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.

Role of the cystine-knot motif at the C-terminus of rat mucin protein Muc2 in dimer formation and secretion

“…The cysteine adjacent to Cys-4 of the knot (Cys-X) played a pivotal role, since its mutation to alanine (construct pRMC-CXA) essentially prevented dimer formation. The Muc2-domaindimerization mechanism thus resembles that of TGF-β2 [25,26], vWF [28] and glial cell-line derived neurotrophic factor [27] in requiring this specific cysteine residue for dimer formation.…”

“…The Cys-X residue of monomeric TGF-β2 is reported to form a stable intermolecular disulphide bond with the equivalent cysteine in a second molecule of TGF-β2 [25,26]. The same residue is responsible for dimer formation of glial cell linederived neurotrophic factor [27]. Absence of this cysteine in the vWF polypeptide prevents dimerization, resulting in type IID von Willebrand disease [28] and mutagenesis of the same cysteine residue in norrin protein disrupts oligomer formation [29].…”

Evidence that a peptide corresponding to the rat Muc2 C‐terminus undergoes disulphide‐mediated dimerization

“…During or mucin polymerization and gel formation. Studies of dimer formashortly after N-glycosylation (presumably in the endoplasmic re-tion in transforming growth factor-β2 [14] and, more recently glial ticulum [41]), the monomers dimerized via disulphide bonds and cell line-derived neurotrophic factor [19], indicate that a likely gave a product having a mobility centred at 150 kDa.…”

“…The termed the 'cystine knot' motif, which is also present and plays protein core (or apomucin) usually consists centrally of a vari-an active role in the dimerization of transforming growth factorable number of imperfect tandem repeats which contribute to β2 [14], nerve-growth factor [15], Norrie disease protein [16, molecular rigidity because of extensive O-glycosylation of their 17], human chorionic gonadotropin [18] and glial cell line-deserine and threonine residues. The flanking end regions of most rived neurotrophic factor [19]. The involvement of the cystine secretory mucins are enriched in cysteine, contain consensus se-knot motif in the initial C-tail to C-tail dimerization of von Wilquences for N-glycosylation, and are presumed to be folded and lebrand factor has been documented using deletion mutants [20].…”

Localization of Disulfide Bonds in the Cystine Knot Domain of Human von Willebrand Factor

“…Complete Reduction with DTT and Alkylation with 4VP-The remaining disulfide bonds in partially reduced and alkylated VWF CK domains or purified fragments thereof were reduced with 50 mM DTT in 0.2 M Tris-HCl, pH 8.5, 6 M guanidine HCl, and alkylated with excess 4VP (21). The modified products were purified by RP-HPLC using a C18 column (2.1 ϫ 150 mm; Vydac/The Separation Group Inc.) and acetonitrile gradient.…”