Understanding Of Underlying The Molecular Mechanism Of Brachydactyly Type A-1

Brachydactyly type A-1 (BDA1) is the first recorded disorder of the autosomal dominant Mendelian trait (1903), which is cited in many genetic and biological textbooks. BDA1 is characterized by pronounced shortening or missing of the middle phalanges. Over 100 familial or sporadic cases from different ethnic groups have so far been reported. However, the real breakthrough in identifying the cause of BDA1 has not taken place until very late last century and early this century, when the Bio-X Center, Shanghai Jiao Tong University and Chinese Academy of Sciences, mapped and cloned the causative gene. We recruited three large families of BDA1 in the previous studies and mapped the locus for BDA1 on chromosome 2q35-q37 where we discovered mutations in Indian Hedgehog (IHH) was the cause for BDA1. In total we identified three heterozygous missense mutations (G283A (E95K), C300A (D100E) and G391A (E131K)) in the region encoding the amino-terminal signaling domain in all affected members of all three large, unrelated families. The three mutant amino acids, which are conserved across all vertebrates and invertebrates studied so far, are predicted to be adjacent on the surface of Ihh. In this work, we attempted to study the functional character of the mutants in order to understand the molecular basis of BDA1.Ihh protein is a secreted morphogen that is essential for endochondral bone development. Ihh is synthesized as a 46 kDa precursor that undergoes autocatalytic cleavage into an active 19 kDa N-terminal fragment (IhhN) which is subsequently modified by attachment of cholesterol and palmitic acid, and a more divergent C-terminal domain (IhhC). The long-range effects of Ihh during embryonic development are facilitated by heparan sulfate proteoglycans (HSPGs) in the surrounding intracellular matrix and a soluble multimeric form. The IhhN product triggers Hh pathway activation by binding to Patched (Ptc), the primary receptor for Hh signaling. The emphasis of this study is to explore the difference of the synthesis, processing, secretion and receptor binding between wild type and mutant Ihh.The eukaryotic constructs of wild type and mutant IHH gene were generated and transfected into ECHO cell line. Consequently, western-blotting analysis for the cell lysates indicated that the intermolecular cleavage and the cholesterol modification of three mutant Ihh were all normal and the IhhN fragment of E95K and D100E was unstable and degraded easily. In addition, the cell membrane location were all normal for three mutant proteins by immunofluorescence and confocal microscope. However, the assay of conditional culture medium implicated that the secretion of E95K and E131K mutants were normal but not D100E. Furthermore, temperature sensitivity analysis suggested that D100E protein was more unstable than E95K in 37℃and all proteins displayed the same stability in 30℃. Anyway, the secretion of D100E was normal in 30℃. Taken together, we speculated that D100E could't secrete because of its degradation in short time.3D structure of ShhN protein indicated that the position of E95K and D100E mutations is in an eight amino acid (8aa) conserved domain. Subsequently, we generated some truncation constructs and western-blotting analysis indicated that the 8aa domain is very important for Ihh protein because the IhhN fragment of T6 and T8 was degraded easily. Interestingly, we found that the IhhN fragment was unstable for E95G and D100N like E95K and D100E mutants and this implicated the mechanism of these two mutants associated with BDA1 may be consistent with E95K and D100E.Affinity chromatography assay implicated that the binding potency between E95K protein and heparin was enhanced and it is possible to result in the variety of the protein diffusion pattern. Gel chromatography assay indicated that the multimeric form of E95K and E131K were consistent with wild type. In addition, we suggested that the palmitoylation of E95K and E131K mutant proteins were normal because it could't form multimer for Hedgehog protein without palmitoylation.The IhhN product triggers Hh pathway activation by binding to Ptc receptor. The induction assay in C3H10T1/2 cell line by Prokaryotic IhhN proteins implicated that the ability of three mutant proteins to induce Hedgehog signaling was significantly impaired compared with wild type, suggesting these mutants may affect interaction with the receptor Ptc either directly or indirectly. Subsequently, the binding assay indicated that the interaction potency of three mutant proteins to Ptc were significantly reduced compared with wild type, their KI value were listed below: E95K = 40.6nM; E131K = 30.5nM; D100E >> 100nM, whereas KD of wild type was 20.6nM.Taken together, the present study analyzed the functional character including protein expression, modification, secretion and receptor binding of three mutants compared with wild type Ihh, and found some differences among them. Thereby, we could understand well the molecular mechanism of BDA1 from these findings.