Recombinant DSP-PP and native HP protein reveal proteolytic activity

Introducton: Dentin sialoprotein (DSP) and phosphophoryn (PP) are the two most abundant noncollagenous proteins in dentin. DSP and PP coding sequences are derived from DSP-PP transcripts. Immunohistochemistry and in situ studies showed that DSP/PP proteins and DSP-PP mRNA expression are tightly associated with dentin mineralization. Mutations in the DSP-PP gene are linked to dentinogenesis imperfecta II and hearing loss while DSP-PP null mouse exhibit hypomineralization and dentin dysplasia. These studies, taken together with the early finding that PP binds Ca++ and can initiate hydroxyapatite formation in vitro, strongly support the assertion that DSP and PP proteins play significant roles in dentin mineralization. However, more recent findings by Godovikova et al. demonstrate DSP-PP mRNA expression occurring not only in teeth, but also in bone, kidney and salivary glands suggesting that the DSP-PP gene may participate in a variety of processes during organogenesis. Objectives: Because DSP-PP240 precursor protein is capable of self-processing to yield both DSP430 and PP240. Methods: To test whether this process could be due to proteolysis, gel purified DSP-PP240 was electrophoresed on a genolytic zymogram gel. We also tested whether native HP protein has proteolytic acitivity. HP is equivalent to isoform PP523, which is derived from the DSP-PP523 transcript. Results: The zymographic gel showed two white bands at 120 kDa (i.e., DSP-PP240) and 33 kDa (i.e., PP240) indicative of gelatinolytic activity. This is the first evidence that DSP-PP and PP possess proteolytic activity. We also found that HP has proteolytic activity. Conclusion: Our current data, demonstrating that DSP-PP240, PP240 and PP523 all exhibit proteolytic activity suggests that these proteins may play important roles in tissue modeling during organ development. This work was supported by NIH DE11442-9 to HHR.