Fates of Mutant DSPP Proteins That Cause Nonsyndromic DGI/DD

Objectives: Nonsyndromic Dentinogenesis Imperfecta (DGI) and the milder Dentin Dysplasia (DD) are both caused by two classes of mutations within the DSPP (dentin sialophosphoprotein) gene. Previously we showed that a loss of one DNA base within the DSPP repeat domain results in a change from a contiguous hydrophilic domain to a long hydrophobic domain and failure to exit the endoplasmic reticulum (ER). Curiously, the change of just one amino acid at the beginning of the DSPP protein also results in ER retention and the DGI phenotype. We hypothesized that the amino-terminal isoleucine-proline-valine (IPV) tripeptide motif enables DSPP binding to a cargo receptor for trafficking out of the ER.
Methods: As a model for DD and DGI, we transfected HEK293 cells with expression constructs and compared the biosynthesis of wildtype and representative mutant forms of DSPP.
Results: As expected, the A15V mutant that retains its short hydrophobic leader sequence was degraded by the ER-associated degradation (ERAD) proteasome pathway. However, the much longer hydrophobic domains caused by the frameshift mutations were not predominantly degraded by the ERAD pathway. Both the frameshift and the IPV mutants traffic inefficiently and were eventually degraded by the lysosomal proteolysis system instead.
Conclusions: Preliminary data suggest that the frameshift and the IPV DSPP mutants traffic inefficiently out of the ER. The energy supplied by the P97/ERAD system may be insufficient to disengage frameshift DSPP’s large hydrophobic domain from the ER membrane. Furthermore, the frameshift and the IPV-motif mutants translocate through different intermediate compartments before arriving at the lysosome for degradation. Using a spectrum of detergents and inhibitors, we are identifying the dual pathways that odontoblasts of DD and DGI patients may use to recognize, move, and destroy mutant DSPP proteins before they can lead to cell death. This research was supported by the Intramural Research Program of the NIH, NIDCR.