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Identification and characterization of the direct interaction between methotrexate (MTX) and high-mobility group box 1 (HMGB1) protein.

Kuroiwa Y, Takakusagi Y, Kusayanagi T, Kuramochi K, Imai T, Hirayama T, Ito I, Yoshida M, Sakaguchi K, Sugawara F - PLoS ONE (2013)

Bottom Line: Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood.These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX.These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Yamazaki, Noda, Chiba, Japan.

ABSTRACT

Background: Methotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood. METHODOLOGY/RESULT: Here, we performed a screening of MTX-binding proteins using T7 phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 phage display screen, we identified T7 phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD) of 0.50±0.03 µM for Al and 0.24 ± 0.01 µM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macrophage-like cells (RAW 264.7), TNF-α release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited in the presence of MTX.

Conclusions/significance: These data demonstrate that HMGB1 is a direct binding protein of MTX. Moreover, binding of MTX to RAGE-binding region in HMGB1 inhibited the HMGB1/RAGE interaction at the molecular and cellular levels. These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.

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Effect of MTX for the HMGB1 binding to DNA or to RAGE.(A) Electrophoretic mobility shift assay (EMSA). The linearized form III pGEM DNA vector (0.5 ng) and AlBj protein was complexed for 1 h at various molar ratios of AlBj/DNA (0–800) in the absence (−) or presence (+) of MTX (1 mM). (B) The relative retarding distance of the DNA band was plotted against the molar ratio (0–800) of AlBj protein to DNA. Relative retardation = Distance from applied well to DNA band/Distance from applied well to control DNA band. N = 3; data represented as mean ± SE. (C–E) Representative SPR sensorgram with global fitting curve between Bj protein and RAGE (purity >90%) immobilized on a CM5 sensor chip. Six or seven different concentrations of Bj protein were injected over the immobilized RAGE for 120 s and then dissociation was monitored for a further 120 s at 30 µl/min. (C, D) Interaction between Bj protein (0.31–10 µM) with immobilized RAGE in the absence (C) or presence (D) of MTX (1 mM). (E) Interaction between MTX (1–63 µM) with immobilized RAGE. (F) Concentration-response curve and Lineweaver-Burk plot between Bj protein and RAGE in the absence (open circle) or presence (filled circle) of MTX (1 mM). Linear equations in Lineweaver-Burk plot are as follows; [MTX (−)]: y = 0.0083x +0.0061 (r2 = 1), KD = 1.35 µM, Rmax = 163, [MTX (+)]: y = 0.0686x +0.0493 (r2 = 0.90), KD = 1.39 µM, Rmax = 33, KI = 142 µM. (G, H) Computer-aided binding model between MTX and HMGB1. (G) Molecular modeling of the MTX binding site in HMGB1 (K81–K164) potentially involved in the interference of HMGB1/RAGE interaction was derived using the NMR structure of fragments associated with previously published data (PDB accession code 2gzk). The predicted binding structure was solved using DS 1.7 with the CDOCKER application program by calculating the binding energy in an aqueous environment. (H) Expansion diagram of the MTX-binding site within part of RAGE-binding region (K149-V175). N92, D157, Y161 and R162 form hydrogen bonds with MTX. The binding energy is −29.31 kcal/mol.
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pone-0063073-g004: Effect of MTX for the HMGB1 binding to DNA or to RAGE.(A) Electrophoretic mobility shift assay (EMSA). The linearized form III pGEM DNA vector (0.5 ng) and AlBj protein was complexed for 1 h at various molar ratios of AlBj/DNA (0–800) in the absence (−) or presence (+) of MTX (1 mM). (B) The relative retarding distance of the DNA band was plotted against the molar ratio (0–800) of AlBj protein to DNA. Relative retardation = Distance from applied well to DNA band/Distance from applied well to control DNA band. N = 3; data represented as mean ± SE. (C–E) Representative SPR sensorgram with global fitting curve between Bj protein and RAGE (purity >90%) immobilized on a CM5 sensor chip. Six or seven different concentrations of Bj protein were injected over the immobilized RAGE for 120 s and then dissociation was monitored for a further 120 s at 30 µl/min. (C, D) Interaction between Bj protein (0.31–10 µM) with immobilized RAGE in the absence (C) or presence (D) of MTX (1 mM). (E) Interaction between MTX (1–63 µM) with immobilized RAGE. (F) Concentration-response curve and Lineweaver-Burk plot between Bj protein and RAGE in the absence (open circle) or presence (filled circle) of MTX (1 mM). Linear equations in Lineweaver-Burk plot are as follows; [MTX (−)]: y = 0.0083x +0.0061 (r2 = 1), KD = 1.35 µM, Rmax = 163, [MTX (+)]: y = 0.0686x +0.0493 (r2 = 0.90), KD = 1.39 µM, Rmax = 33, KI = 142 µM. (G, H) Computer-aided binding model between MTX and HMGB1. (G) Molecular modeling of the MTX binding site in HMGB1 (K81–K164) potentially involved in the interference of HMGB1/RAGE interaction was derived using the NMR structure of fragments associated with previously published data (PDB accession code 2gzk). The predicted binding structure was solved using DS 1.7 with the CDOCKER application program by calculating the binding energy in an aqueous environment. (H) Expansion diagram of the MTX-binding site within part of RAGE-binding region (K149-V175). N92, D157, Y161 and R162 form hydrogen bonds with MTX. The binding energy is −29.31 kcal/mol.

