Chemo-enzymatic synthesis and in vitro cytokine profiling of tailor-made oligofructosides
© Homann et al.; licensee BioMed Central Ltd. 2012
Received: 15 June 2012
Accepted: 16 November 2012
Published: 26 November 2012
It is well known that carbohydrates play fundamental roles in cell signaling and infection processes as well as tumor formation and progression. However, the interaction pathways and cellular receptors targeted by carbohydrates and glycoconjugates remain poorly examined and understood. This lack of research stems, at least to a major part, from accessibility problems of large, branched oligosaccharides.
To test glycan - cell interactions in vitro, a variety of tailored oligosaccharides was synthesized chemo-enzymatically. Glycosyltransferases from the GRAS organisms Bacillus megaterium (SacB) and Aspergillus niger (Suc1) were used in this study. Substrate engineering of these glycosyltransferases generally acting on sucrose leads to the controlled formation of novel tailored di-, tri- and tetrasaccharides. Already industrially used as prebiotics in functional food, the immunogenic potential of novel oligosaccharides was characterized in this study. A differential secretion of CXCL8 and CCL2 was observed upon oligosaccharide co-cultivation with colorectal epithelial Caco-2 cells.
Pure carbohydrates are able to stimulate a cytokine response in human endothelial cells in vitro. The type and amount of cytokine secretion depends on the type of co-cultivated oligosaccharide.
KeywordsOligofructoside Glycosyltransferase Suc1 Aspergillus niger SacB Bacillus megaterium CXCL8 (IL-8) CCL2 (MCP-1) Caco-2
Inflammation processes are essential for the immune system of a host organism attacked by bacteria, viruses or other immunogenic molecules. However, persistent inflammation is a pathologic indication. The intestine, being the largest barrier of the human body to the environment, is under a state of persistent controlled inflammation because of its permanent contact with the gut microbiota. Intestinal epithelial cells release cytokines and chemokines upon external stimulation, e.g. by bacteria and their surface structures . The factors which trigger inflammation and the release or suppression of cytokines and chemokines have been investigated thoroughly over the last decade, but the process is still not fully understood. Clearly, cytokine secretion can be triggered by lipopolysaccharide (LPS) on the surface of Gram-negative bacteria [2, 3] or capsular polysaccharides and lipoteichoic acid from Gram-positive species [4, 5].
Oligo- and polysaccharides containing fructose have been known for several years as prebiotics [6, 7]. Fructose recently was described as a signaling molecule and lead structure for carbohydrates with enhanced antigenicity in HIV vaccination . The extent of the fructan oligo- and polymerization was described as controllable in an enzymatic synthesis process . Fructosyltransferases like inulosucrases and levansucrases which synthesize fructans of various chain lengths are common in many different bacteria including the gut microbiota . The challenges to access large, branched oligosaccharides using chemical synthesis, may be overcome using chemo-enzymatic approaches [11–13]. Sucrose analogues synthesized by SacB from B. megaterium were used as precursors for the synthesis of oligofructosides with the fructosyltransferase Suc1 from A. niger. For the present study, the enzymatic synthesis process was scaled up to yield biological test amounts. The tailored oligofructosides tested in this study are capped by the monosaccharides d-glucose, d-mannose, d-galactose, d-fucose or d-xylose, elongated with fructosyl units under tight control of the degree of polymerization. These oligofructosides are supposed to mimic the structural characteristics of immunogenic carbohydrate patterns of antigens, thus triggering the release of cytokines and/or chemokines.
