Innovative Enzymatic Approach to Resolve Homogalacturonans

Article pubs.acs.org/Biomac

Innovative Enzymatic Approach to Resolve Homogalacturonans Based on their Methylesterification Pattern Marie-Christine Ralet,*,† Martin A. K. Williams,‡,§,∥ Abrisham Tanhatan-Nasseri,† David Ropartz,† Bernard Quéméner,† and Estelle Bonnin† †

INRA, UR1268 Biopolymères Interactions Assemblages, rue de la Géraudière, BP 71627, F-44300 Nantes, France Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand § MacDiarmid Institute for Advanced Materials and Nanotechnology, New Zealand ∥ The Riddet Institute, Palmerston North, New Zealand ‡

ABSTRACT: Three series of model homogalacturonans (HGs) covering a large range of degree of methylesterification (DM) were prepared by chemical and/or enzymatic means. Randomly demethylesterified HGs, HGs containing a few long demethylesterified galacturonic acid stretches, and HGs with numerous but short demethylesterified blocks were recovered. The analysis of the degradation products generated by the action of a purified pectin lyase allowed the definition of two new parameters, the degree of blockiness, and the absolute degree of blockiness of the highly methylesterified stretches (DBMe and DBabsMe, respectively). By combining this information with that obtained by the more traditional endopolygalacturonase digestion, the total proportion of degradable zones for a given DM was measured and was shown to permit a clear differentiation of the three types of HG series over a large range of DM. This double enzymatic approach will be of interest to discriminate industrial pectin samples exhibiting different functionalities and to evaluate pectin fine structure dynamics in vivo in the plant cell wall, where pectin plays a key mechanical role.

■

fied one and can then sequentially remove neighboring methylesters on the HG chain.14,15 Since 8−15 consecutive nonmethylesterified GalA units are required to form a stable calcium-mediated junction zone between two HG chains,16−18 the starting substrate, the final DM achieved, and the demethylesterification process implemented will all play a role in determining the ionic sensitivity and ionotropic gel forming properties of modified pectins.6,7,11,19−22 The relationship between the methylester distribution in HG domains and pectin functionality in the presence of calcium has recently been further detailed. It has been shown that plantPME-demethylesterified pectin samples can produce networks containing different types of junction zones, depending on the length of the nonmethylesterified stretches.23−26 Pectin encompassing small stretches (∼ 10) of consecutive nonmethylesterified GalA units gel through rather short dimeric calcium-chelating junction zones, which leads to the formation of a flexible network architecture. For a similar DM, pectin exhibiting longer stretches of consecutive nonmethylesterified GalA units gel, in contrast, through the formation of bundles consisting of extensively aggregated dimeric junction zones, leading to the formation of a semiflexible network architecture.26 It can be hypothesized that within the cell wall, in which enzymes consistently tailor the amount and distribution of resident methylesters,5,24 different network architectures ful-

INTRODUCTION Plant cells are encapsulated in a cell wall, whose most prominent components are polysaccharides. The latter, jointly with some structural proteins, determine the shape and mechanical properties of plant cells. The fine structure of cell wall polysaccharides governs their functional properties both after extraction and in planta. Pectin is an abundant polysaccharide that is a key component controlling the architecture of dicotyledon primary cell walls and is involved in various cell functions and plant processes.1−4 Pectin molecules are composed of several acidic and neutral structural domains among which homogalacturonan (HG), a linear partly methylesterified α-(1,4)-linked D-galacturonic acid (GalA) homopolymer, is the most abundant. The degree of methylesterification (DM), i.e., the percentage of total GalA residues methylesterified, and the distribution of these methylesters in the HG domains are key features for pectin functionality. Indeed, many of the properties, in particular the gelling capabilities, and biological functions of pectin are closely linked to its ionic binding abilities.1,3,5 The latter are governed not only by the number of methylesters but also by their distribution pattern. Two main patterns of methylester distribution have classically been described: random or blockwise. Fungal-pectin-methylesterases (PMEs) and base treatment lead to a random distribution of methylesters.6−9 Plant-PMEs by contrast demethylesterify pectin in a processive manner, leading to the appearance of demethylesterified stretches or blocks.6−13 These enzymes initiate action on a methylesterified carboxyl group adjacent to a nonmethylesteri© 2012 American Chemical Society

Received: March 1, 2012 Revised: April 10, 2012 Published: April 20, 2012 1615

dx.doi.org/10.1021/bm300329r | Biomacromolecules 2012, 13, 1615−1624

Biomacromolecules

Article

filling different structural and mechanical functions might exist. Parameters that can discriminate between pectin samples exhibiting subtle differences in methylesterification pattern are thus of great interest in order, among other things, to predict the properties of resulting networks. To date, two main parameters have been introduced in order to characterize the presence or absence of demethylesterified stretches: (i) the degree of blockiness (DB), which is amount of nonmethylesterified monomers, dimers, and trimers released by incubation with endopolygalacturonase (endo-PG) divided by the amount of nonmethylesterified GalA present in the pectin sample;8 and (ii) the absolute degree of blockiness (DBabs), which is the amount of nonmethylesterified monomers, dimers and trimers released by endo-PG divided by the total amount of GalA present in the pectin sample (methylesterified GalA included).27 The endo-PG specificity renders the amount and distribution of its oligogalacturonate digestion products methylester-sequence-dependent and simple DB or DBabs measurements often allow a clear differentiation between pectin substrates demethylesterified in a random or blockwise fashion.21,28−30 Differences in the exact sizes of demethylesterified blocks however remains extremely difficult to assess. Although differences in demethylesterified blocklength have been related to the relative proportion of mono-, di-, and trigalacturonic acid liberated in endo-PG digests,28,31 such effects are of limited applicability and similar proportions of mono-, di-, and trigalacturonic acid have been observed in such digests above a critical DBabs value.30 More recently,13 unmethylesterified blocklengths in pectin substrates have been estimated from the lengths of unmethylesterified fragments released from very limited endo-PG treatments. With the aid of evaporative light scattering detection, the longer oligomeric unmethylesterified sections excised by limited treatments could be identified and quantified and serve as an approximation to the distribution of blocklengths in the polymer backbone. Although these studies are a promising line of enquiry, focusing entirely on demethylesterified stretches through the examination of endo-PG hydrolysis products gives only a partial view of HG structure. Herein the highly methylesterified stretches have also been studied through the examination of pectin lyase degradation products.32 Although pectin lyase has been used to qualitatively characterize pectin samples differing in their methylesterification pattern previously,9,33 in the work described herein clear parameters are introduced related to the methylesterified stretches in analogy to those defined previously for unmethylesterified stretches based on endo-PG digestion. Furthermore it is shown using model HGs with tailored patterns of methylesterification22 that by examining both endo-PG and pectin lyase digests HG samples exhibiting only subtle differences in methylesterification pattern can be resolved over a large range of DM.

