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

该提案旨在确定球囊霉素的化学结构，球囊霉素是一种复杂的分子，由生长在土壤中植物根部的一组特殊真菌的成员大量产生。球囊霉素是普遍存在的，丰富的，并表现出对土壤物理性质和碳含量的深刻影响。球囊霉素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，一个显着缺乏知识的形式，结构和变异性的这一重要分子目前存在。通过阐明球囊霉素（及其变体）的化学结构，这一早期探索性研究概念（EAGER）提案将在真菌生物学和土壤科学领域产生潜在的变革性结果。一名博士后科学家和几名本科生也将接受蛋白质化学和真菌生物学方面的复杂技术培训，作为这个令人兴奋和重要项目的一部分。严格意义上的球囊霉素的结构将在根内食根霉的菌丝生物量中以及球囊霉门内的球囊霉属、食根霉属、Claroideoglomus、多样孢子菌属、巨孢子菌属和无梗孢子菌属的代表中进行检查。无菌培养物将在双室系统中生长，以获得无植物菌丝生物量。将对细胞壁糖蛋白组分进行SDS-PAGE和/或2D凝胶电泳，分离的条带/斑点将用于结构解析。将对每个条带/斑点的一部分进行酶促去糖基化，以鉴别N-聚糖的位点，然后进行HPLC以鉴别N-聚糖结构，并进行LC/MS/MS以鉴别一级氨基酸骨架组成。将根据LC/MS/MS裂解模式鉴别O-聚糖位点和组成。每个条带/斑点的第二部分将用于鉴定C末端处GPI锚的出现。我们假定：Rhizophagus intraradices中的狭义球囊霉素不是单一肽，而是由具有不同N-和O-糖基化模式以及GPI锚定位点的多个肽组成（H1）。我们进一步假设（H2），球囊菌门不同属之间的球囊菌蛋白狭义结构的比较将揭示保守肽部分的通用骨架，反映该门中单一的祖先进化起源，糖基化模式和鉴定的氨基酸序列的变化将反映不同球囊菌门谱系内随后不同的进化轨迹。

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.