Biosynthesis and secretion of enamel proteins in the rat incisor.
The synthesis and secretion of enamel proteins (EPs) in rat incisors was examined using cytochemical and biochemical methods. Radioautography after injection of 3H-methionine showed that ameloblasts in the presecretory, secretory, and maturation stages of amelogenesis actively synthesized and secreted proteins. Immunocytochemistry with an antibody to mouse amelogenins revealed the presence of EPs in the protein synthetic and secretory organelles of these cells at all three stages. Labeling was also found in elements of the endosomal/lysosomal compartment. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining of proteins extracted from enamel and enamel organ showed several protein bands. However, transfer to nitrocellulose paper and immunoblotting revealed that most of the proteins recognized by the antibody were situated between approximately 14 and 32 kDa. EPs were further characterized by using lectins to examine their carbohydrate content. Lectin-gold cytochemistry on sections showed the binding of wheat germ agglutinin and Helix pomatia lectin to secretory stage enamel. Lectin blotting indicated that the amelogenins were heterogeneously glycosylated and contained the sugars N-acetyl-glucosamine/N-acetyl-neuraminic acid and N-acetyl-D-galactosamine. Fluorography at 6 and 10 min and 1 h after injection of 35S-methionine revealed four labeled bands in the main amelogenin group near 22, 28, 30, and 32 kDa. A short-lived protein of approximately 58 kDa was also observed primarily in cells. The appearance of labeled proteins in enamel was paralleled by their disappearance from cells and the intensity of the radiolabeled protein bands, both, in enamel and in cells, decreased towards the maturation stage. These data are consistent with the concept that ameloblasts produce multiple amelogenins throughout amelogenesis.
Department of Anatomy, Université de Montréal, Québec, Canada.
A
A
Nanci
J P
JP
Ahluwalia
J R
JR
Pompura
C E
CE
Smith
D000565:Ameloblasts / Q000378:metabolism; D053523:Amelogenin; D000818:Animals; D003746:Dental Enamel Proteins / Q000096:biosynthesis* / Q000378:metabolism; D007150:Immunohistochemistry; D007180:Incisor / Q000254:growth & development / Q000378:metabolism*; D008297:Male; D008854:Microscopy, Electron; D051381:Rats; D011919:Rats, Inbred Strains
D053523:Amelogenin; C507200:Amelx protein, mouse; C507201:Amelx protein, rat; D003746:Dental Enamel Proteins