Mentions: The peptide sequence identified by our T7 phage display screen (K86-V175) overlaps three significant regions for inflammatory activities of HMGB1. One is Box B (K89-Y161) for DNA recognition to enhance intranuclear transcription of cytokines [21], [22], [23]. Second is F88-E107 in Box B, which constitutes the minimal epitope for inducing TNF-α release via TLR4 binding [24], [25], and the third is K149-K175, which plays an important role in binding to RAGE, a specific receptor for HMGB1 on the cell-surface that can facilitate many extracellular events (Figure 3A) [9], [26]. To clarify whether binding of MTX to HMGB1 inhibits the biological events via these regions, we initially performed EMSA [15], [27]. Previously, we reported that AlBj protein, which contains two DNA binding domains (Box A and B) [19], [20], binds to DNA with a dissociation constant (KD) of 0.95±0.37 µM as determined by SPR analysis [15]. However, the binding of AlBj protein to DNA was not inhibited in the presence of MTX at a concentration of 1 mM (Figure 4A, B), despite the stronger affinity of MTX for Box A and B domains over that of AlBj for DNA (Table 1).


Identification and characterization of the direct interaction between methotrexate (MTX) and high-mobility group box 1 (HMGB1) protein.

Kuroiwa Y, Takakusagi Y, Kusayanagi T, Kuramochi K, Imai T, Hirayama T, Ito I, Yoshida M, Sakaguchi K, Sugawara F - PLoS ONE (2013)

Effect of MTX for the HMGB1 binding to DNA or to RAGE.(A) Electrophoretic mobility shift assay (EMSA). The linearized form III pGEM DNA vector (0.5 ng) and AlBj protein was complexed for 1 h at various molar ratios of AlBj/DNA (0–800) in the absence (−) or presence (+) of MTX (1 mM). (B) The relative retarding distance of the DNA band was plotted against the molar ratio (0–800) of AlBj protein to DNA. Relative retardation = Distance from applied well to DNA band/Distance from applied well to control DNA band. N = 3; data represented as mean ± SE. (C–E) Representative SPR sensorgram with global fitting curve between Bj protein and RAGE (purity >90%) immobilized on a CM5 sensor chip. Six or seven different concentrations of Bj protein were injected over the immobilized RAGE for 120 s and then dissociation was monitored for a further 120 s at 30 µl/min. (C, D) Interaction between Bj protein (0.31–10 µM) with immobilized RAGE in the absence (C) or presence (D) of MTX (1 mM). (E) Interaction between MTX (1–63 µM) with immobilized RAGE. (F) Concentration-response curve and Lineweaver-Burk plot between Bj protein and RAGE in the absence (open circle) or presence (filled circle) of MTX (1 mM). Linear equations in Lineweaver-Burk plot are as follows; [MTX (−)]: y = 0.0083x +0.0061 (r2 = 1), KD = 1.35 µM, Rmax = 163, [MTX (+)]: y = 0.0686x +0.0493 (r2 = 0.90), KD = 1.39 µM, Rmax = 33, KI = 142 µM. (G, H) Computer-aided binding model between MTX and HMGB1. (G) Molecular modeling of the MTX binding site in HMGB1 (K81–K164) potentially involved in the interference of HMGB1/RAGE interaction was derived using the NMR structure of fragments associated with previously published data (PDB accession code 2gzk). The predicted binding structure was solved using DS 1.7 with the CDOCKER application program by calculating the binding energy in an aqueous environment. (H) Expansion diagram of the MTX-binding site within part of RAGE-binding region (K149-V175). N92, D157, Y161 and R162 form hydrogen bonds with MTX. The binding energy is −29.31 kcal/mol.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643934&req=5

