Recombinant DSP-PP protein and native HP protein reveal proteolytic activity

Introduction: 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. 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 strongly support the assertion that DSP and PP proteins play significant roles in dentin mineralization. However, more recent findings by Godovikova et al. (2006) 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. Because DSP-PP240 precursor protein is capable of self-processing to yield both DSP430 and PP240 (see Godovikova et al Abstract, AADR 2008), we hypothesize that DSP-PP240 or its processed products have proteolytic activity. Objectives: To test this hypothesis, we examine whether this processing to yield DSP430 and PP240 is due to a protease in the medium or due to DSP-PP240 itself. Methods: We utilized a baculovirus expression system to produce recombinant DSP-PP precursor proteins from a DSP-PP240 cDNA. Gel purified DSP-PP240 was electrophoresed on a genolytic zymogram gel. We also tested whether native dentin extract (DE) and native HP protein have proteolytic activity. HP is equivalent to isoform PP523. 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 and DE have 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.