Characterization of recombinant pig enamelysin activity and cleavage of recombinant pig and mouse amelogenins

Enamelysin (MMP-20) is a tooth-specific matrix metalloproteinase that is in itially expressed by ameloblasts and odontoblasts immediately prior to the onset of dentin mineralization, and continues to be expressed throughout th e secretory stage of amelogenesis. During the secretory stage, enamel prote ins are secreted and rapidly cleaved into a large number of relatively stab le cleavage products. Multiple proteinases are present in the developing en amel matrix, and the precise role of enamelysin in the processing of enamel proteins is unknown. We have expressed, activated, and purified the cataly tic domain of recombinant pig enamelysin, and expressed a recombinant form of the major secreted pig amelogenin rP172. These proteins were incubated t ogether, and the digestion products were analyzed by SDS-PAGE and mass spec trometric analyses. We assigned amelogenin cleavage products by selecting a mong the possible polypeptides having a mass within 2 Daltons of the measur ed values.;The polypeptides identified included the intact protein (amino a cids 2-173), as well as 2-148, 2-136, 2-107, 2-105, 2-63, 2-45, 46-148, 46- 147, 46-107, 46-105, 64-148, 64-147, and 64-136. These fragments of rP172 i nclude virtually all of the major amelogenin cleavage products observed in vivo. We propose that enamelysin is the predominant proteinase that process es enamel proteins during the secretory phase of amelogenesis.