Abstract
Brachydactyly type A1 (BDA1), the first recorded Mendelian autosomal dominant disorder in humans, is characterized by a shortening or absence of the middle phalanges. Heterozygous missense mutations in the Indian Hedgehog (IHH) gene have been identified as a cause of BDA1; however, the biochemical consequences of these mutations are unclear. In this paper, we analyzed three BDA1 mutations (E95K, D100E, and E131K) in the N-terminal fragment of Indian Hedgehog (IhhN). Structural analysis showed that the E95K mutation changes a negatively charged area to a positively charged area in a calcium-binding groove, and that the D100E mutation changes the local tertiary structure. Furthermore, we showed that the E95K and D100E mutations led to a temperature-sensitive and calcium-dependent instability of IhhN, which might contribute to an enhanced intracellular degradation of the mutant proteins via the lysosome. Notably, all three mutations affected Hh binding to the receptor Patched1 (PTC1), reducing its capacity to induce cellular differentiation. We propose that these are common features of the mutations that cause BDA1, affecting the Hh tertiary structure, intracellular fate, binding to the receptor/partners, and binding to extracellular components. The combination of these features alters signaling capacity and range, but the impact is likely to be variable and mutation-dependent. The potential variation in the signaling range is characterized by an enhanced interaction with heparan sulfate for IHH with the E95K mutation, but not the E131K mutation. Taken together, our results suggest that these IHH mutations affect Hh signaling at multiple levels, causing abnormal bone development and abnormal digit formation.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (30800613), the 973 Program (2010CB529600, 2007CB947300), the 863 Program (2009AA022701), the Shanghai Municipal Commission of Science and Technology Program (09DJ1400601), the Natural Science Foundation of Shanghai, China (Grant No. 08ZR1411000), the National Key Project for the Investigation of New Drugs (2008ZX09312-003), the Shanghai Leading Academic Discipline Project (B205), and the General Research Fund of Hong Kong (HKU760608M). The co-author, Xizhi Guo, was supported by the “Pujiang Talent” Project (08PJ1407200).
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(Supplementary information is linked to the online version of the paper on the Cell Research website.)
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Supplementary information, Table S1
Data collection and refinement statistics of IHH structure (PDF 96 kb)
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The subcellular localization of WT and mutant IhhN in the EcR-CHO cells. (PDF 207 kb)
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The stability of WT and mutant IhhN proteins. (PDF 199 kb)
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Ma, G., Yu, J., Xiao, Y. et al. Indian hedgehog mutations causing brachydactyly type A1 impair Hedgehog signal transduction at multiple levels. Cell Res 21, 1343–1357 (2011). https://doi.org/10.1038/cr.2011.76
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DOI: https://doi.org/10.1038/cr.2011.76
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