Glycosylation Flashcards
What are the problems with therapeutic proteins?
Low activity
Rapid Clearance
Immunogenicity
What characteristics do carbohydrates affect?
Solubility Stability In vivo activity Immunogenicity Bioavailability Pharmacokinetics
What two cellular components are involved in glycosylation?
ER and Golgi
What is a glycoform?
All the different forms of a glycoprotein (with differing oligosaccharide structures). No set structure - influenced by cell metabolism, not genome - creates regulatory difficulty
What are the main types of glycosylation?
N Linked
O Linked
What is the main sugar found in yeast?
Mannose
What is N Linked Glycosylation?
Asparagine NH side group binds to sugar via N glycosidic bond
What is the consensus sequence in N Linked Glyco
ASN-X-Ser/Thr (X is any AA except Pro/Asp)
What are the 3 types of N Glycosylation?
Mannose Rich
Hybrid
Complex
What is the pentasaccharide core?
2 GlcNac, 3 Mannose
What are the main enzymes involved in N-linked glycol?
Dolichol Phosphotase Flipase Oligosaccharyltransferase Glucosidase Glucosyltransferase
What is O-lnked glyco?
Attaches to the OH group of either serine/threonine, hydroxylysine, hydroxyproline
For serine/threonine, GalNac attaches using GalNac Transferase
What are the main differences between the 2 types?
N Linked - occurs mid translation of protein, in the ER, using flips, inhibited by tunicamycin (antibiotic), consensus sequence, core structure
O-Linked - occurs once final protein produced - no consensus sequence
How do glycoproteins affect protein function?
- Biological activity (Thermal stability, protease protection, solubility)
- Receptor Binding (EPO)
- Ab Function (Fc region has 2 N-linked glycoproteins)
- Pharmacokinetics (In vivo potency - FSH)
- Folding (CD4)
Discuss examples of therapeutic glycoproteins
hGH - non glycosylated - e.coli - child deficiency, AIDS, operation, sport
Insulin - Novorapid, Lantus (Asn)
Interferon (Type B - Fibroblasts) - Betaseron, Avonex (glycosylated)
