from the precursor glycan. Calnexin and calreticulin (membrane-bound and soluble sugar-binding lectins) bind to the nascent protein vía Glc and act as chaperones for protein folding. Final Glc is hydrolyzed by glucosidase II releasing the protein from it’s chaperone for proper folding. Non-correctly folded proteins are recognized by UDP-glucose glycoprotein glucosyltransferase which adds a Glc to the glycoprotein cyclically so the protein is bound to lectin chaperones until correct folding and transport to the Golgi apparatus. Final glycan structure for all properly folded glycoproteins that enter the Golgi is: Man9GlcNAc2 in eukaryotes.
- Incorrectly folded proteins in the endoplasmic reticulum ER-resident mannosidase (ERManl) hydrolyzes mannose in a significantly slower rate, allowing proteins múltiple rounds of glycosylation to fold properly before ERManl identifies them as unable to fold. Proteins that lost 3 to 4 mannose residues are transported out of the E.R. by the enzyme glycanase. Delivery to the E.R-associated degradation system (ERADs).
- Glycan maturation in the Golgi apparatus Glycan maturation in the Golgi apparatus combines both trimming and adding sugars to diversy the glycans on individual glycoproteins in each cisternae. The final glycan structures can be separated into 3 groups: Complex oligosaccharides (endo-H resistant) 1.1. Trimmed by mannosidase I and II. 1.2. Glycosylated by GlcNAc transferase in a common core región. 1.3. Múltiple Gtfs transfer sugar moieties from sugar nucleotides to build variable GlcNAc chains, galactose (Gal), sialic acid (NANA) and fucose. High mannose oligosaccharides (endo-H sensitive) Do not carry other sugar moieties but some Man residues are often trimmed by mannosidase I. Hybrid oligosaccharides Combination of complex and high mannose oligosaccharides.
- Glycan maturation in the Golgi apparatus Glycan maturation in the Golgi apparatus combines both trimming and adding sugars to diversy the glycans on individual glycoproteins in each cisternae. The final glycan structures can be separated into 3 groups: Complex oligosaccharides (endo-H resistant) 1.1. Trimmed by mannosidase I and II. 1.2. Glycosylated by GlcNAc transferase in a common core región. 1.3. Múltiple Gtfs transfer sugar moieties from sugar nucleotides to build variable GlcNAc chains, galactose (Gal), sialic acid (NANA) and fucose. High mannose oligosaccharides (endo-H sensitive) Do not carry other sugar moieties but some Man residues are often trimmed by mannosidase I. Hybrid oligosaccharides Combination of complex and high mannose oligosaccharides.
- Contrary to N-glycosylation, O-glycosylation occurs post-translationally by adding sugars one at a time to serine or threonine residues inside the Golgi’s apparatus cisternaes. O-glycosylation can also occur on hydroxylysine and hydroxyproline and is essential in the biosynthesis of mucins and other core proteoglycans that form components of the extracellular matrix. O-GalNac mechanism Proteins trafficked into the Golgi are more often glycosylated by N-acetylgalactosamine (GalNac) transferase. These Gtfs are distributed in diferent expresión profiles according to each cisternae of each cell lineage, acting as a quality control. The build-up process for both N-linked and O-linked can be summarized in 4 processes: 1. Initiation 2. Core extensión 3. Elongacion 4. Capping. O-GalNac transferase GALNT attaches an O-GalNac monosaccharide to a serine, threonine or a tyrosine. Resulting glycan can follow on of 8 to 11 known pathways (core 1- to 4- are the most common). Glycan termini is capped with sialic acid and fucose.
- A. Provide shielding from pro-protease convertases in the trans-Golgi network. B. Select proteins of the immune system that have lectin domains that recognize glycans. C. Protect proteins from ectodomain shedding by ADAM-10 and -17. D. Increase peptide-hormones half-life circulation. E. Modulation of interaction with host surface receptors. F. Effective barrier against pathogens. G. Modulate uptake rate of cargo receptors. H. Regulation of leukocyte extravasation.
