Alternative splicing of ameloblastin in enamel formation

牙釉质形成中成釉细胞的选择性剪接

• 批准号: 10361491
• 负责人: Yong-Hee Patricia Chun
• 金额: $ 19.38万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10361491
• 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; 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

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.

项目摘要/摘要 牙齿牙釉质的发育畸形，称为障碍症（AI），是罕见的 继承条件。受AI影响的儿童及其家人因外观毁容而终身遭受了终身的影响， 社交互动，心理抑制，敏感牙齿，龋齿的风险增加和 咬伤，无法咀嚼。牙科治疗需要全口康复和频繁的生活 - 长时间维护。已知所有搪瓷蛋白都会引起AI。 Ameloblastin（AMBN）是第二个 丰富的搪瓷蛋白。 AMBN基因属于分泌钙结合的簇 起源于地下膜基因SPARC的磷蛋白（SCPP）。在进化期间， 选择用于矿化的基因以专门用于搪瓷，牙本质和骨骼。与功能一致 对于SCPP，AMBN是一种无序，磷酸化的，钙结合和富含脯氨酸的酸性蛋白。这 通过替代剪接增加了多种搪瓷蛋白。 AMBN表示为全长和剪接 蛋白质。剪接段由外显子6表示，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，成成木材在不同的 浓度和不同的发展阶段。在分泌阶段，剪接AMBN表示 高于全长AMBN。但是在成熟阶段，剪接的AMBN的表达减少了 长度AMBN表示高于剪接。在AMBN NULL小鼠中，用转基因全长重构 AMBN，存在一个搪瓷层，但是搪瓷层未恢复。这一发现表明 全长AMBN不足以适合适当的牙釉质，而剪接的AMBN可能具有重要功能 搪瓷形成。总体假设是AMBN剪接变体执行明显不同的功能， 取决于搪瓷阶段。在特定的目标1中，我们将确定全阶段的功能 与搪瓷矿化中剪接的AMBN相比，长度为AMBN。在特定目标2中特定于阶段 全长AMBN与片段胶细胞中的剪接AMBN的功能，用于细胞附着和基础层次 决定。对于拟议的研究，临床医生，基因组工程专家团队 为独特的相互作用和新方法组装了生成测序和生物信息学。 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。将通过阶段分析木材细胞，以了解全长和剪接的途径 Ambn。