Monomeric and dimeric GDF-5 show equal type I receptor binding and oligomerization capability and have the same biological activity.
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.
Sieber
Christina
C
Institut für Chemie/Biochemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany.
Plöger
Frank
F
Schwappacher
Raphaela
R
Bechtold
Rolf
R
Hanke
Michael
M
Kawai
Shinji
S
Muraki
Yoshifumi
Y
Katsuura
Mieko
M
Kimura
Michio
M
Rechtman
Maya Mouler
MM
Henis
Yoav I
YI
Pohl
Jens
J
Knaus
Petra
P
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
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