Role of Organic Matrix Molecules in the Formation of Very High Magnesium Calcite

有机基质分子在极高镁方解石形成中的作用

• 批准号: 2104759
• 负责人: Keith Alvares
• 金额: $ 22.5万
• 依托单位: Northwestern University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2104759&HistoricalAwards=false
• 关键词: Role; Organic; Matrix; Molecules; Formation

Non-technical abstract: Mineralized tissues are sophisticated organic-inorganic composites that are assembled bottom-up and exhibit hierarchical architecture. Highly evolved design enables features such as high bone toughness at low weight, self-sharpening teeth, and continuous adaptive remodeling/self-repair. The arrangement of the cellular components of the sea urchin tooth allows continuous growth and regeneration, in response to tooth wear and abrasion during feeding. From a materials perspective, the sea urchin tooth is highly unusual in that it is the only known instance of very high magnesium calcite (VHMC) in the columns that connect the primary and secondary plates to each other. With up to 33% Mg, VHMC is far from equilibrium, much harder than calcite, and cannot be made in the lab. The overarching goal of this proposal is to elucidate the mechanism of formation of VHMC in the sea urchin tooth. The PI previously isolated a set of four proline-alanine rich acidic phosphoproteins (PARPs) that are unique to the tooth biomineral. The central hypothesis of this proposal is that PARPs are involved in VHMC formation. The main objective of the proposed work is to test this hypothesis by a) mapping the distribution of PARPs in the tooth in relation to the columns using immunohistochemistry; b) quantifying the impact of recombinant PARPs on nucleation, growth, and final composition of crystalline calcium carbonate in the presence of magnesium in vitro; and finally, by c) determining the impact of knock down of the most promising candidate in vivo. Poised at the intersection of molecular biology, materials science, and bioengineering, this research has the potential to inform a wealth of new technologies, from bio-inspired and bio-enabled materials to materials for carbon dioxide sequestration. The team will leverage the interdisciplinary potential of this research to train high school, undergraduate, masters and a graduate student from a broad range of backgrounds. Technical abstract:The proposed activities address gaps in the understanding of how living organisms control crystal growth processes, with the long-term objective to develop bio-inspired and bio-enabled materials. The arrangement of the cellular components of the sea urchin tooth allows continuous growth and regeneration, in response to tooth wear and abrasion during feeding. An intriguing aspect of the tooth architecture is the use of both high magnesium calcite in the plates (HMC, Ca1-xMgxCO3, where x ~ 0.13) and very high magnesium calcite (VHMC, x~0.33) in the inter-plate columns. While other mineralized tissues in the sea urchin are comprised of HMC, VHMC is unique to the tooth. VHMC composition is far from equilibrium and there is no known synthesis. The PI previously identified a set of proline-alanine rich acidic phosphoproteins (PARPs) that are unique to the tooth biomineral. The central hypothesis of this proposal is that PARPs are involved in VHMC formation. To test this hypothesis, all 4 PARPs will be recombinantly expressed in a system that will ensure appropriate post-translational modification (phosphorylation). Antibodies will be raised against all recombinant PARPs to determine, using immunohistochemistry, which of them, if any, co-localize with the inter-plate columns, but not the plates. The two most promising candidates will be chosen for in-vitro experiments to determine their impact on nucleation, growth and composition of calcium carbonate in the presence of Mg. Both fully and un-phosphorylated forms will be tested to establish the functional role of phosphorylation. Finally, based on the in-vitro crystallization results, the most promising candidate will be knocked down in the adult sea urchin (using in-vivo morpholinos) and its effect on the composition of the inter-plate columns determined. The results should provide a mechanistic basis for bioinspired synthesis of VHMC, and how the sea urchin synthesizes calcite with a composition very far from equilibrium, thereby improving the properties of the material, and the performance of the tooth dramatically. Elucidating the mechanism of VHMC formation will feed back into the fields of cell and development biology. Complementary to the proposed research objectives, the team will engage undergraduates, a masters and a graduate student in research, and host a high school intern through Northwestern University's Science in Society program.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

非技术摘要：矿化组织是复杂的有机-无机复合材料，自下而上组装，并呈现出层次结构。高度进化的设计实现了低重量下的高骨韧性、自锐牙齿和持续自适应重塑/自修复等功能。海胆牙齿细胞成分的排列允许持续生长和再生，以应对进食期间的牙齿磨损和磨蚀。从材料的角度来看，海胆牙齿是非常不寻常的，因为它是唯一已知的极高镁方解石（VHMC）的例子，在柱中连接初级和次级板块彼此。由于镁含量高达33%，VHMC远未达到平衡，比方解石硬得多，因此无法在实验室中制造。这项建议的首要目标是阐明海胆牙齿中VHMC的形成机制。PI先前分离出一组四种富含脯氨酸-丙氨酸的酸性磷蛋白（PARP），它们是牙齿生物矿物所特有的。该建议的中心假设是PARP参与VHMC的形成。所提出的工作的主要目的是通过以下方式来测试该假设：a）使用免疫组织化学绘制PARP在牙齿中相对于柱的分布; B）在体外存在镁的情况下量化重组PARP对结晶碳酸钙的成核、生长和最终组成的影响;以及最后，通过c）确定体内敲除最有希望的候选物的影响。这项研究处于分子生物学、材料科学和生物工程的交叉点，有可能为大量新技术提供信息，从生物启发和生物激活材料到二氧化碳封存材料。该团队将利用这项研究的跨学科潜力，培养来自广泛背景的高中生，本科生，硕士生和研究生。技术摘要：拟议的活动解决了对活生物体如何控制晶体生长过程的理解方面的差距，其长期目标是开发生物启发和生物激活材料。海胆牙齿细胞成分的排列允许持续生长和再生，以应对进食期间的牙齿磨损和磨蚀。牙齿结构的一个有趣的方面是在板中使用高镁方解石（HMC，Ca 1-xMgxCO 3，其中x ~ 0.13）和在板间柱中使用非常高镁方解石（VHMC，x~0.33）。虽然海胆中的其他矿化组织由HMC组成，但VHMC是牙齿所特有的。VHMC的组成远未达到平衡，并且没有已知的合成方法。PI先前鉴定了一组富含脯氨酸-丙氨酸的酸性磷蛋白（PARP），它们是牙齿生物矿物所特有的。该建议的中心假设是PARP参与VHMC的形成。为了检验这一假设，所有4种PARP将在确保适当的翻译后修饰（磷酸化）的系统中重组表达。将针对所有重组PARP产生抗体，以使用免疫组织化学确定哪些（如果有的话）与板间色谱柱共定位，而不是与板共定位。将选择两种最有希望的候选物进行体外实验，以确定它们对Mg存在下碳酸钙的成核、生长和组成的影响。将对完全磷酸化和未磷酸化形式进行检测，以确定磷酸化的功能作用。最后，基于体外结晶结果，将在成年海胆中敲除最有希望的候选物（使用体内吗啉代），并确定其对板间柱组成的影响。这些结果应该为VHMC的生物启发合成提供机械基础，以及海胆如何合成具有非常远离平衡的组成的方解石，从而显著改善材料的性能和牙齿的性能。阐明VHMC的形成机制将对细胞和发育生物学领域产生重要影响。作为对拟议研究目标的补充，该团队将通过西北大学的社会科学项目，邀请本科生、硕士生和研究生参与研究，并接待一名高中实习生。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。