Elevated mRNA expression and defective processing of cathepsin D in HeLa cells lacking the mannose 6-phosphate pathway
© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies..
Disruption of the mannose 6-phosphate (M-6-P) pathway in HeLa cells by inactivation of the GNPTAB gene, which encodes the α/β subunits of GlcNAc-1-phosphotransferase, results in missorting of newly synthesized lysosomal acid hydrolases to the cell culture media instead of transport to the endolysosomal system. We previously demonstrated that the majority of the lysosomal aspartyl protease, cathepsin D, is secreted in these GNPTAB-/- HeLa cells. However, the intracellular content of cathepsin D in these cells was still greater than that of WT HeLa cells which retained most of the protease, indicating a marked elevation of cathepsin D expression in response to abrogation of the M-6-P pathway. Here, we demonstrate that HeLa cells lacking GlcNAc-1-phosphotransferase show a fivefold increase in cathepsin D mRNA expression over control cells, accounting for the increase in cathepsin D at the protein level. Further, we show that this increase at the mRNA level occurs independent of the transcription factors TFEB and TFE3. The intracellular cathepsin D can still be trafficked to lysosomes in the absence of the M-6-P pathway, but fails to undergo proteolytic processing into the fully mature heavy and light chains. Uptake experiments performed by feeding GNPTAB-/- HeLa cells with various phosphorylated cathepsins reveal that only cathepsin B is capable of partially restoring cleavage, providing evidence for a role for cathepsin B in the proteolytic processing of cathepsin D.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:11 |
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| Enthalten in: |
FEBS open bio - 11(2021), 6 vom: 05. Juni, Seite 1695-1703 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Liu, Lin [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 17.02.2022 Date Revised 17.02.2022 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1002/2211-5463.13169 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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NLM324852673 |
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| 245 | 1 | 0 | |a Elevated mRNA expression and defective processing of cathepsin D in HeLa cells lacking the mannose 6-phosphate pathway |
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| 520 | |a © 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. | ||
| 520 | |a Disruption of the mannose 6-phosphate (M-6-P) pathway in HeLa cells by inactivation of the GNPTAB gene, which encodes the α/β subunits of GlcNAc-1-phosphotransferase, results in missorting of newly synthesized lysosomal acid hydrolases to the cell culture media instead of transport to the endolysosomal system. We previously demonstrated that the majority of the lysosomal aspartyl protease, cathepsin D, is secreted in these GNPTAB-/- HeLa cells. However, the intracellular content of cathepsin D in these cells was still greater than that of WT HeLa cells which retained most of the protease, indicating a marked elevation of cathepsin D expression in response to abrogation of the M-6-P pathway. Here, we demonstrate that HeLa cells lacking GlcNAc-1-phosphotransferase show a fivefold increase in cathepsin D mRNA expression over control cells, accounting for the increase in cathepsin D at the protein level. Further, we show that this increase at the mRNA level occurs independent of the transcription factors TFEB and TFE3. The intracellular cathepsin D can still be trafficked to lysosomes in the absence of the M-6-P pathway, but fails to undergo proteolytic processing into the fully mature heavy and light chains. Uptake experiments performed by feeding GNPTAB-/- HeLa cells with various phosphorylated cathepsins reveal that only cathepsin B is capable of partially restoring cleavage, providing evidence for a role for cathepsin B in the proteolytic processing of cathepsin D | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a GlcNAc-1-phosphotransferase | |
| 650 | 4 | |a cathepsin B | |
| 650 | 4 | |a cathepsin D | |
| 650 | 4 | |a cathepsin L | |
| 650 | 4 | |a lysosomes | |
| 650 | 4 | |a mannose 6-phosphate pathway | |
| 650 | 7 | |a Mannosephosphates |2 NLM | |
| 650 | 7 | |a RNA, Messenger |2 NLM | |
| 650 | 7 | |a mannose-6-phosphate |2 NLM | |
| 650 | 7 | |a 3672-15-9 |2 NLM | |
| 650 | 7 | |a Transferases (Other Substituted Phosphate Groups) |2 NLM | |
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| 650 | 7 | |a GNPTAB protein, human |2 NLM | |
| 650 | 7 | |a EC 2.7.8.15 |2 NLM | |
| 650 | 7 | |a GNPTG protein, human |2 NLM | |
| 650 | 7 | |a EC 2.7.8.17 |2 NLM | |
| 650 | 7 | |a CTSD protein, human |2 NLM | |
| 650 | 7 | |a EC 3.4.23.5 |2 NLM | |
| 650 | 7 | |a Cathepsin D |2 NLM | |
| 650 | 7 | |a EC 3.4.23.5 |2 NLM | |
| 700 | 1 | |a Doray, Balraj |e verfasserin |4 aut | |
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