■

to yield B- and P-series, respectively. A third series quoted BP- was obtained by demethylesterifying selected NaOH-demethylesterified HG samples a further time with plant-PME. HG samples are quoted HGx-By, HGx-Py or HGx-Bx′-Py with B = basic demethylesterification, P = plant-PME demethylesterification, x = DM of ″mother″ HG, x′ = DM achieved after basic demethylesterification and y = final DM achieved (Figure 1).

Figure 1. Nomenclature of the homogalacturonan samples. Characterization of Model Substrates. GalA measurements were performed by the automated m-hydroxydiphenyl method.36 DM was determined by quantifying base-released methanol according to Anthon and Barrett (2004).37 Briefly, 600 μL of HGs solutions (1 mg/ mL) were demethylesterified with 600 μL of 0.2 M NaOH for 1 h at 4 °C. The solution was neutralized with 600 μL of 0.2 M HCl and diluted by adding 1.2 mL of H2O. An aliquot of the solution was used for GalA determination. A total of 100 μL of 200 mM Tris-HCl buffer pH 7, 400 μL (3 mg/mL) of methylbenzothiazolinone-2-hydrazone (MBTH, Sigma M8006−1G), 50 μL of sample or 40 mM NaCl (as a blank), and 20 μL of alcohol oxidase (E.C. 1.1.3.13, Sigma, A2404; 0.02 UI/μL) were mixed in this order to oxidize the released methanol. After addition of the alcohol oxidase, the samples were incubated for 20 min at 30 °C prior to addition of 200 μL of a solution containing 5 mg/mL each of dodecahydrated ferric ammonium sulfate and sulfamic acid. After 20 min at room temperature, 600 μL of H2O was added and the tubes were vortexed vigorously. The absorbance at 620 nm was then measured and quantification carried out with the use of an external calibration curve (generated using methanol standards in the range 0 to 20 μg/mL). DM was calculated as

DM =

MeOH(mg/g)/32 × 100 GalA(mg/g)/176

Enzymes. Endopolygalacturonase II. Endo-PGII [EC 3.2.1.15, Uniprot P26214] was purified from an Aspergillus niger preparation provided by Novozymes (Copenhagen, DK).38 Pectin Lyase. PL [EC 4.2.2.10] was purified from the crude preparation Peclyve of A. niger provided by Lyven (Colombelles, F). A total of 20 mL of crude preparation were dialyzed against ultra pure water at 4 °C for 8 h changing water every 2 h and dialysis tubing every 4 h. The dialyzed enzymatic solution was saturated at 60% ammonium sulfate and centrifuged 15 min at 18000 g at 4 °C. The ammonium sulfate precipitate (AS60) was solubilized in a minimal volume of ultra pure water, dialyzed against ultra pure water at 4 °C for 8 h changing water and dialysis tubing every 2 h, and then dialyzed twice against 500 mL of 50 mM Na-phosphate buffer pH 6 at 4 °C for 90 min. Ammonium sulfate was added to reach a final concentration of 1.5 M. Hydrophobic interaction chromatography (HIC) was performed on an Ä kta Purifier 10 system, driven by Unicorn 3.21 software (GE Healthcare). AS60 (5 × 1 mL) was loaded onto a

EXPERIMENTAL SECTION

Model Homogalacturonans. Three series of model homogalacturonans (B-, P-, and BP-series) were prepared, as fully described in Tanhatan-Nasseri et al.22 Briefly, demethylesterified citrus pectin was hydrolyzed by 0.1 M HCl at 80 °C for 72 h.34 The acid-insoluble fraction containing HGs was resuspended in distilled water, the pH of the suspension was brought to 7 by tetrabutyl ammonium hydroxide, and the resulting solution (HG0-TBA) was extensively dialyzed against distilled water and freeze-dried. Methylation of HG0-TBA was achieved with methyl iodide in dimethyl sulfoxide.35 The highly methylesterified HG (HG96) obtained was demethylesterified by 0.2 M NaOH or by plant-PME [EC 3.1.1.11] (Sigma P5400; 194 U/mg) 1616


