Glycobiology Advance Access originally published online on August 28, 2007
Glycobiology 2007 17(12):1299-1310; doi:10.1093/glycob/cwm088
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Immunological and Structural Properties of a Pectic Polymer from Glinus Oppositifolius
2 Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, Oslo 0316, Norway
3 School of Biosciences, Sutton Bonington campus, University of Nottingham, Loughborough, Leicestershire LE12 5RD, UK
4 Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2 RD, UK
5 Department of Chemistry, Swedish University of Agricultural Sciences, P.O. Box 7015, Uppsala 750 07, Sweden
6 Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane Minato-ku, Tokyo 108-8641, Japan
7 Department of Traditional Medicine, B.P. 1746, Bamako, Mali
8 The Norwegian Institute of Public Health, P.O. Box 4404, Nydalen, Oslo 0403, Norway
9 Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1105, Blindern, Oslo 0317, Norway
1 To whom correspondence should be addressed: Tel: +47-2-28-56-572; Fax: +47-2-28-54-402; e-mail: k.t.inngjerdingen{at}farmasi.uio.no
Received on March 14, 2007; revised on August 13, 2007; accepted on August 13, 2007
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsSupplementary dataFundingConflict of interest statementReferences |
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The aim of this paper was to further elucidate the structure and the immunomodulating properties of the pectic polymer GOA2, previously isolated from Glinus oppositifolius. Enzymatic treatment of GOA2 by endo-
-D-(1
4)-polygalacturonase led to the isolation of three pectic subunits, GOA2-I, GOA2-II, and GOA2-III, in addition to oligogalacturonides. GOA2-I was shown to consist of 1,2-linked Rhap and 1,4-linked GalpA in an approximately 1:1 ratio, and NMR-analysis showed that the monomers were linked together in a strictly alternating manner. The galactose units in GOA2-I were found as terminal-, 1,3-, 1,6-, 1,4-, 1,3,4-, and 1,3,6-linked residues, while the arabinofuranosyl existed mainly as terminal- and 1,5-linked units. A rhamnogalacturonan-I type structure was suggested being the predominant part of GOA2-I. According to linkage analysis GOA2-II and GOA2-III contained glycosidic linkages characteristic for rhamnogalacturonan-II type structures. GOA2 was shown by sedimentation velocity in the analytical ultracentrifuge, to have a broad degree of polydispersity with a mode s20,w value of 
1.9 S, results reinforced by atomic force microscopy measurements. The polydispersity, as manifested by the proportion of material with s20,w > 3 S, decreased significantly with enzyme treatment. The abilities of GOA2, GOA2-I, GOA2-II, and GOA2-III to induce the proliferation of B cells, and to exhibit complement fixing activities were tested. In both test systems, GOA2-I showed significantly greater effects compared to its native pectin GOA2. GOA2-I was in addition shown to exhibit a more potent intestinal immune stimulating activity compared to GOA2. The ability of GOA2 to induce secretion of proinflammatory cytokines was examined. Marked upregulations in mRNA for IL-1ß from rat macrophages and IFN-
from NK cells were found. Key words: Glinus oppositifolius / immunomodulation / pectic polymer / rhamnogalacturonan
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataFundingConflict of interest statementReferences |
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Glinus oppositifolius (L.) Aug. DC. (Aizoaceae) is used in Malian traditional medicine in the treatment of wounds, inflammations, gastric ulcers, malaria, and fever, diseases which are all related to the immune response. A previous study has reported the isolation and partial structural characterization of two pectic polysaccharides from a hot water extract of the aerial parts of G. oppositifolius. The pectic polymers, named GOA1 and GOA2, were shown to exhibit dose-dependent complement fixing activities, and chemotactic properties toward human macrophages, T cells, and NK cells (Inngjerdingen et al. 2005
).
The pectic polysaccharides are a heterogeneous group of complex polymers found in the primary cell wall of all plants, showing substantial diversity with botanical origin. They consist of a number of structurally different regions, which include homogalacturonan (HG), RG-I, and substituted galacturonans, such as rhamnogalacturonan II (RG-II). HGs are composed of linear 1,4-linked -D-galactopyranosyluronic acid (GalpA) residues, in which varying proportions of the acid groups are methyl esterified. RG-I, a highly branched region of a pectin, consists of a backbone of alternating 1,4-linked GalpA and 1,2-linked rhamnopyranosyl (Rhap) units. A fraction of the Rhap residues are branch points for neutral sugar side chains, attached through position 4 of 1,2-linked Rhap in the backbone. The nature of the side chains display a wide structural diversity, both in terms of the constituting sugars, being mainly composed of arabinosyl (Ara) and galactosyl (Gal) residues, and in terms of the glycosidic linkages. The Ara and Gal units may be present as pure arabinans and/or galactans or arabinogalactans. A minor component of plant cell wall is RG-II, which has an extremely complex structure. It has a HG backbone of 1,4-linked GalpA, and is substituted with four different oligosaccharide side chains (Perez et al. 2003; Bui et al. 2006).
A wide range of polysaccharides, extending from homopolymers to highly complex heteropolymers, have been reported to exhibit immunomodulating activities. So far, no clear information has been obtained on how a polysaccharide has to be structurally designed in order to have an optimal inducing effect on specific immune cells. However, for both glucans and pectic polymers, high molecular weight appears to increase the bioactivities, and branching of side chains are needed for optimal activity (Lin 2005). Regarding immunomodulating activities of pectic polysaccharides, there are several reports that the rhamnogalacturonan region of the pectins have more potent activities compared to the corresponding original pectin, and that oligogalacturonides have weak or negligible activities (Yamada and Kiyohara 1999; Paulsen and Barsett 2005).
