Immunochemical Characterization of a Chicken Egg Yolk Antibody to Secretory Forms of Rat Incisor Amelogenin

Abstract

Amelogenins represent the major component of the organic matrix of enamel, and consist of several intact and degraded forms. A precise knowledge of their respective distributions throughout the enamel layer could provide some insight into their functions. To date, no antibody exists that can selectively detect the secretory forms of amelogenin. In this study we used the chicken egg yolk system to generate an antibody to recombinant mouse amelogenin. Immunoblots of whole homogenates from rat incisor enamel organs and enamel showed that the resulting antibody (M179y) recognized proteins corresponding to the five known secretory forms of rat amelogenin. Immunogold cytochemistry demonstrated that reactivity was restricted to ameloblasts and enamel. Secretory forms of amelogenin persisted in significant amounts throughout the enamel layer. The density of labeling was highest over the surface portion of the enamel layer, but enamel growth sites in this region showed a localized paucity of gold particles. Immunoreactivity was lowest over the mid-portion of the layer and increased moderately near the dentino-enamel junction. These results indicate that intact forms of amelogenin probably have a more complex distribution in the enamel layer than was heretofore suspected.

Characterization of Apin, a secreted protein highly expressed in tooth‐associated epithelia

“…For Western blotting, conditioned media and cell extracts were separated on 10% SDS–PAGE gels under reducing conditions and transferred to nitrocellulose. The blots were probed with the rabbit anti‐Apin antiserum at 1:2,500, a chicken anti‐amelogenin purified IgY [Orsini et al, 2001] at 1:1,000, or a mouse monoclonal anti‐actin IgG (clone mAbGEa, Affinity BioReagents) at 1:10,000, all in PBS‐Tween 0.1% with 5% non fat milk. The species‐specific secondary horseradish peroxidase conjugated antibodies (Sigma, St. Louis, MO) were used 1:30,000.…”

A post-classical theory of enamel biomineralization… and why we need one

“…This does not occur properly when proteins associated with the basement membrane, such as collagen 17, 11,12,13 α6/β4 integrin 14,15,16 or laminin-332 17,18,19,20 or proteins associated with the mineralization front, such as enamelin 21 or ameloblastin, 22,23 are defective or missing. 24,25,26 Amelogenin (the most abundant enamel matrix protein) appears to be sparse at the mineralization front 23,27,28 and a thin layer of enamel (∼15 µm vs. ∼110 µm) is deposited in AmelX knockout mice. 29 …”

Distal cis‐regulatory elements are required for tissue‐specific expression of enamelin (Enam)

“…Increasing evidence suggests that each of the enamel proteins has novel properties that may correlate with unique functional roles. Amelogenin, for instance, is relatively absent from the mineralization front where enamel crystal elongate (10), but forms spherical macromolecular assemblies designated ‘nanospheres’ (11) that may space enamel crystals and organize them into parallel arrays (12, 13). Ameloblastin (also called amelin or sheathlin) is thought to maintain rod and inter‐rod boundaries (14, 15) and attach ameloblasts to the enamel surface (6, 16).…”