Modification of superoxide dismutase 1 (SOD1) properties by a GFP tag--implications for research into amyotrophic lateral sclerosis (ALS).
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.


RESULTS
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
Our findings indicate that most, but not all, properties of SOD1 remain the same with a GFP tag.
Stevens
James C
JC
Department of Neurodegenerative Disease, UCL Institute of Neurology, London, United Kingdom.
Chia
Ruth
R
Hendriks
William T
WT
Bros-Facer
Virginie
V
van Minnen
Jan
J
Martin
Joanne E
JE
Jackson
Graham S
GS
Greensmith
Linda
L
Schiavo
Giampietro
G
Fisher
Elizabeth M C
EM
D000690:Amyotrophic Lateral Sclerosis / Q000378:metabolism*; D000818:Animals; D002942:Circular Dichroism; D019281:Dimerization; D015971:Gene Expression Regulation, Enzymologic; D014644:Genetic Variation; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D009046:Motor Neurons / Q000378:metabolism; D009154:Mutation; D016366:Open Reading Frames; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D051381:Rats; D013482:Superoxide Dismutase / Q000378:metabolism*; D000072105:Superoxide Dismutase-1
C000606290:SOD1 protein, human; D049452:Green Fluorescent Proteins; C000606292:Sod1 protein, rat; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1