The pectic polymer GOA1 was after different enzymatic treatments and weak acid hydrolysis suggested to contain a mixture of arabinogalactan type I (AG-I) and type II (AG-II) moieties, both most probably linked to a RG-I backbone. The partial removal of Araf units in the outer chains of the polymer by enzymatic degradation did not influence the intestinal immune stimulating activity or the complement fixing activity. The immunomodulating properties of GOA1 were further shown to include proliferation of B cells, the secretion of IL-1ß by macrophages, in addition to a marked increase of mRNA for IFN- in NK cells (Inngjerdingen et al. 2007).
The aim of this study is to further elucidate the structure and immunomodulating properties of the pectic polymer GOA2, previously isolated from G. oppositifolius. Enzymatic treatment of the pectin to isolate its different structural elements has been performed, and the resulting fractions compared regarding both structure and bioactivity.
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Results |
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Composition of GOA2
The isolation and partial structural characterization of the pectic polymer GOA2 isolated from the aerial parts of G. oppositifolius have been previously described (Inngjerdingen et al. 2005
). The polymer is composed of 45% carbohydrate of which GalpA (66.6 mol%) is the major component (Table I). In addition to GalpA, Rhap (11.4 mol%), Gal (8.3 mol%), Ara (7.2 mol%), and minor amounts of xylose (Xyl), 2-O-methyl xylose (2-O-Me Xyl), fucose (Fuc), 2-O-methyl fucose (2-O-Me Fuc), glucose (Glc), glucuronic acid (GlcA), and 4-O-methyl glucuronic acid (4-O-Me GlcA) are also present. The degree of methyl esterification and acetylation were determined to be 3 and 6%, respectively.
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The carbohydrates determined only accounts for about 45% of the total mass of GOA2. The protein content is 1.5%, as previously reported (Inngjerdingen et al. 2005
), and the phenolic content, expressed as ferulic acid equivalents, was determined to be lesser than 0.05%. The remaining mass is unaccounted for, but some could arise from moisture, inorganic material, or as a consequence of the inaccuracies of the colometric assays. In this study, only the carbohydrate part of the fraction is studied in detail.
Enzymatic treatment of GOA2 by endo--D-(14)-polygalacturonase
Endo-polygalacturonase hydrolyses HG regions of de-esterified GalpA 1,4-linkages. Treating high molecular pectin with the enzyme usually generates RG-I (100 kDa), RG-II (5–10 kDa), and oligogalacturonides with degrees of polymerization between 1 and 5 (O’Neill and York 2003), which can be separated by size exclusion chromatography. The enzyme hydrolysate after the degradation of GOA2 by endo-polygalacturonase was applied to a Bio-Gel P30 column. This led to the isolation of three pectic subunits, GOA2-I, a high molecular weight fraction appearing in the void volume, and two intermediate molecular weight fractions denominated GOA2-II and GOA2-III, in addition to oligogalacturonides in GOA2-IV. The monosaccharide compositions of the different fractions are shown in Table I. GOA2-I consists mainly of Rha (29.1 mol%), GalA (25.3 mol%), Gal (21.9 mol%), and Ara (17.5 mol%). The fractions GOA2-II and GOA2-III contain about 30 mol% GalA, 20 mol% of Rha, 15 mol% of Ara, and 9 mol% of Gal, in addition to 4–5 mol% of Fuc, 2-O-Me Fuc, and 2-O-Me Xyl. The fraction GOA2-IV was analyzed by HPAEC-PAD which showed that the fraction consists of oligomers of GalA, and not monomers (data not shown).
Linkage analysis
According to linkage analysis GOA2 contains glycosidic linkages characteristic for both HG, RG-I, and RG-II (Table II). The distribution of sugar residues and their linkages suggest a RG-I like structure being the predominant part of GOA2-I. The fraction consists of 1,2- and 1,2,4-linked Rhap and 1,4-, 1,2,4- and 1,3,4-linked GalpA in an approximately 1:1 ratio. In GOA2-I, the Gal units present are found as terminal, 1,3-, 1,6-, 1,4-, 1,3,4-, and 1,3,6-linked residues. A complex structure is indicated by the relative high degree of branching, 28.8% of the Galp residues exist in 1,3,4- or 1,3,6-linkages. Arabinose exists mainly as terminal and 1,5-linked units, in addition to smaller amounts of 1,2-, 1,3-, 1,3,5-, and 1,2,3-linked residues. The minor monosaccharides are present as terminal and 1,4-linked GlcA/4-O-Me-GlcA, terminal and 1,4-linked Xyl and terminal Fuc.
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The intermediate fractions GOA2-II and GOA2-III contain similar type and amounts of glycosidic linkages (Table II). According to linkage analysis these fractions contain glycosidic linkages characteristic for RG-II type structures, such as terminal- and 1,2,3-linked Ara, terminal-, 1,2- and 1,3-linked Rha, terminal-linked Gal, 1,3,4-linked Fuc, 1,3'-linked apiose (Api), 1,2-linked GlcA, and 1,4-linked GalpA with branch points in positions 2 and 3. In addition 2-O-Me Fuc and 2-O-Me Xyl, characteristics of RG-II, were shown to be present by monosaccharide analysis. These results reflect the presence of two RG-II populations in GOA2.
NMR
Signals in the 1H-NMR spectra were assigned as completely as possible, based on the monosaccharide composition, linkage analysis, and on literature values (Colquhoun et al. 1990; Nergard et al. 2006). The 1H-NMR of GOA2-I showed resonances corresponding to a C-methyl proton at 
1.27 ppm. H-1 signals were assigned to the Rhap residues ( 5.26 ppm), the GalpA residues ( 5.06 ppm), and the Galp residues ( 4.63 ppm). No coupling between GalA-H-1 and GalA-H-4 was observable by 2D-NMR NOESY of GOA2-I (Supplementary Figure S1), which indicates a negligible amount of HG in the polymer. The NOESY spectrum gave cross peaks due to couplings between GalA-H-4 and Rha-H-1, and GalA-H-1 and Rha-H-2. These results indicate an alternating structure of Rhap and GalpA units in GOA2-I, -GalpA-(12)- -Rhap-(14)- -GalpA-(12)- -Rhap, which is a typical backbone for RG-I regions of pectins (Table III, Supplementary Figure S1).
