r-hPAP has antinociceptive properties <i>in vivo</i>.

<p>Antinociceptive properties of native hPAP, r-hPAP and r-hPAP (N62Q, N188Q) injected intrathecally into wild-type (n = 10) and <i>A₁R</i>^−/− mice (n = 10). Equivalent unit amounts (250 mU/mouse) of native hPAP and r-hPAP were injected. Equivalent protein amounts (0.21 mg/mL) of r-hPAP and r-hPAP (N62Q, N188Q) were injected. Paired t-tests were used to compare responses at each time point to baseline (BL). *p<0.05, **p<0.005, ***p<0.0005. Data are plotted as means ± standard error of the mean (SEM).</p

Antinociceptive effects of r-hPAP in chronic inflammatory pain model.

<p>(A, B) CFA was injected into one hindpaw (CFA-arrow) of wild-type (n = 10) and <i>A₁R^−/−</i> mice (n = 10). r-hPAP (250 mU) was intrathecally injected 1 day later (r-hPAP-arrow). Inflamed and non-inflamed (control) hindpaws were tested for (A) thermal and (B) mechanical sensitivity. Data are plotted as means ± SEM. Paired t-tests were used to compare responses at each time point between genotypes, same paw comparisons. *p<0.05, **p<0.005, ***p<0.0005.</p

r-hPAP does not affect balance or motor function in mice.

<p>Rotarod tests with wild-type (n = 10) and A₁R^−/− mice (n = 10) 24 h before (3 iterations, separated by 40 s) and 24 h after (2 test iterations, separated by 40 s) intrathecal injection of r-hPAP (250 mU/mouse). No significant differences between genotypes or treatment. Data are plotted as means ± SEM. The same mice shown were also tested for thermal sensitivity, to confirm that hPAP injections were successful (as evidenced by a significant thermal antinociceptive effect in wild-type mice injected with hPAP, data not shown).</p

<i>Pichia</i>-derived r-hPAP is secreted and catalytically active.

<p>(A) Western blot of crude cell lysates (Lys) and supernatants (Sup) from <i>P. pastoris</i> X33 untransformed controls and from <i>P. pastoris</i> X33 expressing r-hPAP. Blot probed with anti-hPAP antiserum. (B) DiFMUP fluorometric enzyme assay with concentrated supernatants (secreted fractions) in comparison to native hPAP from human semen. 0.625 µg total protein used per reaction. Data are plotted as an average of duplicate trials ± standard deviation (SD).</p

Antinociceptive effects of r-hPAP in neuropathic pain model.

<p>(A, B) The sural and common peroneal branches of the sciatic nerve were ligated and then transected (injure-arrow) in wild-type (n = 10) and <i>A₁R^−/−</i> mice (n = 10). Six days later, r-hPAP (250 mU) was injected intrathecally. Injured and non-injured (control) hindpaws were tested for (A) thermal and (B) mechanical sensitivity. Data are plotted as means ± SEM. Paired t-tests were used to compare responses at each time point between genotypes, same paw comparisons. *p<0.05, **p<0.005, ***p<0.0005.</p

Expression and activity of N-linked glycosylation mutants.

<p>Western blots of (A) crude cell lysates and (B) crude secreted fractions from the indicated <i>P. pastoris</i> X33 integrants. Blots probed with anti-hPAP antiserum. Equivalent amounts of total protein were loaded in each lane. (C, D) DiFMUP fluorometric enzyme assays of (C) crude cell lysates and (D) crude secreted fractions. Data are plotted as an average of duplicate trials ± SD.</p

Expression and activity of r-hPAP lacking signal peptide(-SP).

<p>(A) Western blot of crude cell lysates from untransformed X33 cells and transformants expressing hPAP with (r-hPAP) or without (-SP) a signal peptide. Blot probed with anti-hPAP antiserum. Equivalent amounts of total protein were loaded in each lane. (B) DiFMUP fluorometric enzyme assay with the indicated crude cell lysates. Data are plotted as an average of duplicate trials ± SD.</p

Location of N-linked asparagines residues relative to the active site of hPAP.

<p>The x-ray crystallographic structure depicts the essential active site residue H12 and the three N-linked residues (highlighted in yellow) in one subunit of native hPAP. Distances were calculated in PyMOL between the alpha carbon of each amino acid. Structure coordinates from PDB #1ND6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032741#pone.0032741-Ortlund1" target="_blank">[18]</a>.</p

O-BT is non-enzymatically converted to thiamine <i>in vitro</i>.

<p>(<b>A</b>) HPLC analysis of chromatographically purified O-BT (1 mg/mL) in methanol and (<b>B</b>) in aqueous solutions buffered at acidic, neutral and alkaline pH values (t = 0 and 30 min, respectively at 37°C). (<b>C</b>) Analyte peak areas for O-BT and thiamine generated <i>in vitro</i>. Data are plotted as the total peak area observed at each time point.</p

PAP dephosphorylates BT in small-to-medium diameter DRG neurons and afferent axon terminals in the spinal cord.

<p>(<b>A–F</b>) Sections from wild-type and <i>Pap^−/−</i> mouse (<b>A,B</b>) DRG and (<b>C–F</b>) spinal cord stained using enzyme histochemistry (6 mM BT at pH 7.0). Scale bar, 50 µm in (<b>B,D</b>), 500 µm in (<b>F</b>).</p