Biomacromolecules

Article

Table 1. Summary of Pectin Lyase Purification fraction

activity

nkat total

activity yield (%)

peclyve

protein yield (%)

specific activity (nkat/mg)

purification rate

100 pectin lyase galactanase endo-PG

12921 1605 152000

100 100 100

pectin lyase galactanase endo-PG

1881 52 728

14.6 3.2 0.5


524 6 345

4.1 0.4 0.2


547 0 0

4.2 0 0

AS60 precipitate

446

1

528

1.2

639

1.4

3600

8.1

12.3

HIC excluded

2.8

AEC 0.3 M NaCl

0.5

Resource ether column (GE Healthcare, 0.86 mL) previously equilibrated with 50 mM Na-phosphate buffer containing 2 M (NH4)2SO4 pH 6. Elution was performed at a flow rate of 1 mL/min using the following gradient: 0−7 mL, 100% B; 7−25 mL, linear gradient 100−0% B; 25 mL, 100% A; with A being 50 mM Naphosphate buffer pH 6 and B being 50 mM Na-phosphate buffer pH 6 containing 2 M (NH4)2SO4. Fractions of 500 μL were collected. HICexcluded fractions were pooled, dialyzed overnight against 1 L of ultra pure water at 4 °C and then dialyzed for 24 h against 50 mM Naacetate buffer pH 5 at 4 °C. Anion exchange chromatography (AEC) was performed on an Ä kta Purifier 10 system driven by Unicorn 3.21 software (GE Healthcare). HIC excluded fractions (5 × 2 mL) were loaded onto a MonoQ column (GE Healthcare, 1 mL) previously equilibrated with 50 mM Na-acetate buffer pH 5. Elution was performed at a flow rate of 1 mL/min using the following gradient: 0− 6 mL, 100% A; 6−26 mL, linear gradient 100 to 50% A; 26−37 mL, linear gradient 50 to 0% A; with A being 50 mM Na-acetate buffer pH 5 and B being 50 mM Na-acetate buffer pH 5 containing 1 M NaCl. Fractions of 500 μL were collected. Fractions eluting at 0.3 M NaCl were pooled and dialyzed against 50 mM Na-acetate buffer pH 5. The different pools obtained along the purification process were analyzed for their pectin lyase, pectate lyase, endo-PG and galactanase activity and for their protein content using BSA as standards.39 To assess the activity of pectin and pectate lyase, raw lemon pectin (Grinsted 3450) and polygalacturonic acid were used as substrates, respectively. Activity was followed using 2.5 mg/mL of substrates in 50 mM Na-acetate buffer pH5 at 40 °C. The reaction was initiated by addition of the enzymatic fraction and the activity measured at 20 and 30 min. The rate of reaction was determined spectrophotometrically by measuring the rate of formation of double bond (CC) by its absorbance at 235 nm (ε = 5500 M−1 cm−1 as the molar absorption coefficient of CC at 235 nm).40 Galactanase and endo-PG activities were assessed in 50 mM Na-acetate buffer pH 5 on potato galactan (2.5 mg/mL) and polygalacturonic acid as substrates (2.5 mg/mL), respectively, by measuring the appearance of reducing ends.41 A purification summary is given in Table 1. Characterization and Quantification of Pectin-Lyase Digest Products. Preparative Low-Pressure Size-Exclusion Chromatography. Fifteen mg of HG samples were solubilized in 5 mL of 50 mM Na-acetate buffer pH5. Thirty μL of pectin lyase (57.6 nkat/mL) were added prior to incubation for 24 h at 40 °C. Digested samples were subsequently concentrated to approximately 1 mL under vacuum and desalted on a Sephadex G10 column (78 × 4.4 cm) eluted with distilled water at 90 mL/h. The elution was monitored using refractometric detection. The resultant desalted enzymatic digests were concentrated to less than 1 mL under vacuum at 40 °C and injected to a chromatographic system constituted of a Biogel P6-extra fine (92 × 5 cm) column and a Biogel-P4- fine (86 × 5 cm) column

mounted in series. Elution was performed using a 100 mM Na-acetate buffer pH 3.6 at a flow rate of 10 mL/h and collected fractions were analyzed for their GalA content.36 Appropriate fractions were pooled and either extensively dialyzed against distilled water (polymeric pools), or concentrated under vacuum at 40 °C to ∼3 mL, desalted on a Sephadex G10 column (78 × 4.4 cm) and concentrated to reach a final GalA concentration of ∼150 μg/mL. In order to prepare large amounts of unsaturated oligogalacturonate standards a HG-P64 pectin lyase digest was chromatographed as described above. Recovered dp-resolved-fractions were desalted and further purified on a Dionex system equipped with a semipreparative Carbopac PA-100 column (250 × 9 mm). The flow rate of the eluant was constant at 3 mL/min. The elution was performed using a linear gradient of 250 mM to 500 mM Na-acetate in 100 mM NaOH (0−20 min). The column was then re-equilibrated with the starting buffer for 30 min. Pulsed amperometry was used for detection. Recovered fractions were neutralized by 100 mM HCl, desalted on a Sephadex G10 column (78 × 4.4 cm) and analyzed for their GalA content.36 High-Performance Anion-Exchange Chromatography. Two mg of the HG sample to be analyzed was solubilized in 2 mL of 50 mM Na-acetate buffer pH 5. Twelve μL of pectin lyase (57.6 nkat/mL) was added and the sample incubated for 24 h at 40 °C. Completed digests were filtered (0.45 μm Millipore) and 20 μL were injected on HPAEC. A Waters system equipped with an analytical Carbopac PA1 column (250 × 2 mm) with pulsed amperometric detection was used. Flow rate was kept constant at 0.25 mL/min. The elution was carried out with four linear gradient phases of Na-acetate in 100 mM NaOH as described in Tanhatan-Nasseri et al.22 The mobile phases were all degassed with helium in order to prevent absorption of carbon dioxide and transformation to carbonate. The column was thermostatted at 20 °C. Chromeleon Software (Dionex) was used for collecting and processing the data. The unsaturated oligogalacturonate standards, whose preparation is described above, were individually injected in order to calculate the response factors for each degree of polymerization that were used to quantify unsaturated oligogalacturonates in HG digests. Analyses were performed in triplicate. Capillary Electrophoresis. Two mg of the HG sample to be analyzed were solubilized in 2 mL of 50 mM Na-acetate buffer pH5. Twelve μL of pectin lyase (57.6 nkat/mL) was added and the sample incubated for 24 h at 40 °C. Completed digests were filtered (0.45 μm Millipore) and freeze-dried. They were subsequently rehydrated in 100 μL of Milli-Q water and run in capillary electrophoresis (CE). Experiments carried out in this work used an automated CE system (HP 3D), equipped with a diode array detector. Electrophoresis was carried out in a fused silica capillary of internal diameter 50 μm and a total length of 46.5 cm (40 cm from inlet to detector). The capillary incorporated an extended light-path detection window (150 μm) and was thermostatically controlled at 25 °C. Phosphate buffer at pH 7.0 1617


