Enamel proteins and proteases in Mmp20 and Klk4 null and double-null mice

Dental enamel forms in two stages. During the secretory stage mineral ribbons lengthen along a mineralization front at the enamel surface (1, 2). By the end of the secretory stage the enamel layer has already reached its final dimensions. In the rat incisor about 9% of the volume and 36% of the weight of the enamel matrix is mineral, which equals only about 14% of the mineral present at eruption (3). During the maturation stage, crystals initially deposited during the secretory stage grow thicker and wider. By the end of the maturation stage the mineral comprises about 70% of the volume and over 90% of the weight of the enamel matrix, which is more highly mineralized than other mineralized tissues, such as bone and dentin. Proteins are abundant in secretory-stage and early maturation-stage enamel, but are virtually absent from the late maturation-stage matrix (4). Proteins secreted during the secretory stage are degraded during both the secretory and maturation stages. Enamel protein cleavage products are reabsorbed by ameloblasts and degraded (4–7). There are two major secreted enamel proteases: matrix metalloproteinase 20 (MMP20) (8) and kallikrein-related peptidase 4 (KLK4) (9). These enzymes are necessary for enamel formation, as mutations in MMP20 (10) and KLK4 (11) cause inherited enamel malformations. A major function of enamel proteases is to facilitate the removal of enamel proteins to free up space within the enamel matrix for the enamel crystallites to grow in width and thickness (12). Several reviews on the roles of proteases in dental enamel formation are available (13–15). Enamel protein cleavage sites have been characterized for proteins that accumulate in secretory-stage pig enamel, and MMP20 is able to catalyze the same amelogenin (16, 17) and ameloblastin (18, 19) cleavages in vitro as occur in vivo. Kallikrein-related peptidase 4 cleaves amelogenin at many sites, but the cleavage pattern is different from that produced by MMP20 (20). The amelogenin C-terminus is highly charged relative to the rest of the protein and increases the affinity of amelogenin for hydroxyapatite (21). Matrix metalloproteinase 20 removes the amelogenin C-terminus, suggesting that MMP20 may be necessary to dissociate amelogenin from the crystals. However, cleavages by KLK4 on the N-terminal side of amelogenin also decrease amelogenin binding to hydroxyapatite in vitro (22). Mmp20 (23, 24) and Klk4 (25) null mice both have dramatic enamel phenotypes in which the hypomineralized enamel undergoes rapid attrition. The enamel in the Mmp20 null mice breaks off at the dentino–enamel junction (DEJ), while the enamel in the Klk4 null mice breaks just above the DEJ, in the deep enamel (26). Mmp20 null mice cover dentin with a rough mineral layer that is generally thin but irregular, and lacks rod and inter-rod organization (27). The enamel in Klk4 null mice has normal thickness and rod organization and is hard at the surface but is progressively less mineralized with depth (27). The enamel layers of both types of null mice retain enamel proteins, but the state of degradation of these proteins has not been characterized. In this study we analyzed the enamel proteins and proteases in wild-type, Mmp20 null, Klk4 null, and Mmp20/Klk4 double-null mouse maxillary first molars during the secretory stage, the maturation stage, and just prior to tooth eruption.