Saccharomyces cerevisiae chitin biosynthesis activation by N-acetylchitooses depends on size and structure of chito-oligosaccharides
© Becker et al; licensee BioMed Central Ltd. 2010
Received: 5 July 2011
Accepted: 27 October 2011
Published: 27 October 2011
To explore chitin synthesis initiation, the effect of addition of exogenous oligosaccharides on in vitro chitin synthesis was studied. Oligosaccharides of various natures and lengths were added to a chitin synthase assay performed on a Saccharomyces cerevisiae membrane fraction.
N-acetylchito-tetra, -penta and -octaoses resulted in 11 to 25% [14C]-GlcNAc incorporation into [14C]-chitin, corresponding to an increase in the initial velocity. The activation appeared specific to N-acetylchitooses as it was not observed with oligosaccharides in other series, such as beta-(1,4), beta-(1,3) or alpha-(1,6) glucooligosaccharides.
The effect induced by the N-acetylchitooses was a saturable phenomenon and did not interfere with free GlcNAc and trypsin which are two known activators of yeast chitin synthase activity in vitro. The magnitude of the activation was dependent on both oligosaccharide concentration and oligosaccharide size.
Chitin, one of the essential fungal cell wall components, is a β-1,4 linked N-acetylglucosamine (GlcNAc) homopolymer. Since it is absent in plants and mammals and has an important structural role in the fungal cell wall, its biosynthesis is recognized as a valuable target for fungicides . Chitin is synthesized by multiple membranous isoenzymes called chitin synthases (CHS) . In Saccharomyces cerevisiae, the organism in which chitin biosynthesis has been most studied, three differentially expressed genes code for three different proteins belonging to the family of glycosyltransferases 2 [3, 4]. CHS1p, is responsible for only 10% of the in vivo chitin pool, but accounts for most of the chitin synthase activity determined in vitro. This activity is enhanced by trypsin and GlcNAc .
CHS are processive glycosyltransferases capable of successively transferring GlcNAc monomers from the UDP-GlcNAc donor to a growing polymer acceptor. Elongation takes place at the non reducing end of the polymer [5, 6]. CHS isoenzymes belong to a subfamily of processive enzymes involved in polysaccharide biosynthesis such as cellulose and hyaluronan synthases [7, 8]. The 3D-structure of a non-processive member of glycosyltransferases family 2 had shed some light on both their donor and acceptor sites . In contrast, due to the lack of structures, donor and acceptor sites have not been delineated in the case of polysaccharide synthases. While the donor sites can be inferred from sequence comparison between processive and non-processives glycosyltransferases, the way polysaccharide synthases accommodate the growing polysaccharide is totally unknown.
As for any polymer, chitin formation is expected to require chain initiation, elongation and termination. Several mechanisms of polysaccharide priming by oligosaccharides, either in a free or conjugated form, have been described, specifically Carbohydrate polymerase GlfT2 from Mycobacterium tuberculosis acts on glycolipid acceptors for the galactan polymer synthesis . Also, experiments on cellulose synthase from cotton fiber led to the proposal of a model in which cellulose synthesis would be initiated by a sitosterol-β-glucoside . Important outcomes of this model were the fact that a certain number of discrete steps should precede the final elongation, and the existence of an oligoglucoside species as a discrete intermediate. Free glycoside utilisation, as primers, are described for other glycosyltransferases, such as β-D-xylosides used for glycosaminoglycans chain biosynthesis on proteoglycan core proteins . Other polysaccharide synthases responsible for the addition of a sugar monomer to oligosaccharide acceptors have been described for hyaluronan , chondroitin sulfate , heparan sulfate , glycogen  and heparosan . Also, high concentrations of GlcNAc were shown to prime N-acetylchitopentaose synthesis by the oligosaccharide synthase NodC  for the synthesis of rhizobial lipo-chitin oligosaccharides (Nod factors). Concerning chitin synthase, a priming by GlcNAc has never been observed and GlcNAc is thought to be an allosteric activator [18–20]. McMurrough and Bartnicky-Garcia tested a range of compounds structurally related to GlcNAc but none successfully substituted for GlcNAc in the allosteric activation of chitin synthesis, although N-acetylchitooses exerted a slight stimulation of substrate incorporation supposed to a different mechanism as GlcNAc .