Biomacromolecules

Article

Figure 2. Size-exclusion chromatography elution patterns of pectin lyase-digested homogalacturonans. A. Base-demethylesterified homogalacturonan of DM 40 (HG96-B40); B. Base-demethylesterified homogalacturonan of DM 69 (HG96-B69); C. Plant-PME-demethylesterified homogalacturonan of DM 36 (HG96-P36); D. Plant-PME-demethylesterified homogalacturonan of DM 64 (HG96-P64). The different methylesterified unsaturated oligogalacturonates (dpDM) present in each SEC fraction are shown in Table 2. dp1

was used as a CE background electrolyte (BGE) and was prepared by mixing 0.2 M Na2HPO4 and 0.2 M NaH2PO4 in appropriate ratios and subsequently reducing the ionic strength to that desired (typically 50 or 90 mM). Detection was carried out using UV absorbance at 235 nm (reporting on the presence of the unsaturated double bond left at the nonreducing end of all pectin-lyase generated fragments). Samples were loaded hydrodynamically (various injection times at 5000 Pa, typically giving injection volumes of the order of 10 nL), and typically electrophoresed across a potential difference of 25 kV. All experiments were carried out at normal polarity (inlet anodic) unless otherwise stated. Mass Spectrometry. Mass spectrometry studies were carried out on the dp resolved fractions separated from the pectin-lyase digests in order to obtain more information on the component oligogalacturonides. 2,5-Dihydroxybenzoic acid (DHB) was purchased from SigmaAldrich Co. (Saint Quentin Fallavier, F). N,N-Dimethylaniline (DMA) was purchased from Fisher Scientific (Fair Lawn, NJ, U.S.A.). Milli-Q water (Millipore, Bedford, MA, U.S.A.) was used in preparation of all solutions. All chemical reagents used were HPLC grade. DMA/DHB was prepared by dissolving 100 mg of DHB in 1 mL of H2O/ acetonitrile/DMA (49/49/2). One μL of sample was directly mixed with 1 μL of matrix solution on a polished steel target plate. Acquisition was performed on an Autoflex III MALDI TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, D) equipped with a Smartbeam laser (355 nm) in positive ion mode with a reflector. Laser power was adapted for each sample. Mass spectra were automatically processed by FlexAnalysis software (Bruker Daltonics, Bremen, D). Characterization and Quantification of endo-PG Digest Products by High-Performance Anion-Exchange Chromatography. Two mg of samples were solubilized in 2 mL of 50 mM Naacetate buffer pH4. Twelve μL of endo-PG (35.5 nkat/mL) were added prior to incubation for 72 h at 40 °C, 6 μL of fresh enzyme being added at t = 24 h and 48 h. Hydrolyzates were filtered (0.45 μm Millipore) and analyzed by HPAEC as described above. Analyses were performed in triplicate. Monomer, dimer and trimer of GalA were used as standards. The degree of blockiness (DB) and absolute degree of blockiness (DBabs) were calculated as follows:

DB =

∑dp3 [oligoGalA saturated] [GalA unmethylesterified]

× 100

dp1

DBabs =

■

∑dp3 [oligoGalA saturated] [GalA total ]

× 100

RESULTS AND DISCUSSION Model HGs. HGs were chemically and/or enzymatically tailored with the aim of controlling the distribution of methylester groups, as described in the Experimental Section (Figure 1) and elsewhere.22 Randomly demethylesterified HGs (B-series) were obtained in the classical manner, by alkali demethylesterification of a very highly methylesterified “mother” HG (HG96). Manipulating the initial DM of randomly methylesterified “mother” HGs prior to enzymatic blockwise-demethylesterification by plant-PME allowed the recovery of two types of blocky HG. Samples containing either a few long demethylesterified blocks of GalAs (P-series) were generated directly from the HG96 mother sample, or alternatively HGs with more numerous but shorter blocks of demethylesterified GalA residues (BP-series) were produced from “mother” HGs of lower DM (HG96-B ≈ 80). These lower DM starting substrates offered the processive PME both a larger number of start points (epitopes required for an initial successful binding) and a greater chance, once blockwise demethylesterification was underway, of encountering already nonmethylesterified residues that increase the chances of detaching from the chain. HGs of moderate to high DM exhibiting subtle, yet potentially functionally important differences in methylesterification patterns, such as “slightly blocky” versus random, have previously proved difficult to differentiate from each other on the basis of their DB and DBabs values, as have HGs of low DM (95

42, 43, 44 52, 53, 54, 55 63, 64, 65, 66 74, 75, 76, 84, 85, 86, 95, 96, 97, 105, 106, 107 116, 117, 118, 126, 127, 128, 136, 137, 138, 139, 147, 148, 149, 157, 158, 159, 1510, 168, 169, 1610, 1611 nd 31, 32, 33

B C D E F A

5 11 7 95 >95 >95 nd 8 >95

42, 53, 64, 75, nd 31,

43, 54, 65, 76,

B C D E F G

13 15 13 6 1 43

>95 >95 >95 nd nd 8

42, 53, 64, 75, nd nd

43, 54, 65, 76,

2

44 55 66 77

32, 33 44 55 66 77

nd not determined. bbase peak for each dp appears in bold.

