Preparing rhamnogalacturonan II domains from seven plant pectins using Penicillium oxalicum degradation and their structural comparison

Accepted Manuscript Title: Preparing rhamnogalacturonan II domains from seven plant pectins using Penicillium oxalicum degradation and their structural comparison Authors: Di Wu, Liangnan Cui, Guang Yang, Xing Ning, Lin Sun, Yifa Zhou PII: DOI: Reference:

S0144-8617(17)31183-9 https://doi.org/10.1016/j.carbpol.2017.10.037 CARP 12886

To appear in: Received date: Revised date: Accepted date:

8-4-2017 28-9-2017 9-10-2017

Please cite this article as: Wu, Di., Cui, Liangnan., Yang, Guang., Ning, Xing., Sun, Lin., & Zhou, Yifa., Preparing rhamnogalacturonan II domains from seven plant pectins using Penicillium oxalicum degradation and their structural comparison.Carbohydrate Polymers https://doi.org/10.1016/j.carbpol.2017.10.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Preparing rhamnogalacturonan II domains from seven plant pectins using Penicillium oxalicum degradation and their structural comparison

Di Wu, Liangnan Cui, Guang Yang, Xing Ning, Lin Sun*, Yifa Zhou*

Jilin Province Key Laboratory for Chemistry and Biology of Natural Drugs in Changbai Mountain, School of Life Sciences, Northeast Normal University, Changchun 130024, PR China *Corresponding author. Tel/Fax: +86 431 85098212; E-mail addresses: [email protected]; [email protected].

Highlight 

Penicillium oxalicum could degrade pectin to prepare RG-II domains.



RG-II domains from 7 kinds of plants were prepared and analyzed.



Side chain B of RG-II domain with DP 10 was discovered in prepared RG-II domains.

1

Abstract

Rhamnogalacturonan II (RG-II) is a complex pectin with diverse pharmaceutical activities. To assess how RG-II functions, the development of methods for its preparation is required. In this paper, pectin from Codonopsis pilosula was used to evaluate the ability of fungi and bacteria to degrade the pectin. We discovered that the fungus Penicillium oxalicum could efficiently lead to the recovery of RG-II domains by degrading the other pectic domains. Further, six pectin fractions from different medical plants were used as the sole carbon source for the growth of Penicillium oxalicum. The major polymeric products remaining after fungus degradation was RG-II domains. Depending of plant source, side chains A differed with respect to their proportion of L-Gal and L-Fuc and to their degree of methyletherification. Side chains B were made of 8 to 10 sugar residues and up to 2 acetyl groups. Overall, our method provides an effective way to prepare RG-II pectin domains for investigating their structure-function relationships.

Key words: Pectin; Rhamnogalacturonan II; Penicillium oxalicum; Degradation.

2

1. Introduction

Rhamnogalacturonan II (RG-II), a pectin domain found in the cell wall of plants, has numerous functions. During evolution, the amount of RG-II increased in vascular plants (Matsunaga et al., 2004). In Chenopodium album, RG-II has been shown to be related to the pore size in the cell wall (Fleischer, O'Neill & Ehwald, 1999). In Arabidopsis thaliana mutants mur1, a replacement of L-Fuc by L-Gal leads to reduced amounts of the RG-II dimer and a dwarfed morphology (O'Neill, Eberhard, Albersheim & Darvill, 2001) or a decrease in cellular attachment (Iwai, Masaoka, Ishii & Satoh, 2002). RG-II cross-linking may influence plant growth by modulating the porosity and mechanical properties of primary cell walls (Seveno et al., 2009). This is also correlated with pollen tube elongation and contributes to the formation of the primary cell wall (Pabst et al., 2013). In addition, RG-II exhibits diverse bioactivities, such as anti-metastatic activity (Park, Park, Hong, Suh & Shin, 2016), reduction in lead absorption (Tahiri et al., 2000), modulation of the intestinal immune system (Yu, Kiyohara, Matsumoto, Yang & Yamada, 2001), and enhancement of macrophage Fc receptor expression (Shin, Kiyohara, Matsumoto & Yamada, 1997). Therefore, RG-II pectin has potential applications in the food and drug industries. The highly conserved RG-II structure is composed of 12 different monosaccharides, including rhamnose (L-Rha), galacturonic acid (D-GalA), glucuronic acid (D-GlcA), galactose (L-Gal), arabinose (L-Ara), apiose (D-Api), fucose (L-Fuc), 2-Me-xylose (2-Me-D-Xyl), 2-Me-fucose (2-Me-L-Fuc), aceric acid

3

(L-AceA),

3-deoxy-D-lyxo-2-heptulosaric

acid

(D-Dha),

and

3-deoxy-D-manno-2-octulosonic acid (D-Kdo). RG-II has a homogalacturonan (HG) backbone with the degree of polymerization (DP) ranging from 8 to 12 sugar residues. The backbone was substituted by five well-defined side chains (i.e. A, B, C, D, E) (Buffetto et al., 2014). A recent study which uncovered the metabolic mechanism of how RG-II is used by the human gut bacterium Bacteroides thetaiotaomicron has revised the structure of RG-II and found a new side chain F (Ndeh et al., 2017). Among these side chains, chain E and F contains only one L-Ara, chain D contains D-Dha and L-Ara, chain C contains D-Kdo and L-Rha residues, chain B contains 6 to 9 sugar residues, and chain A contains 8 sugar residues in which L-Fuc can be replaced by L-Gal. Methyl-etherification and methyl-esterification can be found in side chain A, and acetylation can be found in side chain B. In the plant cell wall, RG-II is covalently attached to HG (Caffall & Mohnen, 2009; Ishii & Matsunaga, 2001). The functions and activities of RG-II are related to their structures. Regardless of how RG-II functions in plant growth or in our body, one first needs to prepare it. Around 70 RG-II fractions from different plants have been reported, and these were prepared by different methods. Some use pectinase alone or in combination with e.g. pectin lyase, Endo-PG, Exo-PG, Pectinex, Ultra SPL, pectinase SS, pectin methylesterase, or rapidase (Chormova, Messenger & Fry, 2014; Hilz, Williams, Doco, Schols & Voragen, 2006; Ishii & Matsunaga, 2001). However, using these approaches, the products are usually a mixture of RG-I, RG-II and oligogalacturonides. In this study, we report the use of Penicillium oxalicum to 4

degrade pectin molecules and produce RG-II domains as major polysaccharides.

2. Materials and Methods

2.1. Materials and microorganisms

Chromatographic

materials

Sephadex

G-75,

Sephadex

G-50

and

DEAE-Sepharose Fast Flow were purchased from GE Healthcare (USA). DEAE-Cellulose (7.5 × 20 cm, Cl-) was provided by Shanghai Chemical Reagent Research Institute (Shanghai, China). All other reagents were of analytical or HPLC grade. Strains from fifteen microorganism were isolated from Changbai Mountain and provided by the Jilin Academy of Agricultural Sciences. Series of plant pectins were isolated from Codonopsis pilosula, Panax ginseng, Veratrum nigrum, Viscum coloratum, Apocynum venetum, Helianthus annuus, Scrophularia ningpoensis by hot water extraction and DEAE-Cellulose fractionation according to the procedure established previously (Zhang et al., 2009). In brief, plant materials were extracted with water at 100oC for 4 h and the solid material was extracted twice again under the same conditions. The extracts were precipitated by 75% ethanol to obtain water-soluble

polysaccharide. The polysaccharide was

then

loaded on a

DEAE-Cellulose column (7.5 × 20 cm, Cl-) pre-equilibrated with distilled water. Neutral polysaccharide was eluted with distilled water, and pectin was eluted by 0.5 M NaCl. 5

2.2. General methods Total carbohydrate content was determined by using the phenol-sulphuric acid method (Dubois, Gilles, Hamilton, Rebers & Smith, 1956). Uronic acid content was determined by using the m-hydroxydiphenyl method (Blumenkrantz & Asboe-Hansen, 1973), with galacturonic acid as the standard. Kdo and Dha were colorimetrically determined using the modified thiobarbituric acid (TBA) method (York, Darvill, McNeil & Albersheim, 1985). Gel-permeation and anion-exchange chromatographies were monitored by assaying the total sugar and uronic acid content.

2.3. Homogeneity and molecular weight determination

Homogeneity and molecular weight were estimated by HPGPC on a TSK-gel G-3000PWXL column (7.8×300 mm, TOSOH, Japan) coupled to a Shimadzu HPLC system (Zhang et al., 2009). The column was pre-calibrated by using standard dextrans (50 KDa, 25 KDa, 12 KDa, 5 KDa and 1 KDa).

2.4. Sugar composition analysis by HPLC

Polysaccharide samples (2 mg) were first methanolysed with anhydrous methanol containing 2 M HCl at 80oC for 16 h and then with 2 M TFA at 120oC for 1 h. The resulting hydrolysates were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) according to the literature and analyzed on a DIKMA Inertsil ODS-3 column (4.6 × 150 mm) connected to a Shimadzu HPLC system (LC-10ATvp pump and SPD-10AVD UV–VIS detector). The PMP derivative (10 μl) was injected, eluted with 6

82.0% PBS (0.1 M, pH 7.0), and 18% acetonitrile (v/v) at a flow rate of 1.0 ml/min and monitored by UV absorbance at 245 nm (Zhang et al., 2009).

2.5. Screening of fungus capable of pectin degradation

A complete synthetic Czapek Dox Medium was prepared first. It contained 0.3% NaNO3, 0.05% KCl, 0.05% MgSO4.7H2O, 0.1% K2HPO4 and 0.001% FeSO4.7H2O. 1% Codonopsis pilosula pectin (named CPPA) as a unique carbon source was added. Strains from fifteen microorganisms were cultured with this medium separately at 28oC, 140 rpm for 6 d. Culture supernatants (200 μl) were removed daily to monitor the degradation process of pectin by HPGPC on a TSK-gel G-3000 PWXL column.