- The analysis of Gtfs orchestrating the glycome is currently more tractable than cataloguing the diversity of the glycan structures produced to predict the cellular glycome. Single cell transcriptome and proteome data clearly offers opportunities to asses the repertoire of expressed glycotransferases in individual cells and use this to estimate the total cellular glycosylation capacities based on known glycosylation pathways, to model glycosylation constraints that may affect glycosylation efficiency and ultimately develop reliable predictions for the structural architecture of the cellular glycome, the nature of the glycoproteomes and the structure of glycans themselves.
- Rainbow atlas of human glycosylation pathway maps with assigned functions of 173 glycosyltransferases. Vertical representation of glycosynthesis based on full gene label names.
- Transcriptionally active glycosylation genes and biosynthetic steps. RNAseq data obtained from 51 human tissues to generate a heatmap of the transcriptional regulation of the 173 glycosyltransferase genes, which produce a prediction for the tissue-level variation of each gene. This tissue heatmap of gene regulation was overlaid upon the rainbow depiction with a blue-red scale for each gene. The blue-red heatmap scale indicates high (red) to low (blue) degree of overall organ variation based on the quantitative RNA sequence.
- Vertical positional overlay of glycotransferases genes known to cause congenital disorders of glycosylation (CDGs) as well as their overlay in several databases (light green). Genes known to underlie CDGs have little overlap with gene activity predicted by GWAS analysis, almost mirroring each other. Genes implicated by GWAS overlap with genes exhibiting high tissue regulation in RNAseq analysis. This findings correlate with prior evidence, suggesting that seemingly rare disorders affect fundamental glycosylation pathways that are found to be highly regulated in tissues, on the opposite, GWAS predicted traits associated with poorly -if none- regulated isoenzymes than more often than not have redundant functions.
- GWAS analysis of mice glycosynthesis pathways based on 115 knockout models.
- Current tool-box for N- and O- glycan functional studies Glycoengineered cells used to express N- or O-glycoproteins with different glycan structures that can be isolated and screened in binding or functional assays. Same studies can be performed on cells grown on 2D culture so that these functions can also be monitored directly. Cells can be harvested and applied in cell-based glycan arrays where specific glycan binding patterns can be decrypted. Cells can be grown in organotypic cultures, allowing for tissue-specific research questions and expression of distinct layers. KI/KO animal models to evaluate glycan functions and their significance in disease models.
- Over 90% IgG mAbs are N-glycosylated (>95% are N- or O- glycosylated). The antibody glycan precursor Glc3Man9GlcNAc2 is transferred onto the antibody polypeptide in the endoplasmic reticulum.
- The glycosylated polypeptide is then transported by COPII to the Golgi apparatus where it’s trimmed and extended into a classical biantennary structure, first into Glc4Man3 precursor then extended by one of four gtfs: FUT8 (fucose), B4GALT1 (galactose), MGAT3 (b-GlcNAc) and ST6GAL1 (sialic acids). Functional studies regarding glycosylation of mAbs are nevertheless obscured. Prior results have shown that total or partial glycan deficiencies will inevitably affect the addition of other sugars. Galactose deficiency: Decrease syalation. B-GalNAc: Decreases fucose levels because of hindrance of the enzyme. Agalactosylation: Produces mannose-binding lectins directly to core 1 GlcNAc. Hypergalactosylation: Drives ADCC vía enhanced FcgRIIIa binding. Afucosylation: Increases ADCC by enhancing FcgRIIIa binding through the FcgRIIIa glycan.