pone-0063073-g004: Effect of MTX for the HMGB1 binding to DNA or to RAGE.(A) Electrophoretic mobility shift assay (EMSA). The linearized form III pGEM DNA vector (0.5 ng) and AlBj protein was complexed for 1 h at various molar ratios of AlBj/DNA (0–800) in the absence (−) or presence (+) of MTX (1 mM). (B) The relative retarding distance of the DNA band was plotted against the molar ratio (0–800) of AlBj protein to DNA. Relative retardation = Distance from applied well to DNA band/Distance from applied well to control DNA band. N = 3; data represented as mean ± SE. (C–E) Representative SPR sensorgram with global fitting curve between Bj protein and RAGE (purity >90%) immobilized on a CM5 sensor chip. Six or seven different concentrations of Bj protein were injected over the immobilized RAGE for 120 s and then dissociation was monitored for a further 120 s at 30 µl/min. (C, D) Interaction between Bj protein (0.31–10 µM) with immobilized RAGE in the absence (C) or presence (D) of MTX (1 mM). (E) Interaction between MTX (1–63 µM) with immobilized RAGE. (F) Concentration-response curve and Lineweaver-Burk plot between Bj protein and RAGE in the absence (open circle) or presence (filled circle) of MTX (1 mM). Linear equations in Lineweaver-Burk plot are as follows; [MTX (−)]: y = 0.0083x +0.0061 (r2 = 1), KD = 1.35 µM, Rmax = 163, [MTX (+)]: y = 0.0686x +0.0493 (r2 = 0.90), KD = 1.39 µM, Rmax = 33, KI = 142 µM. (G, H) Computer-aided binding model between MTX and HMGB1. (G) Molecular modeling of the MTX binding site in HMGB1 (K81–K164) potentially involved in the interference of HMGB1/RAGE interaction was derived using the NMR structure of fragments associated with previously published data (PDB accession code 2gzk). The predicted binding structure was solved using DS 1.7 with the CDOCKER application program by calculating the binding energy in an aqueous environment. (H) Expansion diagram of the MTX-binding site within part of RAGE-binding region (K149-V175). N92, D157, Y161 and R162 form hydrogen bonds with MTX. The binding energy is −29.31 kcal/mol.
Mentions: The peptide sequence identified by our T7 phage display screen (K86-V175) overlaps three significant regions for inflammatory activities of HMGB1. One is Box B (K89-Y161) for DNA recognition to enhance intranuclear transcription of cytokines [21], [22], [23]. Second is F88-E107 in Box B, which constitutes the minimal epitope for inducing TNF-α release via TLR4 binding [24], [25], and the third is K149-K175, which plays an important role in binding to RAGE, a specific receptor for HMGB1 on the cell-surface that can facilitate many extracellular events (Figure 3A) [9], [26]. To clarify whether binding of MTX to HMGB1 inhibits the biological events via these regions, we initially performed EMSA [15], [27]. Previously, we reported that AlBj protein, which contains two DNA binding domains (Box A and B) [19], [20], binds to DNA with a dissociation constant (KD) of 0.95±0.37 µM as determined by SPR analysis [15]. However, the binding of AlBj protein to DNA was not inhibited in the presence of MTX at a concentration of 1 mM (Figure 4A, B), despite the stronger affinity of MTX for Box A and B domains over that of AlBj for DNA (Table 1).

Bottom Line: Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood.These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX.These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Yamazaki, Noda, Chiba, Japan.

ABSTRACT

Background: Methotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood. METHODOLOGY/RESULT: Here, we performed a screening of MTX-binding proteins using T7 phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 phage display screen, we identified T7 phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD) of 0.50±0.03 µM for Al and 0.24 ± 0.01 µM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macrophage-like cells (RAW 264.7), TNF-α release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited in the presence of MTX.

Conclusions/significance: These data demonstrate that HMGB1 is a direct binding protein of MTX. Moreover, binding of MTX to RAGE-binding region in HMGB1 inhibited the HMGB1/RAGE interaction at the molecular and cellular levels. These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.

Show MeSH
Related in: MedlinePlus