2.6. Preparation of products from pectin degraded by Penicillium oxalicum

The fungus Penicillium oxalicum was cultured on the above-mentioned liquid medium at 28oC, 140 rpm for 4 d, with pectins from seven plants. The fermentation liquor was filtered through nylon cloth and centrifuged to remove mycelium. Supernatants were precipitated by addition of four volumes of ethanol and kept overnight at room temperature. After centrifugation, the precipitate was dried by solvent exchange with 95% ethanol and absolute ethanol in succession. The products were further purified by anion exchange and size exclusion chromatography. The fractionation procedure with the products from Codonopsis pilosula pectin (CPPAT) is described below as an example. CPPAT was dissolved in distilled water and applied on DEAE-Cellulose (7.5 × 20 cm, Cl-), pre-equilibrated 7

with distilled water. The column was eluted with distilled water, 0.1 M, 0.2 M and 0.3 M NaCl, repectively, at 13 ml/min. The eluate was detected for the distribution of total sugar. The appropriate fractions were collected, dialyzed against distilled water and lyophilized. Acidic fractions obtained by NaCl elution were further purified by Sephadex G-50 (1.5×100 cm) or Sephadex G-75 (1.5×100 cm) column, eluted with 0.15 M NaCl at 0.15 ml/min. The eluate was collected at 3 ml per tube and detected for the distribution of total sugar and uronic acid. The appropriate fractions were combined, dialyzed, and lyophilized yielding purified RG-II domains. The purification process for the degradation products from other plant pectins was similar to that used for CPPAT.

2.7. Structure analysis of RG-II domain by partial acid hydrolysis.

The side chain A and side chain B of RG-II domains were recovered according to the procedure established previously (Buffetto et al., 2014). RG-II domains were first hydrolyzed by 0.1 M TFA at 40oC for 16 h, and TFA was removed by vacuum rotary evaporation. Hydrolysates were loaded onto DEAE-Sepharose fast flow column (1.0×25 cm, Cl-), pre-equilibrated with 25 mM NaAc (pH 4.5). The column was eluted sequentially by using 25 mM NaAc buffer (40 ml), 0-0.1 M NaCl in 25 mM NaAc (200 ml) and 0.1-1 M NaCl in 25 mM NaAc (40 ml), respectively. The appropriate fractions were collected, desalted, and freeze-dried. The fraction eluted by 0.1 to 1 M NaCl was further hydrolyzed by using 0.1 M TFA at 60oC for 8 h, and TFA was removed by vacuum rotary evaporation. Hydrolysates were loaded onto 8

DEAE-Sepharose fast flow column (1.0×25 cm, Cl-) and eluted by NaAc buffer (40 ml), 0-0.15 M NaCl in 25 mM NaAc (200 ml) and 0.15-1 M NaCl in 25 mM NaAc (40 ml), respectively. The appropriate fractions were collected, desalted by Sephadex G-10, and freeze-dried for ESI-MS analysis.

2.8. ESI-MS analysis

RG-II side chains obtained by partial acid hydrolysis were analyzed by ESI–MS using an amaZon ETD Ion Trap Mass Spectrometer (Bruker Inc., Germany) at a spray voltage of 2 kV (negative mode).

2.9. 13C NMR spectra

13

C NMR spectra were obtained using a Bruker Avance 600 MHz spectrometer

(Bruker Inc., Rheinstetten, Germany) operating at 150 MHz for carbon. Samples (20 mg) were dissolved in D2O (99.8 %, 0.5 ml). Chemical shifts were given in ppm with acetone as the internal chemical shift reference.

3. Results and Discussion

3.1. Degradation of the pectin from Codonopsis pilosula

3.1.1. Screening of microorganisms for pectin degradation potential To obtain microorganisms that can selectively degrade pectin into RG-II domains, we screened fifteen strains of fungi and bacteria isolated from Changbai 9

Mountain using pectin from Codonopsis pilosula (CPPA) as the sole carbon source (Fig. S1). Among them, one filamentous fungus F68 grew and could degrade CPPA efficiently. The F68 degradation of CPPA was monitored by high performance gel-permeation chromatography (HPGPC) (Fig. 1). As can be seen, the high molecular weight portion of CPPA (retention time 9 ~ 13 min, 400 ~ 15 kDa) has disappeared after degradation by F68 for 4 d, indicating that CPPA was degraded by F68. When the degradation time was prolonged to 6 d, similar results were obtained, suggesting that the degradation process was complete within 4 d, and the products were stable under these conditions. We previously identified strain F68 as Penicillium oxalicum (Gao et al., 2012). —: 0 d of culture; ····: 4 d of culture; - - -: 6 d of culture.

3.1.2. Isolation and purification of degradation products Following four days of CPPA degradation by Penicillium oxalicum, the products (named CPPAT) were precipitated by addition of 80% ethanol to remove small molecules and fractionated according to the procedure shown in Fig. S2A. CPPAT was applied onto a DEAE-Cellulose column (7.5 × 20 cm, Cl-), eluted stepwise by using H2O, 0.1, 0.2 and 0.3 M NaCl to yield a neutral fraction CPPAT-H (yield 1.0% in relation to CPPA) and three acidic fractions CPPAT-1 (yield 0.9% in relation to CPPA), CPPAT-2 (yield 6.2% in relation to CPPA), and CPPAT-3 (yield 2.7% in relation to CPPA), respectively (Fig. S2B). Due to low yields, CPPAT-H and CPPAT-1 were not further fractionated. Fractions CPPAT-2 and CPPAT-3 were then purified on 10

a Sephadex G-50 or Sephadex G-75 column to give two homogeneous fractions, CPPAT-2A (Fig. S2C) and CPPAT-3A (Fig. S2D).

