Possible O-linked Glycosylation of Enamelin 23 kDa Cleavage Product
Love, Matthew J.
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Enamel is composed of 95% mineral and 5% water and organic material. Because of its unusually high mineral content, enamel is the hardest substance in the body. Although not exactly the same, dental enamel can best be described as calcium hydroxyapatite (OHAp). Calcium hydroxyapatite crystals are grown outward from the dentinoenamel junction (DEJ). The crystals orient to form enamel rods and interrod enamel. The growing of enamel occurs through a three stage process known as amelogenesis. During the first phase, the pre-secretory stage, cubical epithelial cells polarize into ameloblasts which will serve to regulate amelogenesis. In the second stage, the secretory stage, ameloblasts secrete matrix proteins into the intercellular space. Although the exact role of the matrix proteins is unknown, it is certain that they help facilitate the formation of enamel. As the ameloblasts pull away from the DEJ and excrete more enamel matrix proteins, the OHAp crystals lengthen. In the final stage, the maturation stage, the ameloblasts alter their shape one final time in order to mineralize the fresh laid enamel. Mutations in the genes that encode for enamel matrix proteins can result in a defect known as amelogenesis imperfecta (AI). To date, there are five proteins that each have at least one mutation that will result in AI, and several candidate genes whose mutations have yet to be found. There are still many more genes and mutations that must be discovered before amelogenesis imperfecta can be completely understood. Using freshly sacrificed commercial pigs from Michigan State University's meat laboratory in East Lansing, Michigan and a fractionation method developed at Tsurumi University in Yokohama, Japan enamel matrix proteins were isolated and analyzed for use in better understanding AI. This method was successful in isolating a cleavage product of a known protein, Enamelin, that had never been isolated before. After fractionation by Sorensen buffer, ammonium sulfate, ion exchange chromatography and finally Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) a protein fragment was isolated. Using the Pro-Q Emerald 300 staining kit, it was determined that the protein fraction was also a glycoprotein. With no reasonable places for N-linked glycosylation, it is predicted that the 23 kDa Enamelin cleavage product is an O-linked oligosaccharide with a possible glycosylation site at T598 on the Enamelin amino acid sequence.