Generation of engineered recombinant hepatocyte growth factor cleaved and activated by Genenase I

Hayata, Daichika; Fukuta, Kazuhiro; Matsumoto, Kunio; Adachi, Eri; Hanada, Keita; Adachi, Kenjiro; Nakamura, Toshikazu

doi:10.1016/j.jbiotec.2007.11.006

Cited by 3 publications

(3 citation statements)

References 27 publications

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“…Since the wild-type HGF undergoes spontaneous cleavage by the action of serum-derived proteases during the protein expression in mammalian cells, we prepared engineered HGF where the cleavage site of wild-type HGF (KQLR/V) was mutated to the recognition sequence of Factor Xa (IEGR/V). Similar engineered HGF was previously reported by introducing the Genenase I cleavage site 18 , but we chose Factor Xa to achieve more controlled and efficient cleavage. This mutant HGF (called HGF(Xa) hereafter) would remain single chain (i.e., scHGF(Xa)) during the biosynthesis and purification but can be converted to an active two-chain species (i.e., tcHGF(Xa)) after the treatment with Factor Xa, exposing a new N-terminus at Val495 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Probing conformational and functional states of human hepatocyte growth factor by a panel of monoclonal antibodies

Sakai

Ogasawara

Kaneko

et al. 2016

Sci Rep

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HGF-Met signaling contributes to various biological events by controlling cell migration. Since the abnormal activation of Met receptor causes cancer progression, inhibitors such as neutralizing antibodies are regarded as promising therapeutics. HGF is secreted as a single-chain (sc) precursor and is processed by extracellular proteases to generate disulfide-bonded two-chain (tc) HGF. Although this proteolytic processing of HGF is necessary for its biological activity, exactly how the proteolysis leads to the conversion of HGF to the active form is still unclar due to the lack of structural information. In order to gain insights about this point, we generated 6 antibodies against HGF. All antibodies recognized different epitopes on the native HGF protein and showed distinct effects when tested in a cell-based HGF-Met signaling assay. They included one antibody (t1E4) that strongly blocks Met activation by tcHGF, as well as one antibody (t8E4) exclusively recognizing the active tcHGF but not inactive scHGF. Thus, a panel of anti-HGF antibodies suitable for probing the structural mechanism of HGF activation were obtained.

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Section: Resultsmentioning

confidence: 99%

Probing conformational and functional states of human hepatocyte growth factor by a panel of monoclonal antibodies

Sakai

Ogasawara

Kaneko

et al. 2016

Sci Rep

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“…HGF was originally, like platelet‐derived growth factor (PDGF), isolated from platelets and it has been suggested that it is secreted as an inactive single‐chain glycoprotein precursor pro‐HGF 23. The pro‐HGF forms an active heterodimeric peptide consisting of a 69‐kDa α‐chain (HGFα) and a 34‐kDa β‐chain (HGFβ) after being cleaved by a serum serine protease 23, 34. The functional HGF then binds the corresponding receptor, HGF receptor (c‐MET), via β‐chain 35 to stimulate cell proliferation and migration.…”

Section: Discussionmentioning

confidence: 99%

Hepatocyte growth factor induces hypoxia‐related interleukin‐8 expression in lung adenocarcinoma cells

Chiang

2009

Molecular Carcinogenesis

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Rapid growth of cancer cells often creates insufficient supply of oxygen and nutrients in the tumour nest. The frequent detection of hypoxia-inducible factor (HIF) and interleukin-8 (IL-8) in afflicted tissues suggests that IL-8 expression could be associated with elevated levels of HIF. Recently, we found that hypoxia also upregulated the expression of hepatocyte growth factor (HGF) in lung adenocarcinoma (LAD) cells. However, the relationship between HGF and IL-8 has not been investigated in LAD cells. In this study, we found that HGF induced IL-8 expression in LAD. Interestingly, hypoxia also increased the level of prostaglandin F(2alpha) (PGF(2alpha)), a product of dihydrodiol dehydrogenase (DDH). When expression of DDH was suppressed by siRNA, the levels of PGF(2alpha), HGF and IL-8 were reduced; however, their levels returned to normal after DDH was reintroduced. These data suggest that hypoxia induces biosynthesis of PGF(2alpha), which then activates HGF and IL-8 expression. The results provide a reasonable explanation of how PGF(2alpha), HGF and IL-8 exert their effects on cancer cell metastasis.

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“…Mv1Lu mink lung epithelial cells obtained from RIKEN BioResource Center (Tsukuba, Japan) were used for the cell proliferation assay [46,47]. Mv1Lu cells were cultured in Minimum Essential Medium supplemented with 10% FBS and 1% non-essential amino acids.…”

Section: Cell Proliferation Assaymentioning

confidence: 99%

Dimer Interface in Natural Variant NK1 Is Dispensable for HGF-Dependent Met Receptor Activation

Tahira

Sakai

Sato

et al. 2021

IJMS

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NK1, a splicing variant of hepatocyte growth factor (HGF), binds to and activates Met receptor by forming an NK1 dimer and 2:2 complex with Met. Although the structural mechanism underlying Met activation by HGF remains incompletely resolved, it has been proposed that the NK1 dimer structure participates in this activation. We investigated the NK1 dimer interface’s role in Met activation by HGF. Because N127, V140, and K144 are closely involved in the head-to-tail NK1 dimer formation, mutant NK1 proteins with replacement of these residues by alanine were prepared. In Met tyrosine phosphorylation assays, N127-NK1, V140-NK1, and K144-NK1 showed 8.3%, 23.8%, and 52.2% activity, respectively, compared with wild-type NK1. Although wild-type NK1 promoted cell migration and scattering, N127-NK1, V140-NK1, and K144-NK1 hardly or marginally promoted them, indicating loss of activity of these mutant NK1 proteins to activate Met. In contrast, mutant HGFs (N127-HGF, V140-HGF, and K144-HGF) with the same amino acid replacements as in NK1 induced Met tyrosine phosphorylation and biological responses at levels comparable to those of wild-type HGF. These results indicate that the structural basis responsible for NK1-dependent Met dimer formation and activation differs from, or is at least distinguishable from, the structural basis responsible for HGF-dependent Met activation.

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Generation of engineered recombinant hepatocyte growth factor cleaved and activated by Genenase I

Cited by 3 publications

References 27 publications

Probing conformational and functional states of human hepatocyte growth factor by a panel of monoclonal antibodies

Probing conformational and functional states of human hepatocyte growth factor by a panel of monoclonal antibodies

Hepatocyte growth factor induces hypoxia‐related interleukin‐8 expression in lung adenocarcinoma cells

Dimer Interface in Natural Variant NK1 Is Dispensable for HGF-Dependent Met Receptor Activation

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