Project Details
Projekt
Functional analysis of glycan-binding proteins and glycan transporters
Applicant
Professor Dr. Matthias Höhne
Subject Area
Microbial Ecology and Applied Microbiology
Term
from 2016 to 2024
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 277249973
The subproject A3 aims to elucidate structure-function relationships of SusD-like proteins by identifying their binding polysaccharide substrates and by unraveling molecular mechanisms of substrate binding. In the highly diffusive marine environment, polysaccharide binding and uptake of oligosaccharide fragments is an important detail of polysaccharide utilization, but the interplay between binding and transport proteins and how they contribute to an efficient internalization of glycan fragments in marine bacteria is not well understood. During the first funding period, we have identified a laminarin-binding SusD-like protein from Gramella sp. MAR_2010_102, GM_SusDlam, which specifically binds branched laminarin polymers. We solved the crystal structure of GM_SusDlam and identified key residues for substrate binding. Based on the molecular selectivity of GM_SusDlam for highly branched laminarin, our working hypothesis is that SusD proteins with different selectivity must have developed that enable processing of various types of laminarin structures. Therefore, we aim in the second funding phase to study systematically the mechanisms of processing and uptake of different types of this important marine glycan. We wish to unravel how this SusD specificities correlate with different subclasses of laminarin PULs, which will be investigated in subproject A1. The main function of SusD was currently proposed to be the transport of oligosaccharides to the SusC barrel. Because all of our (and literature-reported) binding analyses of marine SusD-like proteins have been performed using polymeric glycans, we will focus to study binding at the oligosaccharide level. Therefore, we will use structurally different laminarins extracted from model diatoms, and treat them with surface-localized endoglucanases to investigate the nature of oligosaccharides formed at the cell surface in the initial degradation step. By using a limited number of already available and well-characterized laminarin-specific CAZymes we will prepare defined oligosaccharides to investigate the specificities of a collection of SusD-like proteins from different laminarin PUL types. The preparation of fluorescence-labeled oligosaccharides will enable us to demonstrate binding and uptake of oligosaccharides with viable cells. Mutagenesis studies of selected SusD-like proteins will help to correlate different glycan specificities to key residues. This allows the extraction of amino acid sequence fingerprints useful to predict specificities of SusD proteins found in metagenomic samples in the environment by sequence analyses. In addition to our focus on laminarin, we will also characterize binding specificities of SusD-like proteins of PULs involved in degradation of mannan and xylan, which are studied in the consortium by the subprojects A2 and A3.
DFG Programme
Research Units
