title: 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.
abstract: Rat ameloblastin is a recently cloned tooth-specific enamel matrix protein containing 422 amino acid residues. We investigated the expression of this protein during the matrix formation stage of the rat incisor immunohistochemically and immunochemically, using anti-synthetic peptide antibodies that recognize residues 27-47 (Nt), 98-107 (M-1), 224-232 (M-2), 386-399 (M-3), and 406-419 (Ct) of ameloblastin. Immunohistochemical preparations using antibodies Nt and M-1 stained the Golgi apparatus and secretory granules of the secretory ameloblast and the entire thickness of the enamel matrix. Only M-1 intensely stained the peripheral region of the enamel rods. Immunostained protein bands were observed 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 secretion sites, but not deeper enamel layers. Immunostaining using M-2 and 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 apparatus but not the enamel matrix. This antibody recognized a protein band near 55 kD, but none larger. After brefeldin A treatment, immunoreaction of the 55-kD protein band intensified, and dilated cisternae of rER of the secretory ameloblast contained immunoreactive material irrespective of the antibodies used. These data indicate that ameloblastin is 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 polypeptides, some of which concentrate in the prism sheath, and C-terminal polypeptides, which are rapidly degraded and lost from the enamel matrix soon after secretion.
authors.affiliation: Department of Oral Anatomy, Hiroshima University School of Dentistry, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Uchida
authors.forename: C
authors.initials: C
authors.lastname: Murakami
authors.forename: N
authors.initials: N
authors.lastname: Dohi
authors.forename: K
authors.initials: K
authors.lastname: Wakida
authors.forename: T
authors.initials: T
authors.lastname: Satoda
authors.forename: O
authors.initials: O
authors.lastname: Takahashi
mesh_terms: D000565:Ameloblasts / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D015153:Blotting, Western; D020126:Brefeldin A; D003517:Cyclopentanes / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000648:ultrastructure; D003746:Dental Enamel Proteins / Q000276:immunology / Q000378:metabolism*; D004658:Enamel Organ / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D015870:Gene Expression; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D007150:Immunohistochemistry; D007180:Incisor / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D016253:Microscopy, Immunoelectron; D010446:Peptide Fragments / Q000276:immunology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
chemical_list: C098519:Ambn protein, rat; D003517:Cyclopentanes; D003746:Dental Enamel Proteins; D010446:Peptide Fragments; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A