Therapeutic
glycoproteins with exposed
galactose (Gal) residues are cleared rapidly from the bloodstream by
asialoglycoprotein receptors in hepatocytes. Various approaches have been used to increase the content of
sialic acid, which occupies terminal sites of N- or O-linked
glycans and thereby increases the half-life of therapeutic
glycoproteins. We enhanced sialylation of human
erythropoietin (EPO) by genetic engineering of the sialylation pathway in Chinese hamster ovary (CHO) cells. The
enzyme GNE (
uridine diphosphate-
N-acetyl glucosamine 2-
epimerase)/MNK (N-acetyl
mannosamine kinase), which plays a key role in the initial two steps of
sialic acid biosynthesis, is regulated by
cytidine monophosphate (
CMP)-sialic acid through a feedback mechanism. Since
sialuria patient cells fail in regulating
sialic acid biosynthesis by feedback mechanism, various
sialuria-like mutated rat GNEs were established and subjected to in vitro activity assay. GNE/MNK-R263L-R266Q mutant showed 93.6% relative activity compared with wild type and did not display feedback inhibition. Genes for
sialuria-mutated rat GNE/MNK, Chinese hamster
CMP-sialic acid transporter and human α2,3-sialyltransferase (α2,3-ST) were transfected simultaneously into recombinant human (rh) EPO-producing CHO cells.
CMP-sialic acid concentration of engineered cells was significantly (>10-fold) increased by
sialuria-mutated GNE/MNK (R263L-R266Q) expression. The
sialic acid content of rhEPO produced from engineered cells was 43% higher than that of control cells. Ratio of tetra-sialylated
glycan of rhEPO produced from engineered cells was increased ∼32%, but ratios of asialo- and mono-sialylated
glycans were decreased ∼50%, compared with control. These findings indicate that
sialuria-mutated rat GNE/MNK effectively increases the intracellular
CMP-sialic acid level. The newly constructed host CHO cell lines produced more highly sialylated therapeutic
glycoproteins through overexpression of
sialuria-mutated GNE/MNK,
CMP-SAT and α2,3-ST.