The Intermolecular Disulfide Bridge of Human Glial Cell Line-Derived Neurotrophic Factor: Its Selective Reduction and Biological Activity of the Modified Protein

Abstract

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.

Effect of the intermolecular disulfide bond on the conformation and stability of glial cell line-derived neurotrophic factor

“…Sedimentation velocity and equilibrium data clearly demonstrated that C101A GDNF forms a stable non-covalent dimer in solution with a dissociation constant below 1 nM. This is consistent with the observed biological activity of the mutant, which is indistinguishable from the wild-type protein and is also consistent with the activity of GDNF following reduction of the intermolecular disulfide (Hui et al, 1999). These results suggest that the dimerization is a prerequisite for receptor dimerization.…”

“…The disulfide structure of GDNF was recently determined (Hui et al, 1996), showing that GDNF contains the seven conserved cysteine residues that form the intramolecular disulfide knot characteristic of the TGF-β superfamily, although it shares very little sequence homology with TGF-β2 (Lin et al, 1993). In addition, Cys101 of GDNF was shown to be involved in the intermolecular disulfide bond (Hui et al, 1996(Hui et al, , 1999. The three-dimensional structure of GDNF, determined by X-ray crystallography (Eigenbrot and Gerger, 1997), revealed that the dimeric contact in GDNF is significantly different from that found in TGF-β.…”

“…For instance, activin A was reported to be only 1% active compared with the wild-type protein while TGF-β1 showed only 20% activity when the intermolecular disulfide bond was removed (Amatayakul-Chantler et al, 1994;Huesken-Hindi et al, 1994). However, bioactivity appeared to remain unchanged following reduction of the disulfide bond in GDNF (Hui et al, 1999). For most of the homodimeric proteins of the TGF-β family it is unclear whether they remain as non-covalent dimers or become monomeric after the removal of the intermolecular disulfide bond.…”