Elucidation of the disulfide bridge pattern of the recombinant human growth and differentiation factor 5 dimer and the interchain Cys/Ala mutant monomer.
Growth and differentiation factor 5 (GDF5) is involved in many developmental processes such as chondrogenesis and joint and bone formation. A recombinant monomeric human GDF5 mutant rGDF5(C84A) is in vitro as potent as the dimeric native form, and clinical investigations of rGDF5(C84A) are in progress. Native homodimeric GDF5 belongs to the transforming growth factor beta (TGF-beta) superfamily; each monomer contains a cystine knot formed by three intrachain disulfide bridges, and the monomers are connected via an interchain disulfide bridge. The disulfide bridge pattern of recombinant homodimeric rGDF5 was recently elucidated by X-ray diffraction. A combination of proteolytic degradation with thermolysin, separation of the generated fragments by reverse-phase high-performance liquid chromatography (RP-HPLC), and subsequent analyses of the disulfide-linked peptides by electrospray-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, amino acid analysis, and Edman degradation led to the unambiguous identification of the disulfide bridge pattern of the monomeric mutant rGDF5(C84A) and of the homodimeric rGDF5 in solution. The cystine knot of homodimeric rGDF5 exhibits the pattern Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7 (three intrachain disulfide bonds), and the monomers are connected by a single interchain disulfide bridge (Cys4-Cys4) in accordance with other members of the TGF-beta superfamily. The monomeric mutant rGDF5(C84A) exhibits the same cystine knot pattern as homodimeric rGDF5.
Trachsel
Christian
C
Department of Chemistry and Biochemistry, University of Bern, CH-3012 Bern, Switzerland.
Kämpfer
Urs
U
Bechtold
Rolf
R
Schaller
Johann
J
Schürch
Stefan
S
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D055428:Growth Differentiation Factor 5 / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics*
D004220:Disulfides; C525893:GDF5 protein, human; D055428:Growth Differentiation Factor 5; D011994:Recombinant Proteins; D003545:Cysteine; D000409:Alanine