methylesterified GalAs contained within the fine structure of the starting substrate. Fine Structure of the dp-Resolved Digest Fragments. The oligomeric fractions collected from SEC, that had been fractionated purely on the grounds of dp, were further analyzed in order to obtain more information on distribution of their methylesterification within the samples. Mass Spectrometry. MALDI-TOF-MS was applied in positive ion mode and the dp and DM of GalA oligomers present in the different SEC fractions was thereby assessed simultaneously (Table 2). The MS spectrum of the HG96-B69 fraction C (Figure 3A) is fully described for clear illustration of the observed ions. This MS spectrum was characterized by the predominance of sodium adducts representative of unsaturated GalA oligomers of dp5 bearing 2 to 5 methyl groups (m/z 931, 945, 959, and 973). Sodiation of the carboxylic functions of predominant ions (m/z 945 and 959) was observed (m/z 967, 981 for single sodiation of 53 and 54, respectively, and m/z 989 for double sodiation of 53). The major species (54, m/z 959) existed also in the saturated form (m/z 977). This ion relates to oligomers arising from the nonreducing end of HG molecules, thereby bearing no unsaturation.32 Mass spectra of the different SEC fractions were similarly interpreted and results obtained are summarized in Table 2. Although the same digest-derived oligomers of dp 3 to 6 were observed for HG96-B69, HG96-P36 and HG96-P64, their relative intensities differed widely. The relative amounts of the different oligomers present in one fraction cannot be assessed by the intensity of the peaks since ionization rates may vary 1619


Biomacromolecules

Article

Figure 3. MALDI-TOF mass spectra (positive ion mode) of selected pectin lyase-digested homogalacturonans SEC fractions. A. Fraction C from base-demethylesterified homogalacturonan of DM 69 (HG96-B69 C); B. Fraction C from plant-PME-demethylesterified homogalacturonan of DM 36 (HG96-P36 C).

nn‑4 to nn‑8 (dpDM), respectively (Table 2). Oligomers of dp >8 were absent in HG96-P36 and HG96-P64 pectin lyase digests. Capillary Electrophoresis. While completely methylesterified digest fragments do not carry a charge, and as such will not be resolved by electrophoretic techniques, capillary electrophoresis (CE) has been applied to the study of these lyase digests in order to investigate its possible utility in revealing the identity and quantity of the different partially methylesterified species present. Figure 4 shows the electropherograms obtained from the pectin-lyase digests of the starting HG96 substrate, and samples from the (A) P- and (B) B- series. (Note that the digest of the HG96-P75 sample is also included in (B) in order to facilitate a comparison). This is the first time such lyasegenerated digests have been examined by CE and in order to help with the identification of the peaks obtained in the electropherograms, the dp-resolved digest fractions generated by SEC were used (dp 3−6). The putative assignment of all CE

depending on chemical properties of each species. Relative intensities between HG fractions of identical dp can however be compared. This approach was used for the different fractions using the fully methylesterified oligomer nn (dpDM) as internal standard. This is illustrated in Figure 3 for HG96-B69 and HG96-P36 fractions C, which both consist of GalA oligomers of dp 5. Altogether, singly and doubly demethylated oligomers (nn‑1 and nn‑2, respectively) of dp 3 to 6 appeared to be much more frequent in HG96-B69 than in HG96-P36 and HG96P64, in agreement with the overall DM values calculated for the corresponding SEC fractions. Beside oligomers of low dp, the HG96-B69 pectin lyase digest also contained high amounts of oligomers of higher dp, recovered by the SEC experiments as fractions E (dp 7−10) and F (dp 11−16) (Table 2). For these fractions the base peaks consisted of oligomers corresponding to nn‑2 and nn‑3 and to 1620


Biomacromolecules

Article

smaller amount of fragments released from substrates compared with the P-series polymers of the same DM, more species types liberated, and a greater dependence of the speciation on the initial substrate DM. CE clearly offers the potential of quantifying the different partially methylesterified species, providing extra information that might be useful in the more detailed modeling of the enzyme action and this is being pursued in further work. Figure 5A shows the results obtained from the lyase digests of the BP-series compared with that obtained from the mother

Figure 4. Electropherograms obtained (235 nm) from the pectin-lyase digests of the starting HG96 substrate, and samples from the (A) Pand (B) B- series. Based on the investigations involving fractions separated from various complete digests on the grounds of dp, the identity of several key unsaturated species is suggested. The six peaks marked in (A), left to right are proposed to be (i) completely methylated oligomers of any dp; (ii) 65; (iii) 54; (iv) 43; (v) 32; and (vi) 53; and in (B) the three additional peaks, again left to right (i) 64; (ii) 53; 42; (iii) 31.

peaks observed in digests was complex and involved both observing the electrophoretic consequences of further processing of these dp-resolved fractions using a f ungal-PME and an alkali treatment, and running samples against other saturated and unsaturated standards, and will be reported in detail elsewhere (Manuscript in Preparation). The work reported here however clearly shows the potential of the technique for the investigation of the partially methylesterified unsaturated fragments released by the lyase digests. As found in the SEC experiments the results obtained from the P-series substrates show predominantly the same peaks present in all digests (specifically the first six peaks present in both series, assigned in the figure captions) with the amount of these highly methylesterified species reducing as the DM of the substrate is decreased. Although comparisons of the amounts of different species present and their absolute quantitation requires accounting for differential absorption coefficients, the measurement of which is being pursued, the relative amounts of the same species present in different digests can be directly inferred from the comparison of peak areas. All completely methylesterified species can be observed migrating with the electroosmotic flow regardless of dp while the partially methylesterified species can be resolved in this technique. The speciation of these fragments is also seen to be consistent with the results obtained from MS analysis and the average DM of the SEC fractions, with the singly demethylesterified species overwhelmingly dominant and a smaller amount of doubly demethylesterified oligomers detectable. The B series results are also consistent with the previous results showing an overall

