Bgm3039 Glycosylation

Front 1

What proportion of marketed therapeutic proteins are glycoproteins?

Back 1

more than 2/3rds

Front 2

What determines the biological activity of most therapeutic proteins?

Back 2

Its amino acid sequence

Front 3

How many human proteins are glycosylated?

Back 3

50%

Front 4

What does the glycan component of a recombinant glycoprotein affect?

Back 4

1. Pharmacokinetics

2. Bioactivity

3. Secretion

4. In vivo clearance

5. Solubility

6. Recognition

7. Antigenicity

Front 5

What is the difference between glycosylation and glycation?

Back 5

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.

Front 6

Define glycosylation?

Back 6

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.

Front 7

What is a glycoform?

Back 7

Any of several different forms of a glycoprotein having different saccharides attached, or having a different structure. Glycoproteins exist as heterogenous populations of molecules.

Front 8

What are the complications that arise as a result of glycosylation?

Back 8

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.

Front 9

What are the 2 main glycan linkages to proteins?

Back 9

N-glycosidic

O-glycosidic

Front 10

How manydifferent N-glycan linkages have been reported?

Back 10

5

Front 11

What are the 3 types of N-glycans?

Back 11

- High mannose

- Hybrid

- Complex

Front 12

What is the most common N-glycan linkage?

Back 12

N-acetylglucosamine to asparginine

(GlcNacβ1-Asn)

Front 13

How many residues do high mannose oligosaccharides have?

Back 13

3 - 60 mannose residues

arranged as mono- bi- tri- tetra- penta- antennary structures

Front 14

What is the precursor of N-glycans?

Back 14

Lipid (dolichol) linked to a glycan by a pyrophosphate bond

Front 15

What is a dolichol?

Back 15

A polyisoprenoid lipid carrier utilized during the assembly of N-glycans and GPI anchors.

Front 16

How is glycosylation initiated?

Back 16

Theglycosylation is initiated in the ER where the N-glycan precursor is attachedthe the Asn-X-Ser/Thrconsensus region by the enzyme oligosaccaryltransferase.

Front 17

What is an O-glycan?

Back 17

A single N-acetylgalactosamine residue linked to the hydroxyl group of serine or threonine.

Front 18

What is usually the first residue of O-glycans?

Back 18

N-acetylgalactosamine (GalNAc)

Front 19

What is the importance of glycosylation?

Back 19

1. Biological activity

2. Protein folding

3. Receptor binding

4. Antibody effector function

5. Pharmacokinetics, clearance and half-life

Front 20

What change to the biological activity does glycosylation have?

Back 20

- Enhance thermal stability

- Provide protection from proteases

- Improve stability

- Inhibit aggregation of proteins

Front 21

Give an example of the change in antibody effector function from glycosylation?

Back 21

Antibodies with low fucose enhance NK cell activity

Antibodies with high fucose content more effectively recruit polymorphonuclear cells

Front 22

How can glycosylation alter FSH?

Back 22

FSH has a short half life

- Adding glycosylation will create long-acting FSH

- Adding mannose reduces the half life

Front 23

Give some examples of glycosylation in therapeutic proteins?

Back 23

- Growth hormone (hGH)

- Insulin

- Haemophilia B

- Interferons (IFN-α, IFN-β, IFN-γ)

Front 24

How is hGH changed?

Back 24

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)

Front 25

Insulin

Back 25

Insulin was first derived from cows and pigs but caused inflammatory response

Insulin can now be made by recombinant DNA technology in E.coli

Front 26

What causes Haemophilia B?

Back 26

Deficiency of Factor XI

Front 27

What are interferons?

Back 27

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.

Front 28

What are the 3 main types of interferons?

Back 28

B-lymphocytes - IFN-α,

Fibroblasts - IFN-β,

T-lymphocytes - IFN-γ

Front 29

Give some examples of IFN-β glycosylation?

Back 29

- Bateseron is non-glycosylated

- Avonex is glycosylated with 10x higher specificity allowing for lower doses and lower immunogenicity