Evidence for a blockwise distribution of acetyl groups onto homogalacturonans from a commercial sugar beet (Beta vulgaris) pectin
Graphical abstract
The oligogalacturonates generated by three PGs were quantified and their sequences determined. An “overlap method” was used to assess acetyl groups distribution and a blockwise repartition of those onto sugar beet pectin homogalacturonan is proposed.
Introduction
Pectins are amongst the most abundant polysaccharides in many plant primary cell walls. Pectin molecules are mainly composed of four different structural elements: homogalacturonans (HGs), xylogalacturonans (XGAs), type I rhamnogalacturonans (RGs-I) and type II rhamnogalacturonans (RGs-II), which have been extensively described in previous reviews (Voragen et al., 1995, Ridley et al., 2001, Ralet et al., 2002). HGs consist in a repetition of α−(1→4)-linked d-galacturonic acid (GalA) units that can be partly methyl-esterified at C-6 and, in some species, partly acetyl-esterified at O-2 and/or O-3. Many of the properties and biological functions of pectins are believed to be mediated by ionic interactions between HG domains (Ridley et al., 2001, Willats et al., 2001). Not only the degree of esterification, but also the distribution of methyl and acetyl groups onto HGs has a deep impact on those interactions (Kohn et al., 1983, Thibault and Rinaudo, 1985, Ralet et al., 2003). In particular, acetylation of HG domains is well known to strongly alter pectins associative properties (Pippen et al., 1950, Kohn and Furda, 1968, Kohn and Malovikova, 1978, Renard and Jarvis, 1999, Ralet et al., 2003). To get a better understanding of the relationship between the acetylation of pectins and their associative properties, more information about the distribution of acetyl groups onto pectin molecules, and more particularly onto HGs, is necessary. In that purpose, enzymatic degradation of polysaccharides followed by structural analysis of the degradation products proved to be efficient tools (Massiot and Thibault, 1989, Sakamoto and Sakai, 1995, Needs et al., 1998, Perrone et al., 2002, Ralet et al., 2005). Basically, HG is the best substrate for endo-polygalacturonases (poly [1→4-α-d-galacturonide] glycano-hydrolase, EC 3.2.1.15) (PGs), as they hydrolyse 1→4 linkages between two GalA residues (as reviewed by Jayani et al., 2005). They are widely distributed in plants, fungi, yeasts, and bacteria (Fogarty and Kelly, 1983) and display variable tolerance toward methyl- and acetyl-esterification (Chen and Mort, 1996, Benen et al., 1999, Bonnin et al., 2002a, Bonnin et al., 2003, André-Leroux et al., 2005). In a previous work (Ralet et al., 2005), acetylated oligogalacturonates were recovered – after enzymatic hydrolysis of sugar beet pectin using an AnPGI from Aspergillus niger in combination with a pectin-methyl esterase from Aspergillus aculeatus – purified, and structurally characterized by mass spectrometry. A list of oligogalacturonates sequences was obtained but the puzzle of in which order the oligogalacturonates were linked up in the original HG had still to be solved. The distribution of acetyl groups onto HG domains remained thereby speculative.
In the present work, an extensive degradation of commercial acid-extracted sugar beet pectin was carried out with three different PGs, AnPGI and AnPGII from A. niger and FmPG from Fusarium moniliforme, in combination with A. aculeatus pectin-methyl esterase (AaPME). Those PGs are known to display variable tolerance towards methyl and/or acetyl groups (Bonnin et al., 2002a, Bonnin et al., 2003, André-Leroux et al., 2005) so that an “overlap method” could be tentatively used to assess acetyl groups distribution onto HG domains. The oligogalacturonates generated by the three PGs were quantified and their sequences determined by mass spectrometry. A hypothetical HG was constructed using the AnPGI-recovered oligogalacturonates as building blocks and the validity of the model was checked taking into account the hydrolysis products released by the other PGs.
Section snippets
Analysis of hydrolysis products by high-performance anion-exchange chromatography
An acid-extracted commercial sugar beet pectin was extensively hydrolysed by a given PG (AnPGI, AnPGII or FmPG) in combination AaPME. PGs activity is most often reduced by increasing DM (Chen and Mort, 1996, Benen et al., 1999) and combined activity of PG and PME is necessary to extensively degrade pectin HG regions. Around 75% of the methyl groups initially present in sugar beet pectin (DM 62) were removed by AaPME, leading to an overall mean final DM of ∼14. The enzymatic digests were
Conclusion
In the present work, the various tolerance of AnPGI and AnPGII from A. niger and FmPG from F. moniliforme to methyl and acetyl-substituants was used to built a homogalacturonan answering all the structural as well as the enzymatic requirements, i.e. chain length, DM, DAc and cleavage sites. The obtained polymer shows a rather blockwise distribution of acetyl groups with zones of 7--15 contiguous non-acetylated GalA units. As the substrate used in this study was extracted by chemical means,
Enzymes
The pectin methylesterase (PME, UniProt Q12535) from A. aculeatus, AnPGI (UniProt P26213) and AnPGII from A. niger were kindly provided by Novozymes (Bagsvaerd, Denmark). The two PGs were provided as monocomponent enzymes and were further purified before use. The purification of AnPGII was previously described (Bonnin et al., 2002a). AnPGI was purified by anion-exchange chromatography on a Mono Q HR 5/5 column (0.5 × 0.5 cm, GE Healthcare) equilibrated with 20 mM piperazine buffer pH 6 and eluted
Acknowledgements
Mass spectrometry analyses were performed within the “Biopolymers-Interaction-Structural Biology” platform located at the INRA Centre of Nantes. http://www.nantes.inra.fr/plateformes_et_plateaux_techniques/plateforme_bibs.
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