Figure 5. A. Electropherograms obtained (235 nm) from the lyase digests of the BP-series compared with that obtained from the mother sample. B. Resulting digest patterns from three substrates with very similar DM values, but that have been generated from the starting HG96 in quite different ways, one directly with plant-PME, one with alkali treatment and one with limited alkali treatment followed by plant-PME.

sample. The effect of a further round of demethylesterification, this time carried out using the processive PME are clearly seen. While further reducing the still relatively high starting DM (albeit randomly distributed) yields an overall reduction in the amount of low DM digest fragments liberated, the relative amount of the different species clearly becomes closer to that found in the P-series digests, as opposed to those of the Bseries, as more blocky stretches are introduced prior to lyase treatment. Figure 5B shows the resulting digest pattern from three substrates with similar DM values, but that have been generated from the starting HG96 in quite different ways, one directly with plant-PME, one with alkali treatment and one with limited alkali treatment followed by plant-PME. As expected the P-series sample shows the smallest number of distinct species and the largest amount of low-dp material liberated by pectin lyase. The B- and BP- series both show more complex digest patterns but clearly with the B- series producing less overall fragments, again as expected, and also a decrease in the amount of completely methylesterified species released compared with those with one or two methylester groups removed. 1621


Biomacromolecules

Article

Quantification of the dp-Resolved Digest Fragments. HG model substrates from B-, P-, and BP-series were degraded by pectin lyase as described and, in addition to performing preparative SEC, the digests were analyzed by high-performance anion-exchange chromatography in order to quantify the generated oligogalacturonides of different dps. As previously pointed out,32,33,42 not only the DM of the pectic substrates but also the distribution pattern of methylesters influences the activity of pectin lyases. Although the enzyme accepts partially methylesterified substrates,32,33 tolerance for carboxyl groups is rather limited and the products of dp 3−8, which have been characterized here, can be considered as faithful representatives of the highly methylesterified stretches within the HG molecule. After pectin lyase digestion of an ideal substrate (HG96), unsaturated GalA oligomers with dp 3−7 were recovered as major products with unsaturated GalA oligomers of dp2 and dp8 as very minor ones. The overall recovery (mg of recovered unsaturated GalA oligomers/100 mg of GalA initially present in the HG sample) was >92. Saturated GalA oligomers of dp 4 and 5, representing altogether around 5 mg/100 mg of GalA initially present, were also detected. As previously evidenced for HG96-P36 (Figure 3B), those saturated GalA oligomers appeared totally methylesterified by MALDI-TOF-MS analysis and correspond to HG nonreducing ends. The overall recovery of GalA oligomers was >97%, which demonstrates the validity of the response factors used. Degree of Blockiness and Absolute Degree of Blockiness of Highly Methylesterified Stretches (DBMe and DBabsMe). Several parameters have been introduced to characterize the presence or absence of nonmethylesterified blocks among which the degree of blockiness (DB)8 and the absolute degree of blockiness (DBabs)27 are the most widely used. Similarly, in this work, a degree of blockiness and an absolute degree of blockiness of the highly methylesterified stretches (DBMe and DBabsMe, respectively) have been calculated based upon the results of the pectin lyase digestion

Figure 6. Degrees of blockiness as a function of degree of methylesterification (DM) for the three series of homogalacturonans. A. Absolute degree of blockiness of methylesterified zones (DBabsMe); B. Degree of blockiness of methylesterified zones (DBMe); C. Absolute degree of blockiness of nonmethylesterified zones (DBabs) (values from ref 22); D. Degree of blockiness of nonmethylesterified zones (DB) (values from ref 22). Black circle, B-series; white circle, Pseries; black inverted triangle, BP-series. Average of three independent measurements.

HGs from the B-series, obtained by alkaline demethylesterification of the very highly methylesterified HG96, behaved totally differently with a steep decrease in DBabsMe for decreasing DM (Figure 6A). The following linear relationship was established for highly methylesterified substrates with DM values in the range 65−100

dp2

DBMe =

∑dp8 [oligoGalA unsaturated]

DBabsMe =

[GalA methylesterified] dp2 ∑dp8

× 100

[oligoGalA unsaturated] [GalA total]

DBabsMe = (2.44 × DM) − 141.87(R2 > 0.99)

but for DM < ∼45, HGs from the B-series were not substrates for pectin lyase anymore, in good agreement with Mutenda et al.’s findings.33 When methylester groups are randomly distributed, the deleterious effect of the introduced nonmethylesterified GalA units is high since stretches of ideal highly methylesterified substrate become less and less available as the DM decreases.32 In the DM range 65−100, pectin lyase activity was 2.5 times more affected by the DM decrease for a random distribution of methylester groups than for a fully blockwise one. HGs from the BP-series, obtained by plant-PME demethylesterification of HG96-B∼80, exhibited an overall behavior close to that of HGs from the P-series with the following linear relationship describing that data well for the limited DM range studied (48−63) (Figure 6A).

× 100

As illustrated in Figure 6A, HGs from the P-series, obtained by plant-PME demethylesterification of the very highly methylesterified HG96, are close to ideal substrates for pectin lyase with the linear relationship between DBabsMe and DM DBabsMe = (0.98 × DM) − 5.05(R2 = 0.98)

illustrating that essentially all of the methylester groups in these substrates inhabit methylesterified stretches that enable them to be released in low-dp unsaturated digest products. Since HGs from the P-series were also found, as expected, to be close to ideal substrates for endo-PGII (Figure 6C), it can be concluded that HGs from the P-series are constituted of a few long methylesterified stretches (highly degradable by pectin lyase) alternating with a few long unmethylesterified ones (highly degradable by endo-PGII), with the respective length of each of these blocks depending on the DM. This is further confirmed by the fact that their DB and DBMe values are high and do not depend largely upon DM (Figure 6, panels B and D).

