RHAMNOGALACTURONAN-I OLIGOGALACTURONIDE PROFILING BY HPSEC-MS/MS ELUCIDATES RGI STRUCTURE AND THE MODE OF ACTION OF ENZYMES - Archive ouverte HAL Accéder directement au contenu
Communication Dans Un Congrès Année : 2021

RHAMNOGALACTURONAN-I OLIGOGALACTURONIDE PROFILING BY HPSEC-MS/MS ELUCIDATES RGI STRUCTURE AND THE MODE OF ACTION OF ENZYMES

Olivier Habrylo
Sébastien Rigaud
Serge Pilard
Anaïs Guillaume
  • Fonction : Auteur

Résumé

In primary plant cell wall, cellulose microfibrils and hemicelluloses are embedded in a matrix of pectins. Depending on their chemical compositions and structures, four pectic domains can be described in including rhamnogalacturonan I (RGI) [1]. RGI consists of a backbone of repeating diglycosyl units (→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→), partially substituted at O-4 and/or O-3 positions of α-L-Rhap residues with notably side chains of →5)-α-L-arabinans-(1→ and →4)-β-D-galactans-(1→ [2]. Due to its complexity, the determination of RGI fine structure is challenging. We developed a sensitive analytical method based on the chromatographic separation of RGI-derived oligogalacturonides (OG-RGI), combined with accurate determination of their sizes and side-chains patterns using MS/MS [3,4]. This method can reveal the structure of RGI following its hydrolysis by specific enzymes such as rhamnogalacturonan hydrolases (RHG) and rhamnogalacturonan lyases (RGL). The three enzymes used in this study were expressed in Pichia pastoris heterologous system, purified by affinity chromatography, and their biochemical specificities determined. Using simple RGI-rich substrate, modified potato RG and more complex Camelina sativa mucilage, as well as two RHG and one RGL from Aspergillus aculeatinus we build an experimental OG-RGI library of 149 OGs. These OGs vary depending on their degree of polymerization (DP), degree of substitution as well as in their side-chains composition. Using a theoretical database, OG-RGI compounds were identified on the basis of their retention time, m/z and MS2 fragmentation patterns. We further showed that this method allows analyzing the structure of RGI from Arabidopsis cell wall and determining the specificities among multigenic families of fungal rhamnogalacturonan hydrolases. This new approach paves the way, through the analysis of mutants for RGI-degrading enzyme, for analyzing the contribution of RGI in modulating plant development.
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Dates et versions

hal-04409664 , version 1 (22-01-2024)

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Paternité - Pas d'utilisation commerciale - Pas de modification

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  • HAL Id : hal-04409664 , version 1

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Adrien Lemaire, Catalina Duran Garzon, Pauline Trezel, Justine Miternique, Olivier Habrylo, et al.. RHAMNOGALACTURONAN-I OLIGOGALACTURONIDE PROFILING BY HPSEC-MS/MS ELUCIDATES RGI STRUCTURE AND THE MODE OF ACTION OF ENZYMES. The 7th International Conference on Plant Cell Wall Biology -PCWB2021, Jun 2021, Japon, Japan. ⟨hal-04409664⟩
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