Chemo-enzymatic synthesis of novel oligofructosides by substrate engineering of fructosyltransferases
Reaction times and yields for the oligosaccharide synthesis by the fructosyltransferase Suc1 from A. niger
[% mol mol-1]
Oligofructoside-stimulated Caco-2 cells differentially secrete CXCL8 and CCL2
Co-incubation of human epithelial Caco-2 cells with certain types of pure, unconjugated oligofructosides leads to enhanced secretion of CXCL8 and CCL2. CXCL8 is a potent inflammation marker recruiting neutrophils to sites of infection. It is secreted by various cell types including epithelial cells . CCL2 is described as an effective chemoattractor for monocytes from the blood stream . Our results show that CCL2 release can be clearly triggered by the tetrasaccharide 1-nystose and even more enhanced by the nystose analogues Man-Fru3 and Fuc-Fru3. The observed differential cytokine secretion pattern raises questions: Is this stimulating effect dependent on the carbohydrate structure and if so, which structural elements trigger or suppress the release of cytokines and chemokines? Because of the differential secretion pattern and only two significant signals (out of 25 investigated), it was shown that cytokine secretion by Caco-2 cells in this assay is dependent on the oligofructoside type. But why are just 2 significant signals of secreted proteins detectable? One possibility is, that epithelial cells in the intestine only have a restricted repertoire of cytokines to be synthesized and secreted. Another point is, that this cell type is in constant contact with ubiquitous nutrients and commensal gut bacteria. Hence, it may have evolved tolerance against certain alien structures. The intestine being the largest barrier of the human body to the environment, has a special set of immunologically active cells. This area is under a state of persistent controlled inflammation because of its permanent contact with the gut microbiota. Special intestinal macrophages (IMACs) mediate tolerance to beneficial gut bacteria. Perturbations of these processes, like the release of CCL2, inhibit the differentiation of macrophages to IMACs thus leading to (chronic) inflammatory bowel diseases (IBDs) . Intestinal epithelial cells release cytokines and chemokines upon external stimulation, e.g. by bacteria and their surface structures . The factors which trigger inflammation and the release or suppression of cytokines and chemokines have been investigated thoroughly over the last decade, but the process is still not fully understood.
In this study, mannose- and fucose-capped oligofructosides generally evoke the highest increase in CCL2 and CXCL8 release (Figure 4). This might be due to their participation in natural cell-cell communication processes. Fucose often is a branching carbohydrate unit e.g. in the Lewis X motif. This motif is known as immunogenic under certain conditions, e.g. incomplete sialylation. Mannose is part of the core N-glycan structure. Its exposition often leads to the release of cytokines, e.g. CCL2 in mannosidase knock-out mice . Interestingly, the different monosaccharide cap structure of the fructosyl backbone is not the only factor influencing the release of CXCL8 and CCL2, but also the length of the fructosyl backbone. For example, CCL2 secretion is triggered by 1-nystose and its tetrasaccharide analogues Man-Fru3 and Fuc-Fru3 but suppressed by kestose and its analogue Man-Fru2 (Figure 4). Thus, stereochemical and spatial aspects of oligosaccharides obviously have to be considered in terms of cell signalling processes. Recently, it was described that the different shape of bacterial lipopolysaccharide (LPS) determines which receptor is targeted and thus how cell signalling is processed [23–25]. The potential target receptors which are known to act competitively are shown in Additional file 1: Figure S1. The differential secretion of cytokines and thus the induction of an inflammatory response by the interaction of these receptors is still a scientific area with many long-standing questions.
Carbohydrates are ubiquitious structures on the surface of a plethora of different cell types including potentially pathogenic and beneficial gut bacteria. Auto-immune diseases like Crohn`s disease are linked to persistent, pathologic inflammation. As abundant surface structures of host and pathogen cells, carbohydrates may play an important role in the induction of inflammation and tolerance, respectively. Advances in carbohydrate research in combination with cell biology and immunology methods may lead to a detailed understanding of inflammation processes. The pure, tailored carbohydrate structures examined in this study induce such a differential secretion of cytokines in endothelial cells in vitro. Further advances in oligosaccharide synthesis lead broadened possibilities to investigate in vivo inflammation mechanisms of carbohydrate-cell receptor crosstalk. Controlled stimulation of the immune system may be one component towards a successful treatment of auto-immune diseases.
Chemo-enzymatic synthesis of tailored oligofructosides
The fructosyltransferases from the GRAS organisms B. megaterium (SacB) and A. niger (Suc1) were used for the synthesis of a fructosyl-based carbohydrate backbone capped with different types of monosaccharides (glucose, galactose, mannose, fucose and xylose).The oligofructosides were synthesized in two steps. First, sucrose analogues were synthesized by the fructosyltransferase SacB from B. megaterium. After analysis and purification, the elongation reaction was performed by the fructosyltransferase Suc1 from A. niger.
Synthesis and purification of sucrose analogues by the fructosyltransferase SacB from bacillus megaterium
For the synthesis of sucrose analogues, the acceptor monosaccharide was used in a concentration of 1.2 M. The transfructosylation reaction was performed with added sucrose (600 mM) in phosphate buffer after Sörensen (50 mM, pH 6.6). SacB was applied in a final concentration of 10 mg l-1 at 200 rpm and 37°C for 2 h in a 1.5 ml or 15 ml reaction tube. The resulting sucrose analogues were analyzed qualitatively and quantitatively by thin-layer chromatography (TLC, 2.2) and high-performance anion exchange chromatography (HPAEC, 2.3). The purification of the sucrose analogues was performed by a silica column with a carbohydrate-containing mobile phase (60% ethylacetate, 30% isopropanol, 10% water, all v/v). The products were analyzed by TLC and HPAEC.
