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.