The proper post-transcriptional modification of recombinant human erythropoietin (rhEPO) is critical to retain its biological functions, either in vivo or in vitro. The major glycosyltransferases for the determinant of glycosylation patterns of rhEPO are N-acetylglycosaminyltransferase (GnT) and α-1-3/4 fucosyltransferase (Fut). GnT-III expression (388 ± 19.09) in the mouse mammary gland has been shown to be dramatically different from that in CHO cells, although FuT-VIII expression in CHO cells (1970 ± 255.9) is comparable to mouse mammary gland (272 ± 14.8), suggesting that the mammary gland may proceed with the proper glycosylation of rhEPO as shown in CHO cells. To identify this hypothesis and establish the rhEPO bioreactor system for mass production of protein in transgenic animals, we have generated two transgenic mouse lines that express rhEPO in milk. Both lines of transgenic mouse express only rhEPO in the lactating mammary gland, and the protein yield of rhEPO in lactating milk is comparable to that in CHO cells. After determining the protein expression in lactating milk, using three different methods – enzymatic release of oligosaccharide analysis, two-dimensional electrophoresis, and 2-aminobenzamide-labeled analysis – we report that the rhEPO produced by the animal bioreactor system has the proper glycosylation patterns as shown in CHO cell-derived Epoietin α, and has more tetra-acidic oligosaccharide structures than Epoietin α, which is the widely used rhEPO for therapeutic purposes. The in vitro biological property of transgenic mouse milk-derived rhEPO has been tested by measuring luciferase activity in MCF-7 cells, indicating that rhEPO from mammary gland up-regulates the EPO-receptor-mediated STAT5 gene expression in a dose-dependent manner the same as Epoietin α does. In addition, in vivo biological activity demonstrated that direct injection of rhEPO into a mouse vein increases blood components such as RBC and HCT. In light of these findings, we suggest that high levels of tetra-acidic structures observed in transgenic mouse milk-derived rhEPO may be related to the high level of expression of glycosiltransferases (GnT-III and FuT-VIII) in mammary gland; thus the bioreactor system using the mammary gland of a transgenic animal could be a good candidate for production of rhEPO for pharmaceutical purposes.
This work was supported in part by a grant program from RDA(Biogreen21) and Cho-A, Republic of Korea. D.N. Kwon is the recipient of a scholarship from the BK21 program, granted by the Ministry of Education, Korea.
Recombinant human erythropoietin induces intussusceptive microvascular growth in vivo