Fig 1.
HAI-2 species in Caco-2 human enterocytes.
Caco-2 human enterocytes were incubated in PBS as a non-activation control (lanes 1) or a phosphate buffer pH 6.0 for 20 min to induce matriptase zymogen activation (lanes 2). Matriptase species were subsequently immunodepleted from the lysates that had been prepared from Caco-2 cells in which matriptase zymogen activation was induced (lanes 3). The three Caco-2 lysates were then analyzed by immunoblot for HAI-2 species with significant N-glycan branching using HAI-2 mAb DC16 (HAI-2 DC16), HAI-2 species without N-glycan branching using HAI-2 mAb XY9 (HAI-2 XY9), and matriptase species using matriptase mAb M24 (Total MTP). Small quantities of mouse IgG shed from the beads used for immunodepletion can be seen as a band of approximately 150-kDa (lanes 3 in each panel) is indicated by an arrow. The HAI-2 species in Caco-2 cells have been observed in at least 10 independent experiments. The induction of matriptase zymogen activation in Caco-2 cells and the immunodepletion of matriptase species has been carried out at least 3 times. Representative data are shown.
Fig 2.
Prostasin species in Caco-2 human enterocytes.
Caco-2 lysates (lanes 1) were subjected to immunodepletion to remove prostasin species (lanes 2), or HAI-1 species (lanes 3), or HAI-2 species (lanes 4), or both HAI-1 species and HAI-2 species together (lanes 5), as indicated. The lysates were analyzed by immunoblot for HAI-2 species (A.), prostasin species (B.), and HAI-1 species (C.). The identification of the prostasin species has been replicated at least twice and further verified by immunopurification in Fig 3.
Fig 3.
Caco-2 cells constitutively activate prostasin.
A. The prostasin containing species present in Caco-2 cell lysates were separated and purified by sequential immunoaffinity chromatography using HAI-1 mAb M19-Sepharose, HAI-2 mAb DC-16 Sepharose, and then prostasin mAb YL11-Sepharose. The purified prostasin-HAI-1 complexes (lane 1), prostasin-HAI-2 complexes (lane 2), and prostasin monomers (lane 3) were analyzed for prostasin species by immunoblot using the prostasin-specific mAb YL11. B. Purified prostasin monomers (lane 1) were incubated with purified HAI-1 (lane 2) at 37°C for 5 min to allow the formation of prostasin-HAI-1 complexes (lane 3). The samples were then analyzed by western blot for prostasin-containing species. C. Purified prostasin monomers were incubated with a fluorogenic prostasin substrate and the kinetics of AMC release was recorded. Prostasin proteolytic activity assays were conducted at least four times. Representative data are shown. RFU stands for relative fluorescent unit.
Fig 4.
Prostasin and HAI-2 are localized intracellularly with the highest density near the apical surface of intestinal enterocytes.
Tissue sections of human intestine were immunostained with the prostasin-specific mAb YL89 (Prostasin) or the HAI-2-specific mAb DC16 (HAI-2). Sections were also stained with a non-specific mouse IgG antibody as a negative control (Data not shown). Higher magnification images of the staining are presented in lower panels and the insets. Arrows indicate the accumulation of prostasin and HAI-2 right beneath brush border. Cells nuclei were counterstained blue with hematoxylin. The immunohistochemical staining studies with intestinal tissues were at least repeated twice. Representative staining is shown. Scale bar: 25 μm.
Fig 5.
Matriptase and HAI-1 are localized at intercellular junctions between intestinal enterocytes.
Tissue sections of human intestine were immunostained with the matriptase-specific mAb M24 (Matriptase) or the HAI-1-specific mAb M19 (HAI-1). Sections were also stained with a non-specific mouse IgG antibody as a negative control (Data not shown). Higher magnification images of the staining are presented in lower panels and the insets. Arrows indicate the brush border. Arrow heads indicate matriptase-positive cells in the lamina propria. Cells nuclei were counterstained blue with hematoxylin. The immunohistochemical staining studies with intestinal tissues were at least repeated twice. Representative staining is shown. Scale bar: 25 μm.
Fig 6.
Analysis of prostasin, matriptase, HAI-1 and HAI-2 species in human intestine tissue.
Human intestine tissue was used to prepare lysate that were analyzed by western blot for matriptase species (A. lane 1), prostasin species (A. lane 2), HAI-1 species (A. lane 3) and HAI-2 species (B. lane 1). Various amounts of tissue proteins were analyzed for the four proteins examined. The lysate was then immunodepleted by incubation with prostasin-specific mAb YL-11-Sepharose or HAI-2-specific mAb DC16-Sepharose. The immunodepleted lysate was analyzed by western blot for HAI-2 species (B. lanes 2 and 3). Mouse IgG (mIgG) that leaked from the beads, prostasin-HAI-2 complex, and HAI-2 were indicated. Human intestine tissues were examined at least twice with the same expression status observed for the four proteins.
Fig 7.
Analysis of the prostasin and HAI-2 species present in human prostate cancer cells, keratinocytes and mammary epithelial cells.
LNCaP human prostate cancer cells, HaCaT human keratinocytes and 184 A1N4 human mammary epithelial cells were transiently exposed to a pH 6.o buffer to induce matriptase and prostasin zymogen activation. Lysates were prepared and analyzed by western blot for HAI-2 species using the HAI-2 specific mAb DC16 and for prostasin species using the prostasin-specific mAb YL11. Western blot analysis for the prostasin and HAI-2 species in these three cell lines has been conducted multiple times (>5) with the same profile observed. Representative data are shown.