Journal: The Journal of Biological Chemistry

Article Title: Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

doi: 10.1074/jbc.RA119.009716

Figure Lengend Snippet: Recombinant AtMDLs only have residual tautomerase activity, likely due to a sterically-impeded substrate-binding pocket. A and B, tautomerase activity of recombinant HsMIF/AtMDL protein homologs measured by spectrophotometry. A, tautomerase activity of the three 6xHis-tagged AtMDLs was compared with that of 6xHis-tagged HsMIF using HPP as a substrate. Untagged HsMIF was measured for comparison. Data shown are from four to seven independent experiments ± S.D., each performed in triplicate (scatter plot with white circles indicates individual data points). For statistical comparisons, one-way ANOVA between buffer control and the different samples was applied (***, p < 0.005). B, comparison of the tautomerase activity of AtMDL1–6xHis and HsMIF–6xHis using DCME as a substrate. Data shown are from four independent experiments ± S.D., performed in triplicate each (scatter plot with white circles indicates individual data points). For statistical comparisons, one-way ANOVA between buffer control and the different samples was applied (***, p < 0.005). C, multiple sequence alignment of 6xHis-tagged variants of AtMDLs and HsMIF. Amino acid sequences of AtMDL1 (identifier Q9LU69), AtMDL2 (identifier Q9M011), AtMDL3 (identifier Q8LG92), and HsMIF (identifier P14174) were retrieved from the UniProt database and aligned by ClustalW using standard parameters in the Jalview multiple sequence alignment editor desktop application. The amino acid residues forming the tautomerase substrate-binding pocket are highlighted in magenta and red. D, comparative view of predicted 3D structures of 6xHis-tagged HsMIF and the three AtMDLs. Only the monomers are shown for simplicity reasons. Amino acid sequences of the AtMDL proteins were subjected to analysis via the PHYRE2 Fold Recognition server and visualized with PyMOL. The predicted 3D structures (ribbon, surface, and electrostatic surface potential models) of AtMDL1–6xHis, AtMDL2–6xHis, and AtMDL3–6xHis were analyzed compared with the known X-ray–resolved 3D structure of HsMIF–6xHis. The upper and middle panels highlight the location of crucial tautomerase pocket residues in the ribbon structure and on the protein surface (magenta and red). In the lower panel, red and blue, respectively, indicate an excess of negative or positive charges near the surface, and grayish color symbolizes neutral regions. E, comparison of the tautomerase activity of N98K–HsMIF–6xHis and HsMIF–6xHis using HPP as a substrate. Data shown are from three to four independent experiments ± S.D., performed in triplicate each (scatter plot with white circles indicates individual data points). For statistical comparisons, one-way ANOVA between buffer control and the different samples was applied (*, p < 0.05; ****, p < 0.001), as well as comparison between 250 nm HsMIF and 250 nm N98K–HsMIF–6xHis (####, p < 0.001). F, same as E except that DCME was used as a substrate, and HsMIF–6xHis was applied at a concentration of 100 nm (***, p < 0.005; ****, p < 0.001; ####, p < 0.001).

Article Snippet: His-tagged proteins were detected by mouse anti-6xHis tag mAb (Ma1–135; Invitrogen) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary goat anti-mouse IgG (ab6789; Abcam, Cambridge, UK).

Techniques: Recombinant, Activity Assay, Binding Assay, Spectrophotometry, Sequencing, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

doi: 10.1074/jbc.RA119.009716

Figure Lengend Snippet: Comparison of the kinetic tautomerase activity parameters between recombinant His 6 -tagged Hs MIF and At MDL1 Data represent triplicate measurements ± S.D.

Article Snippet: His-tagged proteins were detected by mouse anti-6xHis tag mAb (Ma1–135; Invitrogen) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary goat anti-mouse IgG (ab6789; Abcam, Cambridge, UK).

