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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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Effect of knockdown of single proteases on ABCA3 processing.Expression of lysosomal cathepsins was silenced using siRNA mediated knockdown and ABCA3 cleavage was assessed by Western blotting. Experiments were performed thrice, every time in triplicates. Representative Immunoblots are shown.
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pone.0152594.g005: Effect of knockdown of single proteases on ABCA3 processing.Expression of lysosomal cathepsins was silenced using siRNA mediated knockdown and ABCA3 cleavage was assessed by Western blotting. Experiments were performed thrice, every time in triplicates. Representative Immunoblots are shown.

Mentions: To further verify ABCA3 processing by cathepsins, we used siRNA-mediated knockdown of cathepsin genes. To do this, we applied siRNAs directed against all cysteine proteases of the cathepsin family that had been identified in lysosomes [17]. This included proteases encoded by CTSB, CTSF, CTSH, CTSK, CTSL1, CTSL2, CTSO, and CTSS. As a control, we also used siRNA knockdown of CTSD (Asp protease). Gene knockdown was monitored using quantitative PCR and was considered successful when expression was reduced by more than 70% (data not shown). Immunoblot performed on cell lysates collected 48 h after siRNA transfection showed accumulation of the 190 kDa band as an indicator for inhibition of cleavage only in the case of CTSL1 (Fig 5). The effect was slightly enhanced when knockdown of cathepsin L and cathepsin B was performed simultaneously, indicating a possible additive action of both proteases. When we quantified protein bands to assess the ratio of 190 kDa to 170 kDa ABCA3 bands, we found significant increase of the band ratio in the case of CTSL1 alone and also for the combination of CTSL1 and CTSB (Fig 6). Thus, siRNA knockdown confirms the involvement of cathepsins B and L in the processing of ABCA3 and points to combined action of these proteases.


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)

Effect of knockdown of single proteases on ABCA3 processing.Expression of lysosomal cathepsins was silenced using siRNA mediated knockdown and ABCA3 cleavage was assessed by Western blotting. Experiments were performed thrice, every time in triplicates. Representative Immunoblots are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152594.g005: Effect of knockdown of single proteases on ABCA3 processing.Expression of lysosomal cathepsins was silenced using siRNA mediated knockdown and ABCA3 cleavage was assessed by Western blotting. Experiments were performed thrice, every time in triplicates. Representative Immunoblots are shown.
Mentions: To further verify ABCA3 processing by cathepsins, we used siRNA-mediated knockdown of cathepsin genes. To do this, we applied siRNAs directed against all cysteine proteases of the cathepsin family that had been identified in lysosomes [17]. This included proteases encoded by CTSB, CTSF, CTSH, CTSK, CTSL1, CTSL2, CTSO, and CTSS. As a control, we also used siRNA knockdown of CTSD (Asp protease). Gene knockdown was monitored using quantitative PCR and was considered successful when expression was reduced by more than 70% (data not shown). Immunoblot performed on cell lysates collected 48 h after siRNA transfection showed accumulation of the 190 kDa band as an indicator for inhibition of cleavage only in the case of CTSL1 (Fig 5). The effect was slightly enhanced when knockdown of cathepsin L and cathepsin B was performed simultaneously, indicating a possible additive action of both proteases. When we quantified protein bands to assess the ratio of 190 kDa to 170 kDa ABCA3 bands, we found significant increase of the band ratio in the case of CTSL1 alone and also for the combination of CTSL1 and CTSB (Fig 6). Thus, siRNA knockdown confirms the involvement of cathepsins B and L in the processing of ABCA3 and points to combined action of these proteases.

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