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

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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.