3.1.3. Identification of degradation products The TBA assay is a very sensitive reaction used to identify Kdo and Dha, which are characteristic monosaccharides in RG-II (Yapo, 2011a). Here, the four degradation products of CPPA were assessed by TBA. As shown in Table 1, all three acidic fractions (except for neutral fraction CPPAT-H) contained TBA-positive

constituents,

suggesting

they

were

RG-II

domains.

Monosaccharide composition analysis indicated that CPPAT-1, CPPAT-2A, and CPPAT-3A were mainly composed of GalA, with minor contributions of Rha, Gal, Ara, Fuc and Glc (Table 1). Both CPPAT-2A and CPPAT-3A showed single peaks on HPGPC (Fig. S3). Their molecular weights were estimated to be 5.1 kDa and 6.2 kDa, respectively, consistent with that reported for monomeric RG-II (Buffetto et al., 2014).

3.1.4. Structure analysis by 13C NMR 13

C NMR spectra of CPPAT-2A and CPPAT-3A are shown in Fig. 2. Some

characteristic signals indicate that both fractions are RG-II type pectin (Table S1). (Glushka et al., 2003; Hervé, Gey, Pellerin & Perez, 1999; Vidal et al., 2000).

3.1.5. Structure analysis by partial acid hydrolysis and ESI-MS To further identify the structures of RG-II domains, partial acid hydrolysis and 11

MS analyses, which have previously been used to study RG-II structural features (Buffetto et al., 2014; Pabst et al., 2013), were performed. Based on these methods, CPPAT-2A and CPPAT-3A were first hydrolyzed by using 0.1 M TFA at 40oC for 16 h, followed by separation with DEAE-Sepharose fast flow. Elution profiles were very similar, and two sub-fractions (P1 and P2) were obtained from both CPPAT-2A and CPPAT-3A (Fig. S4A). The P2 fractions were further hydrolyzed under slightly harsher conditions by using 0.1 M TFA at 60oC for 8 h, and DEAE-Sepharose fast flow fractionation of the hydrolysates generated three sub-fractions (P2-1, P2-2 and P2-3) (Fig. S4B). As shown in Fig. 3, negative-ion mode ESI-MS analysis of P1 from CPPAT-2A and CPPAT-3A gave a series of pseudo-molecular ions showing the structure features of side chain B. These ions were consistent with those reported by Buffetto et al. (2014). According to the identification of these ions in their work, side chain B in both CPPAT-2A and CPPAT-3A were nona-saccharides with 0 or 1 acetyl group in CPPAT-2A and 0 to 2 acetyl groups in CPPAT-3A. Other structures corresponding to hepta- and octa-saccharides with or without acetyl decoration were also present. In the MS spectrum of P2-2 from CPPAT-2A, there were ions assigned to non-methylated, mono-methylated and di-methylated forms of side chain A of DP8. Ions assigned to mono- and di-methylated side chains A of DP 8 in which α-L-Fuc was replaced by α-L-Gal were also observed, in accordance with Buffetto et al. (2014) and Pabst et al. (2013). P2-2 from CPPAT-3A only showed two sets of [M-2H]2- ions at m/z 638 and 645, corresponding to non- and mono-methylated side chains A of DP 8. In some 12

plants like Arabidopsis thaliana, Brassica oleracea, Daucus carota, Valerianella locusta, Apium graveolens and Cupressus macrocarpa, both methyl ester and ether groups have been found in side chain A. One or two ether groups were found on β-GalA, and one ester group was located on β-D-GlcA (Pabst et al., 2013). In this paper, the methyl group linkage was identified by de-esterification and ESI-MS. Side chain A was de-esterified by treatment with 0.1 M NaOH. After neutralization, it was further detected by ESI-MS. The side chain A showed the same MS result before and after de-esterification, suggesting all methyl groups were methyl ether groups. In summary, RG-II domains from Codonopsis pilosula contain side chains B of DP 9 decorated with 0 to 2 acetyl groups, and side chains A of DP 8 decorated with 0 to 2 methyl groups and in which L-Fuc residue can be replaced by L-Gal.

3.2. Penicillium oxalicum degradation of pectin from six other plants

In order to verify the ability of Penicillium oxalicum to degrade pectin and prepare RG-II domains, pectin from six other plants were tested. Products were fractionated as described in 3.1.2. Fourteen purified products were obtained and shown to be RG-II domains by using the TBA reaction (Table 2). Among them, nine fractions showed narrow and symmetric peaks on HPGPC (Fig. 4), and their molecular weights were estimated to fall in the range of 3.7 to 6.2 kDa, indicating that they are monomeric RG-II. The HPGPC elution profiles from another five fractions (ALPAT-3A, VCPAT-3A, WHPAT-2A, SNPAT-3A and GLPAT-3A) were asymmetric with a shoulder appearing at 13.0 to 13.5 min. In these fractions, they all contained 13

high molecular weight parts (~10 KDa, RGII dimers) and low molecular weight parts (~5 KDa, RGII monomer). RGII dimers could be converted to RGII monomer with mild acid treatments, as the peak with high molecular weight was disappeared, leaving only a single peak with low molecular weight (Fig. S5). RGII monomer could reform to RGII dimer with borate buffer, showing high molecular weight parts (Fig. S5). Therefore, we speculated that both RGII dimers and monomers were present in these fractions. RG-II type pectin constituted 4.6% to 7.2% (w/w) of the total pectin from these six plants. Even though these values are slightly lower than previously reported (10% w/w, (Pabst et al., 2013)), RG-II from different monocotyledons and dicotyledons has also been found to be less than 5% (w/w) (Yapo, 2011b).

