Synthetic disialyl hexasaccharides protect neonatal rats from necrotizing enterocolitis.

Two novel synthetic α2-6-linked disialyl hexasaccharides, disialyllacto-N-neotetraose (DSLNnT) and α2-6-linked disialyllacto-N-tetraose (DS'LNT), were readily obtained by highly efficient one-pot multienzyme (OPME) reactions. The sequential OPME systems described herein allowed the use of an inexpensive disaccharide and simple monosaccharides to synthesize the desired complex oligosaccharides with high efficiency and selectivity. DSLNnT and DS'LNT were shown to protect neonatal rats from necrotizing enterocolitis (NEC) and are good therapeutic candidates for preclinical experiments and clinical application in treating NEC in preterm infants.

Cloning and characterization of a viral α2-3-sialyltransferase (vST3Gal-I) for the synthesis of sialyl Lewisx.

Sialyl Lewis(x) (SLe(x), Siaα2-3Galß1-4(Fucα1-3)GlcNAcßOR) is an important sialic acid-containing carbohydrate epitope involved in many biological processes such as inflammation and cancer metastasis. In the biosynthetic process of SLe(x), α2-3-sialyltransferase-catalyzed sialylation generally proceeds prior to α1-3-fucosyltransferase-catalyzed fucosylation. For the chemoenzymatic synthesis of SLe(x) containing different sialic acid forms, however, it would be more efficient if diverse sialic acid forms are transferred in the last step to the fucosylated substrate Lewis(x) (Le(x)). An α2-3-sialyltransferase obtained from myxoma virus-infected European rabbit kidney RK13 cells (viral α2-3-sialyltransferase (vST3Gal-I)) was reported to be able to tolerate fucosylated substrate Le(x). Nevertheless, the substrate specificity of the enzyme was only determined using partially purified protein from extracts of cells infected with myxoma virus. Herein we demonstrate that a previously reported multifunctional bacterial enzyme Pasteurella multocida sialyltransferase 1 (PmST1) can also use Le(x) as an acceptor substrate, although at a much lower efficiency compared to nonfucosylated acceptor. In addition, N-terminal 30-amino-acid truncated vST3Gal-I has been successfully cloned and expressed in Escherichia coli Origami™ B(DE3) cells as a fusion protein with an N-terminal maltose binding protein (MBP) and a C-terminal His(6)-tag (MBP-Δ30vST3Gal-I-His(6)). The viral protein has been purified to homogeneity and characterized biochemically. The enzyme is active in a broad pH range varying from 5.0 to 9.0. It does not require a divalent metal for its α2-3-sialyltransferase activity. It has been used in one-pot multienzyme sialylation of Le(x) for the synthesis of SLe(x) containing different sialic acid forms with good yields.

Helicobacter hepaticus Hh0072 gene encodes a novel alpha1-3-fucosyltransferase belonging to CAZy GT11 family.

Lewis x (Le(x)) and sialyl Lewis x (SLe(x))-containing glycans play important roles in numerous physiological and pathological processes. The key enzyme for the final step formation of these Lewis antigens is alpha1-3-fucosyltransferase. Here we report molecular cloning and functional expression of a novel Helicobacter hepaticus alpha1-3-fucosyltransferase (HhFT1) which shows activity towards both non-sialylated and sialylated Type II oligosaccharide acceptor substrates. It is a promising catalyst for enzymatic and chemoenzymatic synthesis of Le(x), sialyl Le(x) and their derivatives. Unlike all other alpha1-3/4-fucosyltransferases characterized so far which belong to Carbohydrate Active Enzyme (CAZy, http://www.cazy.org/) glycosyltransferase family GT10, the HhFT1 shares protein sequence homology with alpha1-2-fucosyltransferases and belongs to CAZy glycosyltransferase family GT11. The HhFT1 is thus the first alpha1-3-fucosyltransferase identified in the GT11 family.

Sialidase substrate specificity studies using chemoenzymatically synthesized sialosides containing C5-modified sialic acids.

para-Nitrophenol-tagged sialyl galactosides containing sialic acid derivatives in which the C5 hydroxyl group of sialic acids was systematically substituted with a hydrogen, a fluorine, a methoxyl or an azido group were successfully synthesized using an efficient chemoenzymatic approach. These compounds were used as valuable probes in high-throughput screening assays to study the importance of the C5 hydroxyl group of sialic acid in the recognition and the cleavage of sialoside substrates by bacterial sialidases.

