title: Glial cell line-derived neurotrophic factor: selective reduction of the intermolecular disulfide linkage and characterization of its disulfide structure.
abstract: Glial cell line-derived neurotrophic factor is a protein known to enhance the survival of dopaminergic neurons against several neurotoxins. It has been shown to have therapeutic potential in the treatment of Parkinson's disease and other neurodegenerative diseases. We have determined the inter- and intramolecular disulfide linkages of the dimeric molecule by a combination of direct peptide analysis and peptide analysis after either partial reduction or partial oxidation of the protein. Under an acidic condition, the interchain disulfide bond was selectively cleaved with tris(2-carboxyethyl)phosphine, revealing that Cys101 was involved in the intermolecular disulfide linkage. Three other disulfides, Cys68-Cys131, Cys72-Cys133, and Cys41-Cys102, were identified as intramolecular linkages. The determined disulfide structure is highly homologous to that of transforming growth factor beta 2. Since one intramolecular disulfide points through a ring consisting of eight amino acid residues based on the similarity with transforming growth factor beta 2, the disulfide-linked peptides were not purified by conventional methods. Only the peptides from an N-terminal region (residues -1 to 37) were liberated by proteolytic treatment with trypsin or endoproteinase Lys-C, resulting in a stable cystine-knot protein.
authors.affiliation: Department of Protein Structure, Amgen, Inc., Thousands Oaks, California 91320-1789, USA.
authors.forename: M
authors.initials: M
authors.lastname: Haniu
authors.forename: J
authors.initials: J
authors.lastname: Hui
authors.forename: Y
authors.initials: Y
authors.lastname: Young
authors.forename: J
authors.initials: J
authors.lastname: Le
authors.forename: V
authors.initials: V
authors.lastname: Katta
authors.forename: R
authors.initials: R
authors.lastname: Lee
authors.forename: G
authors.initials: G
authors.lastname: Shimamoto
authors.forename: M F
authors.initials: MF
authors.lastname: Rohde
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D051100:Glial Cell Line-Derived Neurotrophic Factor; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009457:Neuroglia / Q000737:chemistry*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016212:Transforming Growth Factor beta / Q000737:chemistry
chemical_list: D004220:Disulfides; C495039:GDNF protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D003545:Cysteine