Prostasin Is a Glycosylphosphatidylinositol-anchored Active Serine Protease

Abstract

A recombinant human prostasin serine protease was expressed in several human cell lines. Subcellular fractionation showed that this serine protease is synthesized as a membrane-bound protein while a free-form prostasin is secreted into the culture medium. Prostasin was identified in nuclear and membrane fractions. Membrane-bound prostasin can be released by phosphatidylinositol-specific phospholipase C treatment, or labeled by [ 3 H]ethanolamine, indicating a glycosylphosphatidylinositol anchorage. A prostasin-binding protein was identified in mouse and human seminal vesicle fluid. Both the secreted and the membrane-bound prostasin were able to form a covalently linked 82-kDa complex when incubated with seminal vesicle fluid. The complex formation between prostasin and the prostasin-binding protein was inhibited by a prostasin antibody, heparin, and serine protease inhibitors. Prostasin's serine protease activity was inhibited when bound to the prostasin-binding protein in mouse seminal vesicle fluid. This study indicates that prostasin is an active serine protease in its membrane-bound form.Prostasin is a serine protease discovered in ejaculated human semen in 1994 (1). The molecular mass of prostasin is 40 kDa when examined by SDS-polyacrylamide gel electrophoresis (PAGE) 1 under reducing conditions. Prostasin displays trypsin-like enzymatic activities by hydrolyzing peptidyl fluorogenic substrates such as D-Pro-Phe-Arg-AMC. This trypsinlike enzymatic activity can be inhibited by aprotinin, antipain, leupeptin, and benzamidine. Prostasin is present at high levels in normal human semen (8.61 Ϯ 0.42 g/ml) and in the prostate gland (143.7 Ϯ 15.9 ng/mg). Lower amounts of prostasin can also be detected in other tissues. In the prostate gland, the prostasin protein is present in the epithelial cells as well as in the secretion inside the lumen. The full-length human prostasin mRNA has been deduced (2). The predicted mature prostasin peptide sequence has a potential carboxyl-terminal hydrophobic membrane anchorage domain followed by a short cytoplasmic tail. The translated amino acid residue sequence of prostasin is similar to those of human prostase, testisin, plasma kallikrein, coagulation factor XI, hepsin, plasminogen, and acrosin (2-4). A membrane-bound Xenopus kidney epithelial cell sodium channel-activating protease (CAP1) was found highly homologous to human prostasin, sharing 53% sequence identity at the amino acid level (5). Recently, the mouse counterpart of CAP1, mCAP1, has been cloned from a cortical collecting duct cell line (6). mCAP1 shares 77% amino acid sequence identity with human prostasin.Serine proteases play important roles in a diverse range of the body's normal physiological processes, and they are implicated in various pathological processes such as cardiovascular disorders and cancers (7). The prostate produces a number of serine proteases such as prostate-specific antigen (8), human glandular kallikrein (9), and the most recently discovered prostase (3). Some of these serine proteases are ...

Regulation of the Epithelial Sodium Channel by Serine Proteases in Human Airways

“…Secretion into the prostatic lumen, therefore, required cleavage between the heavy chain domain and the COOH-terminal transmembrane domain. Further work has shown that prostasin, like xCAP1, is a glycosylphosphatidylinositol-anchored protein that may either be secreted or membrane-bound (22). Consistent with our in situ hybridization and library screening data, Yu et al (20) detected prostasin mRNA in the lung by Northern analysis as well as in other epithelialined organs (prostate, liver, salivary gland, kidney, pancreas, and colon).…”

Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis

“…Prostasin, the human ortholog of CAP1, is a glycosylphosphatidylinositol (GPI)-anchored serine protease that activates ENaC (4,8,13,44). Donaldson et al (13) demonstrated that prostasin is expressed in airway epithelium and increases the Na ϩ current by ϳ75% when coexpressed with ENaC in Xenopus oocytes.…”

Prostasin Serine Protease as a Breast Cancer Invasion Marker and a Metastasis Suppressor

“…Expression of prostasin protein in the mixed colony was confirmed by western blot analysis (data not shown). We then further evaluated the expression of prostasin protein in the mixed transfectants by immunocytochemistry using a previously described method (1). The result showed a varying level of prostasin protein expression in the transfected cells, approximately 50% of the cell population produced an intense immunostaining for prostasin (data not shown).…”

“…We have previously shown that membrane-anchored prostasin serine protease was an in vitro suppressor of tumor cell invasion (1)(2)(3). The expression of prostasin is down-regulated in prostate cancers (2), and invasive cancer cell lines of the human prostate and breast (1,3).…”

“…We have previously shown that membrane-anchored prostasin serine protease was an in vitro suppressor of tumor cell invasion (1-3). The expression of prostasin is down-regulated in prostate cancers (2), and invasive cancer cell lines of the human prostate and breast (1,3). For this project, we hypothesized that the down-regulation of prostasin is causal to breast cancer invasion and metastasis in vivo.…”