title: Monomeric and dimeric GDF-5 show equal type I receptor binding and oligomerization capability and have the same biological activity.
abstract: Growth and differentiation factor 5 (GDF-5) is a homodimeric protein stabilized by a single disulfide bridge between cysteine 465 in the respective monomers, as well as by three intramolecular cysteine bridges within each subunit. A mature recombinant human GDF-5 variant with cysteine 465 replaced by alanine (rhGDF-5 C465A) was expressed in E. coli, purified to homogeneity, and chemically renatured. Biochemical analysis showed that this procedure eliminated the sole interchain disulfide bond. Surprisingly, the monomeric variant of rhGDF-5 is as potent in vitro as the dimeric form. This could be confirmed by alkaline phosphatase assays and Smad reporter gene activation. Furthermore, dimeric and monomeric rhGDF-5 show comparable binding to their specific type I receptor, BRIb. Studies on living cells showed that both the dimeric and monomeric rhGDF-5 induce homomeric BRIb and heteromeric BRIb/BRII oligomers. Our results suggest that rhGDF-5 C465A has the same biological activity as rhGDF-5 with respect to binding to, oligomerization of and signaling through the BMP receptor type Ib.
authors.affiliation: Institut für Chemie/Biochemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Sieber
authors.forename: Frank
authors.initials: F
authors.lastname: Plöger
authors.forename: Raphaela
authors.initials: R
authors.lastname: Schwappacher
authors.forename: Rolf
authors.initials: R
authors.lastname: Bechtold
authors.forename: Michael
authors.initials: M
authors.lastname: Hanke
authors.forename: Shinji
authors.initials: S
authors.lastname: Kawai
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Muraki
authors.forename: Mieko
authors.initials: M
authors.lastname: Katsuura
authors.forename: Michio
authors.initials: M
authors.lastname: Kimura
authors.forename: Maya Mouler
authors.initials: MM
authors.lastname: Rechtman
authors.forename: Yoav I
authors.initials: YI
authors.lastname: Henis
authors.forename: Jens
authors.initials: J
authors.lastname: Pohl
authors.forename: Petra
authors.initials: P
authors.lastname: Knaus
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D052005:Bone Morphogenetic Protein Receptors, Type I / Q000378:metabolism*; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops / Q000378:metabolism; D019281:Dimerization; D005455:Fluorescent Antibody Technique; D055428:Growth Differentiation Factor 5; D006801:Humans; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism; D012995:Solubility
chemical_list: D019485:Bone Morphogenetic Proteins; C525893:GDF5 protein, human; D055428:Growth Differentiation Factor 5; D011994:Recombinant Proteins; D051785:Smad Proteins; D052005:Bone Morphogenetic Protein Receptors, Type I; D000469:Alkaline Phosphatase