Techniques: Activity Assay, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

doi: 10.1074/jbc.RA119.009716

Figure Lengend Snippet: AtMDLs share homology with human MIF in the MIF receptor–binding sites, bind to CD74, and activate CXCR4-mediated signaling in a yeast-based reporter system. A, multiple sequence alignment of the AtMDLs, HsMIF, and human CXCL12. Amino acid sequences of AtMDL1 (identifier Q9LU69), AtMDL2 (identifier Q9M011), AtMDL3 (identifier Q8LG92), and HsMIF (identifier P14174) were retrieved from the UniProt database and aligned by ClustalW using standard parameters in the Jalview multiple sequence alignment editor desktop application. The amino acid residues contributing to the site I and II binding interface between HsMIF and CXCR4 (41) or CXCL12 and CXCR4 (90, 91), the binding sites between HsMIF and CD74, and the predicted corresponding regions in the AtMDLs are indicated. Determinants of HsMIF contributing to CXCR2 binding, although not further examined in this study, are indicated for comparison. The degree of homology/identity of the MIF, CXCL12, or AtMDL residues in these regions is highlighted by the following color score: blue, hydrophobic; red, positively charged; magenta, negatively charged; green, polar; pink, cysteine; orange, glycine; yellow, proline; cyan, aromatic; white, unconserved. B, comparison of the in vitro binding capacity between HsMIF–6xHis and MBP–sCD74 with that of the three His-tagged AtMDLs. Binding was measured by an ELISA-type plate-binding assay. BSA, blank PBS buffer (control), and MBP alone served as negative controls as indicated to account for nonspecific binding effects. Wells were coated with BSA (2% w/v), 500 nm HsMIF, and AtMDL1–6xHis, AtMDL2–6xHis, and AtMDL3–6xHis (500 nm), followed by binding of MBP or MBP–sCD74 (500 nm). After signal development, absorbance at 450 nm was measured, and the signals were normalized by setting the absorbance of HsMIF as 1. C, curve for binding of MBP–sCD74 and AtMDL3 using increasing concentrations of MBP–sCD74 as indicated. The data in B and C are displayed as means ± S.D. (n = 3); (scatter plot with white circles indicates individual data points); ns, not significant; ***, p < 0.001; **, p < 0.01. D, CXCR4-mediated signaling in a yeast-based reporter system. In this assay, the Ste2 GPCR of the pheromone-response pathway of S. cerevisiae was substituted by the human CXCR4 receptor. Ligand binding to CXCR4 triggers signaling and expression of the lacZ gene, as assessed by β-gal activity. The concentrations of native (untagged) HsMIF, HsMIF–6xHis, and His-tagged AtMDLs were 20 μm each. The concentration of human CXCL12 was equal to 2 μm. Reporter activity is given as relative luminescence, normalized to the untreated control (Ctrl). Values shown represent means ± S.D. from three independent experiments, in which the activity of each was assessed in technical duplicates (scatter plot with white circles indicates individual data points). Statistical analysis was performed using one-way ANOVA and post-hoc Tukey's HSD test with multiple comparisons. Different letters above the bars denote a statistically significant difference between groups (p < 0.05), and groups showing the same letters are not statistically significantly different from each other.

Article Snippet: His-tagged proteins were detected by mouse anti-6xHis tag mAb (Ma1–135; Invitrogen) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary goat anti-mouse IgG (ab6789; Abcam, Cambridge, UK).

Techniques: Binding Assay, Sequencing, In Vitro, Enzyme-linked Immunosorbent Assay, Ligand Binding Assay, Expressing, Activity Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

doi: 10.1074/jbc.RA119.009716

Figure Lengend Snippet: AtMDLs activate the CXCR4–PI3K/Akt-signaling pathway in human CXCR4-transfected HEK293 cells. A, representative Western blotting indicates Akt phosphorylation (pAkt) at different time intervals as indicated following stimulation with HsMIF–6xHis at a concentration of 16 nm. Ctrl, untreated control sample. Total Akt and actin were analyzed as loading control and for quantification purposes. B, quantification of pAkt band intensities in relation to Akt and actin band intensities according to the Western blot analysis in A. Bar graph represents means ± S.D. of 5–15 experiments (scatter plot with white circles indicates individual data points). Statistical analysis was performed using one-way ANOVA between the untreated control (Ctrl) and the various time points following treatment (*, p < 0.05; ***, p < 0.005). C, E, and G, same as A except that the time-dependent phosphorylation of Akt following treatment with AtMDL1–6xHis, AtMDL2–6xHis, and AtMDL3–6xHis, respectively, was analyzed and compared with the effect of HsMIF–6xHis at 15 min. D, F, and H, same as B, except that the quantification refers to the Western blot analysis in C, E, and G and that data are from 5 to 10 experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.005).

Article Snippet: His-tagged proteins were detected by mouse anti-6xHis tag mAb (Ma1–135; Invitrogen) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary goat anti-mouse IgG (ab6789; Abcam, Cambridge, UK).

