<i>N</i>-linked glycoproteins exert WN1316-mediated cytoprotective activity.

<p>Differentiated SH-SY5Y cells were pretreated with 8 μM WN1316 in DMEM containing 0.2 mg/ml FBS, Con A pass, or Con A elute for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 μM menadione for 4 h. As control experiments, differentiated SH-SY5Y cells were treated with 8 μM WN1316 (as a positive control) or DMSO (as a vehicle control) in DMEM supplemented with 2% FBS (corresponds to a protein concentration of approximately 0.5 mg/ml). The cell viability was calculated by AlamarBlue, and was expressed as a relative value (relative cytoprotective activity; -fold) of the WN1316-treated samples for vehicle control (DMSO) set as 1. Data are expressed as mean ± SD (n = 4). Statistical significance was evaluated by one-way ANOVA (<i>p</i><0.0001) followed by Dunnett’s <i>post hoc</i> test compared with DMSO-treated control (**<i>p</i><0.001, *<i>p</i><0.01).</p

Human WN1316-activating factors exert WN1316-mediated cytoprotective activity.

<p>Differentiated SH-SY5Y cells were treated with 8 μM WN1316 in the presence of purified GST-fusion proteins for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 μM menadione for 4 h. Concentrations of the GST-fusion proteins used are as follows; GST; 4.4 μg/ml, GST-RBP4; 2.2 μg/ml, GST-AHSG; 1.9 μg/ml, GST- SERPINA1; 2.4 μg/ml, GST-ITIH4; 2.3 μg/ml, GST-A1BG; 1.5 μg/ml, GST-HPX; 1.7 μg/ml, GST-CFB; 3.3 μg/ml. As control experiments, differentiated SH-SY5Y cells were treated with 8 μM WN1316 (as a positive control) or DMSO (as a vehicle control) in DMEM supplemented with 2% FBS (corresponds to a protein concentration of approximately 0.5 mg/ml). The cell viability was calculated by AlamarBlue, and was expressed as a relative value (relative cytoprotective activity; -fold) of the WN1316-treated samples for vehicle control (DMSO) set as 1. Data are expressed as mean ± SD (n = 4). Statistical significance was evaluated by one-way ANOVA (<i>p</i><0.0001) followed by Dunnett’s <i>post hoc</i> test compared with GST-RBP4 (**<i>p</i><0.001).</p

Silver staining and Western blot analysis of Blue pass fraction treated with or without glycosidase.

<p>Blue pass fraction was treated with or without EnzMix (removal <i>N</i>- and <i>O</i>-glycans) and PNGaseF (removal of <i>N</i>-glycans) at 37°C for 16 h. The molecular masses and homogeneities of the proteins were analyzed by SDS-PAGE (A) and Western blotting with anti-AHSG antibody as a glycoprotein standard (B). Proteins were visualized by silver staining using a Silver Stain Kit Wako (Wako).</p

Purification of WN1316-activating factors.

<p>Purification of WN1316-activating factors.</p

Deglycosylation does not affect WN1316-mediated cytoprotective activity.

<p>Differentiated SH-SY5Y cells were pretreated with 8 μM WN1316 in DMEM containing 0.28 mg/ml Blue pass treated with or without EnzMix and PNGaseF for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 μM menadione for 4 h. As control experiments, differentiated SH-SY5Y cells were treated with 8 μM WN1316 (as a positive control) or DMSO (as a vehicle control) in DMEM supplemented with 2% FBS (corresponds to a protein concentration of approximately 0.5 mg/ml). The cell viability was calculated by AlamarBlue, and was expressed as a relative value (relative cytoprotective activity; -fold) of the WN1316-treated samples for vehicle control (DMSO) set as 1. Data are expressed as mean ± SD (n = 4). Statistical significance was evaluated by one-way ANOVA (<i>p</i><0.0001) followed by Dunnett’s <i>post hoc</i> test compared with DMSO-treated control (**<i>p</i><0.001).</p

Protein factors in FBS exert WN1316-mediated cytoprotective activity.