3.3. Structural analysis by partial acid hydrolysis and ESI-MS of RG-II domains from six plants

To analyze structural differences between these RG-II fractions, partial acid hydrolysis and ESI-MS analyses were performed. MS demonstrated that these are RG-II domains with chain A and chain B (Table 3). In these various RG-II domains, side chains A were all octa-saccharides with 0 to 2 methyl groups. The presence of methyl groups were detected in all plants except Viscum coloratum. By de-esterification and ESI-MS analysis, all methyl groups were identified to be methyl ether groups. Methylesterification was not detected in these fractions, presumably because it does not exist in these plants or had been removed by enzymes which were produced by the fungus. In some of RG-II fractions, α-L-Fuc was partially replaced 14

by α-L-Gal in side chain A, whereas in VCPAT-1A, VCPAT-2A, VCPAT-3A and VPAT-2A, α-L-Fuc was totally replaced by α-L-Gal (Table 3). In previous studies, replacement of α-L-Fuc by α-L-Gal was only observed in Arabidopsis thaliana mur1 mutant, and this was considered the possible reason for dwarfism of the aerial part (O'Neill, Eberhard, Albersheim & Darvill, 2001). However, in recent years, the L-Gal replacement in side chain A was found in many normal plants, such as Solanum lycopersicum, Nicotiana benthamiana, Arabidopsis thaliana, Brassica oleracea, Daucus carota, Valerianella locusta, Apium graveolens, Cupressus macrocarpa, Asplenium nidus (Pabst et al., 2013). The degree of replacement by L-Gal in these plants ranged from 0.3% to 45%. In our present study, L-Gal replacement was present in side chains A of RG-II from all the plants investigated. In the three RG-II domains (VCPAT-1A, VCPAT-2A and VCPAT-3A) from Viscum coloratum, the L-Fuc residue was nearly absent and was replaced by L-Gal, suggesting that the degree of replacement by L-Gal could reach up to 100%. Side chains B in most of these RG-II domains were non-, mono- or di-acetylated nona-saccharides (Table 3). Side chains B with DP 8 were also found in ALPAT-1A, WHPAT-1A and WHPAT-2A. Unexpectedly, non- or mono-acetylated side chains B with DP 10 were observed in ALPAT-2A. According to previous reports, side chains B of RG-II are composed of 9 sugar residues in red wine, ginseng leaves, and citrus peels. Among them, red wine and ginseng leaves were substituted by 0 to 2 acetyl groups, and citrus peels were found to contain no acetyl groups (Buffetto et al., 2014; Park, Park, Hong, Suh & Shin, 2016; Shin, Kiyohara, Matsumoto & Yamada, 1997). 15

Side chains B contained 8 sugar residues in sycamore, Arabidopsis thaliana and bamboo, and were substituted by 0 to 2 acetyl groups (Kaneko, Ishii & Matsunaga, 1997; Pabst et al., 2013; Whitcombe, O'Neill, Steffan, Albersheim & Darvill, 1995). Side chains B of RG-II in rice is composed of 7 sugar residues, with 0 and 1 acetyl group (Thomas, Darvill & Albersheim, 1989). Side chains B of pectinol AC is composed of 6 sugar residues (Buffetto et al., 2014). Here, most side chains B were composed of 9 sugar residues with 0 to 2 acetyl groups, similar to those found in red wine-derived RG-II. RG-II fractions from Helianthus annuus head were composed of 8 sugar residues with 0 to 1 acetyl group in side chains B, similar to those from Arabidopsis thaliana RG-II. Interestingly, in our study, we report here for the first time the discovery of a novel RG-II domain from Apocynum venetum, ALPAT-2A, that contains 10 sugar residues in side chains B. Among these RG-II domains, some of them had same side chain structures, such as GLPAT-2A, GLPAT-3A and VPAT-3A, or VCPAT-1A and VCPAT-2A (Table 3). Other fractions were different from each other in number of acetyl groups and the degree of polymerization in side chains B, and in the number of methyl ether groups and L-Gal replacement in side chains A. These results indicate that RG-II exhibits small structural variations within an individual plant or different plant species although it is conserved during plant evolution. In ALPAT-2A, we found that side chain B with 10 sugar residues, was different from the other RG-II fractions. MS result with ALPAT-2A side chain B was shown in Fig. 5. As can be seen, ions at m/z 1537 and 1495 were clearly observed and assigned 16

to non- and mono-acetylated side chains B with DP 10. This structure has not previously been reported in RG-II domains. A series of ions at m/z 1405, 1363, 1259, 1217, 1113 and 1071 were also observed and assigned to non- or mono-acetylated side chains B of DP 10 lacking Ara or Rha residues, respectively. Prior to our report, only 6~9 sugar residues had been reported for RG-II side chain B. According to the mass difference, an extra monosaccharide with molecular weight of 180 Da was observed in the side chains B of ALPAT-2A. A detailed investigation to assess the chemical structure of the tenth residue and its position in side chain B is presently in progress in our lab.

