Localization of EMSP1 expression during tooth formation and cloning of mouse cDNA

Enamel Matrix Serine Proteinase 1 (EMSP1) is a proteolytic enzyme that has been isolated from the developing enamel of pig teeth. Its apparent functio n is to degrade the organic matrix in preparation for enamel maturation. Th e expression of EMSP1 has never been investigated in another organism besid es the pig, and EMSP1 expression in the enamel organ has never been specifi cally demonstrated in ameloblasts. Here we report the expression of recombi nant pig EMSP1 (rpEMSP1), the generation of rabbit polyclonal antibodies ag ainst rpEMSP1, the characterization of the antibodies and EMSP1 expression by Western blot and immunohistochemical analyses, the cloning and character ization of a full-length cDNA encoding mouse EMSP1, and the localization of EMSP1 expression in ameloblasts in mouse day 14 first and second molars by in situ hybridization. The full-length mouse EMSP1 cDNA clone has 1237 nuc leotides, excluding the poly(A+) tail, and encodes a preproprotein of 255 a mino acids. Mouse EMSP1 shares 75% amino acid identity with pig EMSP1 and h as three potential N-linked glycosylation sites, two of which are conserved in the pig homologue. Western blot analysis shows that the polyclonal anti bodies are specific for EMSP1 and do not cross-react with trypsin. Immunohi stochemistry of pig incisors shows discrete staining in the surface enamel at the earliest part of the maturation stage. In mouse molars, in situ hybr idization gives a distinct and specific signal in maturation-stage amelobla sts, and in the junctional epithelium following tooth eruption. We conclude that EMSP1 is expressed by pig and mouse ameloblasts during the early matu ration stage of amelogenesis.