Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases

• Published in: PloS one Vol. 11; no. 3; p. e0152594
• Main Authors: Hofmann, Nicole, Galetskiy, Dmitry, Rauch, Daniela, Wittmann, Thomas, Marquardt, Andreas, Griese, Matthias, Zarbock, Ralf
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 31.03.2016; Public Library of Science (PLoS)
• Subjects: Alveoli; Amino Acid Sequence; Apoptosis; ATP-Binding Cassette Transporters - chemistry; ATP-Binding Cassette Transporters - metabolism; Biology and life sciences; Cathepsin B; Cathepsin B - antagonists & inhibitors; Cathepsin B - genetics; Cathepsin B - metabolism; Cathepsin L; Cathepsin L - antagonists & inhibitors; Cathepsin L - genetics; Cathepsin L - metabolism; Cell Line, Tumor; Children; Chromatography, High Pressure Liquid; Cleavage; Enzymes; Gene mutation; Genetic aspects; Humans; Lung diseases; Manufacturers; Mass spectrometry; Mass spectroscopy; Mimicry; Mutation; Peptide Hydrolases - chemistry; Peptide Hydrolases - genetics; Peptide Hydrolases - metabolism; Peptides; Peptides - analysis; Physiological aspects; Proteases; Protein Structure, Tertiary; Proteins; Proteolysis; Proteomics; Respiratory distress syndrome; RNA Interference; RNA, Small Interfering - metabolism; Scientific imaging; siRNA; Substrates; Tandem Mass Spectrometry; Germany; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0152594

ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity. The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS). Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis. We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active. We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.