Amelin extracellular processing and aggregation during rat incisor amelogenesis

Amelin (also known as ameloblastin and sheathlin) is a recently described p rotein that is secreted by ameloblasts during enamel formation. Here, the e xtracellular distribution and processing of amelin during rat incisor amelo genesis were investigated by Western blot probing using anti-recombinant ra t amelin antibodies. In addition. the solubility behaviour and aggregative properties of rat amelin were investigated using a sequential extraction pr ocedure involving (1) extraction with simulated enamel fluid to extract pro teins most likely to be soluble in vivo; (2) extraction with phosphate buff er to desorb proteins bound to enamel crystal surfaces: (3) extraction with sodium dodecyl sulphate (SDS) to extract proteins present as insoluble agg regates; followed by (4) a final acid demineralization step to release any remaining proteins. Proteins immunoreactive to the anti-amelin antibodies w ere detectable in secretory- and transition-stage enamel. Maturation-stage enamel appeared devoid of amelin. The largest immunoreactive protein detect ed migrated at 68 kDa on SDS gels, corresponding to the M-r of nascent amel in. Other immunoreactive bands at 52, 40, 37, 19, 17, 16, 15, 14 and 13 kDa were presumably amelin processing products. The sequential extraction proc edure revealed that the 68-, 53-, 40-, 37- and 13-kDa amelins were complete ly extracted under solution conditions similar to those reported to exist i n vivo. In contrast, the 19-, 17- and 16-kDa amelins were only partially ex tracted, whilst the 15- and 14-kDa amelins could not be extracted with simu lated enamel fluid. A proportion of the remaining 17- and 16-kDa amelins wa s desorbed from the enamel crystals: with phosphate buffer and appeared to have been mineral-bound. The 15- and 16-kDa amelins and the remainder of th e 17-;nd 16-kDa amelins were extracted with SDS only, suggesting that these species were present in vivo as an insoluble aggregate. The results: provi de additional information on amelin processing and degradation. and on how such processing influences the solubility and aggregative properties of ame lin-derived proteins. (C) 2001 Elsevier Science Ltd. All rights reserved.