Differential in vitro and in vivo glycosylation of human erythropoietin expressed in adenovirally transduced mouse mammary epithelial cells Jorge R. Toledo a , Oliberto Sa ´nchez a, * , Raquel Montesino Seguı ´ b , Yaiza Ferna ´ndez Garcı ´a a , Marı ´a P. Rodrı ´guez a , Jose ´ A. Cremata b a Department of Transgenesis and Animal Cloning, Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba b Department of Carbohydrate Chemistry, Center for Genetic Engineering and Biotechnology, PO. Box 6162, Havana 10600, Cuba Received 27 March 2005; received in revised form 10 June 2005; accepted 10 June 2005 Available online 27 June 2005 Abstract The expression of human erythropoietin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce erythropoietin in the milk of transgenic animals resulted in very low expression levels and in a detrimental effect in the health of the founder animals. Here, we show that the direct transduction of the mouse mammary gland with an adenoviral vector carrying the cDNA of erythropoietin promotes its expression in milk at a level as high as 3.5 mg/ml. The recombinant erythropoietin derived from mouse milk showed a different migration and distribution after SDS-PAGE electrophoresis as well as a low in vivo hematopoietic activity. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation compared to with its counterpart produced in CHO and HC11 cell lines. The difference between in vivo and in vitro glycosylation of human erythropoietin expressed in adenovirally transduced mammary epithelial cells suggests that key enzymes in the glycosylation pathway may be insufficient during lactation. Thus, the direct transduction of the mammary epithelium seems to be a powerful tool to express toxic proteins in milk at levels high enough for their physical, chemical and biological characterization before undertaking the generation of a transgenic mammal. D 2005 Elsevier B.V. All rights reserved. Keywords: Adenovirus; Transduction; Milk; Erythropoietin; Glycosylation; Mammary gland 1. Introduction Human erythropoietin (hEPO) is a glycoprotein that stimulates the proliferation and maturation of responsive bone marrow erythroid precursor cells [1]. This hormone is synthesized in the liver during the fetal period and after birth it is synthesized in the kidney [2]. The primary structure of hEPO consists of a 166-amino acid mature protein with a molecular weight of 18,399 Da. However, as a result of post-translational modifications, the carboxy-terminal argi- nine is removed [3] and the three potential N-glycosylation sites at Asn 24, 38 and 83, are occupied. In addition, one O- glycosylation site is always occupied at Ser 126. The effect of glycosylation on hEPO activity is complex since, although unglycosylated hEPO shows full biological activ- ity in vitro, glycosylation is necessary for full activity in vivo. Thus, hEPO produced in E. coli and the deglycosy- lated naturally derived hEPO shows very low activity in animal studies [4–6]. hEPO deficiency is the primary cause of anemia in chronic kidney failure [7–9]. Anemia is associated with an increased risk of mortality and morbidity and has a significant impact on quality of life in chronically ill patients. The treatment with recombinant hEPO has proved to be beneficial, increasing the levels of hemoglobin and hematocrit and consequently, improving the clinical evolution and quality of life of these patients. Production of an in vivo biologically active hEPO requires the biosynthetic machinery of higher cells. There- 0304-4165/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2005.06.007 * Corresponding author. Tel.: +537 2716022; fax: +537 331779. E-mail address: oliberto.sanchez@cigb.edu.cu (O. Sa ´nchez). Biochimica et Biophysica Acta 1726 (2005) 48 – 56 http://www.elsevier.com/locate/bba