Endocytosis of Amelogenins: Full-length, LRAP And TRAP

Objectives:
Amelogenin is the most abundant protein in enamel matrix constituted by a variety of alternatively spliced forms and peptides derived from enzymatic cleavage. During the course of enamel formation peptides are removed from the matrix by ameloblasts to facilitate mineralization of enamel ribbons. Although the process of endocytosis is a major function of ameloblasts, the mechanism is not known. The goal of this study was to determine endocytic pathway of amelogenins into enamel organ epithelium.

Methods:
Localization of transcripts and protein of Rab5, Rab7, and amelogenin was performed on sectioned mandibular molars of 5 and 11 day old mice. For in vitro experiments, LS8 cells were cultured in the presence of full-length recombinant mouse amelogenin (Amel), the splice variant leucine-rich amelogenin peptide (LRAP) or the cleavage product tyrosine rich amelogenin peptide (TRAP). All amelogenins were labeled with Alexa-Fluor-488 and detected with confocal microscopy after a 4-hour incubation. Further, LS8 cells were transduced with Rab5 and Rab 7 siRNA and the uptake of amelogenins was monitored with confocal microscopy.

Results:
During secretory and maturation stages, Rab5 and Rab7 mRNA and protein localized to ameloblasts. Full-length amelogenin, LRAP and TRAP were taken up into LS8 cells and co-localized with Rab5 and Rab7 representing early and late endosomes. Rab molecules are involved in the traffic of vesicles containing proteins destined for secretion, endocytosis or degradation. Knockdown of Rab5 in LS8 cells prevented the uptake of amelogenins.

Conclusions: Rab5 and Rab7 are expressed in secretory and maturation stage ameloblasts and help to pass on the protein content of endocytic vesicles to target organelles. Amelogenins, including full-length, splice variant and cleavage product undergo endocytosis by enamel organ epithelium and are transported through the endocytic machinery via early and late endosomes.