Figure 6
Posttranslational modification of recombinant human GLS2 protein with acetylhypusine. (A) Representative spectrum of a GLS2 peptide with acetylhypusine modification. The peptide KEKKCFPKGVDMMAAL (from amino acid 329 to 344 of the GLS2 sequence) appears as a charge + 3 peptide with a monoisotopic m/z of 661.34753 Da (+0.29 mmu/+0.44 ppm). MH: 1982.02805 Da, including C-5 carbamidomethyl (57.02 Da), M-12 oxidation (15.99 Da) and K4-Acetylhypusine (113.08 Da). (B) Validation of GLS2 hypusination by immunoblot analysis using specific rabbit polyclonal anti-hypusine antibodies. Four different fractions containing affinity-purified recombinant human GLS2 from two different GIP-affinity purifications were analyzed. The anti-hypusine antibodies recognized a band in all four lanes corresponding to the known molecular mass of mature GLS2 protein (29). Electrophoresis was done in 12% polyacrylamide Bis-Tris gel with PageRuler Prestained Protein Ladders (Thermo Scientific) (positions of markers indicated at the right end). (C) Positive and negative controls for hypusination. A whole protein extract from mouse brain and rat prefrontal cortex (20 µg each) were employed as positive controls by revealing a band of the hypusinated eIF5A protein at approx. 17 kDa (lanes 1 and 2). Purified human recombinant GLS256–602 protein expressed in bacteria was chosen as a negative control. It was analyzed in 10% SDS-PAGE gel and revealed with anti-hypusine antibodies (lane 3) or anti-GLS2 antibodies (lane 4). Positions of molecular mass markers are shown at the left end of panel C. Full-length gels and blots are shown in both panels B and C.
