Calbindin28kDa and calmodulin are hyperabundant in rat dental enamel cells. Identification of the protein phosphatase calcineurin as a principal calmodulin target and of a secretion-related role for calbindin28kDa.
Enamel cells are likely to experience heavy demands for intracellular calcium homeostasis during the secretion and hypermineralization of dental enamel. Here, the two major high-affinity calcium-binding proteins in rat enamel epithelium were identified as calbindin28kDa and calmodulin, using a microscale approach. Both proteins were hyperabundant, totalling up to 2% of the soluble protein and surpassing the amounts in cerebellum, the benchmark tissue. Calbindin28kDa and calmodulin accounted for 26% of the total calcium-binding capacity in enamel cell cytosol, under near physiological conditions. Numerous calmodulin-binding proteins were detected with an overlay assay, indicating that calmodulin has multiple major targets in enamel cells. The calcium/calmodulin-regulated protein phosphatase, calcineurin, was identified as a principal calmodulin target constituting 0.1% of the soluble protein. Calmodulin and calcineurin were expressed constitutively, implying continued heavy usage of calcium/calmodulin-based and phosphorylation-based signalling events throughout enamel cell development. Calbindin28kDa, in contrast, was expressed at fourfold higher levels in secretion-phase cells than during the calcium-intensive hypermineralization phase, unexpectedly pointing to an important role associated with secretion. Supporting this notion, immunoblots revealed that 33% of total (SDS-soluble) calbindin28kDa was in the particulate fraction and predominantly associated with the Triton-insoluble cytoskeleton. Solubilisation of cytoskeletal calbindin28kDa required high concentrations of NaCl or urea, indicating the existence of a high-affinity target ligand. The unusual abundance of calmodulin, calbindin28kDa and calcineurin demonstrated here provides the first molecular evidence that enamal cells possess a strong capability for intracellular calcium homeostasis. Since none of these proteins was up-regulated during enamel hypermineralization, it appears that other calcium-binding proteins are primarily involved in the putative transcellular passage of calcium.
Hubbard
M J
MJ
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
D000818:Animals; D064026:Calbindins; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000032:analysis* / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000032:analysis*; D003600:Cytosol / Q000737:chemistry; D003743:Dental Enamel / Q000737:chemistry*; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000032:analysis*; D011817:Rabbits; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000032:analysis* / Q000302:isolation & purification / Q000502:physiology
D064026:Calbindins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D064030:S100 Calcium Binding Protein G; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium