Kazuhiro Fukuta

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.

Induction of hepatocyte growth factor by fucoidan and fucoidan-derived oligosaccharides.

Fucoidan, which is extracted from brown seaweed, is a complex sulphated polysaccharide that is mostly composed of l‐fucose and sulphated ester groups. The structural and anionic characteristics of fucoidan are similar to those of heparin. Heparin stimulates production of hepatocyte growth factor (HGF), which has key roles in tissue regeneration. We have shown that fucoidan and fucoidan‐derived oligosaccharides have similar ability to stimulate production of HGF as heparin and heparin‐derived oligosaccharides. This induction of HGF by heparin or fucoidan and their oligosaccharide derivates occurs primarily at the level of translation, probably via the same mechanism. Fucoidan may thus be useful to protect tissues and organs from various injuries and diseases, via mechanisms involving HGF.

Multiple biological responses are induced by glycosylation-deficient hepatocyte growth factor.

HGF (hepatocyte growth factor), a heterodimeric glycoprotein composed of alpha- and beta-chains, exerts biological activities through the c-Met receptor tyrosine kinase. The alpha-chain has three glycosylation sites, while the beta-chain has two; however, the role of sugar chains on HGF is still unknown. To address the significance of glycosylation of HGF, three different types of glycosylation-deficient HGFs, i.e. non-glycosylated in the alpha-chain, the beta-chain, and in both the alpha- and beta-chains, were respectively expressed in COS-7 cells and then purified from culture supernatants. Unexpectedly, glycosylation-deficient HGFs induced tyrosine phosphorylation of the c-Met receptor and subsequent phosphorylation of ERK (extracellular-signal-regulated kinase) and Akt in rat hepatocytes with the same potency as glycosylated HGF. Consistent with this, glycosylation-deficient HGFs strongly stimulated DNA synthesis of hepatocytes equal to glycosylated HGF. Likewise, glycosylation-deficient HGFs induced cell scattering and branching tubulogenesis in MDCK (Madin-Darby canine kidney) cells, and thus were indistinguishable from glycosylated HGF in biological activities. Glycosylation also did not affect stability, protease sensitivity and tissue distribution, although the plasma clearance of HGF was slightly prolonged by glycosylation deficiency. Glycosylation deficiency resulted in a decrease in post-transcriptional biosynthesis of HGF in the cells, whereas extracellularly secreted HGFs were efficiently activated to a two-chain form. These results indicate that glycosylation influences post-transcriptional biosynthesis of HGF, whereas biological activities and basic physicochemical characteristics are retained, even in completely non-glycosylated HGF. Hence, non-glycosylated HGF is promising as an alternative for glycosylated HGF in clinical applications.

Dissociation of c-Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species.

Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c‐Met. We previously demonstrated that HGF α‐chain hairpin–loop, K1 domain and β‐chain are required for c‐Met signalling. The sequential phosphorylation of tyrosine residues, from c‐Met kinase domain to multidocking regions, is required for HGF‐signalling transduction. Herein, we provide evidence that the disconcerted activation of c‐Met tyrosine regions fails to induce biological functions. When human cells were incubated with ‘mouse HGF’, kinase domain activation (i.e. phospho‐Tyr‐1230/34/35) became evident, but the multidocking site (i.e. Tyr‐1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α‐chain, or of β‐chain, to human c‐Met was lower than that of human HGF, as evidenced by HGF–chimera assay. Notably, only four amino acid positions in HGF α‐chain hairpin–loop and K1 domain and six positions in β‐chain differed between human HGF and mouse HGF. The human‐specific amino acids (such as Gln‐95 in hairpin–loop, Arg‐134 in K1 domain and Cys‐561 in β‐chain) may be important for accurate c‐Met assembly and signalling transduction. Copyright

Characterization of fibroblast growth factor-6 expressed by Chinese hamster ovary cells as a glycosylated mitogen for human vascular endothelial cells.

The gene for fibroblast growth factor (FGF)-6/hst-2 was originally identified by its close homology with the FGF-4/hst-1 gene. Aside from its ability to transform cultured fibroblasts, the characteristics of FGF-6 protein have only been studied using a simple preparation from E. coli. In the present study, we expressed FGF-6 cDNA in CHO cells and characterized the resultant protein. We found that CHO cells secreted several forms of the FGF-6 polypeptide, and that there were multiple N-terminal modifications. The longest form (18-kDa) contained the sequence, SerProAlaGlyAlaArg, as its N-terminus, which was consistent with the signal peptide cleavage site predicted from its primary structure. The core polypeptide was primarily modified by heterogeneous N-glycans that were sialylated to a small degree; among them, biantennary structures were found to predominate. Moreover, possible O-glycosylation was also detected. N-glycosylated FGF-6 potently induced DNA synthesis and proliferation of human vascular endothelial cells, whereas in the absence of N-glycosylation, FGF-6 mitogenicity was substantially diminished. The results clearly indicate that FGF-6 expressed by mammalian cells is a glycosylated mitogen for vascular endothelial cells and further suggests that N-glycosylation plays a key role in determining the mitogenicity of FGF-6.

