Post-translational modification of a chimeric EPO-Fc hormone is more important than its molecular size in defining its in vivo hematopoietic activity

Salgado, ER; Montesino R.; Jimenez, SP; Gonzalez, M.; Hugues F.; Cabezas, OI; Maura-Perez, R; Saavedra P.; Lamazares E.; Salas Burgos A.; Vera, JC; Sánchez O; Toledo JR

Keywords: erythropoietin, Adenovirus vector, N-glycans, Mammary epithelial cell, Milk expression, Hematopoietic activity

Abstract

BACKGROUND: Recombinant erythropoietin (EPO) has been marketed as biopharmaceutical for anemia and chronic renal failure. Long-acting EPO variants that aimed at achieving less frequent dosing have been generated, either by the addition of glycosylation sites or increasing its molecular weight. METHODS: The hEPO cDNA linked to the human IgG Fc fragment was cloned as a single codifying gene on the pAdtrack-CMV vector, yielding the recombinant adenoviral genome. For in vitro and in vivo expression assays cervical cancer cell line (SiHa) and nulliparous goats were used, respectively. The hematopoietic activity of EPO-Fc, expressed as the differential increment of hematocrit was evaluated in B6D2F1 mice. NP-HPLC of the 2AB-labeled N-glycan was carried out to profile analysis. RESULTS: The direct transduction of mammary secretory cells with adenoviral vector is a robust methodology to obtain high levels of EPO of up to 3.5mg/mL in goat's milk. SiHa-derived EPO-Fc showed significant improvement in hematopoietic activity compared to the commercial hEPO counterpart or with the homologous milk-derived EPO-Fc. The role of the molecular weight seemed to be important in enhancing the hematopoietic activity of SiHa-derived EPO-Fc. However, the lack of sialylated multi-antennary glycosylation profile in milk-derived EPO-Fc resulted in lower biological activity. CONCLUSIONS: The low content of tri- or tetra-antennary sialylated N-glycans linked to the chimeric EPO-Fc hormone, expressed in the goat mammary gland epithelial cells, defined its in vivo hematopoietic activity. GENERAL SIGNIFICANCE: The sialylated N-glycan content plays a more significant role in the in vivo biological activity of hEPO than its increased molecular weight.

Más información

Título según WOS: Post-translational modification of a chimeric EPO-Fc hormone is more important than its molecular size in defining its in vivo hematopoietic activity
Título según SCOPUS: Post-translational modification of a chimeric EPO-Fc hormone is more important than its molecular size in defining its in vivo hematopoietic activity
Título de la Revista: Biochimica et Biophysica Acta - General Subjects
Volumen: 1850
Número: 9
Editorial: Elsevier B.V.
Fecha de publicación: 2015
Página de inicio: 1685
Página final: 1693
Idioma: English
DOI:

10.1016/j.bbagen.2015.04.012

Notas: ISI, SCOPUS