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

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Recombinant HGF protein with the engineered factor Xa site is biologically active.(A) A schematic diagram of HGF protein. A disulfied-bond between Cys487 and Cys60427 and the 5 amino-acid sequences in the protease-cleavage sites of wild-type and engineered HGF proteins are indicated. A free cystein residue, Cys561, was mutated to serine (C561S) in some of the recombinant proteins used in this study. (B) Cellular Met activation by HGF, tcHGF(Xa), and scHGF(Xa). EHMES-1 cells were stimulated with indicated concentrations of recombinant HGF protein for 10 min. The cells were fixed and Met activation was detected by anti-phospho-Met (Tyr1234/1235) antibody. The activities were expressed as a relative Met phosphorylation calculated as described in the Method. Data are mean ± SD of six (HGF and tcHGF(Xa)) or four (scHGF(Xa)) independent experiments.
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f1: Recombinant HGF protein with the engineered factor Xa site is biologically active.(A) A schematic diagram of HGF protein. A disulfied-bond between Cys487 and Cys60427 and the 5 amino-acid sequences in the protease-cleavage sites of wild-type and engineered HGF proteins are indicated. A free cystein residue, Cys561, was mutated to serine (C561S) in some of the recombinant proteins used in this study. (B) Cellular Met activation by HGF, tcHGF(Xa), and scHGF(Xa). EHMES-1 cells were stimulated with indicated concentrations of recombinant HGF protein for 10 min. The cells were fixed and Met activation was detected by anti-phospho-Met (Tyr1234/1235) antibody. The activities were expressed as a relative Met phosphorylation calculated as described in the Method. Data are mean ± SD of six (HGF and tcHGF(Xa)) or four (scHGF(Xa)) independent experiments.

Mentions: It is known that the proteolytic processing of HGF is necessary for its capability to activate Met. HGF has a domain organization very similar to plasminogen, a central enzyme in the fibrinolytic system4. Both HGF and plasminogen comprise an N-terminal (N) domain followed by four (in HGF) or five (in plasminogen) consecutive kringle (K) domains, and a C-terminal serine protease (SP) domain (Fig. 1A). The SP domain carries a characteristic catalytic triad comprising His, Ser, and Asp in plasminogen that is responsible for its protease activity, but in HGF His and Ser are not conserved, making it enzymatically nonfunctional4. Upon the biosynthesis and the signal peptide cleavage, HGF is secreted into the extracellular space as a single-chain (sc) HGF, where it is cleaved by extracellular proteases such as HGF activator5 at the linker region between the 4th kringle (K4) and SP. Since this cleavage occurs at Arg494-Val495 bond which is flanked by two disulfide-bonded Cys residues (Cys487 and Cys604), the resulting product is a disulfide-linked two-chain (tc) HGF6. Although both scHGF and tcHGF can bind to Met, only tcHGF can activate Met7.


Probing conformational and functional states of human hepatocyte growth factor by a panel of monoclonal antibodies
Recombinant HGF protein with the engineered factor Xa site is biologically active.(A) A schematic diagram of HGF protein. A disulfied-bond between Cys487 and Cys60427 and the 5 amino-acid sequences in the protease-cleavage sites of wild-type and engineered HGF proteins are indicated. A free cystein residue, Cys561, was mutated to serine (C561S) in some of the recombinant proteins used in this study. (B) Cellular Met activation by HGF, tcHGF(Xa), and scHGF(Xa). EHMES-1 cells were stimulated with indicated concentrations of recombinant HGF protein for 10 min. The cells were fixed and Met activation was detected by anti-phospho-Met (Tyr1234/1235) antibody. The activities were expressed as a relative Met phosphorylation calculated as described in the Method. Data are mean ± SD of six (HGF and tcHGF(Xa)) or four (scHGF(Xa)) independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5017023&req=5

f1: Recombinant HGF protein with the engineered factor Xa site is biologically active.(A) A schematic diagram of HGF protein. A disulfied-bond between Cys487 and Cys60427 and the 5 amino-acid sequences in the protease-cleavage sites of wild-type and engineered HGF proteins are indicated. A free cystein residue, Cys561, was mutated to serine (C561S) in some of the recombinant proteins used in this study. (B) Cellular Met activation by HGF, tcHGF(Xa), and scHGF(Xa). EHMES-1 cells were stimulated with indicated concentrations of recombinant HGF protein for 10 min. The cells were fixed and Met activation was detected by anti-phospho-Met (Tyr1234/1235) antibody. The activities were expressed as a relative Met phosphorylation calculated as described in the Method. Data are mean ± SD of six (HGF and tcHGF(Xa)) or four (scHGF(Xa)) independent experiments.
Mentions: It is known that the proteolytic processing of HGF is necessary for its capability to activate Met. HGF has a domain organization very similar to plasminogen, a central enzyme in the fibrinolytic system4. Both HGF and plasminogen comprise an N-terminal (N) domain followed by four (in HGF) or five (in plasminogen) consecutive kringle (K) domains, and a C-terminal serine protease (SP) domain (Fig. 1A). The SP domain carries a characteristic catalytic triad comprising His, Ser, and Asp in plasminogen that is responsible for its protease activity, but in HGF His and Ser are not conserved, making it enzymatically nonfunctional4. Upon the biosynthesis and the signal peptide cleavage, HGF is secreted into the extracellular space as a single-chain (sc) HGF, where it is cleaved by extracellular proteases such as HGF activator5 at the linker region between the 4th kringle (K4) and SP. Since this cleavage occurs at Arg494-Val495 bond which is flanked by two disulfide-bonded Cys residues (Cys487 and Cys604), the resulting product is a disulfide-linked two-chain (tc) HGF6. Although both scHGF and tcHGF can bind to Met, only tcHGF can activate Met7.

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.