Glycosylation

Question 1

What are glycoproteins?

Answer 1

Proteins with glycans or sugars attached are glycoproteins. Many biopharmaceuticals are glycoproteins. e.g. Rituximab; anti-CD20 mAb

Question 2

Describe glucose.

Answer 2

D-glucose has 6 carbons and can be linear or cyclical. It has an alpha and a beta from depending on the position of OH and H on C1. Beta is more stable due to OH’s being more spread out.

Question 3

Describe the sugars that are produced from glucose as a starting point.

Answer 3

#D-galactose - OH and H of C4 swapped
#D-mannose - OH and H of C2 swapped
#GlcNAc - OH changed to NHCOCH3 at C2
#GalNAc - OH and H of C4 swapped and OH changed to NHCOCH3 at C2
#Sailyic acid - C1--> COO-; C5 --> NHCOCH3; C6 - Glycerol
#Others include D-glucoronic acid, L-fucose, D-xylose

Question 4

How do sugars link together?

Answer 4

Sugars are linked by glycosidic bonds, joined by condensation reaction. Beta1-4 is a common linkage. It requires energy so that precursor is attached to UDP.

Question 5

Describe N-linked glycans.

Answer 5

Use a universal symbol list that doesn’t show linkages but is easy to understand.
Starts with Asn-X-Ser/Thr

Question 6

What type of glycans do different organisms produce?

Answer 6

Yeast - high mannose
Insects - pauci-mannose
plants - xylose
animals - terminal Neu5GC/Neu5AC
humans - terminal Neu5AC

Question 7

Describe O-glycosylation

Answer 7

O-glycosylation occurs and it adds glycans to serine or threonine. The only glycosylation in the cytoplasm in an O-GlcNAc attached to Ser/Thr to regulate function.

Question 8

Why glycosylate?

Answer 8

• provides structural components
• modify protein properties
• directs trafficking of glucoconjugates
• mediating cell adhesion
• mediating cell signalling

Question 9

Describe 2 glycoprotein examples

Answer 9

HIV-1-Env –> sugars to protect it from the immune system.

Human alpha-L-iduronidase –> uses N-glycan to support substrate binding and act as a catalytic module.

Question 10

Describe glycoprotein biosynthesis

Answer 10

Lipid-linker precursors are synthesised:

• dolichol-phosphate sits in the ER membrane close to ribosomes
  -UDP-GlcNAc attaches 2 GlcNAcs to dolichol
  -GDP-Man attaches 4 mannose sugars to the chain
  -it is flipped insie the ER
  -mannose and glucose are made on the outside, flipped inside then transferred
  #En-bloc transfer using N-oligosaccharide transferase
  #Processing using temporal, spatial and sugar recognition as specificity methods.
  #Secretion of delivery to the membrane

Question 11

Describe folding quality control

Answer 11

#unfolded proteins have 3 glucose residues
#2 are trimmed, the final one binds to calnexin and attempts to fold it
#If the correct shape forms the glucose is cleaved and it leaves the ER
#If the wrong shape forms glucose is reattached by glucosyl transferase so it can try again
#eventually a chaperone ubiquinates and ejects it from the ER for degredation

Question 12

What is glycan analysis?

Answer 12

Release glycans using chemicals or enzymes eg PNGase to remove form peptide or endoH is high mannose
Use mass spec or NMR to determine the structures etc

Question 13

What can biopharmaceuticals treat, give examples of protein treatments.

Answer 13

Metabolic disorders, fertility, thrombosis etc
Insulin
Beta-glucocerebrosidase
Interferons
Monoclonal antibodies

Question 14

Describe the pros and cons of CHO cells

Answer 14

CHO cells are becoming more popular than E.coli.
Pros
- safe host: no replication of HIV, influenza, herpes, polio
- suitable for bioreactor, fed-batch
-GRAS; has regulatory approval
-compatible glycosylation
Cons
-growth medium may contain animal-derived materials
-lengthy
-expensive
-glycosylation depends on fermentation conditions

Question 15

What are the key performance criteria?

Answer 15

Titer
Product quality
Continuous system (if possible)
Meet regulations of the industry.

Question 16

Describe the process of making a high titer CHO strain.

Answer 16

Transfection
Selection
Recovery
Amplification
Screening
Expansion
Growth evaluation
Cell banking

Question 17

Describe how plant cells have been used in Israel.

Answer 17

Carrot root cells used to produce glucocerebrosidase

Question 18

Describe Pichia pastoris as a glycoengineering example

Answer 18

• Works in bioreactors

- Deleted yeast-specific modification so human-like glycosylation can occur while maintaining a good activity level.

Question 19

Describe the herceptin example

Answer 19

P.pastoris converts a glycan using knock-outs, new enzymes and adding sugars not normally present.

Question 20

Describe the erythropoietin example

Answer 20

Investigation to see if having more than 3 glycan increased the stability and half-life. –> successful.
Involved changing the sequence but means more red blood cells.