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Homogeneity and solution biophysical data
A summary of the hydrodynamic data for GOA2 and the enzyme-treated materials are given in Table IV. The sedimentation coefficient distribution data (Figure 1A–D) showed all samples to be very polydisperse, with a reduction in polydispersity with increase in treatment. Untreated GOA2 (Figure 1A) showed a mode sedimentation coefficient of
1.9 S and even on a logarithmic scale a pronounced high s20,w tail beyond log(s20,w) = 0.5 (s20,w = 3 S) with material even beyond log(s20,w) = 1.5 (s20,w = 30 S). Enzyme treatment reduced the tail without significantly affecting the position of the mode (Figure 1B–D; Table IV), and in one case (GOA2-II) the software was able to resolve the 1.9 S peak into 1.1 S and 2.5 S peaks. Molecular weight estimates from Size-exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLs) reinforced these estimates (Table IV), the native polymer GOA2 result being influenced by the high s (s > 30 S) material.
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Atomic force microscopy
The above solution data clearly show strong heterogeneity among the pectin fractions. To study this heterogeneity further we use atomic force microscopy (AFM). AFM offers an opportunity to study and directly visualize individual molecules, and images of GOA2 and GOA2-I are presented in Figure 2. As can be seen from the images, both fractions comprise a hierarchy of separate molecules and aggregated species. Based on their histograms evaluated values of the contour lengths for GOA2 and GOA2-I range from <10 to about 100 nm, with a high frequency of the molecules calculated to have contour lengths smaller than 10 nm. As pectin molecules often tangle with each other, it is difficult to give exact statistical data on their chain lengths (Yang et al. 2006
). From Figure 2 it seems that some of the molecules may be branched, which has previously been indicated by AFM images of pectins (Round et al. 2001; Ovodova et al. 2006).
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Complement fixing activity
Figure 3 shows that the rhamnogalacturonan region of GOA2, GOA2-I, inhibits hemolysis of sheep red blood cells in a dose-dependent manner. The polymer possesses the same level of activity as the positive control PMII from Plantago major L. The native polymer GOA2 and the intermediate fractions GOA2-II and GOA-III show very low activities. Any possible contribution of contaminating lipopolysaccharide (LPS) on complement activity was disregarded as a previous study indicated that the amount of LPS present in the polymer GOA2 did not influence the activity observed (Inngjerdingen et al. 2005
).
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B-cell proliferation
The ability of GOA2, GOA2-I, and GOA2-II to induce proliferation of B cells was tested. B cells were purified from rat spleen cell suspensions by positive selection using rat anti-IgG-coated Dynabeads. The cells were labeled with the fluorescent dye CFSE, which allows for the quantifications of cell divisions within a cell population by flow cytometry. Each daughter cell will contain half the amount of CFSE compared to the mother cell. Thus, the fluorescence intensity will decrease in a proliferating cell population. Cells were treated with either medium or increasing concentrations of the pectin fractions for 5 days. B cells showed a high proliferative response toward the positive control LPS (Figure 4). The percentage of cells that had undergone divisions, were based on a gate in the histogram set to define cells that had divided at least once. A dose-dependent induction of B-cell proliferation in response to GOA2-I could be observed (Figure 4), while the native polymer GOA2 and the intermediate fraction GOA2-II did not induce proliferation.
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Intestinal immune stimulating activity
Peyer's patch cells from mice were cultured in the presence of the native polymer GOA2 and the fraction GOA2-I at concentrations of 10 and 100 µg/mL for 5 days in vitro. The resulting cell-free supernatant (conditioned medium) of Peyer's patch cells was used to stimulate bone marrow cells. As can be seen from Figure 5, it is clear that GOA2 does not exhibit any activity at the concentrations tested, while GOA2-I shows potent and statistically significant activity on proliferation of bone marrow cells at both 10 and 100 µg/mL.
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Cytokine secretion by macrophages and NK cells
We examined whether GOA2 induces secretion of proinflammatory cytokines from cells of the innate immune system. The rat macrophage cell line R2-M
and the rat NK cell line RNK-16 (resembling the phenotype of resting NK cells) were treated with GOA2 for 3 days, after which RNA was harvested from the cells and subjected to RT-PCR. There was a marked upregulation (100-fold) in mRNA for the proinflammatory cytokine IL-1ß in R2-M after stimulation with GOA2 (Figure 6A). No differences were observed for IL-6, TNF-, or CD45. In NK cells, we found a marked increase of mRNA for IFN- after GOA2 treatment (Figure 6B). Comparable amounts of mRNA for the positive control CD45 was detected in unstimulated versus GOA2-stimulated samples.
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Discussion |
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Structural features of GOA2
GOA2 contains a native pectic polymer, and the different fractions recovered after treatment with endo-
-D-(14)-polygalacturonase, GOA2-I, GOA2-II, and GOA2-III can be considered as the branched regions of the pectin. GOA2-I was shown to have a RG-I like structure, while GOA2-II and GOA2-III contains monomers and linkages characteristic for RG-II. The GalpA residues present in GOA2-IV are most probably galacturonides released from a HG region in GOA2. We assume that GOA2-I, representing a RG-I moiety, is the predominant branched region in GOA2. In most cell walls from higher plants, RG-I is more abundant than RG-II, representing 10–15% and 2–5% of pectic polysaccharides, respectively (Carpita and McCann 2002; O’Neill and York 2003).
