Reiko Abe

Genetic engineering of CHO cells producing human interferon-γ by transfection of sialyltransferases

Natural human interferon-γ (hIFN-γ) contains mainly biantennary complex-type sugar chains. We previously remodeled the branch structures of N-glycans on hIFN-γ in Chinese hamster ovary (CHO) cells by overexpressing UDP-N-acetylglucosamine: α1,6-D-mannoside β1,6-N-acetylglucosaminyltransferase (GnT-V). Normal CHO cells primarily produced hIFN-γ having biantennary sugar chains, whereas a CHO clone, designated IM4/Vh, transfected with GnT-V, primarily produced hIFN-γ having GlcNAcβ1-6 branched triantennary sugar chains when sialylation was incomplete and an increase in poly-N-acetyllactosamine (Galβ1-4GlcNAcβ1-3)n was observed. In the present study, we introduced mouse Galβ1-3/4GlcNAc-R α2,3-sialyltransferase (ST3Gal IV) and/or rat Galβ1-4GlcNAc-R α2,6-sialyltransferase (ST6Gal I) cDNAs into the IM4/Vh cells to increase the extent of sialylation and to examine the effect of sialyltransferase (ST) type on the linkage of sialic acid. Furthermore, we speculated that sialylation extent might affect the level of poly-N-acetyllactosamine. We isolated four clones expressing different levels of α2,3-ST and/or α2,6-ST. The extent of sialylation of hIFN-γ from the IM4/Vh clone was 61.2%, which increased to about 80% in every ST transfectant. The increase occurred regardless of the type of overexpressed ST, and the proportion of α2,3- and α2,6-sialic acid corresponded to the activity ratio of α2,3-ST to α2,6-ST. Furthermore, the proportion of N-glycans containing poly-N-acetyllactosamine was significantly reduced (less than 10%) in the ST transfectants compared with the parental IM4/Vh clone (22.9%). These results indicated that genetic engineering of STs is highly effective for regulating the terminal structures of sugar chains on recombinant proteins in CHO cells.

Control of Bisecting GlcNAc Addition to N-Linked Sugar Chains

In the present study, experimental control of the formation of bisecting GlcNAc was investigated, and the competition between β-1,4-GalT (UDP-galactose:N-acetylglucosamine β-1,4-galactosyltransferase) and GnT-III (UDP-N-acetylglucosamine:β-d-mannoside β-1,4-N-acetylglucosaminyltransferase) was examined. We isolated a β-1,4-GalT-I single knockout human B cell clone producing monoclonal IgM and several transfectant clones that overexpressed β-1,4-GalT-I or GnT-III. In the β-1,4-GalT-I-single knockout cells, the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased, where β-1,4-GalT activity was reduced to about half that in the parental cells, and GnT-III activity was unaltered. In the β-1,4-GalT-I transfectants, the extent of bisecting GlcNAc addition was reduced although GnT-III activity was not altered significantly. In the GnT-III transfectants, the extent of bisecting GlcNAc addition increased along with the increase in levels of GnT-III activity. The extent of bisecting GlcNAc addition to the sugar chains of IgM was significantly correlated with the level of intracellular β-1,4-GalT activity relative to that of GnT-III. These results were interpreted as indicating that β-1,4-GalT competes with GnT-III for substrate in the cells.

Monitoring biodistribution of glycoproteins with modified sugar chains

Natural human interferon (hIFN)-gamma has mainly biantennary complex-type sugar chains. Previously, we successfully remodeled its sugar chain structure into: (a) highly branched types; or (b) highly sialylated types, by overexpression of: (a) N-acetylglucosaminyltransferase (GnT)-IV and/or GnT-V; or (b) sialyltransferases, in Chinese hamster ovary (CHO) cells. In addition, we prepared asialo hIFN-gammas by treatment with sialidase in vitro. In the present study, we assessed the bioactivity of remodeled hIFN-gamma in terms of antiviral activity, anticellular activity, and biodistribution. Structural changes to the sugar chains did not have a significant influence on the antiviral and anticellular activities of hIFN-gamma, although the attachment of the sugar chain itself affected both activities. However, the biodistribution differed significantly; the number of exposed galactose residues was the major determinant of the specific distribution to the liver and blood clearance rate of hIFN-gamma. This phenomenon was considered to be mediated by the hepatic asialoglycoprotein receptor (ASGP-R), and we showed a linear, not exponential, enhancement of the distribution to the liver with an increase in the number of exposed galactose residues. We also confirmed this tendency using fibroblast growth factor (FGF). Our observation is not the same as the glycoside cluster effect. We thus provide important information on the character of modified recombinant glycoproteins.