Alternative splicing of ameloblastin in enamel formation

项目概要/摘要 牙釉质发育畸形，称为釉质发育异常（AI），是一种罕见的 继承条件。受AI影响的儿童及其家人终身遭受外观受损的痛苦， 社会交往受损，心理压抑，牙齿敏感，龋齿风险增加， 咬合塌陷无法咀嚼牙科治疗需要全口康复和频繁的，生活- 长期维护。所有的牙釉质蛋白都是导致AI的原因。成釉蛋白（Ambn）是第二大 丰富的釉质蛋白。Ambn基因属于分泌型钙结合基因簇， 磷蛋白（SCPP），起源于基底膜的基因。在进化过程中， 选择矿化基因以专门用于釉质、牙本质和骨。与特征一致 对于SCPP，Ambn是一种无序的、磷酸化的、钙结合的、富含脯氨酸的酸性蛋白。的 选择性剪接增加了釉质蛋白的多样性。Ambn表示为全长和剪接 proteins.剪接片段由外显子6表达，由15个高度保守的残基组成， 包括O-糖基化位点和剪接位点。 我们的初步数据表明，成釉细胞表达全长和剪接的Ambn在不同的细胞中， 浓度和不同的发育阶段。在分泌期，剪接的Ambn表达 高于全长Ambn。但在成熟阶段，剪接的Ambn表达减少， 长度Ambn的表达高于剪接。在Ambn敲除小鼠中，用转基因全长 Ambn，存在釉质层，但未恢复釉质层。这一发现表明 全长的Ambn不足以形成适当的釉质，拼接的Ambn可能在牙釉质形成中具有重要功能。 釉质形成总体假设是Ambn剪接变体执行明显不同的功能， 取决于釉质阶段。在具体目标1中，我们将确定完整的阶段特定功能- 长度Ambn相比，拼接Ambn在釉质矿化。在具体目标2中， 全长Ambn与剪接的Ambn在成釉细胞中的细胞附着和基底层的功能将被 测定对于拟议的研究，一个由临床科学家、基因组工程专家、Next- 世代测序和生物信息学被组装用于独特的相互作用和新颖的方法。 高分辨率成像和定量将用于研究釉质表面，厚度，密度 和硬度将分阶段分析成釉细胞，以了解全长和剪接的途径。 大使

PROJECT SUMMARY/ABSTRACT The developmental malformation of dental enamel, known as amelogenesis imperfecta (AI), is a rare inherited condition. Children and their families affected by AI suffer life-long from a disfigured appearance, compromised social interactions, psychological suppression, sensitive teeth, increased risk for caries and bite collapse, inability to masticate. The dental treatment requires full-mouth rehabilitation and frequent, life- long maintenance. All enamel proteins are known to cause AI. Ameloblastin (Ambn) is the second most abundant enamel protein. The Ambn gene belongs to the cluster of secreted calcium-binding phosphoproteins (SCPPs) that originated from the basement membrane gene SPARC. During evolution, genes for mineralization were selected to specialize for enamel, dentin and bone. Consistent with features for SCPPs, Ambn is a disordered, phosphorylated, calcium-binding and proline-rich, acidic protein. The variety of enamel proteins is increased by alternative splicing. Ambn is expressed as full-length and spliced proteins. The spliced segment is expressed by exon 6 and consist of 15 highly conserved residues, including an O-glycosylation site and splice site. Our preliminary data shows that ameloblasts express full-length and spliced Ambn in different concentrations and different developmental stages. During secretory stage, spliced Ambn is expressed higher than full-length Ambn. But at maturation stage, the expression of spliced Ambn reduces and full- length Ambn is expressed higher than spliced. In Ambn null mice, reconstituted with transgenic full-length Ambn, an enamel layer is present, however the enamel layer is not recovered. This finding suggests that full-length Ambn is not sufficient for proper enamel and that spliced Ambn may have an important function in enamel formation. The overall hypothesis is that Ambn splice variants execute distinctly different functions, depending on the enamel stage. In Specific Aim 1 we will determine the stage-specific functions of full- length Ambn compared to spliced Ambn in enamel mineralization. In Specific Aim 2 the stage-specific functions of full-length Ambn vs. spliced Ambn in ameloblasts for cell attachment and basal lamina will be determined. For the proposed studies a team of clinician scientists, experts in Genome Engineering, Next- generation sequencing and bioinformatics was assembled for unique interactions and novel approaches. High resolution imaging and quantification will be applied to study the enamel surface, thickness, density and hardness. Ameloblasts will be analyzed by stage to understand the pathways for full-length and spliced Ambn.