Molecular Analysis of Two Novel Missense Mutations in the GDF5 Proregion That Reduce Protein Activity and Are Associated with Brachydactyly Type C

Abstract

Growth and differentiation factor 5 (GDF5) plays a central role in bone and cartilage development by regulating the proliferation and differentiation of chondrogenic tissue. GDF5 is synthesized as a preproprotein. The biological function of the proregion comprising 354 residues is undefined. We identified two families with a heterozygosity for the novel missense mutations p.T201P or p.L263P located in the proregion of GDF5. The patients presented with dominant brachydactyly type C characterized by the shortening of skeletal elements in the distal extremities. Both mutations gave rise to decreased biological activity in in vitro analyses. The variants reduced the GDF5-induced activation of SMAD signaling by the GDF5 receptors BMPR1A and BMPR1B. Ectopic expression in micromass cultures yielded relatively low protein levels of the variants and showed diminished chondrogenic activity as compared to wild-type GDF5. Interestingly, stimulation of micromass cells with recombinant human proGDF5(T201P) and proGDF5(L263P) revealed their reduced chondrogenic potential compared to the wild-type protein. Limited proteolysis of the mutant recombinant proproteins resulted in a fragment pattern profoundly different from wild-type proGDF5. Modeling of a part of the GDF5 proregion into the known three-dimensional structure of TGFβ1 latency-associated peptide revealed that the homologous positions of both mutations are conserved regions that may be important for the folding of the mature protein or the assembly of dimeric protein complexes. We hypothesize that the missense mutations p.T201P and p.L263P interfere with the protein structure and thereby reduce the amount of fully processed, biologically active GDF5, finally causing the clinical loss of function phenotype.

Biophysical and structural characterization of a folded core domain within the proregion of growth and differentiation factor‐5

“…Point mutations in the proregion of GDF5 can cause skeletal malformations such as brachydactyly C. Two exchanges, T201P and L263P, have recently been identified, and their functional characterization demonstrated a loss of function of the proform in a chicken micromass assay . Notably, the two exchanges are located in a protease‐resistant core domain that has been identified previously .…”

The Fibrodysplasia Ossificans Progressiva (FOP) mutation p.R206H in ACVR1 confers an altered ligand response

“…, mAcvr1 R206H dLBD [22], mBmpr1a (also known as Alk3) and mBmpr1b (also known as Alk6) all in pCS2+ [23]. For generation of the mTgfβr1 c.a.…”

Cystine knot growth factors and their functionally versatile proregions

“…The study revealed that the mutations affected the thermodynamic stabilities of the protein variants. All three exchanges caused a loss of function with regard to chondrogenesis (Stange et al, 2014(Stange et al, , 2015. Another natural amino acid exchange (R380Q) within the proregion of GDF5 had been detected earlier in a patient family diagnosed with Brachydactyly A2, a clinically different malformation of the digits (Ploger et al, 2008).…”

“…The important roles of the proregions during biogenesis of the mature growth factors is highlighted by several reports that describe natural mutations in the proregion of human GDF5 (Table 1). Interestingly, three point mutations were identified that lie within the DNA encoding the core fragment of the GDF5 proregion and led to skeletal malformations in fingers and toes, classified as Brachydactyly C (Stange et al, 2014(Stange et al, , 2015. The biochemical and structural effects of the exchanges have been analysed in the recombinant purified mutant proteins .…”