In order to elucidate the role of oligosaccharides, a systematic study was carried out with preformed oligosaccharides on a chitin synthase assay on S. cerevisiae microsomal preparations. We used oligosaccharides of various degrees of polymerization (DP) and from different structures as well as N-acetylchitooses (β-(1,4) GlcNAc units) or β-(1,4), β-(1,3) or α-(1,6) glucooligosaccharides.
All reagents were purchased from Sigma and Merck Biochemicals, unless indicated otherwise in the text. UDP-N-acetyl[14C]glucosamine was from NEN Life Sciences Products. N-acetylchitooses were either obtained from Dr R. Geremia (CERMAV Grenoble, France) or purchased from Sigma. Both batches were of identical purity (>95%).
Preparation of membrane fractions
Saccharomyces cerevisiae cells (baker's yeast, 10 g) were washed and resuspended in 10 mL of 50 mM Tris-HCl (pH 7.4) - 2.5 mM MgCl2. After addition of glass beads (0.25-0.50 mm, 10 mL), cells were broken by shaking on a vortex for 7 cycles of 30 s alternated with 30 s cooling on ice. After decantation of the solution, the supernatant was recovered, centrifuged at 1000 × g for 10 min and then at 12 000 × g for 10 min to remove mitochondria. Membrane fractions were isolated from this extract by centrifugation at 100 000 × g for 1 h. The membrane pellet was resuspended in approximately 500 μl of 50 mM Tris-HCl (pH 7.4), 30% glycerol. Membrane preparations were conserved at -80 °C at protein concentration around 60 mg/ml, as determined by Bradford protein assay (Biorad) .
Preparation of laminarin and dextran oligosaccharides
Laminarin and dextran (1 g of each) were dissolved in 10 ml 0.1 N HCl and acid hydrolysis performed at ebullition for 4 h. Mixtures were diluted with water, neutralized with Dowex 2 × 8-200 resin (hydroxyl charged) and lyophilized. Oligosaccharides with DP from 3 to 6 were obtained after Bio-gel P2 chromatography purification as previously described for N-acetylchitooses .
Chitin synthase assay
Under our assay conditions, the main enzymatic activity is CHS1p activated by trypsin . At 150 μg of S. cerevisiae membrane fractions containing chitin synthase activity a common standard reaction mix (in a total volume of 50 μl: 5 μl digitonin 2%, 5 μl magnesium acetate 50 mM, 5 μl trypsin 0,2 mg/mL (10000 units/mg protein, Sigma), 2 μl N-acetylglucosamine 1 M, 2.5 μl UDP-N-acetylglucosamine 10 mM, 1 μl UDP-N-acetyl[14C]glucosamine (10 nCi, 288 mCi/mmol) in Tris-HCl buffer 25 mM pH = 7.4) was added. Incubation was performed at 30°C for 10 min, the reaction was stopped with 1 ml of 10% trichloroacetic acid. The radioactivity in the insoluble fraction was counted after filtration through glass-fiber filter (Whatman, GF/C). The soluble fraction containing unincorporated UDP-N-acetyl[14C]glucosamine was discarded. The filter was washed three times with 1 ml of 1 M acetic acid/ethanol: 30/70 and once with 1 ml of 95% ethanol. The filter was transferred to a scintillation vial containing 4 ml of Optiphase Hisafe (Wallac) and the samples were counted in a scintillation counter (LKB 1214 RackBeta).
To determine the effect of adding different oligosaccharides chain lengths to the chitin synthase assay, membrane fractions were preincubated with either β-(1,3), β-(1,4) or α-(1,6) glucooligosaccharides or N-acetylchitoses (from bioses to octaoses) in 25 mM Tris-HCl pH 7.4 for 15 min at 30°C before addition of the common standard reaction mix. Oligosaccharides were used at a concentration of 1 mM unless otherwise stated.