Techniques: Transfection, Western Blot, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

doi: 10.1074/jbc.RA119.009716

Figure Lengend Snippet: Recombinant 6xHis-tagged AtMDLs trigger chemotactic migration of human THP-1 monocytes in a dose-dependent manner. Chemotaxis (referred to here as chemotactic index) of THP-1 monocytes toward HsMIF–6xHis (A), AtMDL1–6xHis (B), AtMDL2–6xHis (C), or AtMDL3–6xHis (D) at the different indicated concentrations. The chemotactic potency was compared with human CXCL12 (at a concentration of 8 nm) serving as a positive control and to buffer control (Ctrl), which also served to normalize treatments to spontaneous (random) migration events. The bar graphs show means ± S.D. of five independent experiments (scatter plot with white circles indicates individual data points). Statistical analyses were performed using one-way ANOVA between the buffer control and the treatment groups at the various doses (*, p < 0.05; **, p < 0.01; ***, p < 0.005).

Article Snippet: His-tagged proteins were detected by mouse anti-6xHis tag mAb (Ma1–135; Invitrogen) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary goat anti-mouse IgG (ab6789; Abcam, Cambridge, UK).

Techniques: Recombinant, Migration, Chemotaxis Assay, Concentration Assay, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

doi: 10.1074/jbc.RA119.009716

Figure Lengend Snippet: AtMDL1–6xHis-triggered monocyte chemotaxis is blocked by the small molecule inhibitors AMD3100 and ISO-1, indicating MIF and CXCR4 specificity. Chemotaxis experiments were performed as shown in Fig. 5 in the absence or presence of the small molecule inhibitors AMD3100 (10 μm) or ISO-1 (100 μm) as indicated. Concentrations of the recombinant proteins were equal to 32 nm for HsMIF–6xHis and AtMDL1–6xHis and 8 nm for CXCL12. Sodium phosphate buffer was used to normalize treatments to spontaneous random migration (control, Ctrl). Bar graph shows means ± S.D. of one of two independently performed experiments carried out as technical triplicates each (for the other experiment see Fig. S5) (scatter plot with white circles indicates individual data points). Statistical analysis was done using one-way ANOVA for comparisons within a group (***, p < 0.005) and paired t test for comparisons between control and the AMD3100 and ISO-1 treatment groups (##, p < 0.01; ###, p < 0.005; NS, not significant).

Article Snippet: His-tagged proteins were detected by mouse anti-6xHis tag mAb (Ma1–135; Invitrogen) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary goat anti-mouse IgG (ab6789; Abcam, Cambridge, UK).

Techniques: Chemotaxis Assay, Recombinant, Migration

Journal: The Journal of Biological Chemistry

Article Title: Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

doi: 10.1074/jbc.RA119.009716

Figure Lengend Snippet: Recombinant 6xHis-tagged AtMDLs dose-dependently trigger chemotactic migration of primary human T cells and desensitize T cells from CXCR4 agonist-triggered chemotaxis. A, recombinant 6xHis-tagged AtMDLs trigger chemotactic migration of primary human T cells in a dose-dependent manner. The chemotactic potency was compared with HsMIF–6xHis and human CXCL12 (at a concentration of 8 nm) serving as a positive control and to buffer control (Ctrl), which also served to normalize treatments to spontaneous (random) migration events. Bar graphs show means ± S.D. of three independent experiments (scatter plot with white circles indicates individual data points). Statistical analyses were performed using one-way ANOVA between the buffer control and the treatment groups at the various doses (*, p < 0.05; **, p < 0.01; ***, p < 0.005). B, recombinant 6xHis-tagged AtMDLs desensitize T cells from chemotaxis elicited by CXCL12 or HsMIF–6xHis. Data are the same as in A, except that T cells in the upper chamber were preincubated with the His-tagged AtMDLs for 2 h (+), before being subjected to chemoattractant exposure (CXCL12 or HsMIF–6xHis) in the lower chamber. Control (Ctrl) incubations were performed without chemoattractant in the lower chamber (random migration). Bar graphs show means ± S.D. of three experiments (scatter plot with white circles indicates individual data points). Statistical analyses were performed using one-way ANOVA between control and CXCL12 or HsMIF (###, p < 0.005; ####, p < 0.001) and between CXCL12 or MIF with and without pre-treatment with AtMDLs (****, p < 0.001).

Article Snippet: His-tagged proteins were detected by mouse anti-6xHis tag mAb (Ma1–135; Invitrogen) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary goat anti-mouse IgG (ab6789; Abcam, Cambridge, UK).

Techniques: Recombinant, Migration, Chemotaxis Assay, Concentration Assay, Positive Control