Chemoenzymatic synthesis of N-linked neoglycoproteins through a chitinase-catalyzed transglycosylation

Abstract

A novel application of the Bacillus sp. chitinase for the chemoenzymatic synthesis of N-linked neoglycoproteins is described. Three chitinases with different molecular size were purified from the crude chitinase preparation. The purified chitinases were evaluated for their hydrolytic and transglycosylation activity. One chitinase with a molecular size of 100 kDa (Chi100) was identified to be the one with highest transglycosylation/hydrolysis ratio. Chi100 could effectively recognize LacNAc-oxazoline and Manalpha1,3Glcbeta1,4GlcNAc-oxazoline as the donor substrate to glycosylate Asn-linked GlcNAc, while it was unable to recognize Manbeta1,4GlcNAc and Man(3)GlcNAc-oxazolines as the donor substrates. The chitinase-catalyzed transglycosylation was successfully extended to the remodeling of ribonuclease B to afford neoglycoproteins. Although the yield needs to be optimized, the chitinase-catalyzed transglycosylation provides a potentially useful tool for the synthesis of neoglycoproteins carrying novel N-linked oligosaccharides.

Figure 1. Purification of Chitinase from Bacillus sp. through Size Exclusion Chromatography

Fractions of the highest chitinase activity were analyzed on a 10% SDS-PAGE electrophoresis. M, protein marker, T, chitinase mixtures before purification. Lane 5, Chi100; Lane 7, Chi60; Lane 10, Chi40.

Figure 2. Fluorogenic assays for the chitinases activity

The hydrolytic activity was assayed by using the fluorogenic substrate (GlcNAc)₂-MU (1) (A) and the transglycosylation activity was determined by a coupled assay using GlcNAc-MU (4) as the acceptor (B).

Figure 3. Assay for the hydrolysis (A) and transglycosylation activity (B) of Chi100 (■), Chi60 (●), Chi40 (♦) and controls (□)

For hydrolysis (A), the reaction was carried out at 37°C using (GlcNAc)₂-MU (1) (0.1 mM) as the sole substrate for the chitinases (0.5 µg). The amounts of the releases MU were determined by fluorescence spectrometry (Arbitrary Unit), and (GlcNAc)₂-MU without the addition of the enzymes was used as a control. For transglycosylation (B), the reaction was carried out using LacNAc oxazoline (3) (0.4mM) as the glycosyl donor and GlcNAc-MU (4) (1mM) as the acceptor, which were incubated with the respective chitinase (0.5 µg) in a phosphate buffer (50 mM, pH 6.5) at 30°C. The product Gal-(GlcNAc)₂MU (5), once formed, would be quickly hydrolyzed by the same chitinase and the released MU was then determined by fluorescence (Arbitrary Unit). GlcNAc-MU with enzymes only was used as a control

Figure 4. The ESI MS spectra and HPLC profile of the synthetic neoglycoproteins

A, the mixture of Gal(GlcNAc)₂-RB (16) and GlcNAc-RB; B, the purified neoglycoprotein ManGlc(GlcNAc)₂-RB (17); C, the HPLC profile of a typical transglycosylation reaction between oxazoline 13 and GlcNAc-RB (15). The starting material GlcNAc-RB was marked as S (t_R = 22.7 min) and the newly formed product Manα1,3Glcβ1,4GlcNAcβ1,4GlcNAc-RB (17) was marked as P (t_R = 21.4 min) (see the experimental part for the details of HPLC condition).

Scheme 1. GlcNAc-Asn as the acceptor for chitinase-catalyzed transglycosylation

Reaction conditions: the enzymatic reaction was performed in a phosphate buffer (50 mM, pH 6.5) at 30°C. The reaction was monitored by HPLC.

Scheme 2. Synthesis of neoglycoproteins by the chitinase-catalyzed transglycosylation

Reaction conditions: the enzymatic reaction was performed in a phosphate buffer (50 mM, pH 6.5) at 30°C. The reaction was monitored by HPLC.