Complete sequencing and oxidative modification of manganese superoxide dismutase in medulloblastoma cells.
Manganese superoxide dismutase (Mn-SOD or SOD2) is a key antioxidant enzyme and was assigned several roles in tumor biology. Working on medulloblastoma cell line DAOY, we identified two spots as Mn-SODs. Because of the proposed pivotal role of this enzyme in oncobiology, we decided to completely sequence the proteins and to determine PTMs. Proteins extracted from DAOY cells were run on 2-DE, multienzyme digestions were carried out and peptides were analyzed by MALDI-TOF/TOF, Qq-TOF and the ion trap using both the CID and ETD principles. Both protein expression forms were completely sequenced and revealed identical protein sequences. Histidines His30 and His31 were oxidized in one protein, whereas tryptophan oxidation (Trp-186) was observed in both. Histidine oxidation was not only indicated by the mass shift of the peptide but also by specific spectra of 2-oxo-histidine and a previously described intermediate (His+14). Complete sequencing of the two Mn-SOD expression forms unambiguously characterizes this enzyme from a tumor cell line providing evidence that can be used for generation of antibodies and allowing conformational studies. The findings of different PTMs in the same gel represent Mn-SOD oxidative states, while oxidative modification of His30 and 31 may even reflect decreased Mn-SOD activity.
John
Julius Paul Pradeep
JPP
Department of Pediatrics, Medical University of Vienna, Vienna, Austria.
Pollak
Arnold
A
Department of Pediatrics, Medical University of Vienna, Vienna, Austria.
Lubec
Gert
G
Department of Pediatrics, Medical University of Vienna, Vienna, Austria.
D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D006639:Histidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008527:Medulloblastoma / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D010446:Peptide Fragments; C083725:2-oxohistidine; D006639:Histidine; D014364:Tryptophan; D013482:Superoxide Dismutase
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