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Modification of superoxide dismutase 1 (SOD1) properties by a GFP tag--implications for research into amyotrophic
James C Stevens1, Ruth Chia, William T Hendriks
1Department of Neurodegenerative Disease, UCL Institute of Neurology, London, United Kingdom.
Background:
Since the discovery that mutations in the enzyme SOD1 are causative in human amyotrophic lateral sclerosis (ALS), many strategies have been employed to elucidate the toxic properties of this ubiquitously expressed mutant protein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins, and whether they behave similarly to their untagged SOD1 counterparts.
Methodology/Principal Findings:
Here we compare the physicochemical properties of SOD1 and the effects of GFP-tagging on its intracellular behaviour. Immunostaining demonstrated that SOD1 alone and GFP-SOD1 have an indistinguishable intracellular distribution in PC12 cells. Cultured primary motor neurons expressing GFP or GFP-SOD1 showed identical patterns of cytoplasmic expression and of movement within the axon. However, GFP tagging of SOD1 was found to alter some of the intrinsic properties of SOD1, including stability and specific activity. Evaluation of wildtype and mutant SOD1, tagged at either the N- or C-terminus with GFP, in PC12 cells demonstrated that some chimaeric proteins were degraded to the individual proteins, SOD1 and GFP.
Conclusions/Significance:
Our findings indicate that most, but not all, properties of SOD1 remain the same with a GFP tag.
Insights
Green fluorescent protein (GFP) tagging of superoxide dismutase 1 (SOD1) largely preserves its intracellular behavior but alters some biochemical properties, including stability and activity. This finding is crucial for understanding SOD1 in amyotrophic lateral sclerosis (ALS) research.
Area of Science:
- Biochemistry
- Cell Biology
- Neuroscience
Background:
- Mutations in superoxide dismutase 1 (SOD1) are linked to amyotrophic lateral sclerosis (ALS).
- Green fluorescent protein (GFP)-tagged SOD1 is used to study protein localization, but its biochemical properties are not well understood.
- Investigating the impact of GFP-tagging on SOD1 is essential for accurate research.
Purpose of the Study:
- To compare the physicochemical properties of SOD1 and GFP-tagged SOD1.
- To assess the intracellular behavior of GFP-SOD1 in neuronal cells.
- To determine if GFP-tagging affects SOD1 stability and activity.
Main Methods:
- Immunostaining in PC12 cells to compare SOD1 and GFP-SOD1 distribution.
- Analysis of cytoplasmic expression and axonal transport in cultured primary motor neurons.
- Evaluation of SOD1 and GFP-SOD1 stability and specific activity.
- Assessment of chimaeric protein degradation in PC12 cells.
Main Results:
- SOD1 and GFP-SOD1 exhibited indistinguishable intracellular distribution in PC12 cells.
- Expression patterns and axonal movement were identical for GFP and GFP-SOD1 in motor neurons.
- GFP tagging altered SOD1's intrinsic properties, affecting its stability and specific activity.
- Some GFP-SOD1 chimaeric proteins degraded into separate SOD1 and GFP components.
Conclusions:
- GFP tagging generally preserves SOD1's intracellular behavior.
- Biochemical properties such as stability and activity are affected by GFP-tagging.
- While useful for localization studies, the biochemical alterations of GFP-SOD1 must be considered in ALS research.
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