An alternating sequence of Rhap and GalpA in the rhamnogalacturonan backbone of GOA2-I is suggested based on NMR-analysis. In RG-I backbones GalpA units can be acetylated in positions 2 or 3, but glycosidic ramifications are mainly reported to take place on the Rhap residues. A single ß-D-GlcpA residue linked to position 3 of about 2% of the GalpA in the backbone of a RG-I isolated from sugar-beet have however been reported (Renard et al. 1999). According to linkage analysis there is a low degree of branching on the GalpA units in the backbone of GOA2-I (Table II), and whether these units are acetylated or have terminal GlcA/4-O-Me GlcA residues attached is not determined. The Rhap units in the backbone of GOA2-I are carrying side chains through position 4 in about 15% of the residues. This is some what lesser than what is usual for RG-I, normally the substituted Rhap units are in the range of 20–80% of the backbone rhamnose (O’Neill and York 2003). The findings of both 1,3-, 1,4-, and 1,6-linked Galp units in GOA2-I indicates that arabinogalactan type I (AG-I) and type II (AG-II) are present in the RG-I side chains. AG-Is are Araf-substituted derivatives of linear 1,4-linked ß-D-Galp units. Araf and Galp residues may form stubs linked via position 3 along the main chains. The AG-II structural units present in GOA2-I most probably consist of inner chains composed of 1,3-linked Galp units to which galactose residues are substituted in position 6 by shorter outer side chains consisting of 1,6-linked Galp residues. Some of these outer side chains are probably substituted by terminally linked Araf, as the amount of terminal Araf residues is higher than the amount of branched Araf residues (Table II). We further suggest pure arabinans with a 1,5-linked backbone to be attached via a 1,4-linked galactose unit to the rhamnogalacturonan backbone of GOA2-I, with small oligoarabinans attached to the 1,5-linked arabinan backbones in position 3. It is possible that the arabinans could be attached to the arabinogalactan moieties, but as the presence of branched galactose residues is low, we propose that the arabinans are not a part of the AG-II side chains. Pure arabinans indirectly linked to the rhamnogalacturonan backbone through small galactans have previously been reported by Strasser and Amadò (2001). As the ratio of the side chain monomers Gal and Ara to the backbone monomers Rhap and GalpA is relative small, 0.8:1, the side chains in GOA2-I are probably quite short and not very complex in nature. It is still unclear whether AG-II structural units are part of RG-I complexes, but co-elution suggests they are covalently linked in GOA2-I. The length of the RG-I sections in the pectin backbones is not known, but it has been estimated that a RG-I backbone could be a few hundred residues in length (Renard et al. 1999).
The weight average Mw estimated for the intermediate fractions GOA2-II and GOA2-III containing structural residues typical for RG-II were estimated to be <20,000 g/mol but because of the broad polydispersity and small size (near the lower molecular limit for SEC-MALLs) – as confirmed by the sedimentation velocity data – a more definite value could not be obtained. Interestingly molecular weights for other RG-II molecules, have been reported to be in the range of 5000–10,000 g/mol (O’Neill et al. 2004). In general, the molecular weights of pectins are difficult to measure accurately because of the presence of heterogeneous groups along with branched and smooth regions (Daas et al. 2001).
The structure of RG-II has been shown to be virtually the same in every plant analyzed hitherto, the polymer consists of a HG backbone of roughly seven to nine residues bearing five oligosaccharide side chains (O’Neill et al. 2004). The content of 1,4-linked GalpA in GOA2-II and GOA2-III is probably due to backbones of RG-II moieties. Besides the linear 1,4-linked GalpA, the branched residues 1,2,4- and 1,3,4-GalpA are present in both fractions. Several differently linked Rha residues are expected in RG-II molecules, and linkage analysis revealed the presence of terminal-, 1,2-, and 1,3-linked rhamnose in GOA2-II and GOA2-III. Other characteristic constituents of RG-II like 1,2-linked GlcA, 1,2,3-linked Ara, 1,3,4-linked Fuc, 1,2,4-linked Gal, 1,3'-linked Api, 2-O-Me Fuc, and 2-O-Me Xyl were also identified in the fractions. The native polymer GOA2 has previously been analyzed for the presence of RG-II by the thiobarbituric acid-assay (Inngjerdingen et al. 2005). A negative reaction was observed, indicating that the monosaccharides 3-deoxy-D-manno-2-octulosonic acid (Kdo) and 3-deoxy-D-lyxo-2-heptulosaric acid (Dha), and thereby RG-II, was not present in the fraction. This might be due to too low concentrations of the monomers to be detected in the assay. The method for linkage analysis used in this work is known to destroy Kdo and Dha, and it was therefore not possible to identify them together with the other constituents.
Both RG-I and RG-II might be attached to a HG backbone in the native GOA2, but whether RG-I occurs in the same chain as RG-II or whether the domains are attached to HG at the reducing or nonreducing end is, as for other studied pectins, not known (Perez et al. 2003). From the AFM images of GOA2 and GOA-I, both linear and branched molecules seems to be present (Figure 2). The branched structures might however be caused by overlapping of molecules, and further analysis would be necessary to prove the presence of branching. The contour lengths of the molecules in the fractions ranged from <10 to 100 nm, which seems to be reasonable in comparison with previously reported values of pectins. Histogram contour lengths of pectins isolated from tomatoes and peaches, ranged from 30 to 390 and 10 to 330 nm, respectively (Round et al. 2001). Morris et al. (2003) have suggested that a future approach in investigating the pectin structure would be to label specific sites, such as RG-I- or HG- regions, by using inactivated enzymes which bind to their specific binding linkages, and then map their location along individual blocks.
