Alternative splicing of ameloblastin in enamel formation

项目摘要/摘要 牙釉质发育畸形，称为釉质发生不全，是一种罕见的畸形。 遗传性疾病。受人工智能影响的儿童及其家人一生都会遭受毁容的痛苦， 社会交往受损，心理压抑，牙齿敏感，患龋齿的风险增加 咬合塌陷，无法咀嚼。牙科治疗需要全口康复和频繁的生活- 长时间的维护。所有的釉质蛋白都是已知的引起人工智能的因素。成釉蛋白(AmBN)是第二大类 丰富的釉质蛋白。AmBN基因属于分泌型钙结合簇 源于基底膜基因SPARC的磷酸蛋白(SCPP)。在进化过程中， 矿化基因被选择专门用于牙釉质、牙本质和骨骼。与功能一致 对于SCPP来说，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质。这个 通过选择性剪接增加了釉质蛋白的种类。AMBN表示为全长和拼接 蛋白质。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。 我们的初步数据显示，成釉细胞在不同的细胞中表达全长和拼接的AMBN 浓度和不同发育阶段。在分泌阶段，剪接的AMBN被表达 比全长AMBN还高。但在成熟期，剪接的AMBN的表达减少，并完全- 长度AmBN的表达高于剪接。在AMBN缺失小鼠中，用转基因全长重组 AMBN，有釉质层存在，但釉质层未恢复。这一发现表明 全长AMBN不足以形成适当的牙釉质，而拼接的AMBN可能在 釉质形成。总体假设是AMBN剪接变异体执行明显不同的功能， 这取决于牙釉质阶段。在具体目标1中，我们将确定FULL的阶段特定功能- 在牙釉质矿化中，长度安培与拼接安培的比较。具体目标2阶段具体目标 在成釉细胞中，全长AMBN和剪接AMBN对细胞附着和基底膜的作用将是 下定决心。对于拟议的研究，由临床科学家、基因组工程专家组成的团队，下一步- 世代测序和生物信息学被组合在一起，以实现独特的相互作用和新的方法。 高分辨率成像和定量将用于研究牙釉质的表面、厚度、密度 和硬度。成釉细胞将被分阶段分析，以了解全长和拼接的途径 安本。

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.