Limits...
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.

Show MeSH

Related in: MedlinePlus

Synthesis of bio-MTX (3).The reaction gave bio-MTX 3a and 3b as a 1∶1 mixture.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3643934&req=5

pone-0063073-g001: Synthesis of bio-MTX (3).The reaction gave bio-MTX 3a and 3b as a 1∶1 mixture.

Mentions: A biotinylated derivative of MTX (bio-MTX) was synthesized according to previous reports (Figure 1) [10], [11]. MTX (1) (10.0 mg, 0.022 mmol) and 5-(biotinamido) pentylamine (2) (10.8 mg, 0.033 mmol) in DMSO (0.4 mL) was added to a solution of WSC (4.5 mg, 0.037 mmol) followed by DMAP (3.4 mg, 0.022 mmol). The mixture was then stirred overnight at room temperature. The resulting solution was concentrated in vacuo and the reaction products purified by reverse phase column chromatography (Wakogel® 100C18, Wako Pure Chemical Industries, Ltd., Osaka, Japan) with 0.1 M NH4HCO3 aq. and MeOH to give a mixture of bio-MTX (3a, 3b) in about 65% yield as a 1∶1 ratio. 1H NMR spectra were recorded on a Bruker 600 MHz spectrometer (Avance DRX-600) using a mixture of DMSO-d6 and D2O as a solvent. Chemical shifts are expressed as δ (ppm) relative to residual solvent resonance (CHD2SOCD3), and coupling constants (J) are expressed in Hertz. Mass spectra (MS) were obtained on an Applied Biosystems mass spectrometer (APIQSTAR pulsar i) under high resolution conditions using (poly)ethylene glycol as an internal standard.


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)

Synthesis of bio-MTX (3).The reaction gave bio-MTX 3a and 3b as a 1∶1 mixture.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063073-g001: Synthesis of bio-MTX (3).The reaction gave bio-MTX 3a and 3b as a 1∶1 mixture.
Mentions: A biotinylated derivative of MTX (bio-MTX) was synthesized according to previous reports (Figure 1) [10], [11]. MTX (1) (10.0 mg, 0.022 mmol) and 5-(biotinamido) pentylamine (2) (10.8 mg, 0.033 mmol) in DMSO (0.4 mL) was added to a solution of WSC (4.5 mg, 0.037 mmol) followed by DMAP (3.4 mg, 0.022 mmol). The mixture was then stirred overnight at room temperature. The resulting solution was concentrated in vacuo and the reaction products purified by reverse phase column chromatography (Wakogel® 100C18, Wako Pure Chemical Industries, Ltd., Osaka, Japan) with 0.1 M NH4HCO3 aq. and MeOH to give a mixture of bio-MTX (3a, 3b) in about 65% yield as a 1∶1 ratio. 1H NMR spectra were recorded on a Bruker 600 MHz spectrometer (Avance DRX-600) using a mixture of DMSO-d6 and D2O as a solvent. Chemical shifts are expressed as δ (ppm) relative to residual solvent resonance (CHD2SOCD3), and coupling constants (J) are expressed in Hertz. Mass spectra (MS) were obtained on an Applied Biosystems mass spectrometer (APIQSTAR pulsar i) under high resolution conditions using (poly)ethylene glycol as an internal standard.

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