The intermolecular disulfide bridge of human glial cell line-derived neurotrophic factor: its selective reduction and biological activity of the modified protein.
Recombinant human glial cell line-derived neurotrophic factor has been implicated to have therapeutic potential in the treatment of neurodegenerative diseases. The mature protein is a single polypeptide of 134 amino acid residues and functions as a disulfide-linked dimer. Reduction of the protein with dithiothreitol at pH 7.0 and in the absence of denaturant showed that the single intermolecular cystine bridge was reduced preferentially. Direct alkylation of the generated free sulfhydryl group using iodoacetamide or iodoacetate without denaturant was incomplete. Unfolding the protein in 6 M guanidine hydrochloride prior to the modification showed rapid disulfide scrambling. However, the sulfhydryl-modifying reagent N-ethylmaleimide was able to label quantitatively the free cysteinyl residue in the absence of any added chaotropic agent. By a combination of peptide mapping, Edman degradation, and mass spectrometric analysis, the labeled residue was identified to be Cys101, hence verifying the location of the intermolecular disulfide bond. The modified protein behaved as a noncovalent dimer when chromatographed through a Superdex 75 column under nondenaturing conditions and was comparable in biological activity to an unmodified control sample. The results therefore indicate that the intermolecular disulfide bridge of the protein is not essential for its biological function.
Hui
J O
JO
Department of Protein Structure, Amgen, Inc., Thousand Oaks, California 91320, USA.
Woo
G
G
Chow
D T
DT
Katta
V
V
Osslund
T
T
Haniu
M
M
D000478:Alkylation; D000595:Amino Acid Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D004220:Disulfides / Q000737:chemistry*; D051100:Glial Cell Line-Derived Neurotrophic Factor; D006801:Humans; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; C495039:GDNF protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins