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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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Identification of MTX-binding protein by T7 phage display.(A) Elution titer and recovery ratio after each round of biopanning. Elution titer ratio = Number of eluted phage particles (pfu/ml) from test well/Number of eluted phage particles (pfu/ml) from the MTX-non-immobilized control well. Recovery ratio (%) = [Number of the eluted phage particles (pfu/ml)/Number of input phage particles (pfu/ml)]×100. Pfu: plaque forming unit. (B) Sequence homology between human HMGB1 and the coding polypeptide of MTX-binding T7 phage particle. The recovered sequence encodes a polypeptide that is 100% identical to a portion of human HMGB1 (K86-V175). (C) The specific affinity of HMGB1-displaying T7 phage to MTX. Isolated HMGB1 (K86-V175)-displaying monoclonal T7 phage or control T7 phage (no cDNA insert) stocks were individually allowed to interact with immobilized MTX. Bound phages were then eluted using buffer containing an excess (200 µM) of MTX [MTX (+)] or no MTX [MTX (−)]. The number of eluted phage particles was counted. N = 3; data represented as mean ± SE.
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pone-0063073-g002: Identification of MTX-binding protein by T7 phage display.(A) Elution titer and recovery ratio after each round of biopanning. Elution titer ratio = Number of eluted phage particles (pfu/ml) from test well/Number of eluted phage particles (pfu/ml) from the MTX-non-immobilized control well. Recovery ratio (%) = [Number of the eluted phage particles (pfu/ml)/Number of input phage particles (pfu/ml)]×100. Pfu: plaque forming unit. (B) Sequence homology between human HMGB1 and the coding polypeptide of MTX-binding T7 phage particle. The recovered sequence encodes a polypeptide that is 100% identical to a portion of human HMGB1 (K86-V175). (C) The specific affinity of HMGB1-displaying T7 phage to MTX. Isolated HMGB1 (K86-V175)-displaying monoclonal T7 phage or control T7 phage (no cDNA insert) stocks were individually allowed to interact with immobilized MTX. Bound phages were then eluted using buffer containing an excess (200 µM) of MTX [MTX (+)] or no MTX [MTX (−)]. The number of eluted phage particles was counted. N = 3; data represented as mean ± SE.

Mentions: Bj (amino acid residues F88-K181 of HMGB1), Al (G1-K87) and AlBj (G1-K181) proteins (Figure 2A) were individually engineered for heterologous expression in E. coli JM109 using pTrcHis vector (Invitrogen, Carlsbad, CA). In each case, the corresponding recombinant protein included an N-terminal His tag (6×His residues) [15]. The transformant was grown in LB medium containing carbenicillin at 37°C and heterologous gene expression was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The cell culture was continued for 3 h before harvesting by centrifugation. Each cell pellet was suspended in purification buffer (20 mM Tris-HCl, 500 mM NaCl, 10 mM 2-mercaptoethanol, pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mg/ml of lysozyme. Cells were disrupted by sonication and the cell-free extract was then clarified by centrifugation at 20400 g. The soluble fraction was filtered and loaded onto a HisTrap HP column (1 ml) (GE Healthcare, Amersham, UK) equilibrated in purification buffer containing 50 mM imidazole using a FPLC system (ÄKTA explorer, GE Healthcare). After washing the column, bound protein was eluted using a gradient of 50 mM (A) to 250 mM imidazole (B). The eluted recombinant protein was then desalted using a PD10 column (GE Healthcare). The purity was analyzed by SDS-PAGE using 15% separation gel. The bands were stained with CBB.


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)

Identification of MTX-binding protein by T7 phage display.(A) Elution titer and recovery ratio after each round of biopanning. Elution titer ratio = Number of eluted phage particles (pfu/ml) from test well/Number of eluted phage particles (pfu/ml) from the MTX-non-immobilized control well. Recovery ratio (%) = [Number of the eluted phage particles (pfu/ml)/Number of input phage particles (pfu/ml)]×100. Pfu: plaque forming unit. (B) Sequence homology between human HMGB1 and the coding polypeptide of MTX-binding T7 phage particle. The recovered sequence encodes a polypeptide that is 100% identical to a portion of human HMGB1 (K86-V175). (C) The specific affinity of HMGB1-displaying T7 phage to MTX. Isolated HMGB1 (K86-V175)-displaying monoclonal T7 phage or control T7 phage (no cDNA insert) stocks were individually allowed to interact with immobilized MTX. Bound phages were then eluted using buffer containing an excess (200 µM) of MTX [MTX (+)] or no MTX [MTX (−)]. The number of eluted phage particles was counted. N = 3; data represented as mean ± SE.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643934&req=5

pone-0063073-g002: Identification of MTX-binding protein by T7 phage display.(A) Elution titer and recovery ratio after each round of biopanning. Elution titer ratio = Number of eluted phage particles (pfu/ml) from test well/Number of eluted phage particles (pfu/ml) from the MTX-non-immobilized control well. Recovery ratio (%) = [Number of the eluted phage particles (pfu/ml)/Number of input phage particles (pfu/ml)]×100. Pfu: plaque forming unit. (B) Sequence homology between human HMGB1 and the coding polypeptide of MTX-binding T7 phage particle. The recovered sequence encodes a polypeptide that is 100% identical to a portion of human HMGB1 (K86-V175). (C) The specific affinity of HMGB1-displaying T7 phage to MTX. Isolated HMGB1 (K86-V175)-displaying monoclonal T7 phage or control T7 phage (no cDNA insert) stocks were individually allowed to interact with immobilized MTX. Bound phages were then eluted using buffer containing an excess (200 µM) of MTX [MTX (+)] or no MTX [MTX (−)]. The number of eluted phage particles was counted. N = 3; data represented as mean ± SE.
Mentions: Bj (amino acid residues F88-K181 of HMGB1), Al (G1-K87) and AlBj (G1-K181) proteins (Figure 2A) were individually engineered for heterologous expression in E. coli JM109 using pTrcHis vector (Invitrogen, Carlsbad, CA). In each case, the corresponding recombinant protein included an N-terminal His tag (6×His residues) [15]. The transformant was grown in LB medium containing carbenicillin at 37°C and heterologous gene expression was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The cell culture was continued for 3 h before harvesting by centrifugation. Each cell pellet was suspended in purification buffer (20 mM Tris-HCl, 500 mM NaCl, 10 mM 2-mercaptoethanol, pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mg/ml of lysozyme. Cells were disrupted by sonication and the cell-free extract was then clarified by centrifugation at 20400 g. The soluble fraction was filtered and loaded onto a HisTrap HP column (1 ml) (GE Healthcare, Amersham, UK) equilibrated in purification buffer containing 50 mM imidazole using a FPLC system (ÄKTA explorer, GE Healthcare). After washing the column, bound protein was eluted using a gradient of 50 mM (A) to 250 mM imidazole (B). The eluted recombinant protein was then desalted using a PD10 column (GE Healthcare). The purity was analyzed by SDS-PAGE using 15% separation gel. The bands were stained with CBB.

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