Structure–activity relations
Among the structural moieties of the pectic polymer in GOA2, the RG-I region GOA2-I seems to be responsible for both B-cell proliferating and complement fixing activity, in addition to the proliferation of bone marrow cells through the intestinal immune system. GOA2-I showed a significantly higher effect in the test systems compared to the native polymer fraction GOA2. The fractions GOA2-II and GOA2-III, containing RG-II type structures, were not active in the complement fixing assay or able to induce proliferation of B cells at the range of concentrations tested.
The complement fixation test has been widely used for location of possible immunomodulating compounds of polysaccharide nature. The complement system plays an important role as a primary defense against bacterial and viral infections, and is a critical effector pathway in both adaptive and innate immunity. Regarding pectic polymers and effects on the complement system, there are several reports that the RG-I region of pectins have more potent complement fixing activities compared to the corresponding original pectin. As oligogalacturonides, and neutral oligosaccharides originating from side chains of pectins have been reported to lack bioactivity, it has been postulated that the activities may be due to a combination of the rhamnogalacturonan core and the neutral sugar chains (Yamada and Kiyohara 1999; Nergard et al. 2005). The complement fixation test used here does not differentiate between activation and inhibition of the system, but shows that the complement system is affected by the presence of certain polysaccharides. However, the positive control PMII, a native pectic polymer, has previously been shown to be a complement activator, both in the classical and alternative pathway (Michaelsen et al. 2000).
Previously Nergard et al. (2006) have reported that there appears to be a positive correlation between a high complement fixing activity and B-cell proliferating activity. This also seems to be the case for the polymer fraction GOA2-I, which exhibits a potent activity in both test systems. The branched region of pectins has also been reported to be responsible for proliferation of B cells induced by pectic polymers. The branched region of a pectic polymer isolated from Bupleurum falcatum has been regarded as the possible structural unit for the recognition of carbohydrate receptors on B cells (Sakurai et al. 1999). The antigenic epitopes were characterized to be 6-linked galactosyl chains containing terminal GlcA or 4-O-Me-GlcA, which were substituted to 1,3-linked galactosyl chains. As proliferation of the B cells was induced by GOA2-I alone, we propose that pectins might function as thymus-independent antigens in a similar fashion as LPS.
Decoctions of G. oppositifolius are often taken by oral administration, and it is therefore a possibility that the clinical effect is expressed through the intestinal immune system. Peyer's patches are the important lymphoid organ in the intestine, and are known as inductive sites for IgA production (Hong et al. 1998). As for complement fixing activity and induction of B cells, the RG-I region GOA2-I seems to be responsible for the intestinal immune stimulating activity of the polymer GOA2. An acidic polysaccharide, ALR-b, purified from rhizomes of Atractylodes lancea, has been reported to exhibit modulating activity on Peyer's patches in intestinal immune system (Yu et al. 2001a). Enzymatic treatment of ALR-b with endo-polygalacturonase led to the isolation of RG-I and RG-II structural moieties, in addition to oligogalacturonides. In contradiction to the results seen for GOA2, the RG-I region in ALR-b did not show enhanced activity compared to its native pectin. However, the RG-II polymer was significantly more potent at modulating the intestinal immune system than the native polymer. Due to a low amount of material the RG-II structural moiety of GOA2, GOA2-II, was not elucidated for its effect on Peyer's patches. A polysaccharide consisting mainly of arabinogalactan type II, ALR-5IIa-1–1, purified from rhizomes of Atractylodes lancea, has also been reported to exhibit modulating activity on Peyer's patches in intestinal immune system. The 1,3,6-linked galactan moiety was suggested to play an important role for the expression of activity (Yu et al. 2001b). The effect seen for GOA2-I may be caused by its arabinogalactan type II side chains, which seems to be more complex compared to the RG-I polymer isolated from ALR-b.
Cytokine secretion by macrophages and NK cells can influence the outcome of wound healing or early infections. It was therefore of interest to examine the ability of GOA2 to induce secretion of proinflammatory cytokines from these cells. Marked upregulation in mRNA for the proinflammatory cytokine IL-1ß in R2-M and for IFN- in NK cells were found. IL-1ß is mainly produced by blood monocytes, and mediates the generation of acute phase proteins important in the early host response to infections. Thus GOA2 might be able to boost the early responses by macrophages, by the secretion of IL-1ß. However, we have not studied the actual secretion of cytokine protein, which may have a different pattern than mRNA generation. IFN- is the principal cytokine released by activated NK cells, normally in response to IL-12 released from macrophages or dendritic cells, and is important for initiating a proper T-cell response. GOA2 has previously been shown to induce modest chemotaxis of IL-2-activated human NK cells (Inngjerdingen et al. 2005), and we have also determined that is has no effect on the cytolytic behavior of NK cells (data not shown). Collectively, these results might indicate that GOA2 principally plays a role for cytokine production in NK cells. However, the detection of released cytokines remains to be examined.
This study illustrates that the pectic polymer GOA2 isolated from G. oppositifolius has many characteristics in common with pectins of other sources, consisting of HG regions and branched regions, and that the bioactivities are expressed by the rhamnogalacturonan I (RG-I) region. The presence of immunomodulating polysaccharides in the Malian medicinal plant can at least partly relate to their medical effects. However, further studies are essential in order to determine the structure–activity relations of the branched region of GOA2-I.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataFundingConflict of interest statementReferences |
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Materials
The aerial parts of G. oppositifolius (L.) Aug. DC. (Aizoaceae) were collected in Gourma, Mali, in 1998. The plant was identified by Professor Drissa Diallo, Department of Traditional Medicine (DMT), Bamako, Mali, and a voucher specimen is deposited in the herbarium at DMT. The pectic polysaccharide fraction GOA2 was isolated from the aerial parts of G. oppositifolius as previously described (Inngjerdingen et al. 2005
).
