Identification and Characterization of the Direct Interaction between Methotrexate (MTX) and High-Mobility Group Box 1 (HMGB1) Protein

Yuki Kuroiwa, Yoichi Takakusagi, Tomoe Kusayanagi, Kouji Kuramochi, Takahiko Imai, Tomoko Hirayama, Ichiaki Ito, Michiteru Yoshida, Kengo Sakaguchi, Fumio Sugawara

Research output: Contribution to journalArticle

24 Scopus citations

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.

Original languageEnglish (US)
Article numbere63073
JournalPLoS One
Volume8
Issue number5
DOIs
StatePublished - May 3 2013
Externally publishedYes

    Fingerprint

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Kuroiwa, Y., Takakusagi, Y., Kusayanagi, T., Kuramochi, K., Imai, T., Hirayama, T., Ito, I., Yoshida, M., Sakaguchi, K., & Sugawara, F. (2013). Identification and Characterization of the Direct Interaction between Methotrexate (MTX) and High-Mobility Group Box 1 (HMGB1) Protein. PLoS One, 8(5), [e63073]. https://doi.org/10.1371/journal.pone.0063073