DBabsMe = DM − 20

It can be seen that the plant-PME demethylesterification mode applied to the parent HG from the BP-series samples (HG96-B ≈ 80) preserved the existing highly methylesterified segments much more than a random demethylesterification 1622


Biomacromolecules

Article

would have. HGs from the BP-series however contain only relatively short nonmethylesterified stretches, as evidenced by their limited degradation by endo-PGII (Figure 6, panels B and D). These are interspersed with long highly methylesterified stretches, as evidenced by their significant degradation by pectin lyase (Figure 6A and Figure 6C). Products obtained after degradation by endo-PGII (dp 1−3) and pectin lyase (dp 2−8) can be considered as mirror images of each other, with endo-PGII degradation products reporting on the blockiness of lowly methylesterified zones of the molecule, and pectin lyase degradation products on the highly methylesterified ones. It seems reasonable to suggest then that the total proportion of zones degradable by endo-PGII and by pectin lyase will be highly sensitive to the intramolecular pattern of DM and may discriminate between pectin samples hitherto difficult to distinguish, such as the P- and BP- series investigated herein. Indeed, the total proportion of degradable zones appears radically different depending on the methylester distribution (Figure 7).

DBabs + DBabsMe = 99.66 − (3.41 × DM) + (0.035 × DM2)(R2 > 0.99)

With the results from the P- and B- series providing confidence in the methodology the results from the HGs originating from the BP-series were plotted in the same manner. It is clear that indeed these substrates exhibit an intermediate behavior and in contrast from previously suggested discriminators monitoring the sum of DBabs and DBabsMe could clearly differentiate these HGs from the two other series studied.

■

CONCLUSION HGs with engineered distributions of methylester groups were prepared using chemical and enzymatic means.22 As shown schematically in Figure 8, three main series were obtained with

Figure 7. Sum of absolute degree of blockiness of methylesterified zones and absolute degree of blockiness of nonmethylesterified zones (DBabsMe + DBabs) as a function of degree of methylesterification (DM) for the three series of homogalacturonans. Black circle, B-series; white circle, P-series; black inverted triangle, BP-series. Average of three independent measurements.

The following linear relationship between the total proportion of degradable zones and DM could be established for the data from the digests of the P-series substrates DBabs + DBabsMe = (0.35 × DM) + 59.88(R2 = 0.85)

demonstrating that a high proportion of HG molecules in the P-series substrates were degradable either by endo-PGII, or by pectin lyase, but with this proportion decreasing slowly with decreasing DM. This decrease with DM simply reflects the fact that the amount of dp 2−8 unsaturated oligomers released for a completely methylesterified HG and the amount of dp 1−3 saturated oligomers released from a totally nonmethylesterified substrate of the same length are not the same. In contrast, HGs from the B-series encompassed a low total proportion of degradable zones, with a mimimum when the DM was close to 50, as expected. Above that DM the amount of lyase-generated fragments increases and the total rises; below that, the amount of endo-PG digest products increases and again the total rises. The following polynomial relationship was found to reasonably capture this dependence:

Figure 8. Schematic representation of methylesterified GalA (white circle) and nonmethylesterified GalA (black circle) distribution for the three series of homogalacturonans and the respective “mother” homogalacturonans they arise from.

respect to their methylester distributions: (i) a random (Bseries), (ii) chains containing long methylesterified stretches interspersed with long unmethylesterified ones (P-series), and (iii) HGs with long methylesterified stretches interspersed with short unmethylesterified ones (BP-series). The amounts of endo-PGII degradation products (dp 1−3) were similar for random or slightly blocky HGs of DM > 55, and so were the amounts of pectin lyase degradation products (dp 2−8) 1623


Biomacromolecules

Article

(22) Tanhatan-Nasseri, A.; Crépeau, M.-J.; Thibault, J.-F.; Ralet, M.C. Carbohydr. Polym. 2011, 86, 1236−1243. (23) Williams, M. A. K.; Vincent, R. R.; Pinder, D. N.; Hemar, Y. J. Non-Newtonian Fluid Mech. 2008, 149, 63−70. (24) Vincent, R. R.; Cucheval, A.; Hemar, Y.; Williams, M. A. K. Eur. Phys. J. E. 2009, 28, 79−87. (25) Vincent, R. R.; Williams, M. A. K. Carbohydr. Res. 2009, 344, 1863−1871. (26) Schuster, E.; Cucheval, A.; Lundin, L.; Williams, M. A. K. Biomacromolecules 2011, 12, 2583−2590. (27) Guillotin, S. E.; Bakx, E. J.; Boulenguer, P.; Mazoyer, J.; Schols, H. A.; Voragen, A. G. J. Carbohydr. Polym. 2005, 60, 391−398. (28) Dass, P. J. H.; Voragen, A. G. J.; Schols, H. A. Carbohydr. Res. 2000, 326, 120−129. (29) Fraeye, I.; Doungla, E.; Duvetter, T.; Moldenaers, P.; Van Loey, A.; Hendrickx, M. Food Hydrocolloids 2009, 23, 2069−2077. (30) Ngouémazong, D. E.; Tengweh, F. F.; Duvetter, T.; Fraeye, I.; Van Loey, A.; Moldenaers, P.; Hendrickx, M. Food Hydrocolloids 2011, 25, 434−443. (31) Daas, P. J. H.; Voragen, A. G. J.; Schols, H. A. Biopolymers 2001, 58, 195−203. (32) van Alebeek, G.-J. W. M.; Christensen, T. M. I. E.; Schols, H. A.; Mikkelsen, J. D.; Voragen, A. G. J. J. Biol. Chem. 2002, 277, 25929− 25936. (33) Mutenda, K. E.; Körner, R.; Christensen, T. M. I. E.; Mikkelsen, J. D.; Roepstorff, P. Carbohydr. Res. 2002, 337, 1217−1227. (34) Thibault, J.-F.; Renard, C. M. G. C.; Axelos, M. A. V.; Roger, P.; Crépeau, M.-J. Carbohydr. Res. 1993, 238, 271−286. (35) Renard, C. M. G. C.; Jarvis, M. C. Carbohydr. Polym. 1999, 39, 201−207. (36) Thibault, J.-F. Lebensm-Wiss.−Technol. 1979, 12, 247−251. (37) Anthon, G. E.; Barrett, D. M. J. Agric. Food. Chem. 2004, 52, 3749−3753. (38) Bonnin, E.; Le Goff, A.; Körner, R.; Vigouroux, J.; Roepstorff, P.; Thibault, J.-F. Biochim. Biophys. Acta 2002, 1596, 83−94. (39) Bradford, M. M. Anal. Biochem. 1976, 72, 248−254. (40) Voragen, A. G. J.; Rombouts, F. M.; Pilnik, W. Lebensm-Wiss.− Technol. 1971, 4, 126−128. (41) Nelson, N. J. Biol. Chem. 1944, 153, 375−380. (42) Voragen, A. G. J. Ph.D. thesis; Wageningen, The Netherlands, 1972.

