Synthesis of heparosan oligosaccharides by Pasteurella multocida PmHS2 single-action transferases

Abstract

Pasteurella multocida heparosan synthase PmHS2 is a dual action glycosyltransferase that catalyzes the polymerization of heparosan polymers in a non-processive manner. The two PmHS2 single-action transferases, obtained previously by site-directed mutagenesis, have been immobilized on Ni(II)-nitrilotriacetic acid agarose during the purification step. A detailed study of the polymerization process in the presence of non-equal amounts of PmHS2 single-action transferases revealed that the glucuronyl transferase (PmHS2-GlcUA(+)) is the limiting catalyst in the polymerization process. Using experimental design, it was determined that the N-acetylglucosaminyl transferase (PmHS2-GlcNAc(+)) plays an important role in the control of heparosan chain elongation depending on the number of heparosan chains and the UDP-sugar concentrations present in the reaction mixture. Furthermore, for the first time, the synthesis of heparosan oligosaccharides alternately using PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) is reported. It was shown that the synthesis of heparosan oligosaccharides by PmHS2 single-action transferases do not require the presence of template molecules in the reaction mixture.

Fig. 1

Schematic representation of the procedure for the step-by-step elongation of heparosan oligosaccharides. The first step is the initiation of the heparosan chain (GlcUA-GlcNAc-UDP) by immobilized PmHS2-GlcUA⁺ (column A) in the presence of UDP-GlcUA and UDP-GlcNAc, which was identified as the acceptor molecule (Chavaroche et al. 2011). Prior to be incubated with immobilized PmHS2-GlcNAc⁺ (column B), the reaction mixture was heat treated (65 °C/15 min) to prevent residual enzyme activity. Successive incubation cycles of the reaction mixture (UDP-sugars and oligosaccharide acceptors [heparosan]-GlcNAc-UDP) enable to synthesize heparosan oligosaccharides of a defined chain length. The oligosaccharides still remain the UDP-group belonging to the first UDP-GlcNAc acceptor molecule as determined by MALDI-TOF MS analysis

Fig. 2

Storage stability of immobilized and PD10-purified PmHS2 single-action transferases. The PmHS2 single-action transferases (25–30 μg/mL of each transferase) were incubated together for 24 h in the presence of 5 mM of each UDP-sugar (closed square immobilized and open square PD10-purified PmHS2 single-action transferases). The amount of UDP-sugar converted after 24 h of incubation at 32 °C was quantified by the coupled enzyme assay. The polymerization activity of the fresh immobilized and PD10-purified enzymes (t = 0) was set as 100%

Fig. 3

Influence of each PmHS2 single-action transferase on the overall polymerization activity. Non-equal amount of freshly immobilized PmHS2-GlcUA⁺ and PmHS2-GlcNAc⁺ (ranging from 3 to 48 μg/mL) were mixed together and were incubated for 4 h in the presence of 1 mM (a) and 5 mM (b) of each UDP-sugar (UDP-GlcUA and UDP-GlcNAc). The amount of UDP-sugars converted after 4 h of incubation was determined by the coupled enzyme assay. The polymerization activity observed in the presence of equal amount of PmHS2-GlcUA⁺ and PmHS2-GlcNAc⁺ (12 μg/mL of each transferase, E1/E2 = 1) was set at 100% relative activity. In all the experiments, E2 was set as a constant (12 μg/mL) and E1 varied from 3 to 48 μg/mL. These results have been obtained from triplicate experiments; the standard deviation was ±3–10%

Fig. 4

Agarose gel of heparosan polymers synthesized in the presence of non-equal amount of immobilized PmHS2 single-action transferases. The reaction mixtures were incubated for 4 h in the presence of 5 mM of each UDP-sugar (UDP-GlcUA and UDP-GlcNAc) and non-equal concentration of PmHS2-GlcUA⁺ and PmHS2-GlcNAc⁺ (E1/E2 ratio ranging from 0.25 to 4, in which E1 = 3 to 48 μg/mL and E2 = 12 μg/mL, respectively)

Fig. 5

Contour plot visualization of the statistical analysis of the PmHS2 single-action transferase polymerization activity with respect to the polymer molecular weight. The reaction mixtures were prepared according to a fractional factorial design with four variables and three levels (3^{4 − 1}) for each variable (immobilized PmHS2-GlcUA⁺ and immobilized PmHS2-GlcNAc⁺ (micrograms per milliliter), UDP-GlcUA, and UDP-GlcNAc (millimolars)). After 4 h of incubation at 32 °C, the heparosan average molecular mass (kilodaltons, presented in the scale at the right of each graph) was determined by high-performance size exclusion chromatography. The data were visualized using the mathematical software package Matlab

Fig. 6

High-performance anion exchange chromatography analysis of the reaction mixtures obtained after different incubation steps in the presence of immobilized PmHS2-GlcUA⁺ or PmHS2-GlcNAc⁺. Heparosan disaccharides (DP2) were released from the PmHS2-GlcUA⁺ column (column A; 0.5 mg PmHS2-GlcUA⁺/mL reaction, incubation step 1), heparosan trisaccharide (DP3) from the PmHS2-GlcNAc⁺ column (column B; 0.5 mg PmHS2-GlcNAc⁺/mL reaction, incubation step 2), and a mixture of heparosan disaccharides (DP2) and tetrasaccharides (DP4) from the PmHS2-GlcUA⁺ column (incubation step 3). UMP, UDP-GlcNAc, UDP, and UDP-GlcUA (not shown) eluted after 21.1, 23.5, 26.0, and 33.6 min, respectively. The annotation DP_X stands for “degree of polymerization X,” in which X represents the number of monosaccharide units

Fig. 7

High-performance anion exchange chromatography analysis of the reaction mixture released from the PmHS2-GlcUA⁺ column (column A). Even numbered heparosan oligosaccharide mixture from disaccharides up to octasaccharides was synthesized by the immobilized PmHS2 single-action transferases (0.5 mg/mL PmHS2-GlcUA⁺ and 0.5 mg/mL PmHS2-GlcNAc⁺), as shown in Fig. 1. The reaction mixture was obtained after nine successive incubation cycles (last incubation cycle on column A). The annotation DP_X stands for “degree of polymerization X”, in which X represents the number of monosaccharide units