SYNTHESIS, SECRETION, DEGRADATION, AND FATE OF AMELOBLASTIN DURING THE MATRIX FORMATION STAGE OF THE RAT INCISOR AS SHOWN BY IMMUNOCYTOCHEMISTRY AND IMMUNOCHEMISTRY USING REGION-SPECIFIC ANTIBODIES

Rat ameloblastin is a recently cloned tooth-specific enamel matrix pro tein containing 422 amino acid residues. We investigated the expressio n of this protein during the matrix formation stage of the rat incisor immunohistochemically and immunochemically, using anti-synthetic pept ide antibodies that recognize residues 27-47 (Nt), 98-107 (M-1), 224-2 32 (M-2), 386-399 (M-3), and 406-419 (Ct) of ameloblastin. Immunohisto chemical preparations using antibodies Nt and M-1 stained the Golgi ap paratus and secretory granules of the secretory ameloblast and the ent ire thickness of the enamel matrix. Only M-1 intensely stained the per ipheral region of the enamel rods. Immunostained protein bands were ob served near 65, 55, and below 22 kD. Immunohistochemical preparations using antibodies M-2 and Ct stained the Golgi apparatus and secretory granules of the ameloblast and the immature enamel adjacent to the sec retion sites, but not deeper enamel layers. Immunostaining using M-2 a nd Ct revealed protein bands near 65 and 40-56 kD, and 65, 55, 48, 36, and 25 kD, respectively. M-3 stained the cis side of the Golgi appara tus but not the enamel matrix. This antibody recognized a protein band near 55 kD, but none larger. After brefeldin A treatment, immunoreact ion of the 55-kD protein band intensified, and dilated cisternae of rE R of the secretory ameloblast contained immunoreactive material irresp ective of the antibodies used. These data indicate that ameloblastin i s synthesized as a 55-kD core protein and then is post-translationally modified with O-linked oligosaccharides to become the 65-kD secretory form. Initial cleavages of the 65-kD protein generate N-terminal poly peptides, some of which concentrate in the prism sheath, and C-termina l polypeptides, which are rapidly degraded and lost from the enamel ma trix soon after secretion.