Promotion of N-linked glycan terminal sialilation of recombinant equine chorionic gonadotrophin (reCG) through exogenous expression of ST6GAL1 protein (ST6 beta-galactoside alpha-2,6-sialyltransferase)

Abstract

Equine chorionic gonadotrophin (eCG) is a glycoprotein hormone synthesized by placental cells during the beginning of the mare gestational period. An important eCG biological activity is its capability to promote ovulation in some mammals due to its molecular similarity with the luteinizing hormone (LH). Due to this biological property, this hormone is often used in cattle's reproductive processes by the livestock sector. Currently, eCG is obtained by purification from blood drawn from pregnant mares. This method presents low efficiency and reproducibility, in addition to its utilization being questionable due to ethical issues. Therefore, the demand for an in vitro production of eCG is evident, with the establishment of a controlled and more efficient production method. The recombinant eCG (reCG) was developed in our laboratory and its production was established in the CHO-DG44 (Chinese hamster ovary cells) cell lineage cultured in suspension, under Good Manufacturers Practice (cGMP). In vivo experiments to test the biological activity of the recombinant hormone showed that it displays a lower activity in comparison with the natural eCG. It is believed that this is mainly due to differences in the glycosylation profile, which can significantly change reCG's molecular pharmacokinetics. CHO-DG44 cells do not express the ST6GAL1 (ST6 beta-galactosídeo alpha-2,6-sialiltransferase) enzyme, which is responsible for the alpha-2,6 chemical bound between the N-acetylneuraminic sialic acid (Neu5Ac) to a galactose, present at the extremity of N-linked oligosaccharides chains. In this project, we propose to transduce the ST6GAL1 recombinant minigene, which encodes the ST6GAL1 enzyme, into CHO DG44 cells which already produce reCG, aiming to obtain a recombinant hormone displaying a glycosylation profile and biological activity similar to that of its natural counterpart.