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29 Cards in this Set
- Front
- Back
What proportion of marketed therapeutic proteins are glycoproteins? |
more than 2/3rds |
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What determines the biological activity of most therapeutic proteins? |
Its amino acid sequence |
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How many human proteins are glycosylated? |
50% |
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What does the glycan component of a recombinant glycoprotein affect? |
1. Pharmacokinetics 2. Bioactivity 3. Secretion 4. In vivo clearance 5. Solubility 6. Recognition 7. Antigenicity |
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What is the difference between glycosylation and glycation? |
Glycosylation is enzyme-mediated ATP-dependent attachment of oligosaccharides to protein molecules at defined sites on the target molecule. Glycation is the result of the typically covalent bonding of a sugar molecule, such as glucose or fructose, to a protein without the controlling action of an enzyme. |
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Define glycosylation? |
Enzyme-mediated ATP-dependent attachment of oligosaccharides to protein molecules at defined sites on the target molecule during synthesis and processing through the ER and Golgi apparatus. |
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What is a glycoform? |
Any of several different forms of a glycoprotein having different saccharides attached, or having a different structure. Glycoproteins exist as heterogenous populations of molecules. |
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What are the complications that arise as a result of glycosylation? |
Glycolsylation is not pre determined by and template, like DNA is for proteins. Therefore a variety of structures are possible for any one protein. This presents difficulty to the industrial production and for regulatory approval of therapeutic glycoproteins. |
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What are the 2 main glycan linkages to proteins? |
N-glycosidic O-glycosidic |
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How manydifferent N-glycan linkages have been reported? |
5 |
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What are the 3 types of N-glycans? |
- High mannose - Hybrid - Complex |
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What is the most common N-glycan linkage? |
N-acetylglucosamine to asparginine (GlcNacβ1-Asn) |
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How many residues do high mannose oligosaccharides have? |
3 - 60 mannose residues arranged as mono- bi- tri- tetra- penta- antennary structures |
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What is the precursor of N-glycans? |
Lipid (dolichol) linked to a glycan by a pyrophosphate bond |
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What is a dolichol? |
A polyisoprenoid lipid carrier utilized during the assembly of N-glycans and GPI anchors. |
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How is glycosylation initiated? |
Theglycosylation is initiated in the ER where the N-glycan precursor is attachedthe the Asn-X-Ser/Thrconsensus region by the enzyme oligosaccaryltransferase. |
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What is an O-glycan? |
A single N-acetylgalactosamine residue linked to the hydroxyl group of serine or threonine. |
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What is usually the first residue of O-glycans? |
N-acetylgalactosamine (GalNAc) |
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What is the importance of glycosylation? |
1. Biological activity 2. Protein folding 3. Receptor binding 4. Antibody effector function 5. Pharmacokinetics, clearance and half-life |
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What change to the biological activity does glycosylation have? |
- Enhance thermal stability - Provide protection from proteases - Improve stability - Inhibit aggregation of proteins |
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Give an example of the change in antibody effector function from glycosylation? |
Antibodies with low fucose enhance NK cell activity Antibodies with high fucose content more effectively recruit polymorphonuclear cells |
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How can glycosylation alter FSH? |
FSH has a short half life - Adding glycosylation will create long-acting FSH - Adding mannose reduces the half life |
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Give some examples of glycosylation in therapeutic proteins? |
- Growth hormone (hGH) - Insulin - Haemophilia B - Interferons (IFN-α, IFN-β, IFN-γ) |
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How is hGH changed? |
Its principle form is a 191aa non-glycosylated protein of 22kDa; glycosylation made a recombinant hGH and allowed therapeutic use in - children with hGH deficiency - enhancing post-surgical repair in adults - slows wasting in AIDS - used by athletes (but now banned) |
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Insulin |
Insulin was first derived from cows and pigs but caused inflammatory response Insulin can now be made by recombinant DNA technology in E.coli |
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What causes Haemophilia B? |
Deficiency of Factor XI |
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What are interferons? |
Signalling proteins made and released by host cells in the presence of pathogens, such as viruses, parasites and tumour cells. They are antiviral and retard the growth of tumour cells. |
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What are the 3 main types of interferons? |
B-lymphocytes - IFN-α, Fibroblasts - IFN-β, T-lymphocytes - IFN-γ |
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Give some examples of IFN-β glycosylation? |
- Bateseron is non-glycosylated - Avonex is glycosylated with 10x higher specificity allowing for lower doses and lower immunogenicity |