Identification of an amelin isoform located in axons

A new axonal isoform of amelin, an analogue of the erythrocyte spectrin binding protein termed protein 4.1 has been identified in mouse brain. This new isoform has a molecular weight of 93 kDa, and migrates to a more acidic pH (pH 7.5-8.0) than the previously described amelin E (pH 8.5) on two dimensional NEPHGE-SDS PAGE. The 93 kDa protein looks nearly identical to amelin E on two dimensional chymotryptic iodopeptide mapping, and both share partial homology with rbc protein 4.1. The new isoform is located in axons, and the soma of neurons in mouse cerebellum, while amelin E is located in neuronal soma and dendrites. The axonal amelin antibody detects a 97 kDa protein in embryonic tissue which diminishes during development; and a 93 kDa protein which is first seen at postnatal day 1 of mouse brain ontogeny, increasing constantly to its adult concentrations. This time course of expression is quite different than amelin E, which is present at embryonic day 15 and diminishes constantly reaching its lowest concentration in the adult brain. We hypothesize that axonal amelin and amelin E may play important roles in the interaction of brain spectrin(240/235) and brain spectrin(240/235E) with f-actin and neuronal membranes.     

Ameloblastin Fusion Protein Enhances Pulpal Healing and Dentin Formation in Porcine Teeth

Ameloblastin (Ambn, also named “amelin” or “sheathlin”) is a protein participating in enamel formation and mesenchymal-ectodermal interaction during early dentin formation in developing teeth. Experiments have demonstrated an association between Ambn expression and healing of acute pulp wounds. The purpose of this study was to investigate if local application of recombinant fusion Ambn (rAmbn) could influence reparative dentin formation in pulpotomized teeth. In this randomized, double-blinded study, pulpotomy was performed in 28 lower central incisors in 17 adult miniature pigs. Following the surgical procedure, the exposed pulp tissue was covered either with rAmbn or with calcium hydroxide. After 2, 4, or 8 weeks, the teeth were extracted and examined by histomorphometry and immunohistochemistry using antibodies against porcine ameloblastin, collagen type I, and dentin sialoprotein (DSP). In rAmbn-treated teeth, a substantial amount of newly formed reparative dentin was observed at the application site, completely bridging the pulpal wound. Dentin formation was also observed in calcium hydroxide-treated teeth; however, the amount of reparative dentin was significantly smaller (P < 0.001) than after rAmbn treatment. Immunohistochemistry confirmed that the new hard tissue formed was similar to dentin. This is the first time a direct link between ameloblastin and dentin formation has been made in vivo. The results suggest potential for rAmbn as a biologically active pulp-dressing agent for enhanced pulpal wound healing and reparative dentin formation after pulpotomy procedures.     

Amelin and synapsin I are 4.1 related spectrin binding proteins in brain

How do synaptic vesicles move towards the presynaptic plasma membrane, fuse with that membrane, and release their contents during synaptic transmission? The answers to these questions at the molecular level are just beginning to be understood. Synapsin I is a neuron specific phosphoprotein that is associated with the cytoplasmic surface of synaptic vesicles. During synaptic transmission, the translocation of the synaptic vesicles to the presynaptic membrane of the neuron is thought to be mediated through changes in the phosphorylation state of synapsin I. It has been suggested that synapsin I is a spectrin binding protein related to the erythrocyte cytoskeletal protein 4.1, which binds to the terminal ends of the erythrocyte spectrin tetramer. The interaction of synapsin I (through brain spectrin) with the neuronal cytoskeleton may be essential for regulating the movement of synaptic vesicles towards the presynaptic plasma membrane. In addition, we have identified another protein in brain that is immunologically and structurally more closely related to erythrocyte 4.1 than is synapsin I. This protein, termed amelin, is localized in the cell body and dendrites of the neuron, whereas synapsin I is found exclusively in the synaptic terminals, suggesting that there is a family of erythrocyte 4.1 related proteins present in brain with distinct subcellular distribution and functions.