EPO

Question 1

What are 4 structural characteristics of EPO?

Answer 1

165 aa
2 disulfide bonds
3 N-glycosylation sites
1 O-glycolystaion site

Question 2

What does EPO stimulate?

Answer 2

Survival, proliferation, differentiation of erthryocyte precursors

Question 3

Where is EPO produced in adults?

Answer 3

Kidneys in peritubular fibroblasts in renal cortex

Question 4

Where is EPO produced in the fetus?

Answer 4

Liver

Question 5

How does hypoxia cause EPO production?

Answer 5

HIF-1B/HIF-2a binds to HRE

Question 6

What are the 2 proline residues found in HIF2a C terminus?

Answer 6

Pro405
Pro531

Question 7

What are the proline residues hydroxylated by?

Answer 7

alpha ketoglutarate requiring dixoygenases in O2

Question 8

What is HIF’s asparagine residue hydroxylated by?

Answer 8

FIH

Question 9

What does EPO promote in bone marrow?

Answer 9

Proliferation & differentiation of erythrocytic progenitors like CFU-Es)

Question 10

What are characteristics of EpoR?

Answer 10

59 kDA glycoprotein (484 aa & 1 N-glycan) that acts as a homodimer

Question 11

What does Epo binding cause?

Answer 11

Intracellular activation of EpoR-associated JAK-2 -> tyrosine phosphorylation

Question 12

What receptor family is EpoR a part of?

Answer 12

type 1 cytokine receptor

Question 13

What is signal transduction through EpoR intiated by?

Answer 13

Ligand binding -> dimerization/reorientation of EpoR monomers

Question 14

Explain the steps following EpoR activation?

Answer 14

JAK2 phosphorylates Tyr residues in EpoR -> docking sites for signalling molecules with phosphotyrosine binding motifs -> STAT5

Question 15

When is the action of EPO terminated?

Answer 15

When EpoR is dephosphorylated by SHP-1 -> complex is internalized

Question 16

What is recombinant human EPO used for?

Answer 16

Treatment of different types of anemia

Question 17

What do Epoietins have to be produced in and why?

Answer 17

mammalian cells (CHO) as EPO is a glycoprotein

Question 18

What is a structural difference in Epoietins to endogenous EPO?

Answer 18

glycans have minor differences

Question 19

What 3 things influence glycosylation?

Answer 19

host cell species & clone
culturing process
purification steps

Question 20

What does the major N-glycan structure of urinary hEPO consist of?

Answer 20

fucose-containing sialylated tetra-antennary glycan

Question 21

What is the in-vivo half-life of glycoproteins determined by?

Answer 21

Sialic acid capping in terminal sugar on N-linked glycans

Question 22

What is responsible for the biological activity of rhEPO in vivo?

Answer 22

Terminal sialylation level

Question 23

What do sialylations mask?

Answer 23

Penultimate galactose residues -> captured by asialoglycoprotein receptors in hepatocytes -> degradation

Question 24

What are 3 pros of glycosylations?

Answer 24

Protect protein from proteolysis
enhance overall solubilty
modulate receptor binding

Question 25

How is glycosylation optimized?

Answer 25

Engineering of EPO Engineering of production hosts culture condition optimization

Question 26

What's an example of hyperglcosylation?

Answer 26

Darbepoetin alfa endowed with 2 extra N-glycans -> 22 sialic acid residues -> increase in plasma half-life & enhanced in vivo activity

Question 27

Whats an example of PEGylation?

Answer 27

ehEPO to PEG -> CERA -> half life of 130hrs

Question 28

What are 3 target points for enhanced sialylation?

Answer 28

IC level of nucleotide sugar CMP-sialic acid transportation of CMP-sialic acid to Golgi complex sialylation of glycans

Question 29

What are 3 modes of culture production?

Answer 29

batch fed-batch perfusion

Question 30

What is used for large-scale production of recombined glycoproteins?

Answer 30

fed-batch & perfusion

Question 31

What can affect the culture performance & glycosylation profile?

Answer 31

culture temp pH

Question 32

What does high conc. of ammonia alter?

Answer 32

Glycosylation pattern of rhEPO by decreasing terminal sialylation, content of O-linked glycan & tetra-antennary of N-glycan

Question 33

What affects the incidence & degree of immunogenicity in Epoietins?

Answer 33

product-specific & patient specific parameters

Question 34

What are 5 modification in place of glycans in SEP?

Answer 34

chemoselective linker hydrophilic spacer consisting of 1 polymer repat unit core structure with 4 branch points linear polymer with 12 repeat units attached to each branch point -ve charge-control unit at end of each linear polymer

Question 35

What are 3 yeilds of SEP?

Answer 35

solid phase peptide synthesis 15-30% oximation 30-50% NCL 40-70% over 100mg of SEP obtained

Question 36

What is SPPS?

Answer 36

stepwise assembly based on repetitive process resulting in elongation of a polymer-bound peptide chain by successive aa additions until peptide of desired sequence & length has been synthesized

Question 37

What are 3 benefits of using E.coli for EPO engineering?

Answer 37

High protei yield low cost no post-translational glycosylation however non-glycosylated proteins are less stable & prone to aggregation

Question 38

What is used for chem-selective conjugation of defined carbohydrate moieties?

Answer 38

copper-catalyzed azidde-alkyne cycloaddition

Question 39

What was found after Plk was introduced to glycosylated EPO variants?

Answer 39

Biologically active successful induction of differentiation into erythrocyte precursor glycans remain intact after CuAAC

Question 40

What are 5 advantages of the Staudinger Phosphite Reaction?

Answer 40

site-specific modification simple reaction without toxic additives tolerant against denaturing salts & high temps can be performed in semi-purified samples results in coupling of branched PEG -> improved protection

Question 41

What does a single short-branched-PEG chain enhance?

Answer 41

resistance against proteolysis unsepcific aggregation

Question 42

What are structural features of human interferon-gamma?

Answer 42

143 aa dimeric structure no disulfide bonds

Question 43

What is human interferon gamma used for?

Answer 43

critical for innate & adaptive immunity approved for chronic granulomatous disease & malignant osteoetrosis anticancer applications

Question 44

What happens after ligandbing to IFN-yR?

Answer 44

JAk2 autophosphorylated -> activated -> JAk1 transphorylation

Question 45

What are 3 signalling molecules activated by IFN-y?

Answer 45

ICAM-1 MIG iNOS

Question 46

What is used for semi-synthesis of homogenous hIFy glycocongugates and why?

Answer 46

PoxF -> high protein yields & reproducible incorporation experiments

Question 47

What are the 6 steps in semi-synthesis?

Answer 47

recovery from inclusion bodies affinity chromotography refolding size exclusion chromotography CuAAc size exclusion chromotography

Question 48

What was the result of using the nnAA PoxF?

Answer 48

too disruptive for scaffold of hIFy triazole linker formed after CuAAC is not tolerated cytokines are not robust model proteins