<p>(A) Effect of FBS on WN1316-induced cytoprotection. Differentiated SH-SY5Y cells were treated with 8 μM WN1316 or DMSO for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 μM menadione for 4 h. The cell viability was measured by AlamarBlue assay, and was expressed as a relative value (relative cytoprotective activity; -fold) of the WN1316-treated samples for vehicle control (DMSO) set as 1. Data are expressed as mean ± SD (n = 4). Statistical significance was evaluated by one-way ANOVA (<i>p</i><0.0001) followed by Dunnett’s <i>post hoc</i> test compared with DMSO-treated control (**<i>p</i><0.001). (B) Effect of heat-denatured FBS on the anti-oxidative stress activity of WN1316. Heat denaturation of FBS was performed at 65°C for 30 min and chilled ice-cold (denatured FBS), and heat denatured FBS was renatured by the incubation at 4°C for 16 h (renatured FBS). Differentiated SH-SY5Y cells were incubated with 8 μM WN1316 or DMSO for 3 h in DMEM supplemented with 10% FBS, 10% denatured FBS, or 10% renatured FBS, followed by 12 h of chase incubation without the compound, and then treated with 40 μM menadione for 4 h. The cell viability was calculated by AlamarBlue assay, and was expressed as a relative value (relative cytoprotective activity; -fold) of the WN1316-treated samples for vehicle control (DMSO) set as 1. Data are expressed as mean ± SD (n = 4). Statistical significance was evaluated by one-way ANOVA (<i>p</i><0.0001) followed by Dunnett’s <i>post hoc</i> test compared with DMSO-treated control (**<i>p</i><0.001).</p

Representative 2DE gel image of high WN1316 active E1 fraction separated by the Bio-Scale Mini CHT Type I column.

<p>Proteins (300 μg) were separated by isoelectric focusing using Immobiline DryStrip of pH 4–7, followed by 9–18% gradient SDS-PAGE gels. Proteins were visualized by SYPRO Ruby staining. Proteins were identified by MALDI-TOF/TOF mass spectrometry. The identified spots are numbered and labeled with circles. Spot numbers refer to numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186227#pone.0186227.t002" target="_blank">Table 2</a>.</p

SDS-PAGE profiles of human WN1316-activating factor candidates synthesized in the cell-free system.

<p>The reaction mixture of GST protein synthesis (crude) and the GST protein after glutathione-Sepharose 4B column purification (puri.) were resolved on a 5–20% gradient SDS–PAGE gel, and gel was stained with Coomassie blue. <i>Black arrowheads</i> indicate synthesized GST fusion WN1316-activating factors. <i>White arrowhead</i> indicates GST used as a negative control.</p

A novel function of <i>N</i>-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection

<div><p>Therapeutic agents to the central nervous system (CNS) need to be efficiently delivered to the target site of action at appropriate therapeutic levels. However, a limited number of effective drugs for the treatment of neurological diseases has been developed thus far. Further, the pharmacological mechanisms by which such therapeutic agents can protect neurons from cell death have not been fully understood. We have previously reported the novel small-molecule compound, 2-[mesityl(methyl)amino]-N-[4-(pyridin-2-yl)-1H-imidazol-2-yl] acetamide trihydrochloride (WN1316), as a unique neuroprotectant against oxidative injury and a highly promising remedy for the treatment of amyotrophic lateral sclerosis (ALS). One of the remarkable characteristics of WN1316 is that its efficacious doses in ALS mouse models are much less than those against oxidative injury in cultured human neuronal cells. It is also noted that the WN1316 cytoprotective activity observed in cultured cells is totally dependent upon the addition of fetal bovine serum in culture medium. These findings led us to postulate some serum factors being tightly linked to the WN1316 efficacy. In this study, we sieved through fetal bovine serum proteins and identified two <i>N</i>-linked glycoproteins, alpha-2-HS-glycoprotein (AHSG) and hemopexin (HPX), requisites to exert the WN1316 cytoprotective activity against oxidative injury in neuronal cells <i>in vitro</i>. Notably, the removal of glycan chains from these molecules did not affect the WN1316 cytoprotective activity. Thus, two glycoproteins, AHSG and HPX, represent a pivotal glycoprotein of the cytoprotective activity for WN1316, showing a concrete evidence for the novel glycan-independent function of serum glycoproteins in neuroprotective drug efficacy.</p></div

The WN1316 treatment prolongs the survival interval after onset in ALS(SOD1^H46R) mice.

<p>The Kaplan-Meier curves demonstrate the probability of survival interval of vehicle control and WN1316 (1 µg/kg, 10 µg/kg and 100 µg/kg)-treated ALS(SOD1^H46R) mice. The average onset of ALS(SOD1^H46R) mice was 125.1±2.4 days (n = 104). Survival intervals in WN1316-treated groups at doses of 1 µg/kg (43.8±5.5 days, n = 26), 10 µg/kg (43.9±4.4 days, n = 26) and 100 µg/kg (45.9±6.0 days, n = 26) were significantly longer than that in vehicle group (36.6±6.2 days, n = 26) (<i>p</i><0.05 by log-rank test). These data are expressed as mean ± SD.</p