Structure of the catalytic domain of matriptase in complex with the 1^st Kunitz domain of HAI-1.

<p>The crystal structure of the complex was solved by Zhao et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094267#pone.0094267-Zhao1" target="_blank">[49]</a> and the atomic coordinates used for the figure were obtained from the Protein Data Bank (code 4ISO). The 1^st Kunitz domain of HAI-1 is shown in gray with the Tyr280 residue (magenta), and the cysteines (red) involved in disulfide bonds. The catalytic domain of the matriptase is represented in black with the cysteines (red) involved in disulfide bonds. <i>Insert</i>: substitution of the Tyr280 (magenta) in the KD1 of HAI-1with a cysteine pointing its side chain towards the Cys283.</p

Tissue distribution of mRNA expression of <i>Spint2</i> and membrane-bound serine proteases.

<p>Quantitative RT-PCRs were performed on selected organs from three wild-type adult mice. From stomach to distal colon, tissues were scraped to get fractions enriched in mucosal cells. Each gene was assessed in duplicates in two independent experiments. Results are expressed as arbitrary units (A.U.) based on standard dilution curves (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094267#s2" target="_blank">Material and Methods</a>).</p

Validation of the functional assay using ENaC as a reporter gene.

<p><b>A</b>, Representative recordings of amiloride-sensitive current (I_Na⁺) in the presence (filled bars) or absence of trypsin (5 μg/ml), in <i>Xenopus</i> oocytes injected with 0.11 ng/subunit ENaC alone (left panel), with ENaC and 0.25 ng tmprss13 (middle panel) and with ENaC, tmprss13 and 1.5 ng spint2 cRNA (right panel). 10 μM amiloride was used to block the ENaC-mediated current. <b>B</b>, Effects of increasing the amounts of injected tmprss13 and enteropeptidase cRNAs on I_Na⁺. I_Na⁺ was measured in oocytes injected with ENaC with/without of tmprss13 or enteropeptidase as indicated. I_Na⁺ was measured without (black bars) or with trypsin (5 μg/ml) perfused extracellularly (white bars) as a positive control for ENaC activation. <b>C</b>, Effects of increasing the amounts of injected spint2 cRNA to prevent the tmprss13- or enteropeptidase-mediated increase in I_Na⁺ (left and right panels, respectively). <b>D</b>, Effect of spint2 on I_Na⁺. I_Na⁺ was measured 12, 24 and 30 hours after injection (left, middle and right panels, respectively) in three independent experiments. n = 6-9 measured oocytes per condition from 2 different batches for each experiment. Data are means ± SEM; *, p<0.05/**, p<0.01 compared to ENaC alone or ENaC + protease (as indicated) after two-way repeated measure ANOVA followed by Dunnett's multiple comparisons test.</p

Functional analysis of interactions between HAI-2 (wt and mutants) and membrane-bound serine proteases.

<p><b>A</b>, ENaC-mediated sodium currents (I_Na⁺) were measured in <i>Xenopus</i> oocytes injected with ENaC with/without candidate serine protease and HAI-2 (wild-type or mutants Y68C and Y163C) as indicated. I_Na⁺ was measured without (black bars) or with trypsin (5 μg/ml) perfused extracellularly (white bars) as a positive control for ENaC activation. n≥15 measured oocytes per condition from at least 2 different animals. Each protease was tested in at least two independent experiments. Data are means ± SEM. <b>B</b>, Relative trypsin-mediated increase in I_Na⁺ was calculated by dividing, for each oocyte from experiments of panel A, I_Na⁺ after treatment with trypsin by I_Na⁺ before treatment with trypsin. Data are means ± SEM. */#/°, p<0.05, **/##/°°, p<0.01, ***/###/°°°, p<0.001, compared to ENaC alone, ENaC + protease or ENaC + protease + HAI-2 WT respectively after One-way ANOVA followed by Tukey's multiple comparisons test.</p

Increase in ENaC activity by membrane-bound serine proteases.

<p>Values were obtained from experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094267#pone-0094267-g003" target="_blank">figure 3</a>. Effect of trypsin is indicated in parentheses. Data are means ± SEM.</p

Effect of HAI-2 Y68S and Y163S mutations on tmprss13 activity.

<p><b>A</b>, ENaC-mediated sodium currents (I_Na⁺), were measured in <i>Xenopus</i> oocytes injected with ENaC, tmprss13 and HAI-2 (wild-type or mutant). I_Na⁺ was measured without (black bars) and with trypsin (5 μg/ml) perfused extracellularly (white bars) as a positive control for ENaC activation. n≥14 measured oocytes from 4 different animals performed in two independent experiments. Data are means ± SEM. <b>B</b>, Relative trypsin-mediated increase in I_Na⁺ was calculated by dividing, for each oocyte from experiments of panel A, I_Na⁺ after treatment with trypsin by I_Na⁺ before treatment with trpysin. Data are means ± SEM. ***, p<0.001, compared to ENaC alone, after One-way ANOVA followed by Tukey's multiple comparisons test.</p