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.

该提案旨在确定肾小球素的化学结构，肾小球的化学结构是由特殊的真菌成员大量生产的复杂分子，该真菌在土壤中生长在植物根部。肾小球无处不在，丰富，并且对土壤物理特性和碳含量产生了深远的影响。肾小球素的生产和降解也可能影响土壤的生育能力和长期碳储量。令人惊讶的是，目前存在有关该重要分子的形式，结构和可变性的显着知识。通过阐明肾小球素（及其变体）的化学结构，这一早期探索性研究（急切）提案的概念将在真菌生物学和土壤科学领域产生潜在的变化结果。作为这个令人兴奋和重要的项目的一部分，一位博士后科学家和几位本科生也将接受蛋白质化学和真菌生物学技术的精致技术的培训。肾小球胶体内部的结构将在根瘤菌内部的菌丝生物量以及glomeromycota内的肾小球，根瘤菌，claroideoglomus，diversispora，gigaspora和acaulospora的代表中进行检查。轴突培养物将在双隔室系统中生长，以获得无植物菌丝生物量。细胞壁糖蛋白分数将经过SDS-PAGE和/或2D凝胶电泳，分离的带/斑点将用于结构阐明。每个条带/点的一部分将是酶上的脱糖基化，以鉴定N-聚糖的位点，然后用HPLC识别N-聚糖结构，以及LC/MS/MS，以识别主要的氨基酸骨干骨组成。 O-Glycan位点和组成将从LC/MS/MS片段化模式中识别出来。每个频段/斑点的第二个部分将用于识别在C端在C端的GPI锚定。我们假设这是：根瘤菌内glomalin sensu stricto不是单个肽，而是由多种具有不同N-和O-糖基化模式的肽组成，以及GPI锚定位点（H1）。我们进一步假设（H2），在各种肾小球属属之间的​​肾小球素sensu严格结构的比较将揭示出反映在Phylum中单个祖先进化起源的保守肽部分的通用骨架，并且鉴定出糖基化模式和氨基酸序列的变化随后在各种肾小球谱系中进行了不同的进化轨迹。