Chitin digestion by chitinase
[14C]-chitin, prepared as described above, was digested by Streptomyces Griseus chitinase (5.10-3U, 0.7 U/mg, Sigma) at 37°C for 24 h in phosphate buffer 2 mM pH = 6. Samples were boiled for 5 min and 1/5 of the sample was applied to TLC plates. Samples were analysed on precoated TLC plates silica gel 60, Merck. The chromatogram was developed with 1-propanol/water/ammonium hydroxide 30% (70:30:1.5, vol/vol/vol). Radioactive spots were detected by means of an automatic TLC-linear analyzer (LB 2821 Berthold).
Effect of GlcNAc and trypsin
The N-acetylchitose activation effect was tested in absence, or at low (1 mM) GlcNAc concentration as described above. Alternatively, activation by trypsin was carried out before polymerization. In this case, membrane fractions were preincubated at 30°C with trypsin from bovine pancreas (1 μg, 10000 units/mg protein, Sigma) in 25 mM Tris-HCl pH 7.4. After 15 min, soybean trypsin inhibitor (2 μg, Sigma) was added and incubation followed for 15 min.
Results and Discussion
Catalytic activity of CHS
Per cent activation of [14C]-GlcNAc incorporation into chitin polymer in presence of oligosaccharides from different lengths and structures.
13,3% ± 2
2% ± 1,1
10% ± 1,5
1,6% ± 1,2
2% ± 1,3
17,3% ± 0,8
0,5% ± 0,5
1,5% ± 1,2
23,3% ± 1,1
1% ± 0,8
2,1% ± 1,5
3% ± 2
24,4% ± 1,2
Effect of oligosaccharide structure and length on chitin synthesis
N-acetylchito-bi and - trioses in the CHS test mixture had little effect on chitin synthesis (enhancement around 11%), and these results were poorly reproducible (Table 1). By contrast, N-acetylchito-tetra, - penta and - octaoses clearly stimulated GlcNAc incorporation from UDP-[14C]-GlcNAc into chitin, reaching up to 25% enhancement for the N-acetylchitooctaoses (Table 1). A clear-cut effect of the oligosaccharide size on the extent of activation was observed.
Oligosaccharides from other carbohydrate series such as laminari-(β-(1,3-gluco)), dextro-(α-(1,6-gluco))- or cello-(β-(1,4-gluco))-oligosaccharides did not stimulate [14C]-GlcNAc unit incorporation into [14C]-chitin (Table 1). The lack of effect of oligosaccharides from other series argues in favor of a specific effect of GlcNAc oligomers. Moreover, in order to achieve acceptable activation reproducibility, the N-acetylchitooses must be of at least 4 residues.
Effect of chitooligosaccharides versus chitin synthase activators
Interference between the effect of N-acetylchitooses and activation by trypsin was also studied. In standard test conditions, microsomal preparations were activated by trypsin at the beginning of the reaction. In a parallel protocol, a preactivation of the microsomal preparation by trypsin was performed, then terminated by addition of trypsin inhibitor. Initial velocity in the presence of N-acetylchitooses clearly reached same value, whether or not trypsinolysis occured before or during chitin polymerization (Figure 3B). This result excluded a possible effect of chitin synthase protection by N-acetylchitooses against overdigestion by trypsin.
Effect of oligosaccharide concentration on chitin synthase activity
In this paper, we have shown that the addition of N-acetylchitooses to a standard chitin synthase assay resulted in an increase of initial velocity of GlcNAc unit incorporation, specifically a 25% enhancement for N-acetylchitooctaoses. The effect induced by N-acetylchitooses is different from other known activators such as trypsin and exogenous GlcNAc. The activating effect of N-acetylchitoses on chitin synthesis was described as a saturable phenomenon dependant of N-acetylchitoose structure and length.
This paper is dedicated to the memory of our colleague, Dr Anne Vidal-Cros, who passed away in 2006. AVC developed the study of chitin synthases in the laboratory and she assisted on the first design of these experiments. We are grateful to Prof. M. Boccara, Université Pierre et Marie Curie, Paris, for helpful discussions and Dr R. Geremia, Dr E. Samain, CERMAV Grenoble, for the kind gift of chitooligosaccharides.
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