Analysis of carbohydrate content and composition
Neutral sugars and uronic acids were after methanolysis of the polymers converted into trimethylsilyl glycoside derivatives and analyzed by capillary gas chromatography on a Carlo Erba Instruments (Milan, Italy) 6000 Vega Series 2 chromatograph with an ICU 600 programmer (Chambers and Clamp 1971; Barsett et al. 1992), in order to determine the composition and content of carbohydrate in the different polymer fractions. Mannitol as internal standard was included throughout the total procedure.
Degree of acetyl- and methyl esterification
The degree of esterification with methanol and/or acetic acid was determined by saponification followed by methanol and acetic acid separation by high performance liquid chromatography (HPLC) on a C18 column and quantification by refractometry (Levigne et al. 2002). The polymer (5 mg) was saponified by suspension in 0.5 mL of a solution containing 10 mM CuSO4 and 25 mM isopropanol as internal standard; 0.5 mL of 1 M NaOH was added to achieve saponification. The reaction mixture was left at 4°C for 1 h before centrifugation at room temperature. The supernatant was purified by the aid of a 1 mL syringe equipped with a Maxi-clean IC-H device (Alltech, IL) prior to injection on a C18 endcapped column (Superspher 100RP-18, 250 x 4 mm). Elution was carried out with 4 mM H2SO4 at 0.7 mL/min, with refractometric detection. The alkaline saponification has to be performed in a heterogeneous system, copper ions being able to insolubilize pectins was used for this purpose.
Monosaccharide linkage analysis
Linkage elucidation was performed by methylation studies. Prior to methylation, the uronic acids of the polymer fractions were reduced to primary alcohols on the polymer level. To distinguish between reduced uronic acids and the corresponding neutral sugars in GC–MS, sodium bordeuteride was used. Carboxyl esters were first reduced with sodium borodeuteride in imidazole buffer to generate 6,6-dideuteriosugars. The free uronic acids were activated with a carbodiimide and reduced with sodium borodeuteride (Kim and Carpita 1992). After reduction of the polymers, methylation was carried out after the method of Ciucanu and Kerek (1984). The methylation procedure was followed by GC–MS analysis of the derived partially methylated alditol acetates using a Fisons GC 8065 (Fisons Instruments, San Carlos, CA) on a SPB-1 fused silica capillary column (30 m x 0.20 mm i.d.) with film thickness 0.20 µm. E.I. mass spectra were obtained using a Hewlett-Packard (Palo Alto, CA) Mass Selective Detector 5970 with a Hewlett-Packard GC. The injector temperature was 250°C, the detector temperature 300°C and the column temperature 80°C when injected, then increased with 30°C/min to 170°C, followed by 0.5°C/min to 200°C and then 30°C/min to 300°C. Data were processed with Fisons Masslab software. The compound at each peak was characterized by an interpretation of the characteristic mass spectra and retention times in relation to standard sugar derivatives. Effective carbon-response factors were applied for quantification (Sweet et al. 1975).
Determination of phenolic content
The quantitative determination of total phenols was performed with the Folin-Ciocalteu reagent (Singleton and Rossi 1965) with ferulic acid as standard reference. Four hundred microliters of lyophilized sample dissolved in water (three replicates) were added the same amount of Fiolin-Ciocalteu's phenol reagent (1:2 in water, Merck, Kebo, Switzerland), mixed and left for 3 min at room temperature. Four hundred microliters of 1 M Na2CO3 was added, the tubes were mixed and allowed to stand for 1 h. The absorbance was measured at 750 nm in a Helios Epsilon Spectrophotmeter (Thermo Spectronic, Waltham, MA). The standard curve was plotted using ferulic acid. The total phenolic content was determined as ferulic acid equivalents (FA/sample) x 100%.
Degradation by endo--D-(14)-polygalacturonase
The polymer GOA2 (500 mg) was dissolved in 50 mL of 0.05 M NaOH for de-esterification, and left for 24 h at 0°C. The solution was neutralized by adding a few drops of acetic acid. The de-esterified sample (5 mg/mL) in 50 mM acetate buffer, pH 5.0, was treated with endo--D-(14)-polygalacturonase from Aspergillus japonicus (430 units/mg protein, EC 3.2.1.15
[EC]
) (Sigma, St. Louis, MO) at 30°C. One unit of the enzymatic solution liberates 1.0 µmole of galacturonic acid from polygalacturonic acid per minute. The hydrolysis proceeded until it was complete (26 h). This was determined by the increase of reducing sugars in a reaction mixture using dinitrosalicylic acid (DNS) (Miller 1959, modified by Knutsen 1991). The reaction was terminated by heating at 100°C. The de-esterified and partially hydrolyzed material was fractionated by size exclusion chromatography on a BioGel P30 (2.5 x 90 cm, Bio-Rad Laboratories, Richmond, CA) column. The column was coupled to a Peristaltic pump P-3 (Pharmacia, Uppsala, Sweden) and a Pharmacia LKB FRAC 100 fraction collector and eluted with 50 mM acetate buffer, pH 5.0, at 30 mL/h. The carbohydrate profile obtained was determined using the phenol sulfuric acid assay, and the relevant fractions pooled (Dubois et al. 1956).
NMR
The 2D NMR NOESY, ROESY, and HMBC spectra of GOA2-I dissolved in D2O at 70°C was obtained on a Bruker DRX-600 spectrometer (Bruker, Fällanden, Switzerland). The chemical shift of D2O (H 4.30) was used a reference for 1H-NMR.
Sedimentation velocity in the analytical ultracentrifuge
Sedimentation velocity experiments were performed using an Optima XL-I analytical ultracentrifuge (Beckman Instruments, Palo Alto, CA). Reference solvent (400 µl) and sample solution (380 µl) were injected into the solvent and sample channels of 12 mm carbon filled centerpieces, and loaded into a 8-hole titanium rotor. Samples were centrifuged at 40,000 rpm and 20.0°C. Data were analyzed using the least squares g*(s) method in the algorithm SEDFIT (Schuck and Rossmanith 2000; Harding 2005). s20,b values were then corrected to standard solvent conditions (density and viscosity of water at 20.0°C) to yield s20,w (S) using the computer algorithm SEDNTERP based on Laue (1992).
