SUMMARY 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.

Our previous immunohistochemical study showed that in the porcine immature enamel, 13-17 kDa non-amelogenins were preferentially localized in the prism sheath. In this study, we revealed that the N-terminal amino acid sequence of a 15 kDa protein, one of the main components of the 13-17 kDa non-amelogenins, was VPAFPRQPGTHGVASL-, which shows no homology to those of known enamel proteins. In immunochemical analysis of porcine immature enamel, antibodies against a synthetic peptide containing this sequence reacted with many proteins of different molecular weights, especially with 62 kDa, 40 kDa and 10-19 kDa proteins. Of these proteins, the 62 kDa protein of the highest molecular weight was found only in newly formed enamel. In immunohistochemical preparations of the porcine tooth germs, the immunoreactivity of immature enamel just beneath the nonsecretory face

Our previous report identified 27- and 29-kDa calcium-binding proteins in porcine immature dental enamel. In this study we revealed that the N-terminal amino acid sequences of the two proteins were identical: LLANPXGXIPNLARGPAGRSRGPPG. The sequence matches a portion of the amino acid sequence of the porcine sheath protein, sheathlin. Porcine tooth germs were investigated immunochemically and immunohistochemically using specific antibodies raised against synthetic peptide that included residues 13-25 of this sequence. The affinity-purified antibodies reacted with several proteins extracted from newly formed immature enamel in immunochemical analyses, especially protein bands migrating at 62, 35-45, 29, and 27 kDa in SDS-polyacrylamide gels. The largest protein detected was a weak band near 70 kDa. In immunochemical analyses of proteins extracted from the inner (old) immature enamel, the antibody reacted faintly with the 27- and 29-kDa proteins. In immunohistochemical preparations, the Golgi apparatus and secretory granules of the secretory ameloblast, and the surface layer of immature enamel showed immunoreactivity. The immunoreactivity of immature enamel just beneath the secretory face of the Tomes' process was intense. No immunoreactivity was found in the Golgi apparatus of the maturation ameloblast. These results suggest that the 70-kDa protein, whose degradation might be very fast, is the parent protein of the 27- and 29-kDa proteins.

0: Sheath proteins: synthesis. secretion, degradation and fate informing enamel. Eur J Oral Sci 1998; 106 (suppl 1): 308 3 14. © Eur J Oral Sci , 1 998We investigated expression of ameloblasti n and sheathlin , recently cloned enamel matrix proteins from the rat a nd pig, in forming enamel immunocytochemically and immunochemically, using region-specific antibodies. The results obtained from the rat and pig were essentially the same. Antibodies wh ich recogni ze the N-terminal region stained the secretory machinery of the secretory ameloblast and the enti re thickness of the enamel matrix. especia ll y the peripheral region of the enamel rod . Tmmunosta ined protein bands were observed near 65 or 70 kDa and below 20 kDa . C-terminal-specific antibodies stained the secretory machinery of the ameloblast and the immature enamel adjacent to the secretion sites. lmmunostained protein band s were found ranging from 25 to 70 kDa. A ntibodies which recogn ize a region in the protein just prior to the C-terminal region stained the cis-side of the Golgi apparatus but not the enamel matrix. Immunostained protein bands were observed of about 55 kDa . These resu lts suggest that post-translational and post-secretory modifications of ameloblast in and sheath lin a re simi la r to each other, and further showed that their cleaved N-terminal polypeptides concentra te in the prism sheath. We propose that sheathlin and ameloblast in share the same role in amelogenesis and should be classified as sheath proteins. Copyriglu Eur J Oral Sci I 998 EUROPEAN JOURNAL OF ORAL SCIENCES

No abstract