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

Abstract

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.

Figure 1. Schematic description of wild-type and glycosylation-deficient mutants of HGF expressed in COS-7 cells

N-linked and O-linked sugar chains are indicated by diamonds and circles respectively. Amino acids are numbered from the N-terminal methionine of the secretion signal sequence.

Figure 2. SDS/PAGE of purified glycosylation-deficient HGFs

(A) SDS/PAGE of enzymatically deglycosylated HGF. Purified glycosylated HGF (CHO-HGF-WT) was digested with a combination of glycosidases under native or denatured conditions, and subjected to SDS/PAGE (4–20% gradient gel) under reducing conditions. The gel was stained with Coomassie Brilliant Blue. Purified preparations of HGF-αβNG and glycopeptidase F were loaded for comparison. Neuraminidase and O-glycanase added to the reaction mixture were undetectable at concentrations used here (not shown). (B) SDS/PAGE of purified HGF-WT and glycosylation-deficient HGF mutants. Each preparation (1 μg of each) was separated by SDS/PAGE (4–20% gradient gel) under reducing conditions and stained with Coomassie Brilliant Blue.

Figure 3. Influence of glycosylation deficiency on expression of HGF in COS-7 cells

(A) Change in extracellular levels of glycosylated and glycosylation-deficient HGFs. The concentration of HGF secreted into the culture medium was measured using ELISA. (B) Northern blot analysis of HGF mRNA expression. Total RNA was prepared from COS-7 cells 2 days after transfection of each expression vector and was electrophoresed. RNA was blotted on to a nylon membrane and hybridized with a specific probe for human HGF mRNA. (C) Change in intracellular levels of glycosylated and glycosylation-deficient HGFs. Cell lysates were prepared from COS-7 cells, and the concentration of HGF was measured by ELISA. (D) Western blot analysis of intracellular HGF. Lysates of COS-7 cells were prepared 24 h after transfection of each expression vector. Intracellular HGF was precipitated with heparin–Sepharose from the lysates, and was subjected to SDS/PAGE under reducing conditions. Proteins were electroblotted on to a PVDF membrane, and HGF was detected using an anti-human HGF polyclonal antibody.

Figure 4. Mitogenic activity of glycosylated and glycosylation-deficient HGFs

Mitogenic activity of glycosylated and glycosylation-deficient HGFs was determined by measuring DNA synthesis of rat hepatocytes in primary culture. Cells were stimulated with HGF for 18 h and pulse-labelled with [³H]thymidine for 6 h.

Figure 5. Morphogenic activity of glycosylated and glycosylation-deficient HGFs

Morphogenic activity of glycosylated and glycosylation-deficient HGFs was measured by evaluating the formation of branching tubes in MDCK cells. The cells were cultured in a collagen gel for 5 days in the absence or presence of HGF. Scale bar, 200 μm.

Figure 6. Activation of c-Met receptor, ERK and Akt by glycosylated and glycosylation-deficient HGFs

(A) Tyrosine-phosphorylation of the c-Met receptor induced by glycosylated and glycosylation-deficient HGFs. HGF was added to subconfluent cultures of adult rat hepatocytes, and cells were lysed with lysis buffer 10 min later. The c-Met receptor was immunoprecipitated and subjected to SDS/PAGE under reducing conditions. Proteins were electroblotted on to a PVDF membrane, and phosphorylated tyrosine (pY) and total c-Met were detected with anti-phosphotyrosine antibody and anti-c-Met antibody, respectively. (B, C) Phosphorylation of ERK (B) and Akt (C). HGF was added to subconfluent cultures of adult rat hepatocytes, and cells were lysed with lysis buffer 10 min later. Total lysates (40 μg) were separated by SDS/PAGE under reducing conditions, and were electroblotted on to a PVDF membrane. Phosphorylated ERK, total ERK, phosphorylated Akt and total Akt were detected with anti-phospho-ERK (Thr-202/Tyr-204) antibody, anti-ERK antibody, anti-phospho-Akt (Ser-473) antibody and anti-Akt antibody respectively.

Figure 7. Changes in plasma levels and organ distribution of glycosylated and non-glycosylated HGFs in mice

(A) Changes in plasma levels of ¹²⁵I-labelled HGFs. Glycosylated (CHO-HGF-WT and HGF-WT) and non-glycosylated HGF (HGF-αβNG) were radiolabelled with ¹²⁵I, and intravenously injected into mice. Radioactivities in plasma samples are expressed as a percentage of the dose. Each value represents the means±S.D. of measurements using three mice. (B) Organ distribution of ¹²⁵I-labelled HGFs. Radioactivity per whole organ was measured 15 min after intravenous injection of ¹²⁵I-labelled HGFs and expressed as a percentage of each dose. Each value represents the means±S.D. of measurements using three mice.