Dentin Sialophosphoprotein (DSPP) Preprotein Signal Peptide Removal and Dentinogenesis Imperfecta (DGI)

About This Grant

Non-syndromic dentinogenesis imperfecta (DGI) is the most common type of inherited dentin disorder. It is caused by mutations in one allele of the dentin sialophosphoprotein (DSPP) gene that encodes a secreted protein. DSPP is synthesized as a preprotein with an amino-terminal sequence, called “signal peptide (SP)”. The SP is essential for targeting of the nascent polypeptide chain to the endoplasmic reticulum (ER) – the entry of the secretory pathway. The SP is removed by an ER membrane-associated signal peptidase complex (SPC) once it has completed its function. The SP removal is required for release of the mature proteins from the ER membrane and for secretion of the mature proteins. Most disease-causing missense mutations and intronic mutations affect the proximal mature region of DSPP preprotein – primarily the first three amino acid residues, i.e., “Isoleucine-Proline-Valine (IPV)” motif, of the mature DSPP. These mutations are together referred to as “IPV mutations”, and the resulting preprotein variants as “IPV mutant DSPP preprotein variants”. However, the molecular pathogenesis caused by these IPV mutations is not well understood. The PI has previously reported that DsppP19L mice, expressing a mutant P19L-DSPP, manifest dental defects associated with an accumulation of P19L-DSPP protein within the Dspp-expressing cells, and that the mutant P19L-DSPP protein is accumulated within the ER when expressed in in vitro cultured cells. Further, new data obtained by the PI show that 1) a significant amount of the mutant P19L-DSPP protein is still successfully processed and secreted to the dentin matrix; 2) the intracellularly accumulated P19L-DSP (DSP, dentin sialoprotein, the N- terminal fragment of DSPP) is associated with the membrane; and 3) a small molecular compound that has been shown to stimulate bacterial Type I signal peptidase dramatically promotes secretion of the mutant P19L- DSP variant in in vitro cultured cells. These findings lead to the novel hypothesis that the IPV mutant DSPP preprotein variants bind to SPC through their SP and proximal mature region as a non-cleavable competitive inhibitor, not only blocking SPC from processing the SP of the bound mutant DSPP preprotein but also preventing SPC from processing normal DSPP preprotein. Three aims are proposed to test this hypothesis: Aim 1 will determine if the disease-causing missense mutations that affect the proximal mature region of DSPP preprotein lead to a membrane association and accumulation of the resulting IPV mutant DSPP preprotein variants within the ER through their uncleaved SPs; Aim 2 will determine if the disease-causing intronic mutations that affect the proximal mature region of DSPP preprotein cause similar molecular pathogenesis as the missense mutations; and Aim 3 will determine if the IPV mutant DSPP preprotein variants bind to and inhibit SPC. Successful completion of the proposed research will not only provide new insights into the molecular pathogenesis underlying DGI caused by the IPV mutant DSPP preprotein variants but will also lead to identification of molecular compounds that may help prevent the dental defects in DGI patients in the future.