- IgG antibodies have a single N-linked glycan attached at asparagine 297 (Asn297) in the CH2 domain of each heavy chain which impacts confirmation through glycan-protein and glycan-glycan interactions. The shape of the Fc región in mAbs defines the capacity of the antibody to interact with innate immune Fc receptors, which direct antibody functionality. Humans have six classical Fc receptors (Fc𝝲RI, Fc𝝲RIIa, Fc𝝲RIIb, Fc𝝲RIIc, Fc𝝲RIIIa, and Fc𝝲RIIIb), found in varied combinations on all immune cells.
- Fc domains also interact with complement proteins (C1q and mannose-binding lectin), other C-type lectin receptors with effector and signaling functions, as well as the neonatal FcRn receptor involved in antibody recycling. Collectively, these interactions regulate numerous actions ranging from innate and adaptive immune cell responses (ADCP, ADCC, CDCC) as well as anti-inflammatory activity. Humans possess four IgG subclasses and 36 possible antibody glycans, of which over 30 have been observed by mass spectrometry. The potential combinatorial diversity of the constant domains thus allows up to 144 possible combinations of Fc regions for each specificity and, theoretically, 144 functional states. To further increase this proteomic diversity, IgG antibodies can be glycosylated asymmetrically, leading to a striking total of 288 possible efector combinations for IgG only (70% of IgG mAbs are asymmetrically glycosylated).
- Glycosylation is actively modulated during inflammatory response, physiologically many cellular pathways shape antibody glycosylation such as: Glycosyltransferase/glycosidase expression. Monosaccharide availability. Hormones. Golgi’s pH. Golgi’s organization. Availability of vesicular transport machinery. Age. Pregnancy. Sex.
- Rheumatoid arthritis Agalactosylated antibodies are present before early symptoms causing synovitis by accumulating within joints, activating FcgRIIa, TNF-alpha and IL-6. HIV Infection shows dramatic and persistent changes in antibody glycosylation. Hypergammaglobulinemia immune activation is linked to an enrichment of agalactosylated antibodies. Contrary to RA where galactosylation levels normalize after flares resolve, HIV-infected patients with controlled antiretroviral therapy maintain their profile over time. These data suggests that B cells are permanently altered. Furthermore, following vaccination with an HIV experimental vaccine across the globe, patients show nearly identical glycosylation profiles despite differences in the total glycosylation, suggesting pathogen-specific antibodies are regulated independently of overall inflammatory changes on total circulating antibodies. These studies evidence immunological control in antibody glycosylation. This control is likely driven by regulation of antigen-specific B cells through antigen exposure at priming and recall.
- Since each IgG posses only one N-glycosylated glycan per Fc independently of the IgG subclass. Fc glycan edition will now be increasingly useful to obtain desired targeted aims (modulation of pro- or anti-inflammatory states by glycoengineered hybridomas). As an example, recently developped glycoengineered IgG antibodies for cancer therapy eliminated fucose on the Fc glycans in order to improve efficacy in killing tumours.
- Since each IgG posses only one N-glycosylated glycan per Fc independently of the IgG subclass. Fc glycan edition will now be increasingly useful to obtain desired targeted aims (modulation of pro- or anti-inflammatory states by glycoengineered hybridomas). As an example, recently developped glycoengineered IgG antibodies for cancer therapy eliminated fucose on the Fc glycans in order to improve efficacy in killing tumours.
- Since each IgG posses only one N-glycosylated glycan per Fc independently of the IgG subclass. Fc glycan edition will now be increasingly useful to obtain desired targeted aims (modulation of pro- or anti-inflammatory states by glycoengineered hybridomas). As an example, recently developped glycoengineered IgG antibodies for cancer therapy eliminated fucose on the Fc glycans in order to improve efficacy in killing tumours.
- Since each IgG posses only one N-glycosylated glycan per Fc independently of the IgG subclass. Fc glycan edition will now be increasingly useful to obtain desired targeted aims (modulation of pro- or anti-inflammatory states by glycoengineered hybridomas). As an example, recently developped glycoengineered IgG antibodies for cancer therapy eliminated fucose on the Fc glycans in order to improve efficacy in killing tumours.