Geschäftsstelle des SPP 1623
Tel.: 030 94793 180
Fax: 030 94793 188
Robert-Rössle Str. 10, Geb. 81
The treatment of cancer is one of the most urgent and important challenges in medicine and society. Human Erythropoietin (EPO) promises great potential for attenuating negative side effects of chemotherapies and therefore to improve patients’ prognosis. EPO is a globular glycoprotein that plays a crucial role in the maturation of erythroid cells from precursors. It consists in a single domain with 165 amino acids in the mature form, and it contains 3 N-linked glycosylation sites at the positions Asn24, Asn35 and Asn83 and one O-linked glycosylation site (Ser126). The glycans linked to EPO have a marginal effect on the binding to the receptor, but they are determinant for maintaining the protein in circulation thus preventing blood clearance. Moreover, they enhance the solubility of the protein due to their polar character and negatively charged sialic acids and mask potential immunogenic sites on the protein sequence. Both from the pharmacological and from the biochemical side it is essential to create stable EPO variants with homogeneous glycosylation patterns. Current synthesis approaches are limited either by poor yields or by the high heterogeneity of attached sugars.
In our approach, recombinant non-glycosylated EPO will be produced in E. coli. We propose to exploit different non-natural amino acids to (i) improve the stability of non-glycosylated EPO and (ii) to provide orthogonal sites for the chemo-selective conjugation of carbohydrate moieties by “click” chemistry. EPO variants with fluorinated amino acids and complex tetra-antennary glycans or combinations thereof will be synthesized and studies will be conducted to correlate the amino acids and sugars with the stability of these analogs against thermal unfolding, aggregation, and proteolysis as well as with their bio-distribution and erythropoietic potential. These results should help to elucidate the interaction between glycans and proteins that is still poorly understood. In the long term, the gained insights from this project and the developed molecular “toolbox” may open up ways to increase the efficiency of EPO administration and guide future approaches for the synthesis and systematic optimization of therapeutic and other proteins with post-translational modifications.