<i>Drosophila</i> Tracheal-prostasin and human prostasin show functional similarity.

(A) Schema showing the protein domain organization of Drosophila Tpr and human prostasin. The signal peptides (sp; blue), conserved disulfide bridges (S-S), the activation cleavage sites (V), the catalytic protease domains (green) and the GPI anchor (red) are indicated. (B, C) Bright field light microscopic images of stage 17 tprD1/tprD1 (B) and tprD1,btl-Gal4/tprD1,UAS-tpr (C) mutant embryos. (D, E) Whole-mount antibody stainings of stage 16 tpr::RFP embryos stained with anti-RFP (D, E) and anti-Spectrin (E) antibodies. Tpr::RFP (green) is restricted to the tracheal system (D) and localized in the tracheal lumen (E). Spectrin (magenta) outlines the tracheal cells (E). (F, G) Transmission electron microscopic images of stage 17 wild-type (F) and tprD1/tprD1 mutant (G) tracheal aECM. The taenidial folds of the tracheal aECM are associated with the apical side of tracheal cells in wild-type (F), while the taenidial folds are detached from tracheal cells in tpr mutant embryos (asterisks in G). (H, I) Whole-mount antibody stainings of dpy::YFP/dpy::YFP (H) and tprD1,dpy::YFP/tprD1,dpy::YFP mutant (I) embryos at stage 17 stained with FITC labelled CBP (green) and anti-GFP antibodies (magenta). Chitin is cleared from the tracheal lumen of wild-type (H) and tpr mutant (I) embryos. Dpy::YFP is cleared from the tracheal lumen of wild-type embryos (H), while Dpy::YFP degradation is incomplete in tpr mutant embryos (I). (J-M) Bright field light microscopic images of stage 17 (J, L) and confocal LSM images of whole mount antibody stainings of stage 16 (K, M) tprD1,btl-Gal4/tprD1,UAS-prostasin (J, K) and tprD1,btl-Gal4/tprD1,UAS-prostasin; UAS-HAI-2/+ (L, M) mutant embryos with anti-prostasin (green) and anti-Spectrin (magenta) antibodies. tpr mutant embryos expressing prostasin in the trachea lack tracheal gas filling (arrow in J) and show prostasin localization in tracheal cells (green in K). In contrast, tracheal co-expression of prostasin and HAI-2 in tpr mutant embryos facilitates gas filling of the tracheal system (arrow in L) and prostasin localizes to the tracheal lumen (green in M). (N) Purified Tpr-Flag or zymogen locked TprZL-Flag were incubated with buffer, Np-Strep or matriptase-Strep and samples were analysed by western blotting and immunostained using anti-Flag antibody. Tpr-Flag is detectable as a fragment of approximately 60 kDa that is processed to a fragment of approximately 40 kDa by incubation with purified Np-Strep or purified matriptase-Strep. Proteolytic processing occurs at the zymogen activation site, since zymogen locked TprZL-Flag is resistant to cleavage by Np-Strep and matriptase-Strep. Scale bars correspond to 1 μm in (F, G) to 10 μm in (E, H, I, K, M) and to 50 μm in (D).