Microbial transglutaminase-mediated formation of erythropoietin-polyester conjugates

Erythropoietin (EPO) is a glycoprotein hormone that has been used to treat anemia in patients with chronic kidney disease and in cancer patients who are receiving chemotherapy. Here, we investigated the accessibility of the glutamine (Gln, Q) residues of recombinant human erythropoietin (rHuEPO) towards a thermoresistant variant microbial transglutaminase (mTGase), TG¹⁶ with the aim of developing novel rHuEPO conjugates that may potentially enhance its biological efficacy. As a model bioconjugation, we studied the reactivity of rHuEPO towards TG¹⁶ with a low molar mass amine group containing substrate, monodansyl cadaverine (MDC). The reactions were carried out at a T_m of 54.3 °C, the transition temperature of rHuEPO. Characterization by SDS-PAGE and mass spectrometry confirmed the conjugates formation. Then, we examined the conjugation of rHuEPO with a biodegradable and biocompatible polyester, poly(D-sorbitol adipate) (PDSA). To achieve this, PDSA was enzymatically synthesized using lipase B from Candida antartica (CAL-B), chemically modified with side chains having free primary amine (NH₂) groups that can be acyl acceptor substrate of TG¹⁶, thoroughly characterized by ¹H NMR spectroscopy, and then applied for the TG¹⁶-mediated conjugation reaction with rHuEPO. rHuEPO conjugates generated by this approach were identified by SDS-PAGE proving that the amine-grafted PDSA is accepted as a substrate for TG¹⁶. The successful conjugation was further verified by the detection of high molar mass fluorescent bands after labelling of amine-grafted PDSA with rhodamine B-isothiocyanate. Overall, this enzymatic procedure is considered as an effective approach to prepare biodegradable rHuEPO-polymer conjugates even in the presence of N- and O-glycans.