A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells.
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A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells. / Partanen, Sanna; Storch, Stephan; Löffler, Hans-Gerhard; Hasilik, Andrej; Tyynelä, Jaana; Braulke, Thomas.
In: BIOCHEM J, Vol. 369, No. 1, 1, 2003, p. 55-62.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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T1 - A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells.
AU - Partanen, Sanna
AU - Storch, Stephan
AU - Löffler, Hans-Gerhard
AU - Hasilik, Andrej
AU - Tyynelä, Jaana
AU - Braulke, Thomas
PY - 2003
Y1 - 2003
N2 - The substitution of an active-site aspartic acid residue by asparagine in the lysosomal protease cathepsin D (CTSD) results in a loss of enzyme activity and severe cerebrocortical atrophy in a novel form of neuronal ceroid lipofuscinosis in sheep [Tyynelä, Sohar, Sleat, Gin, Donnelly, Baumann, Haltia and Lobel (2000) EMBO J. 19, 2786-2792]. In the present study we have introduced the corresponding mutation by replacing aspartic acid residue 293 with asparagine (D293N) into the mouse CTSD cDNA to analyse its effect on synthesis, transport and stability in transfected HEK-293 cells. The complete inactivation of mutant D293N mouse CTSD was confirmed by a newly developed fluorimetric quantification system. Moreover, in the heterologous overexpression systems used, mutant D293N mouse CTSD was apparently unstable and proteolytically modified during early steps of the secretory pathway, resulting in a loss of mass by about 1 kDa. In the affected sheep, the endogenous mutant enzyme was stable but also showed the shift in its molecular mass. In HEK-293 cells, the transport of the mutant D293N mouse CTSD to the lysosome was delayed and associated with a low secretion rate compared with wild-type CTSD. These data suggest that the mutation may result in a conformational change which affects stability, processing and transport of the enzyme.
AB - The substitution of an active-site aspartic acid residue by asparagine in the lysosomal protease cathepsin D (CTSD) results in a loss of enzyme activity and severe cerebrocortical atrophy in a novel form of neuronal ceroid lipofuscinosis in sheep [Tyynelä, Sohar, Sleat, Gin, Donnelly, Baumann, Haltia and Lobel (2000) EMBO J. 19, 2786-2792]. In the present study we have introduced the corresponding mutation by replacing aspartic acid residue 293 with asparagine (D293N) into the mouse CTSD cDNA to analyse its effect on synthesis, transport and stability in transfected HEK-293 cells. The complete inactivation of mutant D293N mouse CTSD was confirmed by a newly developed fluorimetric quantification system. Moreover, in the heterologous overexpression systems used, mutant D293N mouse CTSD was apparently unstable and proteolytically modified during early steps of the secretory pathway, resulting in a loss of mass by about 1 kDa. In the affected sheep, the endogenous mutant enzyme was stable but also showed the shift in its molecular mass. In HEK-293 cells, the transport of the mutant D293N mouse CTSD to the lysosome was delayed and associated with a low secretion rate compared with wild-type CTSD. These data suggest that the mutation may result in a conformational change which affects stability, processing and transport of the enzyme.
M3 - SCORING: Zeitschriftenaufsatz
VL - 369
SP - 55
EP - 62
JO - BIOCHEM J
JF - BIOCHEM J
SN - 0264-6021
IS - 1
M1 - 1
ER -
