Document Detail


Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells.
MedLine Citation:
PMID:  22582115     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Shilpa Nimishakavi; Marina Besprozvannaya; Wilfred W Raymond; Charles S Craik; Dieter C Gruenert; George H Caughey
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-05-11
Journal Detail:
Title:  American journal of physiology. Lung cellular and molecular physiology     Volume:  303     ISSN:  1522-1504     ISO Abbreviation:  Am. J. Physiol. Lung Cell Mol. Physiol.     Publication Date:  2012 Jul 
Date Detail:
Created Date:  2012-07-16     Completed Date:  2012-09-20     Revised Date:  2013-07-17    
Medline Journal Info:
Nlm Unique ID:  100901229     Medline TA:  Am J Physiol Lung Cell Mol Physiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  L97-106     Citation Subset:  IM    
Affiliation:
Cardiovascular Research Institute, San Francisco, CA, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Aprotinin / chemistry,  pharmacology
Cell Culture Techniques
Cell Line
Cell Membrane / drug effects,  enzymology*
Cell Polarity
Cystic Fibrosis / genetics,  pathology
Cystic Fibrosis Transmembrane Conductance Regulator / genetics,  metabolism
Electric Impedance
Epithelial Cells / drug effects,  enzymology*,  physiology
GPI-Linked Proteins / chemistry,  metabolism
Humans
Oligopeptides / chemistry
Proteolysis
Recombinant Proteins / chemistry,  metabolism
Sequence Deletion
Serine Endopeptidases / chemistry,  immunology,  metabolism*
Serine Proteinase Inhibitors / chemistry,  pharmacology
Single-Chain Antibodies / chemistry,  pharmacology
Substrate Specificity
Grant Support
ID/Acronym/Agency:
P01 HL024136/HL/NHLBI NIH HHS; P01 HL024136/HL/NHLBI NIH HHS; R01 CA128765/CA/NCI NIH HHS; R01CA128765/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/GPI-Linked Proteins; 0/Oligopeptides; 0/Recombinant Proteins; 0/Serine Proteinase Inhibitors; 0/Single-Chain Antibodies; 126880-72-6/Cystic Fibrosis Transmembrane Conductance Regulator; 9087-70-1/Aprotinin; EC 3.4.21.-/Serine Endopeptidases; EC 3.4.21.-/prostasin; EC 3.4.21.109/ST14 protein, human
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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