Synthesis and purification of 1-kestose, 1-nystose and analogues by the fructosyltransferase Suc1 from aspergillus Niger
The subsequent synthesis step of 1-kestose, 1-nystose and their analogues was performed by the fructosyltransferase Suc1 from A. niger as described previously with the sucrose analogues synthesized by SacB from B. subtilis . Briefly, the supernatant of a cultivation of A. niger SKAN1015 was used in a dilution of 1:50 (v/v). The Suc1 dilution was mixed with 500 mM of the sucrose analogue to be converted in Sörensen`s phosphate buffer (50 mM, pH 5.6). The reaction was performed at 45°C and 200 rpm. The reaction time depends on the desired oligofructoside to be synthesized .The purification of 1-kestose, 1-nystose and their analogues was performed by size exclusion chromatography. An open chromatography gel filtration system was used (Biogel, Bio-Rad) and degassed water containing the carbohydrates to be separated as mobile phase.
Analysis of carbohydrates by thin-layer chromatography (TLC)
The sample was diluted to a total carbohydrate concentration of 1–3 g l-1. 3 μl of the sample was applied on a TLC plate (TLC aluminium foil coated with silica 60, 20 x 20 cm with concentration zone, Merck). After drying the TLC was run in a TLC chamber equilibrated with the mobile phase. After 45 min the plate was dried and again incubated for 45 min. The staining of the carbohydrates was performed by a short dive into the developing solution (sulfuric acid 5% (v/v) N-(1-naphtyl) ethylendiamine dihydrochloride 0.3% (w/v) in methanol) and incubation at 150°C for 5 min. An appropriate standard has to be applied each time (here: glucose 0.1 g l-1, fructose 0.1 g l-1, sucrose 0.1 g l-1, 1-kestose 0.1 g l-1, 1-nystose 0.1 g l-1).
Analysis of carbohydrates by high-performance anion exchange chromatography (HPAEC)
HPAEC eluent gradient program
0 - 5 min
0% 1 M NaAc
5 - 25 min
to 25% 1 M NaAc
25 - 30 min
to 50% 1 M NaAc
30 - 35 min
50% 1 M NaAc
35 - 37 min
to 0% 1 M NaAc
37 - 60 min
0% 1 M NaAc
Co-cultivation of Caco-2 cells with tailor-made oligofructosides
Caco-2 cells were cultivated in Dulbecco`s modified Eagle`s medium (DMEM)/HamsF12 (Gibco) supplied with 10% fetal calf serum (FCS) and 200 μg l-1 ampicillin at 37°C and 5% CO2. At 80% confluency, cells were split in a ratio of 1:10. For the oligofructoside assay, Caco-2 cells at 80% confluence were cultivated in 24-well dishes (Gibco). The split ratio was 1:10 and each well was supplied with the carbohydrate to be tested in a concentration of 25 μM. After 48 h, from each well a sample of the media supernatant was collected for cytokine analysis.
Cytokine and chemokine detection assay
For the oligofructoside assay, Caco-2 cells at 80% confluence were split as described and cultivated in 24-well dishes (Biochrom). Each well was supplied with the oligofructoside to be tested (final concentration 25 μM). After 48 h (80% confluence) the supernatant medium was collected for cytokine analysis. The assay was performed with a 25-plex human cytokine analysis kit according to the manufacturer`s instructions (Biosource, Invitrogen). Briefly, the supernatant medium was incubated with antibody-functionalized beads and detected with biotinylated secondary antibodies. Streptavidin-R-phycoerythrin was used as fluorescence marker. The final analysis was performed by the luminex system which recognizes spectral properties of the beads and quantifies the bead load by the specific fluorescence intensity. 25 cytokines were analyzed in parallel per sample (Eotaxin, GM-CSF, IFN-α, IFN-γ, IL-1RA, IL- 1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40/p70, IL-13, IL-15, IL-17, IP-10, MCP-1, MIG, MIP-1α, MIP-1β, RANTES, TNF-α) according to the manufacturer`s instructions (luminex system, Qiagen).
This work was performed within the Collaborative Research Centre (SFB) 578 supported by the German Research Foundation (DFG). The authors gratefully acknowledge excellent support with the luminex analysis system by Bastian Pasche (Helmholtz-Centre for Infection Research, Germany).
This publication was funded by the German Research Foundation (DFG) and the University of Wuerzburg in the funding programme Open Access Publishing.
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