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Human Molecular Genetics Advance Access published online on January 18, 2008
Human Molecular Genetics, doi:10.1093/hmg/ddn012
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Brachydactyly Type A2 Associated with a Defect in proGDF5 Processing

Frank Plöger1,#, Petra Seemann2,#, Mareen Schmidt-von Kegler2,3, Katarina Lehmann3, Jörg Seidel4, Klaus W. Kjaer5, Jens Pohl1 and Stefan Mundlos2,3,6,*

1 Biopharm GmbH, 69115 Heidelberg, Germany 2 Max-Planck-Institut für Molekulare Genetik, Research Group Development & Disease, 14195 Berlin, Germany, 3 Institut für Medizinische Genetik, Universitätsmedizin Berlin, Charité, 13353 Berlin, Germany 4 Klinik für Kinder-und Jugendmedizin, SRH Wald-Klinikum, 07548 Gera, Germany 5 Wilhelm Johannsen Centre for Functional Genome Research, Institute of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark 6 Berlin Center for Regenerative Therapies (BCRT), 13353 Berlin, Germany

* To whom correspondence should be addressed Corresponding author Stefan Mundlos Institut für Medizinische Genetik Charité, Universitätsmedizin Berlin Augustenburger Platz 1 13353 Berlin Germany telephone +49 30 450 569-121 FAX +49 30 450 569-915 email address stefan.mundlos{at}charite.de

Received October 15, 2007; Revised December 7, 2007; Accepted January 16, 2008

We investigated a family with a brachydactyly type A2 and identified a heterozygous arginine to glutamine (R380Q) substitution in the Growth/Differentiation Factor 5 (GDF5) in all affected individuals. The observed mutation is located at the processing site of the protein, at which the GDF5 precursor is thought to be cleaved releasing the mature molecule from the prodomain. In order to test the effect of the mutation, we generated the GDF5-R380Q mutant and a cleavage-resistant proGDF5 mutant (R380A/R381A) in vitro. Both mutants were secreted from chicken micromass cultures, but showed diminished biological activity. Western blot analyses showed that wt GDF5 was processed by the chicken micromass cells, whereas the mutants were not, indicating that the mutations interfere with processing and that this leads to a strong reduction of biological activity. To test the requirements for GDF5 processing in vitro we produced recombinant human (rh) proGDF5 wild-type protein in E. coli. The results show that unprocessed (rh) proGDF5 is virtually inactive but can be proteolytically activated by different enzymes such as trypsin, furin and MMP3. (rh) proGDF5 could thus be used as a locally administered depot form with retarded release of activity. In contrast to mature rhGDF5, (rh) proGDF5 shows a high solubility at physiological pH, a characteristic that might be useful for therapeutic applications.

# These authors contributed equally to this work


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