An antibody-based affinity chromatography tool to assess Cu, Zn superoxide dismutase (SOD) G93A structural complexity in vivo.
'Conformational diseases' are a group of diverse disorders that have been associated with misfolding of specific proteins, leading to their aggregation in particular cell tissues. Despite their relevance, the mechanisms involved in neurodegenerative processes remains poorly understood. Mutations in Cu,Zn superoxide dismutase (SOD1) are implicated in death of motor neurons in amyotrophic lateral sclerosis. Among others, the SOD1(G93A) mutation is known to weaken the structure and this could lead to conformational variations of the protein. As an approach to understand the tissue-specific propensity of protein aggregation, we developed an experimental procedure allowing rapid extraction of variants of human SOD1 (hSOD1) produced in different tissues. Using an antibody-based affinity chromatography procedure enzymatically active hSOD was extracted, indicating preservation of its native conformation. Analysis of the eluted fractions of hSOD extracted from the brain and liver of transgenic hSOD(G93A) rats provided evidence about heterodimers rSOD-hSOD(G93A) formation in both extracts. Moreover, when characterized by 2-DE and MALDI-TOF/TOF MS, the extracted hSOD(G93A) showed a complex profile suggesting the existence of various covalent modifications of the enzyme in both tissues. Thus, this method should allow following post-translational modifications of hSOD1 produced in various tissues.
Palacios
Florencia
F
Sección Bioquímica-Biología Molecular, Facultad de Ciencias, UdelaR, Montevideo, Uruguay.
Cota
Germán
G
Horjales
Sofía
S
Lima
Analía
A
Battistoni
Julio
J
Sotelo-Silveira
José
J
Marín
Mónica
M
D000818:Animals; D055910:Antibodies, Immobilized / Q000378:metabolism*; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007163:Immunosorbent Techniques; D008099:Liver / Q000201:enzymology; D011487:Protein Conformation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D055647:Rats, Transgenic; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016622:Silver Staining; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D055910:Antibodies, Immobilized; D011994:Recombinant Proteins; C109415:SOD1 G93A protein; D013482:Superoxide Dismutase