Abstract
Transforming Growth Factor – beta (TGFβ) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.
Highlights
The Transforming Growth Factor-beta (TGFb) superfamily ligands encompass several subfamilies consisting of TGFb, Bone Morphogenetic Proteins (BMPs), Activin and Inhibin, Growth and Differentiation Factors (GDFs), Nodal, and Mullerian Inhibiting Substance (MIS)
At the end of Segment 5, LYLD of BMP-2 is equivalent to LYYD of Activin-bA (Figure 1A, blue box), for which LYYD was taken as the consensus sequence
To avoid the potential complication in the refolding process by these extra disulfide bond, Segment 1 of Activin-bA was removed from the final pool of segments, it consists of 11 Segments (Segments 1 through 6 of BMP-2 and Segments 2 through 6 of Activin-bA)
Summary
The Transforming Growth Factor-beta (TGFb) superfamily ligands encompass several subfamilies consisting of TGFb, Bone Morphogenetic Proteins (BMPs), Activin and Inhibin, Growth and Differentiation Factors (GDFs), Nodal, and Mullerian Inhibiting Substance (MIS). Stretching outward structurally from the centrally located ‘cystine knot’ of the dimer are mainly 4 beta strands forming 2 curved fingers. The functional subunit for the TGFb superfamily can exist both as homo- and hetero-dimers in vivo [10,11,12,13] Some family members, such as GDF9 and BMP15, lack the cysteine required to form the inter-disulfide bond, yet they are still functional and able to form stable dimers [14]
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