TY - JOUR
T1 - Elevated mRNA expression and defective processing of cathepsin D in HeLa cells lacking the mannose 6-phosphate pathway
AU - Liu, Lin
AU - Doray, Balraj
N1 - Publisher Copyright:
© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
PY - 2021/6
Y1 - 2021/6
N2 - 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.
AB - 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.
KW - GlcNAc-1-phosphotransferase
KW - cathepsin B
KW - cathepsin D
KW - cathepsin L
KW - lysosomes
KW - mannose 6-phosphate pathway
UR - http://www.scopus.com/inward/record.url?scp=85105032624&partnerID=8YFLogxK
U2 - 10.1002/2211-5463.13169
DO - 10.1002/2211-5463.13169
M3 - Article
C2 - 33932147
AN - SCOPUS:85105032624
SN - 2211-5463
VL - 11
SP - 1695
EP - 1703
JO - FEBS Open Bio
JF - FEBS Open Bio
IS - 6
ER -