Pharmacokinetics and pharmacodynamics following intravenous administration of recombinant human hepatocyte growth factor in rats with renal injury.

Background/Aim: Hepatocyte growth factor (HGF) plays a role in the regeneration and protection of the kidney, but little information is available concerning the pharmacokinetics of therapeutic treatment with HGF. In this study, HGF was administered after the onset of renal injury, and pharmacokinetic analysis was performed simultaneously with an efficacious dose. Methods: For the study of pharmacodynamics, recombinant human HGF was intravenously administered to rats with glycerol-induced acute kidney injury (AKI). In the pharmacokinetic study, rats subjected to glycerol injection or renal ischemia-reperfusion were used as models of AKI, and rats subjected to 5/6 nephrectomy were used as models of chronic kidney disease (CKD). Results: After intravenous administration of HGF at doses of 0.5-2.0 mg/kg, the elevation of blood urea nitrogen was suppressed, indicating that HGF had a pharmacodynamic effect. However, no significant difference was seen in the pharmacokinetic parameters such as clearance, distribution volume and half-life between the normal, AKI and CKD groups. Conclusion: The intravenous administration of HGF after the onset of renal dysfunction exerted a pharmacological effect on AKI, and renal injury did not affect the clearance of plasma HGF. This unaffected profile may serve as a base for the safety of HGF during therapeutic administration.

Different reactivities of enzyme-linked immunosorbent assays for hepatocyte growth factor

BACKGROUND Hepatocyte growth factor (HGF) plays diverse roles in organ development, tissue regeneration, and tumor progression. Measurement of HGF concentrations in blood and tissues using enzyme-linked immunosorbent assay (ELISA) is a simple and easy way to understand the significance of HGF in tissue regeneration, pathophysiology, and diagnosis. METHODS We evaluated 3 ELISA kits from different sources, referred to herein as kits A, B, and C for convenience. RESULTS We found that the concentrations of human HGF determined using ELISA vary significantly depending on the source of the ELISA kit. Kits A and B detected both single-chain pro-HGF and 2-chain mature HGF, but kit C detected only 2-chain HGF. A difference in reactivity was also detected during analysis of plasma samples. When rat plasma collected 4 h after subcutaneous administration of human HGF was analyzed, the HGF concentration determined using kit B was remarkably higher than those obtained using kits A and C. Results of a biological assay and Western blot analysis indicated that kit B detects even degraded HGF, by which the HGF concentrations determined using kit B were significantly overestimated. CONCLUSIONS This information serves as a guide for the selection of ELISA kits for human HGF.

Pharmacokinetic Modeling of Hepatocyte Growth Factor in Experimental Animals and Humans

Hepatocyte growth factor (HGF) is under development for treatment of renal failure. This study was designed to clarify changes in HGF pharmacokinetics in renal failure and to establish a pharmacokinetic model applicable to single and repeated doses. The plasma concentration profile in mice with glycerol-induced acute renal failure was similar to that in normal mice, indicating a minimal contribution of kidney to systemic clearance of HGF. Nevertheless, accumulation of fluorescein-4-isocyanate-labeled HGF in renal tubules in both cases suggests the occurrence of efficient endocytosis of HGF in kidney. A pharmacokinetic model including plasma and liver compartments was constructed, incorporating both high- and low-affinity receptors for association and subsequent endocytosis of HGF because HGF is eliminated via specific receptor c-Met and heparin-like substance. The model well explained the plasma concentration profiles at all doses examined after bolus injection in animals and humans, and those during infusion in rodents. It includes externalization of receptors, which is negatively regulated by HGF, and can explain the gradual increase in trough concentration during repeated dosing in monkeys. Overall pharmacokinetic profiles of HGF are governed by at least two receptors and are well described by this pharmacokinetic model, which should assist in safe management of clinical trials.

Recombinant human hepatocyte growth factor (HGF), but not rat HGF, elicits glomerular injury and albuminuria in normal rats via an immune complex‐dependent mechanism

1. Hepatocyte growth factor (HGF) has the therapeutic potential to improve renal fibrosis and proteinuria in rodents with chronic kidney disease. In contrast, long‐term administration of human HGF to normal rats reportedly elicits proteinuria. Thus, the role of HGF during proteinuria remains contentious. The aim of the present study was to demonstrate that human HGF is antigenic to rodents and that immune complex formation causes proteinuria.