EPO + IFNy Flashcards
what role do structural disulphide bonds play in the function of EPO?
the two structural disulphide bonds stabilise the 3D conformation of EPO, essential for its biological activity in promoting erythropoiesis
where are the two disulphide bonds in EPO?
Cys7-Cys161 and Cys29-Cys33
how does hypoxia influence EPO production?
hypoxia triggers EPO production through the activation of HIF-1beta/HIF-2alpha transcription factor complex which binds to HRE in the EPO gene
what transcription factors are involved in EPO production in hypoxia?
HIF-1beta
HIF-2alpha
HRE
how does EPO binding activate the EPO receptor?
EPO binding induces the dimerisation of EpoR, activating JAK-2, which phosphorylates tyrosine residues, triggering downstream signalling pathways like JAK/STAT
what are the differences between first-generation and second-generation recombinant EPO products?
1st gen mimics endogenous EPO, 2nd gen are hyperglycosylated for increased half-life but lower receptor binding affinity
what is an example of a second-generation rhEPO?
darbepoetin alpha
what is an example of a first-generation rhEPO?
epoetin alpha
what is the role of sialylation in in vivo activity of rhEPO?
it prevents rapid clearance by masking galactose residues which would otherwise be recognised by asialoglycoprotein receptors in the liver, thus prolonging EPOs half-life
what is the significance of hyperglycosylation in darbepoetin alpha compared to standard EPO?
it adds extra N-linked glycans, increasing sialic acid content which extends the half-life of the protein in circulation
how does PEGylation affect the PK of EPO?
it extends its half-life by increasing its size, reducing renal clearance, and enhancing its resistance to proteolysis as seen in CERA
why are mammalian cells, such as CHO, used for production of recombinant EPO?
because EPO is a glycoprotein, and these cells can perform complex glycosylations that are essential for its biological activity and proper folding
what is the impact of glycosylation micro-heterogeneity on the therapeutic activity of recombinant EPO?
it can lead to variability in the PK and biological activity of EPO as different glycoforms can have different half-lives and receptor affinities
what are the clinical applications of recombinant EPO and what types of anaemia does it treat?
recombinant EPO is used to treat anaemia associated with CKD, chemotherapy and certain HIV treatments by stimulating erythropoesis
how does the presence of CMP-sialic acid in the Golgi apparatus influence EPO glycosylation>
this is the donor molecule for sialylation. Its availability in the Golgi is critical for proper terminal glycosylation of EPO, affecting its stability and half-life
what is the principal signalling pathway activated by EPO binding to EpoR?
JAK/STAT
what are the main strategies used to enhance the sialylation of recombinant EPO in production?
genetic engineering of host cells to over-express sialyltransferases and optimising culture conditions to enhance the availability of CMP-sialic acid
what is the role of Src homology phosphatase-1 (SHP-1) in terminating EPO signalling?
SHP-1 dephosphorylates tyrosine residues on EpoR and associated signalling molecules, terminating teh signal and preventing overactivation of the erythropoietic response
how does ammonia accumulation in cell culture affect the glycosylation pattern of rhEPO?
high ammonia levels can reduce terminal sialylation and alter glycan branching, which decreases the half-life and activity of rhEPO
what is the molecular weight of EPO
30.5kDa
how many amino acids does EPO have?
165
what does HIF stand for?
Hypoxia Inducible Factor
what does HIF do in EPO production?
it binds to HRE in low oxygen conditions (hypoxia) which stabilises HIFa and allows for EPO transcription. Under normal oxygen conditions, HIFa is hydroxylated, leading to its degradation preventing EPO production
what is the effect of removing N-linked glycans on the in vivo half-life of rhEPO?
reduces half-life from 7-8 hours to 2 minutes