Pectin is mainly composed of HG, RG-I, RG-II and arabinogalactan (AG) domains (Ridley, O'Neill & Mohnen, 2001; Yapo, 2011a). RG-II could be prepared mostly in two ways. Fruit wine, especially red wine, has been reported as a major source of RG-II which could be prepared directly by ethanol precipitation and chromatography separation (Hilz, Williams, Doco, Schols & Voragen, 2006; Hwang & Shin, 2011; Pellerin, Doco, Vidal, Williams, Brillouet & O'Neill, 1996). To prepare RG-II from the pectin isolated from plants, the pectin needs to be treated with pectinase, such as polygalacturonase or pectin lyase, which can specially degrade HG in pectin, remaining RG-I, AG and RG-II as products (Fleischer, O'Neill & Ehwald, 1999; Kaneko, Ishii & Matsunaga, 1997; Matsunaga et al., 2004). The mixture of different pectin domains could be separated by a combination of ion-exchange and size-exclusion chromatography. Driselase is an efficient complex enzyme which can degrade pectin to produce arabinogalactan-proteins and RG-II as products (Chormova, Messenger & Fry, 2014; Matoh, Ishigaki, Ohno & Azuma, 1993). Similar with 17

Driselase, Penicillium oxalicum was found in this paper to efficiently degrade pectin, with RG-II domain as major product and minor neutral polysaccharide as contaminant. The purification of RG-II from the products of both Driselase and Penicillium oxalicum would be easier than from those of polygalacturonase or pectin lyase. Thus, we found another way to prepare RG-II domain by Penicillium oxalicum. This method avoids the purification of enzymes and is comparable to fruit wine fermentation.

. Conclusions

In the present study, we screened a strain of the fungus Penicillium oxalicum for degradation of Codonopsis pilosula pectin into RG-II domains. The degradation ability of Penicillium oxalicum was demonstrated using pectin from six other plants as the sole carbon source. Fourteen purified RG-II domains were obtained. The structural features of side chains A and B in these RG-II samples were identified and compared. Side chains B from most plants were found to contain 8 to 9 residues with 0 to 2 acetyl groups. Side chain B of one RG-II fraction from Apocynum venetum was the exception because it is surprisingly composed of 10 residues. Side chains A in most plants contain both L-Fuc and L-Gal residues of DP 8, with 0 to 2 methyl ether groups. In Viscum coloratum, however, the L-Fuc residue in side chain A was absent and fully replaced by L-Gal. Our study shows that RG-II is structurally diverse among different plants due to variations in side chains A and B. Our results have significant impact in refining a structural model for RG-II and studying RG-II structure-activity relationships.

18

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 31470798, 31500274), the Scientific and Technologic Foundation of Jilin Province (20140101122JC).

Reference

Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54(2), 484-489. Buffetto, F., Ropartz, D., Zhang, X. J., Gilbert, H. J., Guillon, F., & Ralet, M. C. (2014). Recovery and fine structure variability of RGII sub-domains in wine (Vitis vinifera Merlot). Annals of botany, 114(6), 1327-1337. Caffall, K. H., & Mohnen, D. (2009). The structure, function, and biosynthesis of plant cell wall pectic polysaccharides. Carbohydrate Research, 344(14), 1879-1900. Chormova, D., Messenger, D. J., & Fry, S. C. (2014). Boron bridging of rhamnogalacturonan-II, monitored by gel electrophoresis, occurs during polysaccharide synthesis and secretion but not post-secretion. The Plant Journal, 77(4), 534-546. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956).

19

Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350-356. Fleischer, A., O'Neill, M. A., & Ehwald, R. (1999). The pore size of non-graminaceous plant cell walls is rapidly decreased by borate ester cross-linking of the pectic polysaccharide rhamnogalacturonan II. Plant Physiology, 121(3), 829-838. Gao, J., Xu, W., Fang, Q., Liang, F., Jin, R., Wu, D., Tai, G., & Zhou, Y. (2012). Efficient

biotransformation

for

preparation

of

pharmaceutically

active

ginsenoside Compound K by Penicillium oxalicum sp. 68. Annals of Microbiology, 63(1), 139-149. Glushka, J. N., Terrell, M., York, W. S., O'Neill, M. A., Gucwa, A., Darvill, A. G., Albersheim, P., & Prestegard, J. H. (2003). Primary structure of the 2-O-methyl-α-l-fucose-containing side chain of the pectic polysaccharide, rhamnogalacturonan II. Carbohydrate Research, 338(4), 341-352. Hervé Du Penhoat, C., Gey, C., Pellerin, P., & Perez, S. (1999). An NMR solution study of the mega-oligosaccharide, rhamnogalacturonan II. Journal of Biomolecular NMR, 14(3), 253-271. Hilz, H., Williams, P., Doco, T., Schols, H. A., & Voragen, A. G. J. (2006). The pectic polysaccharide rhamnogalacturonan II is present as a dimer in pectic populations of bilberries and black currants in muro and in juice. Carbohydrate Polymers, 65(4), 521-528. Hwang, Y. C., & Shin, K. S. (2011). Isolation and characterization of 20

