Transient Accumulation of Proteins At Interrod and Rod Enamel Growth Sites

Abstract

Conceptually, there should be a brief interval in time when newly secreted proteins "pile up" at secretory sites just outside the membrane of ameloblasts. Indeed, previous cytochemical studies have suggested that glycosylated and/or sulfated glycoproteins accumulate at enamel growth sites. Colloidal gold lectin cytochemistry and immunocytochemistry with antibodies to enamel proteins and phosphoserine, combined with cycloheximide and brefeldin A to inhibit protein synthesis and secretion, were applied to characterize the distribution of newly formed proteins at enamel interrod and rod growth sites. Although enamel growth sites show a "rarefied" appearance, the results indicate that one or more subclasses of enamel proteins accumulate near the cell surface at sites where elongation of enamel crystallites contributes to thickening of the enamel layer. These proteins are glycosylated and/or phosphorylated and, at least in the case of the glycosylated ones, are rapidly processed after they are released extracellularly. In contrast, immunolabeling for amelogenins is generally weaker near the cell surface and more intense at a short distance away from the site where crystallites elongate. The data suggest that the enamel proteins accumulating at growth sites likely belong to the non-amelogenin category and play a transient role in promoting the lengthening of crystallites. It is concluded that areas near the ameloblast membrane where certain enamel proteins accumulate in fact constitute the equivalent of a mineralization front.

Immunocytochemical Study of Amelogenin Deposition during the Early Odontogenesis of Molars in Alendronate-treated Newborn Rats

“…Even though it has been reported that odontoblasts express amelogenin mRNA (Veis et al 2000;Oida et al 2002) and that amelogenin is expressed in relatively small amounts in a temporal-dependent pattern by rodent odontoblasts (Papagerakis et al 2003), the odontoblasts were never labeled in either the control or treated specimens, thus confirming previous studies with the same antibody (Nanci et al 1996;Bosshardt and Nanci 1997;Arana-Chavez and Nanci 2001). At least 14 isoforms are translated by an alternative splicing that takes place during amelogenin processing (Tompkins et al 2005b).…”

“…Unosmicated sections were directly processed for immunolabeling. Grids were incubated with a chicken egg yolk anti-rat 24-kDa amelogenin antibody (Nanci et al 1996) diluted 1:150 for 5 hr and then incubated with a rabbit anti-chicken IgG secondary antibody (Sigma; St Louis, MO), diluted 1:2000 for l hr. The sites of antibody-antigen binding were revealed with a protein A-gold complex, prepared as described in Bendayan (1995).…”

Comparative Immunochemical Analyses of the Developmental Expression and Distribution of Ameloblastin and Amelogenin in Rat Incisors

“…The organic phase of developing enamel in optimally fixed teeth appears morphologically homogeneous by electron microscopy. This suggests that constituent amelogenins and various anionic proteins may be uniformly spread, as intact proteins or fragments, throughout the thickness of the enamel layer, albeit in decreasing total bulk amounts forward in time as enamel matures (discussed in Nanci et al 1996). There are several lines of evidence suggesting that this is not the case.…”

“…There are several lines of evidence suggesting that this is not the case. Both newly secreted matrix proteins present at the forming surface of the enamel layer and partially degraded molecular forms located deeper into it can be concentrated at, or relatively missing from, specific sites within the layer (Smith et al 1989a,b;Kogaya 1994;Nanci et al 1996;Murakami et al 1997;Uchida et al 1991aUchida et al ,b,1997. The most dramatic differences in intraenamel protein concentrations have been observed for nonamelogenins such as ameloblastin and enamelin, for which newly secreted forms are present in high concentration at the enamel surface near ameloblasts (Hu et al 1997a,b;Murakami et al 1997;Uchida et al 1997).…”

Localized Infusion of Tunicamycin in Rat Hemimandibles: Alteration of the Basal Lamina Associated with Maturation Stage Ameloblasts

“…Briefly, for the amelogenin class, sections were transferred either on a drop of a chicken egg yolk antibody (Gassmann et al 1990) raised against 24-kD rat amelogenin (AMELy) Nanci et al 1996b), diluted 1:150 for 3 hr, or against mouse recombinant 179 amelogenin isoform (M179y) (Orsini et al in press) diluted 1:100 for 3 hr. They were washed with PBS, refloated on PBS-Oval, and then incubated for 1 hr with a rabbit anti-chicken IgG antibody (diluted 1:2000) (Cappel Research Products; Scarborough, ON, Canada).…”