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

SPR analysis of the interaction between MTX and HMGB1.(A) Full length map of HMGB1 and truncated recombinant versions of the protein engineered in E. coli. The displayed sequence of the MTX-binding T7 phage particle (K86-V175) includes the Box B domain (K89-Y161), TLR4-binding domain (F88-E107) and part of the RAGE-binding domain (K149-V175). (B) SDS-PAGE of AlBj, Al and Bj proteins after purification by affinity chromatography. The bands were stained with CBB. (C, D) Representative SPR sensorgram with a global fitting curve between bio-MTX and Al (C) or Bj (D). Solutions containing various concentrations of Al (0.31–5 µM) or Bj protein (0.16–2.5 µM) were injected over the immobilized MTX-biotin on a SA sensor chip for 120 s and then dissociation was monitored for a further 120 s at a flow rate of 30 µl/min. Response curves were generated by subtraction of the background signals generated simultaneously on the control flow cell (bio-MTX-non-immobilized cell) and the injection of vehicle (0 µM analyte). (E) Concentration-response curve between bio-MTX and AlBj obtained from SPR analysis. Rmax = 66. (F) Scatchard-plot analysis of AlBj binding to MTX. (G) Hill-plot analysis of AlBj binding. Hill coefficients n = 1.1. RU: resonance unit. 1 RU = 1 pg/mm2.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3643934&req=5

pone-0063073-g003: SPR analysis of the interaction between MTX and HMGB1.(A) Full length map of HMGB1 and truncated recombinant versions of the protein engineered in E. coli. The displayed sequence of the MTX-binding T7 phage particle (K86-V175) includes the Box B domain (K89-Y161), TLR4-binding domain (F88-E107) and part of the RAGE-binding domain (K149-V175). (B) SDS-PAGE of AlBj, Al and Bj proteins after purification by affinity chromatography. The bands were stained with CBB. (C, D) Representative SPR sensorgram with a global fitting curve between bio-MTX and Al (C) or Bj (D). Solutions containing various concentrations of Al (0.31–5 µM) or Bj protein (0.16–2.5 µM) were injected over the immobilized MTX-biotin on a SA sensor chip for 120 s and then dissociation was monitored for a further 120 s at a flow rate of 30 µl/min. Response curves were generated by subtraction of the background signals generated simultaneously on the control flow cell (bio-MTX-non-immobilized cell) and the injection of vehicle (0 µM analyte). (E) Concentration-response curve between bio-MTX and AlBj obtained from SPR analysis. Rmax = 66. (F) Scatchard-plot analysis of AlBj binding to MTX. (G) Hill-plot analysis of AlBj binding. Hill coefficients n = 1.1. RU: resonance unit. 1 RU = 1 pg/mm2.

Mentions: To further demonstrate the specific affinity of MTX for HMGB1 and characterize the affinity status, real-time binding of MTX to truncated HMGB1 proteins was monitored in vitro by surface plasmon resonance (SPR) analysis [16]. After immobilizing biotinylated MTX (bio-MTX) on the surface of a sensor chip SA (GE Healthcare), various concentrations of truncated histidine-tagged segments of HMGB1 (Figure 3A), purified by an affinity chromatography procedure as a single band on SDS-PAGE with CBB staining (Figure 3B), were injected over the immobilized bio-MTX to measure the respective binding affinities.


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)

SPR analysis of the interaction between MTX and HMGB1.(A) Full length map of HMGB1 and truncated recombinant versions of the protein engineered in E. coli. The displayed sequence of the MTX-binding T7 phage particle (K86-V175) includes the Box B domain (K89-Y161), TLR4-binding domain (F88-E107) and part of the RAGE-binding domain (K149-V175). (B) SDS-PAGE of AlBj, Al and Bj proteins after purification by affinity chromatography. The bands were stained with CBB. (C, D) Representative SPR sensorgram with a global fitting curve between bio-MTX and Al (C) or Bj (D). Solutions containing various concentrations of Al (0.31–5 µM) or Bj protein (0.16–2.5 µM) were injected over the immobilized MTX-biotin on a SA sensor chip for 120 s and then dissociation was monitored for a further 120 s at a flow rate of 30 µl/min. Response curves were generated by subtraction of the background signals generated simultaneously on the control flow cell (bio-MTX-non-immobilized cell) and the injection of vehicle (0 µM analyte). (E) Concentration-response curve between bio-MTX and AlBj obtained from SPR analysis. Rmax = 66. (F) Scatchard-plot analysis of AlBj binding to MTX. (G) Hill-plot analysis of AlBj binding. Hill coefficients n = 1.1. RU: resonance unit. 1 RU = 1 pg/mm2.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063073-g003: SPR analysis of the interaction between MTX and HMGB1.(A) Full length map of HMGB1 and truncated recombinant versions of the protein engineered in E. coli. The displayed sequence of the MTX-binding T7 phage particle (K86-V175) includes the Box B domain (K89-Y161), TLR4-binding domain (F88-E107) and part of the RAGE-binding domain (K149-V175). (B) SDS-PAGE of AlBj, Al and Bj proteins after purification by affinity chromatography. The bands were stained with CBB. (C, D) Representative SPR sensorgram with a global fitting curve between bio-MTX and Al (C) or Bj (D). Solutions containing various concentrations of Al (0.31–5 µM) or Bj protein (0.16–2.5 µM) were injected over the immobilized MTX-biotin on a SA sensor chip for 120 s and then dissociation was monitored for a further 120 s at a flow rate of 30 µl/min. Response curves were generated by subtraction of the background signals generated simultaneously on the control flow cell (bio-MTX-non-immobilized cell) and the injection of vehicle (0 µM analyte). (E) Concentration-response curve between bio-MTX and AlBj obtained from SPR analysis. Rmax = 66. (F) Scatchard-plot analysis of AlBj binding to MTX. (G) Hill-plot analysis of AlBj binding. Hill coefficients n = 1.1. RU: resonance unit. 1 RU = 1 pg/mm2.
Mentions: To further demonstrate the specific affinity of MTX for HMGB1 and characterize the affinity status, real-time binding of MTX to truncated HMGB1 proteins was monitored in vitro by surface plasmon resonance (SPR) analysis [16]. After immobilizing biotinylated MTX (bio-MTX) on the surface of a sensor chip SA (GE Healthcare), various concentrations of truncated histidine-tagged segments of HMGB1 (Figure 3A), purified by an affinity chromatography procedure as a single band on SDS-PAGE with CBB staining (Figure 3B), were injected over the immobilized bio-MTX to measure the respective binding affinities.

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