DMY inhibits T cells proliferation <i>ex</i><i>vivo</i> and T cells infiltration into mouse liver tissues.

<p>(A) Splenic CD4⁺ T cells were purified from male ICR mouse and stimulated with vehicle alone or anti-CD3/anti-CD28 mAbs plus 0, 25, 50, or 100 μM of DMY for 48 h. [³H]Thymidine incorporation in T cells was determined. (B) The concentration of IL-2 in cultural media of T cells was determined by ELISA. (C) Immunohistochemistry analysis of mouse liver tissues against CD4 mAb. Data are mean ± SEM, <i>n</i> = 3. Means without a common letter differ, <i>P</i> < 0.05.</p

Working hypothesis of the metabolic mechanisms of LPS/D-GalN– induced hepatic toxicity and protection by DMY.

<p>Symbol circle and square represent the relative metabolite changes in the LPS/D-GalN−challenged group and the DMY treatment group, respectively. The decrease, increase and no difference in levels with statistical significance are presented in green, red and tan, respectively.</p

The effect of DMY treatment on NF-κB and MAPK signaling pathway in LPS−stimulated macrophages.

<p>Cells were pre-treated with 50 μM DMY for 1 h, then with LPS treatment for 2, 4, and 8 h. Western blot analysis of (A) Phospho- and total IKK, IκBα, JNK1/2 and ERK1/2 levels. (B) Immunofluorescence staining of nuclear translocation of NF-κB(p65) protein. Cells were stained with DAPI (nuclear marker, blue) and rabbit anti-p65 antibody (red).</p

The effect of DMY treatment on Jak/STAT signaling pathway in LPS−stimulated macrophages.

<p>Cells were pre-treated with 50 μM DMY for 1 h, then with LPS treatment for 2, 4, and 8 h. Western blot analysis of (A) Phospho- and total Jak2 and STAT1 levels, (B) Total STAT3, phospho-STAT3, and SOCS3 levels and the levels of STAT3 and phospho-STAT3 in nuclear and cytosolic fractions. PARP and α-tubulin were used as internal control of nuclear and cytosolic proteins, respectively.</p

Immunohistochemistry of liver tissues from LPS/D-GalN–challenged mice with or without DMY pretreatment.

<p>Immunofluorescence staining and quantification of DMY inhibiting STAT3 and F4/80 (macrophage) infiltration (A) and Ly6G (neutrophil) infiltration (B) in LPS/D-GalN−treated mouse liver. Data are mean ± SEM, <i>n</i> = 4. Means without a common letter differ, <i>P</i> < 0.05.</p

Protective effects of DMY on LPS/D-GalN−induced acute liver dysfunction in mice.

<p>Mice were pretreated with DMY (1 and 10 mg/kg) for three consecutive days, then LPS/D-GalN for 8 h. (A) Serum levels of AST and ALT with or without treatment. Data are mean ± SEM, <i>n</i> = 6. Means without a common letter differ, <i>P</i> < 0.05. (B) Hematoxylin and eosin staining of mouse livers. (C) TUNEL assay of apoptosis in mouse liver with LPS/D-GalN challenge or DMY treatment. Representative image of each treatment group is shown. Brownish cells are TUNEL-positive apoptotic cells. (D) Survival rate of LPS/D-GalN-challenged mice with or without DMY-pretreatment. ^*,#<i>P</i> < 0.05, significant differences within treatment groups and the LPS/D-GalN group (log rank test).</p

Comparative metabolomic analysis of mouse serum.

<p>Score plots (A, C) and corresponding loading plots (B, D) for PCA of UPLC/QTOF MS data from mice treated with vehicle control, LPS/D-GalN, and LPS/D-GalN–challenged mice pretreated with DMY1 and DMY10 (<i>n</i> = 3 in each group). <i>Dashed </i><i>circles</i> group pairs of samples from LPS/D-GalN–challenged group vs. other treatment groups. The ions most responsible for the variance of the score plots are indicated by their distance from the origin. The metabolites are labeled according their retention times in the chromatogram and <i>m</i>/<i>z</i> values. </p

Kinetic properties of native (nLcc4) and Endo H-deglycosylated lcc4 (dLcc4)^a.

<p>^<i>a</i> The enzymatic reaction was performed at the respective optimal temperatures and pHs as indicated.</p><p>^<i>b</i> One unit of enzyme activity was defined as the amount of enzyme that oxidizes 1 μmol of substrate per minute under optimal reaction conditions.</p><p>Kinetic properties of native (nLcc4) and Endo H-deglycosylated lcc4 (dLcc4)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120601#t003fn001" target="_blank">^a</a>.</p

Structural and Functional Roles of Glycosylation in Fungal Laccase from <i>Lentinus</i> sp.

<div><p>Laccases are multi-copper oxidases that catalyze the oxidation of various organic and inorganic compounds by reducing O₂ to water. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated nLcc4) isolated from a white-rot fungus <i>Lentinus</i> sp. nLcc4 is composed of three cupredoxin-like domains D1-D3 each folded into a Greek key <i>β</i>-barrel topology. T1 and T2/T3 copper binding sites and three <i>N</i>-glycosylated sites at Asn⁷⁵, Asn²³⁸, and Asn⁴⁵⁸ were elucidated. Initial rate kinetic analysis revealed that the <i>k</i>_cat, <i>K</i>_<i>m</i>, and <i>k</i>_cat/<i>K</i>_<i>m</i> of nLcc4 with substrate ABTS were 3,382 <i>s</i>^-1, 65.0 ± 6.5 μM, and 52 <i>s</i>^-1μM^-1, respectively; and the values with lignosulfonic acid determined using isothermal titration calorimetry were 0.234 <i>s</i>^-1, 56.7 ± 3.2 μM, and 0.004 <i>s</i>^-1μM^-1, respectively. Endo H-deglycosylated nLcc4 (dLcc4), with only one GlcNAc residue remaining at each of the three <i>N</i>-glycosylation sites in the enzyme, exhibited similar kinetic efficiency and thermal stability to that of nLcc4. The isolated <i>Lcc4</i> gene contains an open reading frame of 1563 bp with a deduced polypeptide of 521 amino acid residues including a predicted signaling peptide of 21 residues at the <i>N</i>-terminus. Recombinant wild-type Lcc4 and mutant enzymes N75D, N238D and N458D were expressed in <i>Pichia pastoris</i> cells to evaluate the effect on enzyme activity by single glycosylation site deficiency. The mutant enzymes secreted in the cultural media of <i>P</i>. <i>pastoris</i> cells were observed to maintain only 4-50% of the activity of the wild-type laccase. Molecular dynamics simulations analyses of various states of (de-)glycosylation in nLcc support the kinetic results and suggest that the local H-bond networks between the domain connecting loop D2-D3 and the glycan moieties play a crucial role in the laccase activity. This study provides new insights into the role of glycosylation in the structure and function of a Basidiomycete fungal laccase.</p></div

Expression profile of recombinant wild-type and mutant forms of Lcc4 in the cultural media of <i>P</i>. <i>pastoris</i> X33 cells.

<p>(A) Time course of yeast host cell growth and protein expression and concentration. (B) CBR stained gel and zymogram of cultural media. For zymogram analysis, the snapshots of SDS gel were immersed in 2 mM ABTS solution for 20 and 60 s, respectively. Protein marker (M); vector (lane 1); wild-type (lane 2); N75D (lane 3); N162D (lane 4); N238D (lane 5); N458D (lane 6). (C) Relative laccase specific activity in the cultural medium of yeast cells transformed with vector only, wt, N75D, N162D, N238D, and N48D gene construct.</p