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

Hofmann N, Galetskiy D, Rauch D, Wittmann T, Marquardt A, Griese M, Zarbock R - PLoS ONE (2016)

Bottom Line: 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.We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L.

View Article: PubMed Central - PubMed

Affiliation: German Centre for Lung Research, Dr. von Hauner Children's Hospital, Ludwig-Maximilians University, 80337, Munich, Germany.

ABSTRACT

Rationale: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

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Related in: MedlinePlus

Mutation of potential Cathepsin cleavage sites.Amino acids forming potential cleavage sites were replaced by alanine residues using site directed mutagenesis. Lane 1: cells transfected with empty vector; lane 2: wild type ABCA3; lane 3: deletion of the first 174 amino acids of ABCA3; lane 4: 173LK174 > AA.
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pone.0152594.g008: Mutation of potential Cathepsin cleavage sites.Amino acids forming potential cleavage sites were replaced by alanine residues using site directed mutagenesis. Lane 1: cells transfected with empty vector; lane 2: wild type ABCA3; lane 3: deletion of the first 174 amino acids of ABCA3; lane 4: 173LK174 > AA.

Mentions: Having identified cathepsin L as the major protease responsible for cleavage of ABCA3 as well as its cleavage site, we aimed to abolish cleavage by abrogating substrate recognition. Since it is known that both amino acid residues that precede the actual cleavage site are crucial for substrate recognition, we replaced Leu173 and Lys174 of ABCA3 with alanine residues. This operation resulted in prominent accumulation of the upper ABCA3 band (Fig 8), indicating inhibition of processing and confirming correct identification of the cleavage site.


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

Hofmann N, Galetskiy D, Rauch D, Wittmann T, Marquardt A, Griese M, Zarbock R - PLoS ONE (2016)

Mutation of potential Cathepsin cleavage sites.Amino acids forming potential cleavage sites were replaced by alanine residues using site directed mutagenesis. Lane 1: cells transfected with empty vector; lane 2: wild type ABCA3; lane 3: deletion of the first 174 amino acids of ABCA3; lane 4: 173LK174 > AA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816274&req=5

pone.0152594.g008: Mutation of potential Cathepsin cleavage sites.Amino acids forming potential cleavage sites were replaced by alanine residues using site directed mutagenesis. Lane 1: cells transfected with empty vector; lane 2: wild type ABCA3; lane 3: deletion of the first 174 amino acids of ABCA3; lane 4: 173LK174 > AA.
Mentions: Having identified cathepsin L as the major protease responsible for cleavage of ABCA3 as well as its cleavage site, we aimed to abolish cleavage by abrogating substrate recognition. Since it is known that both amino acid residues that precede the actual cleavage site are crucial for substrate recognition, we replaced Leu173 and Lys174 of ABCA3 with alanine residues. This operation resulted in prominent accumulation of the upper ABCA3 band (Fig 8), indicating inhibition of processing and confirming correct identification of the cleavage site.

Bottom Line: 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.We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L.

View Article: PubMed Central - PubMed

Affiliation: German Centre for Lung Research, Dr. von Hauner Children's Hospital, Ludwig-Maximilians University, 80337, Munich, Germany.

ABSTRACT

Rationale: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus