Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion.

Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants.

Analysis of different de-esterification mechanisms for pectin by enzymatic fingerprinting using endopectin lyase and endopolygalacturonase II from A. niger.

A series of pectins with different distribution patterns of methyl ester groups was produced by treatment with either plant (p-PME) or fungal pectin methyl esterases (f-PME) and compared with those obtained by base catalysed de-esterification. The products generated by digestion of these pectins with either endopectin lyase (PL) or endopolygalacturonase II (PG II) from Aspergillus niger were analysed using matrix assisted laser desorption ionisation mass spectrometry (MALDIMS) and high-performance anion-exchange chromatography with pulsed amperometric or UV detection (HPAEC-PAD/UV). Time course analysis using MALDIMS was used to identify the most preferred substrate for each enzyme. For PL, this was shown to be fully methyl esterified HG whereas for PG II, long regions of HG without any methyl esterification, as produced by p-PME was the optimal substrate. The blockwise de-esterification caused by p-PME treatment gave a decrease of partly methylated oligomers in PL fingerprints, which did not effect the relative composition of partly methylated oligomers. PG II fingerprints showed a constant increase of monomers and oligomers without any methyl ester groups with decreasing degree of esterification (DE), but almost no change in the concentration of partly methylated compounds. PL fingerprints of f-PME and chemically treated pectins showed decreasing amounts of partly methyl esterified oligomers with decreasing DE, together with a relative shift towards longer oligomers. PG II fingerprints were characterised by an increase of partly methylated and not methylated oligomers with decreasing DE. But differences were also seen between these two forms of homogenous de-esterification. Introduction of a certain pattern of methyl ester distribution caused by selective removal of certain methyl ester groups by f-PME is the most reasonable explanation for the detected differences.

Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation.

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.

Quantification of the amount of galacturonic acid residues in blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and endo-polygalacturonase II from Aspergillus niger.

A method to determine the amount of galacturonic acid in blocksequence (BS) in pectin homogalacturonan (HG) is described. The method is based on a combination of endopolygalacturonase II (endo-PG II) and exopolygalacturonase (exo-PG) digestion followed by quantification of the liberated galacturonic acid monomer. The amount of monomers released is directly related to the amount of non-esterified galacturonic acid units located between two other non-esterified galacturonic acids units on the HG chain. The amount released for exo-PG digestion only corresponds to the BS located at the non-reducing end of the polymer. The difference between total- and exo-BS was calculated to be the amount of endo-BS located either within or on the reducing end of the HG. Three series of model pectins obtained by de-esterification of a high-ester pectin with either plant pectin methyl-esterase (p-PME, P-series), fungal pectin methyl-esterase (f-PME, F-series) and chemical de-esterification using base (B-series) were analysed and compared with a fully de-esterified pectic acid sample obtained from the same raw material. Clear differences for the increase of the amounts of blocksequence could be seen between de-esterification of the P- and F-series samples supporting a blockwise and a homogenous de-esterification mechanism, respectively. f-PME and base treatment showed only minor differences in the increase of galacturonic acid units in BS, despite differences seen in their methyl-esterification pattern. Differences between the amounts of galacturonic acid located in exo- and endo-BS, provided evidence for the need of a certain start side or blocklength for p-PME to de-esterify blockwise.

Sequencing of partially methyl-esterified oligogalacturonates by tandem mass spectrometry and its use to determine pectinase specificities.

Complex mixtures of acidic oligosaccharides were produced by enzymatic digestion of partially methyl-esterified pectin with Aspergillus niger pectin lyase, endopolygalacturonase II, and exopolygalacturonase. To determine the specificities of these pectolytic enzymes toward non-esterified and methyl-esterified galacturonic acid residues, we have studied the methyl esterification patterns of selected oligomers in unseparated pectin digests. Collision-induced dissociation in a nanoelectrospray ionization ion trap mass spectrometer was used to locate methyl-esterified galacturonic acid residues in oligomers up to a degree of polymerization of 10. Analysis of the methyl esterification patterns gave insight into the substrate specificities of these pectolytic enzymes. Isomeric fragment ions containing the reducing and nonreducing ends were differentiated by 18O-labeling of the reducing end.

Characterization of enzymatic pectin digests by matrix-assisted laser desorption/ionization mass spectrometry.

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the characterization of partially methyl-esterified enzymatic pectin digests is described. The sensitivities of several matrices, positive and negative ion modes and desalting techniques for these acidic oligosaccharides were compared. The most favorable results were obtained with a thin-layer preparation of a mixture of 2,4,6-trihydroxyacetophenone and nitrocellulose in the negative ion mode. Results are presented demonstrating the sensitive characterization of separated and unseparated high-ester pectin digests obtained after complete digestion using Aspergillus niger pectin lyase and the analysis of digests after chemical modification. In the case of unseparated digests, the analysis of methylation patterns is demonstrated. Oligomers with a degree of polymerization up to 40 were detected after enrichment of large oligomers by propan-2-ol precipitation.

Isozyme-specific transition state inhibitors for the trypanosomal nucleoside hydrolases.

Protozoan parasites lack de novo purine biosynthesis and require purine salvage from the host. Nucleoside hydrolases are involved in nucleoside salvage and are not found in mammals, making them protozoan-specific targets for inhibitor design. Several protozoan nucleoside hydrolase isozymes with distinct substrate specificities have been characterized. Novel substituted iminoribitols have been synthesized to resemble the transition state structure of the nonspecific inosine-uridine nucleoside hydrolase from Crithidia fasciculata (IU-nucleoside hydrolase). These inhibitors have been characterized for this enzyme and for a purine-specific nucleoside hydrolase (IAG-nucleoside hydrolase) from Trypanosoma brucei brucei. Inhibitors which provide nanomolar inhibition constants for IU-nucleoside hydrolase exhibit micromolar inhibition constants for the IAG-enzyme. For example, p-bromophenyliminoribitol inhibits the IU- and IAG-enzymes with dissociation constants of 28 nM and 190 microM, respectively. Substrate specificity, the action of transition state inhibitors and the pH-dependence of the kinetic constants establish that the catalytic mechanisms and transition state structures are fundamentally different for the IU- and IAG-isozymes. The finding is remarkable since these isozymes share significant homology at the catalytic sites and both use inosine as a preferred substrate. The specificity of the transition state analogues indicates that logically-designed transition state inhibitors are isozyme-specific, with (Km/Ki IU-nucleoside hydrolase)/(Km/Ki IAG-nucleoside hydrolase) values up to 39,000. The mechanism of the differential inhibition is based on the relative leaving group activation and ribosyl-oxocarbenium-forming abilities of these enzymes. In addition to providing isozyme-specific inhibitors, the novel molecules described here have diagnostic value for the nature of the transition states for N-ribohydrolase enzymes.

Conversion of aldonic acids to their corresponding 2-keto-3-deoxy-analogs by the non-carbohydrate enzyme dihydroxy acid dehydratase (DHAD).

Aldonic acids containing four to six carbon atoms were tested as potential substrates of dihydroxy acid dehydratase (DHAD), an enzyme from the biosynthetic pathway of branched chain amino acids. Novel assay systems for observing the course of DHAD catalysed reactions were developed in order to adapt the enzyme to extended practical applications. Kinetic studies for the new substrates (12/13) as well as inhibitor kinetics for the substrate analogue 3-deoxy-aldonic acids (25, 27 and 29) were performed. These gave indications for the restrictions of substrate modifications and contributed to the understanding of the individual effects. Finally L-threonic acid (12) and D-erythronic acid (13) could be successfully applied as substrates for DHAD and this led to the chemoenzymatic synthesis of their 2-keto-3-deoxy-analogue (20) in a preparative scale.