Synthesis of neo-glycosylated human erythropoietin and investigation into interaction with the erythropoietin receptor

  • Dominique Richardson

Student thesis: Phd


Erythropoietin (EPO) is the glycoprotein hormone responsible for regulating the production of erythrocytes. EPO also plays a cytoprotective role in a variety of tissues, including the brain and heart, by preventing apoptosis of healthy cells after ischemic injury (tissue protection). Recombinant human EPO (rhEPO) used clinically to treat anaemia is unsuitable for administration as a tissue protective agent due to the adverse side effects associated with overstimulation of erythropoiesis. As such, there have been a number of attempts to develop a tissue protective EPO derivative by eliminating erythropoietic function. Reported here is the synthesis of neo-glycosylated human erythropoietin and investigation into the effect this modification has on the interaction of EPO with its erythropoietic receptor (EPOR)2. In order to develop a non-erythropoietic EPO derivative, cysteine residues were introduced into the EPO sequence to act as chemical modification sites. EPO variants were then glycosylated at the cysteine residues using a one-step synthesis with alpha-mannosyl iodoacetamide before analysis by ELISA. Residues located within the two binding sites of EPO were targeted for mutation, and a total of 13 EPO variants were generated and expressed from E. coli. Upon expression and purification, EPO cysteine variants were glycosylated with alpha-mannosyl iodoacetamide. Reaction of single EPO variants yielded a mixture of unmodified and mono-glycosylated EPO species. Reaction of the double cysteine EPO variants yielded a mixture of three species; the unmodified EPO, the mono-glycosylated EPO and the di-glycosylated EPO. To determine the effect of this glycosylation on the EPO-(EPOR)2 interactions, an ELISA based assay was developed. Initially, all EPO variants were screened by ELISA for WT-like binding to the (EPOR)2. Of the 13 variants screened only the K45C+K97C EPO variant was used in the proof-of-concept study. Comparison of the WT EPO before and after reaction with the alpha-mannosyl iodoacetamide on the ELISA showed no inhibition of the EPO-(EPOR)2 interactions while comparison of the K45C+K97C EPO before and after reaction showed a statistically significant difference in the EPO-(EPOR)2 binding. This outcome indicates that it is possible to inhibit the EPO-(EPOR)2 binding by introducing non-natural glycosylation sites in to the EPO sequence
Date of Award31 Dec 2014
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorSabine Flitsch (Supervisor)

Cite this