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Utilizing the activation mechanism of serine proteases to engineer hepatocyte growth factor into a Met antagonist.

ABSTRACT: Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase Met, is secreted as single chain pro-HGF that lacks signaling activity. Pro-HGF acquires functional competence upon cleavage between R494 and V495, generating a disulfide-linked alpha/beta-heterodimer, where the beta-chain of HGF (HGF beta) has a serine protease fold that lacks enzymatic activity. We show that, like serine proteases, insertion of the newly formed N terminus in the beta-chain is critical for activity, here by allosterically stabilizing interactions with Met. The HGF beta crystal structure shows that V495 inserts into the "activation pocket" near the Met binding site where the positively charged N terminus forms a salt bridge with the negatively charged D672, and the V495 side chain has hydrophobic interactions with main- and side-chain residues. Full-length two-chain HGF mutants designed to interrupt these interactions (D672N, V495G, V495A, G498I, and G498V) displayed <10% activity in Met receptor phosphorylation, cell migration, and proliferation assays. Impaired signaling of full-length mutants correlated with >50-fold decreases in Met binding of the low-affinity HGF beta domain alone bearing the same mutations and further correlated with impaired N-terminal insertion. Because high-affinity binding resides in the HGF alpha-chain, full-length mutants maintained normal Met binding and efficiently inhibited HGF-mediated Met activation. Conversion of HGF from agonist to antagonist was achieved by as little as removal of two methyl groups (V495A) or a single charge (D672N). Thus, although serine proteases and HGF have quite distinct functions in proteolysis and Met signal transduction, respectively, they share a similar activation mechanism.

SUBMITTER: Kirchhofer D

PROVIDER: S-EPMC1828710 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:MET, the receptor for hepatocyte growth factor (HGF), is involved in wide variety of biological events and its aberrant activation is implicated in the pathogenesis of cancer. Especially, MET signaling is associated with resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), key drugs in therapy of non-small cell lung cancer. MET has 11 potential N-glycosylation sites, however, the site-specific roles of N-glycans have not been elucidated. Here, we report that N-glycans regulate proteolytic processing of MET and HGF induced MET signaling, site specifically. N-glycosylation inhibitors suppressed processing and trafficking of endogenous MET in H1975 and EBC-1 lung cancer cells and exogenous MET in CHO cells. We purified the recombinant extracellular domain of human MET and determined the site-specific N-glycan structures and occupancy using nanoflow liquid chromatography-electrospray ionization mass spectrometry. The results indicated that most sites were fully glycosylated and the dominant population were complex-type which were rich in sialic acids and core fucoses. To examine the effects of N-glycan deletion of MET, we prepared endogenous-MET-knockout Flp-In CHO cells and transfected with series of N-glycan deletion mutants of MET. It was found that several N-glycans are implicated in processing of MET. It was also suggested that the N-glycans of -subunit of MET positively regulate HGF signaling, and the N-glycans of β-subunit negatively regulate HGF signaling. In all-N-glycan deletion mutant, processing and signaling were significantly suppressed. It was observed that the cell proliferation rate was suppressed in all-N-glycan deletion mutant, although the cell surface expression levels of MET were not completely diminished. We examined the HGF biding affinity for the recombinant extracellular domain of MET and found that peptide N-glycosidase F treatment did not affect the ligand binding affinity. It was suggested that N-glycans of MET affect the status and the function of the receptor site-specifically.

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Utilizing the activation mechanism of serine proteases to engineer hepatocyte growth factor into a Met antagonist.

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