SEC-MALLs
SEC-MALLs (Harding et al. 1991; Wyatt 1992) was performed using an HPLC pump (Model PU-1580, Jasco Corporation, Tokyo, Japan) and a Rheodyne injection valve (Model 7125, Rheodyne, St. Louis, MO) fitted with a 100 µL loop. Analytical fractionation was carried out using a series of SEC columns TSK 46000PW and TSK G3000PW protected by a similarly packed guard column (Tosoh Bioscience, Tokyo, Japan). The intensity of scattered light was detected using a Dawn DSP multi-angle laser light scattering photometer and concentration was determined using an Optilab 903 interferometric refractometer (both instruments from Wyatt Technology, Santa Barbara, CA) with phosphate buffered saline (PBS) at pH 7.0 (I = 0.1 M) as mobile phase. The SEC-MALLs system was calibrated overnight at flow rate of 0.8 mL/min at room temperature. Purified samples were dissolved in PBS (pH 7.0, I = 0.1 M), and filtered through 0.45 µm filters (Whatman, Maidstone, UK) and injected (100 µL) at a flow rate of 0.8 mL/min. Repeated injections were made for each sample for reproducibility in the measurements. Signals from the light scattering photometer and the refractometer were captured and analyzed based on the Debye model (Wyatt 1992) using the ASTRATM (for Windows XP) software supplied by the manufacturer. A refractive index increment (dn/dc) for pectins of 0.146 (Chapman et al. 1987; Theisen et al. 2000) was used.
Atomic Force Microscopy
For imaging, the pectin fractions were dispersed into distilled water to a concentration of 1 mg/mL, and further diluted to 10 µg/mL. Aliquots (10 µL) of the diluted samples were deposited onto sheets of mica, and allowed to dry under ambient conditions before imaging by AFM in air. AFM imaging was performed using a Multi-Mode Atomic Force Microscope (Veeco Instruments, Rochester, NY) with a Nanoscope IIIa controller, operated in tapping mode. Silicon cantilevers (Olympus, Tokyo, Japan) with spring constant of about 40 N/m were employed for all images. Most images were acquired at a scan speed of 5000 nm/s (2.5 Hz over a 1000 nm x 1000 nm area), although some smaller scans were obtained at 2500 nm/s (2.5 Hz over a 500 nm x 500 nm area). Analysis of feature areas and lengths were made using the software SPIP (Image Metrology, Hørsholm, Denmark).
Complement fixation assay
The polymers effect on human complement was measured by complement consumption and degree of lysis of antibody sensitized sheep red blood cells by the residual complement (Method A in Michaelsen et al. 2000). A pectic polysaccharide, PMII, from P. major L. was used as a positive control (Samuelsen et al. 1996). Inhibition of hemolysis induced by the test sample was calculated by the formula: [(Acontrol – Atest)/Acontrol] x 100%.
B-cell proliferation
A total of 8- to 12-week-old rats of the PVG.7B strain (which possesses a ‘nonimmunogenic’ CD45 allotype, RT7b, but is otherwise interchangeable with the standard PVG strain RT7a) have been maintained at the Institute of Basic Medical Sciences for more than 20 generations. Rats were maintained under conventional conditions and regularly screened for common pathogens. The animals were housed in compliance with guidelines set by the Experimental Animal Board under the Ministry of Agriculture of Norway. Peripheral blood mononuclear cells were isolated from rat spleen cell suspensions by layering onto Lymphoprep (Nycomed, Norway) and spinning for 20 min at 1800 rpm (170 x g). B cells were isolated by positive selection using sheep anti-rat IgG Dynabeads. Cells were harvested and incubated overnight in complete RPMI (cRPMI; RPMI 1640, 10% FCS, 5 x 10–5 M 2-ME, L-glutamine, and antibiotics) in order to induce release of the Dynabeads from the cells prior to functional analysis. The cells were screened by flow cytometry, and were more than 90% positive for the B-cell marker CD19. The purified B cells were suspended in 2% FCS in PBS at 1 x 107 cells/mL. Cells were preheated for 1 min at 37°C, then stained with 5 µM CFSE (Moleculare Probes, Carlsbad, CA) for 10 min at 37°C. The cells were washed twice in PBS, and then resuspended at 2 x 106 cells/mL in cRPMI. One hundred microliters (2 x 105 cells) were seeded into flat bottom 96-wells and 100 µL of medium or samples were added. The cells were harvested after 5 days, and analyzed by flow cytometry.
Intestinal immune system modulating activity
Specific-pathogen-free C3H/HeJ female mice were purchased from SLC (Shizuoka, Japan) and used at 6–8 weeks of age. The mice were maintained under specific pathogen-free conditions and given free access to standard laboratory chow (CE-2, CLEA, Inc., Japan) and water. The procedure from the Prime Minister's Office of Japan (No. 6 of March 27, 1980) for the care and use of laboratory animals was followed. The experiments were conducted in accordance with the Guidelines for Animal use and Experimentation of the Kitasato Institute, Tokyo. The intestinal immune system modulating activity was measured as proliferation of bone marrow cells as stimulated by the conditioned medium of Peyer's patch cells (Hong et al. 1998). Briefly, C3H/HeJ mice were sacrificed by cervical dislocation and their small intestines were exposed on sheets of clean paper. The Peyer's patches were carefully dissected out using fine scissors from the wall of the small intestine, and placed in ice-cold RPMI-1640 medium in a flat-bottomed Petri dish. The Peyer's patch cells were dispersed by tapping gently with a rubber rod on a 150-gauge sterile stainless sieve. The cell suspensions were passed through a 200-gauge sterile stainless sieve, washed three times with RPMI-1640, and then resuspended in the same medium at a density of 2 x 106 cells/mL. Two hundred microliters of the cell suspension were cultured with 20 µL of test samples (final concentration of 10 and 100 µg/mL) in a 96-well flat bottom microtiter plate for 5 days at 37°C in a humified atmosphere of 5% CO2–95% air. ALR-5IIa (1 mg/mL) from the rhizomes of Atractylodes lancea DC. Yu et al. (1998) was used as positive control and RPMI-1640 as blank. The experiment was performed in triplicates. The resulting culture supernatants (conditioned medium) were used for stimulation of bone marrow cells. Bone marrow cells were obtained from the femora of C3H/HeJ mice. The mice were sacrificed by cervical dislocation, the femora were excised and flushed of bone marrow cells using a 23-gauge needle and then suspended in RPMI 1640 supplemented with 5% FBS (RPMI-FBS). The cells were washed and resuspended in RPMI-FBS at a density of 2.5 x 105 cells/mL. The resulting culture supernatant (10 µL) of Peyer's patch cells and 90 µL of medium (RPMI-FBS) was incubated with 100 µL of bone marrow cell suspension (2.5 x 105 cells/mL) for 6 days in a humified atmosphere of 5% CO2–95% air in order to evaluate the ability for the growth of bone marrow cells. Proliferation of bone marrow cells was measured by the Alamar BlueTM reduction assay. At 8 h prior to culture termination, 20 µL of Alamar BlueTM solution (Alamar Bio-Sciences Inc., Sacramento, CA) was added to each well, and the cells were then continuously cultured. To count cell numbers, the fluorescence intensity was measured by Fluoroskan II (Labosystems, Tokyo, Japan) at an excitation wavelength of 544 nm and emission wavelength of 590 nm. The delta soft II (Ver 4.13 FL, BioMetallics, Inc., Princeton, NJ) was used for data management. The results are expressed as the mean ± SE. The difference between the control and the treatment in these experiments was tested for statistical significance by Student's t-test. A value of P < 0.05 was considered to indicate statistical significance.
Measurement of cytokine mRNA by RT-PCR
RNK-16 cells (a rat leukemic NK cell line) or R2-M (a rat macrophage cell line), were suspended in complete RPMI medium and stimulated overnight at 37°C with 100 µg/mL of GOA2 or medium alone. Total RNA was isolated with TriReagent (Sigma-Aldrich), and cDNA was generated with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) according to manufacturers protocol. PCRs were performed on a GeneAmp PCR thermocycler (Applied Biosystems, Princeton, NJ) using hot start for 3 min at 94°C. Dynazyme polymerase (Finnzymes, Espoo, Finland) was added at 80°C. The following upper and lower primers, respectively, were used: IL-1ß, 5'-TGAAAGCTCTCCACCTCAATGGAC-3' and 5'-TGCAGCCATCTTTAGGAAGACACG-3' (Tm 58°C, 40 cycles);
IL-6, 5'-TCTGGAGTTCCGTTTCTACCTGG-3' and 5'-CATAGCACACTAGGTTTGCCGAG-3' (Tm 55°C, 40 cycles); TNF-, 5'-AGCACAGAAAGCATGATCCGAG-3' and 5'-CCTGGTATGAAGTGGCAAATCG-3' (Tm 55°C, 39 cycles); IFN-, 5'-GTTACTGCCAAGGCACACTCATTGAAAGCC-3' and 5'-TCAGCACCGACTCCTTTTCCGCTTCCTTAGGC-3' (Tm 53°C, 40 cycles); CD45, 5'-CGGGGTTGTTCTGTG CTCTGTTC-3', and 5'-CTTTGCTGTCTTCCTGGGCTTTGT-3' (Tm 67°C, 30 cycles). PCR products were resolved by agarose gel electrophoresis (1% Tris–borate–EDTA).
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Supplementary data |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataFundingConflict of interest statementReferences |
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Supplementary data for this article is available online at www.glycob.oxfordjournals.org.
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataFundingConflict of interest statementReferences |
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The first author acknowledges receiving grants from the Norwegian Research Council and the Bonnevie's Foundation to study in Japan, and from COST (European Co-operation in the Field of Scientific and Technical Research) to perform SEC/MALLS and AFM studies at the University of Nottingham. This project is a part of the NUFU project PRO 22/2002.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataFundingConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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The authors are indebted to Finn Tønnesen, School of Pharmacy, University of Oslo, for performing the GC–MS analysis, Ellen H. Cohen for running the HPAEC-PAD, and Torunn H. Aslaksen Liljebäck for performing and analyzing the data on the degree of esterifaction.
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Abbreviations |
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AFM, atomic force microscopy; AG-I, arabinogalactan typeI; AG-II, arabinogalactan type II; Api, apiose; Ara, arabinose; FBS, fetal bovine serum; Fuc, fucose; Gal, galactose; GalA, galacturonic acid; Glc, glucose; GlcA, glucuronic acid; GC, gas chromatography; HG, homogalacturonan; HPAec, high performance anion exchange chromatography; HPLC, high performance liquid chromatography; LPS, lipopolysaccharide; MALLS, matrix assisted laser desorption ionization; Man, mannose; MS, mass spectroscopy; PAD, pulsed amperometric detection; PBMC, peripheral blood mononuclear cells; PBS, Dulbecco's phosphate buffered saline; PMII, pectin fraction from the leaves of P. major L. (positive control); RG-I, rhamnogalacturonan I; RG-II, rhamnogalacturonan II; Rha, rhamnose; SEC, size-exclusion chromatography; SRBC, sheep red blood cells; Xyl, xylose
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