In vitro synthesis of heparosan using recombinant Pasteurella multocida heparosan synthase PmHS2

Abstract

In vertebrates and bacteria, heparosan the precursor of heparin is synthesized by glycosyltransferases via the stepwise addition of UDP-N-acetylglucosamine and UDP-glucuronic acid. As heparin-like molecules represent a great interest in the pharmaceutical area, the cryptic Pasteurella multocida heparosan synthase PmHS2 found to catalyze heparosan synthesis using substrate analogs has been studied. In this paper, we report an efficient way to purify PmHS2 and to maintain its activity stable during 6 months storage at −80 °C using His-tag purification and a desalting step. In the presence of 1 mM of each nucleotide sugar, purified PmHS2 synthesized polymers up to an average molecular weight of 130 kDa. With 5 mM of UDP-GlcUA and 5 mM of UDP-GlcNAc, an optimal specific activity, from 3 to 6 h of incubation, was found to be about 0.145 nmol/μg/min, and polymers up to an average of 102 kDa were synthesized in 24 h. In this study, we show that the chain length distribution of heparosan polymers can be controlled by change of the initial nucleotide sugar concentration. It was observed that low substrate concentration favors the formation of high molecular weight heparosan polymer with a low polydispersity while high substrate concentration did the opposite. Similarities in the polymerization mechanism between PmHS2, PmHS1, and PmHAS are discussed.

Analysis of the Polymerization Initiation and Activity of Pasteurella multocida Heparosan Synthase PmHS2, an Enzyme with Glycosyltransferase and UDP-sugar Hydrolase Activity

“…Just as observed with PmHS2 (6, 15), PmHS2-GlcUA ϩ and PmHS2-GlcNAc ϩ incubated together, in the absence of heparosan templates, were able to synthesize heparosan polymers. In addition, the PmHS2 single action transferases synthesize, in the presence of low UDP-sugar concentrations, high molecular weight heparosan polymers and thus depict the same polymerization behavior as PmHS2 (15). During the incubation period from 2 to 8 h, the specific activity of the single action transferases was around 0.06 nmol of UDP-sugar converted/g of protein/min, whereas the specific activity of PmHS2 was around 0.12 nmol of UDP-sugar converted/g of protein/min.…”

“…It was reported previously that PmHS2 initiates and elongates simultaneously heparosan polymers (15); therefore, the presence of these products suggests that PmHS2 synthesized new heparosan polymers (25 kDa) and also elongated the heparosan templates, resulting in longer polymers (65 kDa). After 6 h of incubation, the difference between the molecular masses of the two polymer groups was less visible; it resulted in an increase of the overall polydispersity.…”

“…The destaining was carried out in pure water as we described previously (15). In addition, Novex 20% Tris borate-EDTA (TBE) polyacrylamide gel (Invitrogen) were used, depending on the experiment.…”

“…Proteins-The expression and the purification of the recombinant proteins (PmHS2-GlcUA ϩ , PmHS2-GlcNAc ϩ , and PmHS2-nul) was done in the same way as described previously for PmHS2 (15).…”

“…Selected hyaluronan molecular mass markers (Hyalose) of 30, 160, and 262 kDa, respectively, were used for calibration. For each HPSEC analysis, the molecular weight and the size distribution (polydispersity index) of the samples were determined by fitting the elution time of the hyaluronan molecular weight markers to a logarithmic model (15).…”