Saccharomyces cerevisiae chitin biosynthesis activation by N-acetylchitooses depends on size and structure of chito-oligosaccharides

Abstract

Background: 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.

Findings: 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.

Conclusions: 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.

Figure 1

Thin layer chromatography analysis of [¹⁴C]-chitin, produced in vitro by S. cerevisiae membrane fractionation. The CHS reaction product was digested by S. Griseus chitinase and analysed on silica 60 TLC plates as described in Materials and Methods. Samples were loaded onto the TLC plate before (panel A) or after 24 h incubation with commercial chitinase (panel B). In panel C the reference sample containing commercial [¹⁴C]-GlcNAc was applied.

Figure 2

Time course of chitin synthesis in presence or absence of N-acetylchitopentaoses. Kinetics of two chitin synthase assays following preincubation with (open boxes, solid line) or without (black circles, dotted line) 1 mM N-acetylchitopentaoses. The linear part of the curve (up to 12 min) is shown.

Figure 3

Activation by N-acetylchitooses is independent on Glc-NAc and trypsin. (A) [¹⁴C]-chitin was prepared and quantified as described, except that Glc-NAc was present at 40 mM (grey bars), 1 mM (black bars) or absent (hatched bars) in the incubation mixture. (B) [¹⁴C]-Glc-NAc incorporation into chitin measured after two different conditions of CHS activation by trypsin: during (standard conditions, grey bars), or before (black bars) chitin polymerization. Results in presence or absence of N-acetylchitopentaoses are shown.

Figure 4

Effect of N-acetylchitoose concentration on GlcNAc incorporation. Different concentrations of N-acetylchito-tetra (A), -penta (B), -octaoses (C) were preincubated with membrane fractions before initiating the CHS reaction. GlcNAc incorporation into chitin versus N-acetylchitoose concentration is shown. AC₅₀ corresponds to half-activating N-acetylchitoose concentration.

Figure 5

N-acetylchitooses units number influence half-activation concentration. Half-activating N-acetylchitoose concentration was determined from Fig. 4 for N-acetylchito-tetra, -penta and -octaoses. DP = degree of polymerization.