Figure 1.
The reaction gave bio-MTX 3a and 3b as a 1∶1 mixture.
Figure 2.
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.
Figure 3.
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.
Table 1.
Kinetic parameters of each ligand-analyte interaction.
Figure 4.
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.
Figure 5.
Effect of MTX on the truncated HMGB1 (Bj)-elicited TNF-α release and mitogenic activity in RAW 264.7 cells.
(A) Bj protein-dependent TNF-α release. RAW 264.7 cells, in a 24-well culture dish format, were stimulated with the indicated concentrations of Bj protein for 6 h. The amount of TNF-α released into the conditioned medium was then determined by ELISA. N ≥2. (B) Influence of MTX alone for TNF-α release. RAW 264.7 cells were stimulated with 0–10 µM of MTX for 6 h. N ≥3. (C) Inhibition of Bj protein-elicited TNF-α release, in a 24-well culture dish format, stimulated with 0.5 µM of Bj protein for 6 h in the presence of 0–10 µM MTX. N ≥3. (D) Bj protein-elicited mitogenic activity for 10 h. Results are given in terms of relative cell growth. N ≥3. (E) Cell growth in the presence or absence of Bj protein (0.5 µM), or MTX (100 µM). N ≥3. (F) Time course of MTX cytotoxicity. RAW 264.7 cell growth was elucidated using the WST-8 cell proliferation assay and is shown as relative cell growth (%). Data represented as mean ± SE. *P<0.05, ****P<0.001.