immuno-stimulating polysaccharide in Korean black raspberry wine. Journal of the Korean Society for Applied Biological Chemistry, 54(4), 591-599. Ishii, T., & Matsunaga, T. (2001). Pectic polysaccharide rhamnogalacturonan II is covalently linked to homogalacturonan. Phytochemistry, 57(6), 969-974. Iwai, H., Masaoka, N., Ishii, T., & Satoh, S. (2002). A pectin glucuronyltransferase gene is essential for intercellular attachment in the plant meristem. Proceedings of the National Academy of Sciences, 99(25), 16319-16324. Kaneko, S., Ishii, T., & Matsunaga, T. (1997). A boron-rhamnogalacturonan-II complex from bamboo shoot cell walls. Phytochemistry, 44(2), 243-248. Matsunaga, T., Ishii, T., Matsumoto, S., Higuchi, M., Darvill, A., Albersheim, P., & O'Neill, M. A. (2004). Occurrence of the primary cell wall polysaccharide rhamnogalacturonan

II

in

pteridophytes,

lycophytes,

and

bryophytes.

Implications for the evolution of vascular plants. Plant Physiologyl, 134(1), 339-351. Matoh, T., Ishigaki, K., Ohno, K., Azuma, J., 1993. Isolation and Characterization of a Boron-Polysaccharide Complex from Radish Roots. Plant Cell Physiology 34(4), 639-642. Ndeh, D., Rogowski, A., Cartmell, A., Luis, A. S., Baslé, A., Gray, J., Venditto, I., Briggs, J., Zhang, X. Y., Labourel, A., Terrapon, N., Buffetto, F., Nepogodiev, S., Xiao, Y., Field, R. A., Zhu, Y. P., O’Neill, M. A., Urbanowicz, B. R., York, W. S., Davies, G. J., Abbott, D. W., Ralet, M. C., Martens, E. C., Henrissat, B., Gilbert, H. J. (2017) Complex pectin metabolism by gut bacteria reveals novel catalytic 21

functions. Nature, 544, 65–70. O'Neill, M. A., Eberhard, S., Albersheim, P., & Darvill, A. G. (2001). Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth. Science, 294(5543), 846-849. Pabst, M., Fischl, R. M., Brecker, L., Morelle, W., Fauland, A., Kofeler, H., Altmann, F., & Leonard, R. (2013). Rhamnogalacturonan II structure shows variation in the side chains monosaccharide composition and methylation status within and across different plant species. The Plant Journal, 76(1), 61-72. Park, H. R., Park, S. B., Hong, H. D., Suh, H. J., & Shin, K. S. (2016). Structural elucidation of anti-metastatic rhamnogalacturonan II from the pectinase digest of citrus peels (Citrus unshiu). International Journal of Biological Macromolecules, 94(Pt A), 161-169. Pellerin, P., Doco, T., Vidal, S., Williams, P., Brillouet, J. M., & O'Neill, M. A. (1996). Structural characterization of red wine rhamnogalacturonan II. Carbohydrate Research, 290(2), 183-197. Ridley, B. L., O'Neill, M. A., & Mohnen, D. (2001). Pectins: Structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry, 57(6), 929-967. Seveno, M., Voxeur, A., Rihouey, C., Wu, A. M., Ishii, T., Chevalier, C., Ralet, M. C., Driouich, A., Marchant, A., & Lerouge, P. (2009). Structural characterisation of the pectic polysaccharide rhamnogalacturonan II using an acidic fingerprinting methodology. Planta, 230(5), 947-957. Shin, K. S., Kiyohara, H., Matsumoto, T., & Yamada, H. (1997). Rhamnogalacturonan 22

II from the leaves of Panax ginseng C.A. Meyer as a macrophage fc receptor expression-enhancing polysaccharide. Carbohydrate Research, 300(3), 239-249. Tahiri, M., Pellerin, P., Tressol, J. C., Doco, T., Pépin, D., Rayssiguier, Y., & Coudray, C. (2000). The rhamnogalacturonan-II dimer decreases intestinal absorption and tissue accumulation of lead in rats. Journal of Nutrition, 130(2), 249-253. Thomas, J. R., Darvill, A. G., & Albersheim, P. (1989). Isolation and structural characterization of the pectic polysaccharide rhamnogalacturonan II from walls of suspension-cultured rice cells. Carbohydrate Research, 185(2), 261-277. Vidal, S., Doco, T., Williams, P., Pellerin, P., York, W. S., O’Neill, M. A., Glushka, J., Darvill, A. G., & Albersheim, P. (2000). Structural characterization of the pectic polysaccharide rhamnogalacturonan II: evidence for the backbone location of the aceric acid-containing oligoglycosyl side chain. Carbohydrate Research, 326(4), 277-294. Whitcombe, A. J., O'Neill, M. A., Steffan, W., Albersheim, P., & Darvill, A. G. (1995). Structural characterization of the pectic polysaccharide, rhamnogalacturonan-II. Carbohydrate Research, 271(1), 15-29. Yapo, B. M. (2011a). Pectic substances: From simple pectic polysaccharides to complex pectins—A new hypothetical model. Carbohydrate Polymers, 86(2), 373-385. Yapo, B. M. (2011b). Pectin Rhamnogalacturonan II: On the “Small Stem with Four Branches” in the Primary Cell Walls of Plants. International Journal of Carbohydrate Chemistry, 2011, 1-11. 23

York, W. S., Darvill,

A. G., McNeil,

3-deoxy-d-manno-2-octulosonic

acid

M.,

& Albersheim, P. (1985).