obtained from slightly blocky and highly blocky HGs of DM < 55. However, combining the two enzymatic approaches and thereby quantifying the total proportion of degradable zones, by endo-PGII and by pectin lyase, allowed clear differentiation between the three HG series (Figure 8). This double enzymatic approach will be useful not only for discriminating industrial pectin samples exhibiting different functionalities21 but also for obtaining further insights into pectin fine structure dynamics in vivo either at different developmental stages or following specific mutations.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We thank Jacqueline Vigouroux and Marie-Jeanne Crépeau for their excellent technical assistance. REFERENCES

(1) Ridley, B. L.; O’Neill, M. A.; Mohnen, D. Phytochemistry 2001, 57, 929−967. (2) Willats, W. G. T.; McCartney, L.; Mackie, W.; Knox, J. P. Plant Mol. Biol. 2001, 47, 9−27. (3) Willats, W. G. T.; Knox, J. P.; Mikkelsen, J. D. Trends Food Sci. Technol. 2006, 17, 97−104. (4) Mohnen, D. Curr. Opin. Plant Biol. 2008, 11, 266−277. (5) Willats, W. G. T.; Orfila, C.; Limberg, G.; Buchholt, H. C.; van Alebeek, G. J. W. M.; Voragen, A. G. J.; Marcus, S. E.; Christensen, T. M. I. E.; Mikkelsen, J. D.; Murray, B. S.; Knox, J. P. J. Biol. Chem. 2001, 76, 19404−19413. (6) Kohn, R.; Markovic, O.; Machova, E. Collect. Czech. Chem. Commun. 1983, 48, 790−797. (7) Thibault, J.-F.; Rinaudo, M. Biopolymers 1985, 24, 2131−2143. (8) Daas, P. J. H.; Meyer-Hansen, K.; Schols, H. A.; De Ruiter, G. A.; Voragen, A. G. J. Carbohydr. Res. 1999, 326, 120−129. (9) Limberg, G.; Körner, R.; Buchholt, H. C.; Christensen, T. M. I. E.; Roepstorff, P.; Mikkelsen, J. D. Carbohydr. Res. 2000, 327, 293− 307. (10) Denès, J.-M.; Baron, A.; Renard, C. M. G. C.; Péan, C.; Drilleau, J.-F. Carbohydr. Res. 2000, 327, 385−393. (11) Ralet, M.-C.; Dronnet, V.; Buchholt, H. C.; Thibault, J.-F. Carbohydr. Res. 2001, 336, 117−125. (12) Savary, B. J.; Hotchkiss, A. T.; Cameron, R. G. J. Agric. Food Chem. 2002, 50, 3553−3558. (13) Cameron, R. G.; Luzio, G. A.; Goodner, K.; Williams, M. A. K. Carbohydr. Polym. 2008, 71, 287−299. (14) Sajjaanatakul, T.; Pitifer, L. A. In The Chemistry and Technology of Pectin; Walter, R. H., Ed.; Academic Press: New York, 1991; pp 135−156. (15) Fries, M.; Ihrig, J.; Brocklehurst, K.; Shevchik, V. E.; Pickersgill, R. W. EMBO J. 2007, 26, 3879−3887. (16) Benen, J. A. E.; Kester, H. C. M.; Visser, J. Eur. J. Biochem. 1999, 259, 577−585. (17) Chen, E. M. W.; Mort, A. J. Carbohydr. Polym. 1996, 29, 129− 136. (18) Luzio, G. A.; Cameron, R. G. Carbohydr. Polym. 2008, 71, 300− 309. (19) Löfgren, C.; Guillotin, S.; Evenbratt, H.; Schols, H. A.; Hermansson, A.-M. Biomacromolecules 2005, 6, 646−652. (20) Slavov, A.; Garnier, C.; Crépeau, M.-J.; Durand, S.; Thibault, J.F.; Bonnin, E. Carbohydr. Polym. 2009, 77, 876−884. (21) Ström, A.; Ribelles, P.; Lundin, L.; Norton, I.; Morris, E. R.; Williams, M. A. K. Biomacromolecules 2007, 8, 2668−2674. 1624


Innovative Enzymatic Approach to Resolve Homogalacturonans

Recommend Documents