Wong SH , Yen YC , Li SY , Yang JJ .

MOST 107-2320-B-040-027-MY3 Ministry of Science and Technology

	Figure 1. Sequence analysis of TMPRSS3 genomic DNA variants in 230 patients with nonsyndromic hearing loss. Three variants of the TMPRSS3 gene were found in this study, three heterozygous missence variants: (A) c.239G>A, (B) c.551T>C, and (C) c.1253C>T. (D) Schematic representation of the domain structure of the TMPRSS3 protein with indication of the variants (black triangle). N: N-terminal domain; TM: transmembrane domains; LDLAR: low density lipoprotein receptor class A; SRCR: scavenger receptor cysteine rich; C: C-terminal domain.
	Figure 2. Alignment of the amino acid sequences of TMPRSS3 proteins of various species. The (A) p.Arg80, (B) p.Leu184, and (C) p.Ala418 residues, which were variants in this study, are indicated in bold. Arg (R) at codon 80, Leu (L) at codon 184, and Ala (A) at codon 418 are identical among all species compared.
	Figure 3. Assay of the catalytic activity of TMPRSS3 by site-specific proteolysis (sGASP). Transformants expressing both the STE13-substrate-invertase fusion protein and wild type (WT) or variants of the STE13-TMPRSS3 protein, including (A) TMPRSS3WT, (B) TMPRSS3R80H, (C) TMPRSS3L184S, (D) TMPRSS3A418V, and (E) TMPRSS3W251C, were plated on non-selective (glucose-YPD (yeast extract peptone dextrose)) and selective (sucrose-YPS (yeast extract peptone sucrose)) plates. TMPRSS3W251C mutant was as a positive control. (F) Quantitative assay of growth rate of yeast colony on sucrose plates. They were calculated by dividing the number of colony on sucrose plates by the number of colony on glucose plates (n = 5). ***: Asterisks denote the significant p-value <0.001.; STE13: Ste13p (Gene ID: 854394).
	Figure 4. Assay of the Na+ currents of TMPRSS3 by electrophysiological measurements in Xenopus oocytes. The amiloride-sensitive current (INa) was measured in the presence of 120 μM of Na+ in Frog Ringer with 5 μM amiloride hydrochloride at a holding potential of 0 mV. (A) Due to the absence of any capped RNA (cRNA) as a negative control, the Na+ current was not produced and measured. (B) Co-injected cRNA of TMPRSS3WT and rat epithelial sodium channel (ENaC) α, β, and γ subunit into Xenopus oocytes, resulting in increased Na+ currents up to 5000–6000 nA (C) In contrast, the Na+ current was decreased to about 2000 nA in the amiloride hydrochloride treatment.
	Figure 5. Functional expression of TMPRSS3 wild-type and mutant in Xenopus oocytes. Oocytes were injected with rat ENaC subunit in the presence of either (A) water (H2O), (B) TMPRSS3WT, and TMPRSS3 missense mutant, including (C) TMPRSS3R80H, (D) TMPRSS3L184S, (E) TMPRSS3A418V, and (F) TMPRSS3W251C. Water was used as a negative control. (G) All of the four missense mutant Na+ currents were similar to water, wherein there was no evident Na+ current and under 1000 nA in the quantitative analysis. In contrast, oocytes with the TMPRSS3WT emerging Na+ current were 5550 ± 1463 nA. (n ≥ 10).
	Figure 6. Serine protease trypsin can activate ENaC to produce Na+ current in Xenopus oocytes. The three rat ENaC subunits and one of water, WT, or mutant TMPRSS3 were co-injected into Xenopus oocytes. After injection, the oocytes were treated with trypsin and then Na+ currents were recorded. (A) After trypsin treatment, the ENaC was significant activated, producing Na+ currents in the water (H2O) group. (B) In the TMPRSS3WT group, whether there was a trypsin treatment or not did not affect the ENaC activation and the Na+ currents. (C–F) All of the four missense mutation groups were very similar to the water group, and ENaC could be activated, increasing the Na+ currents. (G) In the quantitative analysis, 10 Xenopus oocytes were treated by trypsin and measured by ENaC-mediated INa in each group. (n ≥10).
	Figure 7. Analysis of the effects of the TMPRSS3 mutant proteins on TMPRSS3WT by co-expression studies in the sGASP system and Xenopus oocytes. (A) Yeast (KSY01 strain) was transformed with substrate and equal amounts expression plasmids of TMPRSS3WT and TMPRSS3 mutants, as indicated and plated on YPD and YPS plates. Quantitative assay of growth rate was calculated by dividing the number of colony on YPS plates by the number of colony on YPD plates. All values are expressed as the mean ± SD of duplicate determinations (n = 5). (B) Rat ENaC subunit and equal amounts of TMPRSS3WT and TMPRSS3 mutant cRNA co-injected into Xenopus oocytes for electrophysiological ratios were calculated (n ≥ 10). These results indicated that the Na+ currents were under 2000 nA in the four heterozygous mutations group. Simultaneously, the ratios of Na+ currents were calculated by dividing the currents of TMPRSS3 mutant by the currents of TMPRSS3WT. All of the four heterozygous mutation ratios were less than 40% function of TMPRSS3WT. (C) After trypsin treatment, the ENaC was significant activated, producing Na+ currents in the heterozygous mutation type.

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