(KDO)

is

a

component

of

rhamnogalacturonan II, a pectic polysaccharide in the primary cell walls of plants. Carbohydrate Research, 138(1), 109-126. Yu, K. W., Kiyohara, H., Matsumoto, T., Yang, H. C., & Yamada, H. (2001). Characterization of pectic polysaccharides having intestinal immune system modulating activity from rhizomes of Atractylodes lancea DC. Carbohydrate Polymers, 46(2), 125-134. Zhang, X., Yu, L., Bi, H., Li, X., Ni, W., Han, H., Li, N., Wang, B., Zhou, Y., & Tai, G. (2009).

Total

fractionation

and

characterization

of

the

water-soluble

polysaccharides isolated from Panax ginseng C. A. Meyer. Carbohydrate Polymers, 77(3), 544-552.

24

Fig. 1. Monitoring of CPPA degradation with the culture of strain F68 by HPGPC.

Fig. 2. 13C NMR spectra of (A) CPPAT-2A; (B) anomeric region between 90 and 112 25

ppm of CPPAT-2A shown in detail; (C) CPPAT-3A.

Fig. 3. ESI-MS analysis of RG-II side chains A and B in CPPAT-2A and CPPAT-3A (negative mode).

26

Fig. 4. HPGPC elution profiles of purified RG-II domains from six plants.

Fig. 5. ESI-MS analysis of side chain B in ALPAT-2A (negative mode). “B” in all the peak assignments represent for the normal side chain B of DP 9.

27

Table 1 Structural features of degradation products of CPPA. Yield

Mw

TBA Monosaccharide composition (mol %)

Fraction

(g%)a (KDa) testb

GalA Rha

Gal

CPPAT-H

1.0

--

-

--

--

30.3 8.8

38.6 --

19.0

CPPAT-1

0.9

--

+

63.4

9.0

10.0 9.0

2.7

1.2

1.4

CPPAT-2A 3.3

5.1

+

54.0

8.8

14.5 12.9 3.7

5.9

--

CPPAT-3A 0.8

6.2

+

44.7

24.7 7.9

6.7

--

a

Yields in relation to native pectin CPPA;

b

Ara

Glc

10.1 5.2

Fuc Man

-: negative result indicating no RG-II

domain; +: positive result indicating the presence of RG-II domain.

Table 2 Plant sources, nomenclatures, yields and molecular weights of RG-II domains. Total Fraction Plant source

TBA

Yield

Mw

Organ

yield name

test

a

b

(w/w %)

(KDa) b

(w/w%) Apocynum Venetum

leaves

ALPAT-1A

+

2.4

ALPAT-2A

+

4.4

5.6

ALPAT-3A

+

0.4

11.5&7.6

28

7.2

4.1

Panax

stem &

GLPAT-2A

+

3.4

Ginseng

leaves

GLPAT-3A

+

1.4

Scrophularia

root

SNPAT-2A

+

3.7

SNPAT-3A

+

1.6

VCPAT-1A

+

0.4

VCPAT-2A

+

4.0

4.6

VCPAT-3A

+

0.5

9.3&5.6

WHPAT-1A

+

4.8

WHPAT-2A

+

0.6

VPAT-2A

+

3.0

VPAT-3A

+

1.6

ningpoensis Viscum

leaves

Coloratum

Helianthus

head

Annuus Veratrum

root

Nigrum a

4.8

5.6 9.3&6.2

5.3

6.2 9.3&5.1

4.9

5.4

3.7

5.1 10.3&6.2

4.6

4.6 5.1

+: Positive result indicating of RG-II domain. b Yield in relation to native pectin.

Table 3 Structural features of chain A and Chain B in different RG-II domains. Side chain A

Side chain B

RG-II domain DP

Methyl

L-Gal 29

DP

Acetyl

group

replacement

Group

ALPAT-1A

8

0 or 2

L-Fuc/ L-Gal

8

0 , 1 or 2

ALPAT-2A

8

0 or 2

L-Fuc/ L-Gal

10

0 or 1

ALPAT-3A

8

0 or 1

L-Fuc/ L-Gal

9

0 , 1 or 2

GLPAT-2A

8

0 or 1

L-Fuc/ L-Gal

9

0 , 1 or 2

GLPAT-3A

8

0 or 1

L-Fuc/ L-Gal

9

0 , 1 or 2

SNPAT-2A

8

0 or 2

L-Fuc/ L-Gal

9

0, 1 or 2

SNPAT-3A

8

1

L-Fuc/ L-Gal

9

0, 1 or 2

VCPAT-1A

8

0

L-Gal

9

0 or 1

VCPAT-2A

8

0

L-Gal

9

0 or 1

VCPAT-3A

8

0

L-Gal

9

0, 1 or 2

WHPAT-1A

8

0

L-Fuc/ L-Gal

8

0 or 1

WHPAT-2A

8

0 or 1

L-Fuc/ L-Gal

8

0 or 1

VPAT-2A

8

0 or 1

L-Gal

9

0 or 1

VPAT-3A

8

0 or 1

L-Fuc/ L-Gal

9

0, 1 or 2

3.4. Structure analysis of side chain B in ALPAT-2A

30