title: Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells.
abstract: Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na(+) channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o-) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o- epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.
authors.affiliation: Cardiovascular Research Institute, San Francisco, CA, USA.
authors.forename: Shilpa
authors.initials: S
authors.lastname: Nimishakavi
authors.forename: Marina
authors.initials: M
authors.lastname: Besprozvannaya
authors.forename: Wilfred W
authors.initials: WW
authors.lastname: Raymond
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.forename: Dieter C
authors.initials: DC
authors.lastname: Gruenert
authors.forename: George H
authors.initials: GH
authors.lastname: Caughey
mesh_terms: D000595:Amino Acid Sequence; D007611:Aprotinin / Q000737:chemistry / Q000494:pharmacology; D018929:Cell Culture Techniques; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D016764:Cell Polarity; D003550:Cystic Fibrosis / Q000235:genetics / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism; D017097:Electric Impedance; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D058851:GPI-Linked Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009842:Oligopeptides / Q000737:chemistry; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000494:pharmacology; D013379:Substrate Specificity; D000099271:Prostasin
chemical_list: D007611:Aprotinin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D058851:GPI-Linked Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D057127:Single-Chain Antibodies; D000099271:Prostasin; C485596:ST14 protein, human