EAGER: Eludicidation of the structure of glomalin protein produced by root-associated soil fungi

EAGER：阐明与根相关的土壤真菌产生的球囊霉素蛋白的结构

• 批准号: 1649441
• 负责人: Mostafa Elshahed
• 金额: $ 14.37万
• 依托单位: Oklahoma State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1649441&HistoricalAwards=false
• 关键词: EAGER; Eludicidation; structure; glomalin; protein

The proposal aims to identify the chemical structure of glomalin, a complex molecule produced in large quantities by members of a special group of fungi that grows on plant roots in soil. Glomalin is ubiquitous, abundant, and exhibits a profound impact on soil physical properties and carbon content. Production and degradation of glomalin could also impact the fertility and long-term carbon storage potential of soil. Surprisingly, a remarkable lack of knowledge concerning the form, structure and variability of this important molecule currently exists. By elucidating the chemical structure of glomalin (and its variants), this early concept for exploratory research (EAGER) proposal will yield potentially transformative results in the fields of fungal biology and soil science. One postdoctoral scientist and several undergraduate students will also be trained on sophisticated techniques in protein chemistry and fungal biology as part of this exciting and important project. The structure of glomalin sensu stricto will be examined in the hyphal biomass of Rhizophagus intraradices as well as in representatives of the genera Glomus, Rhizophagus, Claroideoglomus, Diversispora, Gigaspora, and Acaulospora within the Glomeromycota. Axenic cultures will be grown in a dual compartment system to obtain plant-free hyphal biomass. The cell wall glycoprotein fraction will be subjected to SDS-PAGE and/or 2D gel electrophoresis and the separated bands/spots will be used for structural elucidation. A fraction of each band/spot will be enzymatically deglycosylated to identify the site of the N-glycan followed by HPLC to identify the N-glycan structure, and LC/MS/MS to identify the primary amino acid backbone composition. The O-glycan site and composition will be identified from the LC/MS/MS fragmentation pattern. A second fraction of each band/spot will be used for identifying the occurrence of a GPI-anchor at the C-terminus. We hypothesize that: glomalin sensu stricto in Rhizophagus intraradices is not a single peptide but will consist of multiple peptides with distinct N-, and O-glycosylation patterns, as well as GPI anchoring sites (H1). We further hypothesize (H2) that comparison of glomalin sensu stricto structures between various Glomeromycota genera will reveal a universal backbone of conserved peptide moieties reflecting a single ancestral evolutionary origin in the phylum, and that variations in glycosylation patterns and amino acid sequences identified will reflect the subsequent distinct evolutionary trajectories within various Glomeromycota lineages.

该提案旨在确定格鲁马林的化学结构，这是一种复杂的分子，由生长在土壤植物根上的一种特殊真菌的成员大量产生。格洛马林无处不在，含量丰富，对土壤物理性质和碳含量有深远的影响。麦角灵的产生和降解也会影响土壤的肥力和长期碳储存潜力。令人惊讶的是，关于这种重要分子的形式、结构和可变性的知识目前仍然存在。通过阐明格鲁马林(及其变种)的化学结构，这一探索性研究的早期概念(EIGHER)提议将在真菌生物学和土壤科学领域产生潜在的变革性结果。作为这一激动人心的重要项目的一部分，一名博士后科学家和几名本科生还将接受蛋白质化学和真菌生物学方面的复杂技术培训。将在根腐病菌的菌丝生物量以及球孢菌门中的球孢菌属、根腐菌属、球孢子菌属、双孢子菌属、巨孢子菌属和无菌孢子菌的代表中研究狭义球孢菌素的结构。无菌培养将在双室系统中培养，以获得无植物的菌丝生物量。细胞壁糖蛋白部分将经过SDS-PAGE和/或2D凝胶电泳，分离的条带/斑点将用于结构分析。每条带/斑点的一小部分将被酶去糖基化以确定N-糖链的位置，然后用高效液相色谱来确定N-糖链的结构，并用LC/MS/MS来确定主要的氨基酸骨架成分。O-葡聚糖的位置和组成将从LC/MS/MS碎裂模式中确定。每个带/点的第二部分将用于识别在C端出现GPI锚定。我们推测：内生根吸虫中的谷氨酸甘氨酯感觉不是单一的多肽，而是由多个具有不同N-和O-糖基化模式的多肽以及GPI锚定位点(H1)组成的。我们进一步假设(H2)，不同的球菌门之间的球蛋白感觉严格结构的比较将揭示出反映该门中单一祖先进化起源的保守的多肽部分的普遍骨架，并且所识别的糖基化模式和氨基酸序列的变化将反映随后在不同的球菌门中的不同的进化轨迹。