Alternative splicing of ameloblastin in enamel formation

牙釉质形成中成釉细胞的选择性剪接

基本信息

批准号：
10195786
负责人：
Yong-Hee Patricia Chun
金额：
$ 23.19万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10195786
关键词：
Affect Alternative Splicing Ameloblasts Amelogenesis Imperfecta Amino Acid Sequence Amino Acids Appearance Architecture Basal Cell Basal lamina Basement membrane Bioinformatics Bite CRISPR/Cas technology Calcium Binding Cell-Matrix Junction Cells Child Complement Data Defect Dental Dental Care Dental Enamel Dental caries Dentin Development Disease Enamel Formation Enamel Organ Epithelial Evolution Excision Exons Failure Family Gene Mutation Genes Genome engineering Hardness Histology Immunohistochemistry In Situ Hybridization Individual Inherited Ion Transport Knockout Mice Length Life Maintenance Minerals Morphology Mouth Rehabilitation Mus Mutate Pathway interactions Phase Phosphoproteins Process Proline Protein Splicing Proteins RNA Splicing Risk Rod Scientist Site Social Interaction Splice-Site Mutation Surface Thick Thinness Tissues Tooth structure Transgenic Organisms Variant ameloblastin bioinformatics tool bone density glycosylation high resolution imaging malformation mineralization next generation sequencing novel novel strategies psychologic reconstitution single cell sequencing tooth surface transcriptome

项目摘要

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.

项目概要/摘要牙釉质发育畸形，称为釉质形成不全（AI），是一种罕见的疾病。遗传条件。受人工智能影响的儿童及其家人将终生遭受容貌毁损之苦，社交互动受损、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、终生的治疗。维护时间长。已知所有牙釉质蛋白都会导致人工智能。成釉素 (Ambn) 是第二大丰富的牙釉质蛋白质。 Ambn 基因属于分泌性钙结合基因簇磷蛋白（SCPP）源自基底膜基因 SPARC。在进化过程中，选择矿化基因专门针对牙釉质、牙本质和骨骼。与特征相符对于 SCPP，Ambn 是一种无序、磷酸化、钙结合且富含脯氨酸的酸性蛋白质。这选择性剪接增加了牙釉质蛋白的多样性。 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。我们的初步数据表明，成釉细胞以不同的方式表达全长和剪接的 Ambn 浓度和不同的发育阶段。在分泌阶段，剪接的 Ambn 被表达高于全长 Ambn。但在成熟阶段，剪接的 Ambn 的表达量降低并充分表达。 Ambn 的长度表示为高于拼接长度。在 Ambn 无效小鼠中，用转基因全长重组 Ambn，存在牙釉质层，但牙釉质层未恢复。这一发现表明全长 Ambn 不足以形成适当的牙釉质，拼接的 Ambn 可能在以下方面具有重要功能：牙釉质的形成。总体假设是 Ambn 剪接变体执行明显不同的功能，取决于牙釉质阶段。在具体目标 1 中，我们将确定全阶段特定功能牙釉质矿化中 Ambn 的长度与拼接 Ambn 的比较。在特定目标 2 中，特定阶段全长 Ambn 与剪接 Ambn 在成釉细胞中对于细胞附着和基底层的功能将是决定。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，Next- 世代测序和生物信息学的结合是为了实现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牙釉质表面、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安本。