Biochemical characterization of Helicobacter pylori α1-3-fucosyltransferase and its application in the synthesis of fucosylated human milk oligosaccharides.

Fucosylated human milk oligosaccharides (HMOs) have important biological functions. Enzymatic synthesis of such compounds requires robust fucosyltransferases. A C-terminal 66-amino acid truncated version of Helicobacter pylori α1-3-fucosyltransferase (Hp3FT) is a good candidate. Hp3FT was biochemically characterized to identify optimal conditions for enzymatic synthesis of fucosides. While N-acetyllactosamine (LacNAc) and lactose were both suitable acceptors, the former is preferred. At a low guanosine 5'-diphospho-ß-L-fucose (GDP-Fuc) to acceptor ratio, Hp3FT selectively fucosylated LacNAc. Based on these enzymatic characteristics, diverse fucosylated HMOs, including 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III, lacto-N-neofucopentaose (LNnFP) V, lacto-N-neodifucohexaose (LNnDFH) II, difuco- and trifuco-para-lacto-N-neohexaose (DF-paraLNnH and TF-para-LNnH), were synthesized enzymatically by varying the ratio of the donor and acceptor as well as controlling the order of multiple glycosyltransferase-catalyzed reactions.

A Photobacterium sp. α2-6-sialyltransferase (Psp2,6ST) mutant with an increased expression level and improved activities in sialylating Tn antigens.

In order to improve the catalytic efficiency of recombinant Photobacterium sp. JT-ISH-224 α2-6-sialyltransferase Psp2,6ST(15-501)-His6 in sialylating α-GalNAc-containing acceptors for the synthesis of tumor-associated carbohydrate antigens sialyl Tn (STn), protein crystal structure-based mutagenesis studies were carried out. Among several mutants obtained by altering the residues close to the acceptor substrate binding pocket, mutant A366G was shown to improve the sialyltransferase activity of Psp2,6ST(15-501)-His6 toward α-GalNAc-containing acceptors by 21-115% without significantly affecting its sialylation activity to ß-galactosides. Furthermore, the expression level was improved from 18-40 mg L(-1) for the wild-type enzyme to 72-110 mg L(-1) for the A366G mutant. In situ generation of CMP-sialic acid in a one-pot two-enzyme system was shown effective in overcoming the high donor hydrolysis of the enzyme. Mutant A366G performed better than the wild-type Psp2,6ST(15-501)-His6 for synthesizing Neu5Acα2-6GalNAcαOSer/Thr STn antigens.

Efficient Chemoenzymatic Synthesis of an N-glycan Isomer Library.

Quantification, characterization and biofunctional studies of N-glycans on proteins remain challenging tasks due to complexity, diversity and low abundance of these glycans. The availability of structurally defined N-glycans (especially isomers) libraries is essential to help on solving these tasks. We reported herein an efficient chemoenzymatic strategy, namely Core Synthesis/Enzymatic Extension (CSEE), for rapid production of diverse N-glycans. Starting with 5 chemically prepared building blocks, 8 N-glycan core structures containing one or two terminal N-acetyl-D-glucosamine (GlcNAc) residue(s) were chemically synthesized via consistent use of oligosaccharyl thioethers as glycosylation donors in the convergent fragment coupling strategy. Each of these core structures was then extended to 5 to 15 N-glycan sequences by enzymatic reactions catalyzed by 4 robust glycosyltransferases. Success in synthesizing N-glycans with Neu5Gc and core-fucosylation further expanded the ability of enzymatic extension. High performance liquid chromatography with an amide column enabled rapid and efficient purification (>98% purity) of N-glycans in milligram scales. A total of 73 N-glycans (63 isomers) were successfully prepared and characterized by MS2 and NMR. The CSEE strategy provides a practical approach for "mass production" of structurally defined N-glycans, which are important standards and probes for Glycoscience.

Highly efficient one-pot multienzyme (OPME) synthesis of glycans with fluorous-tag assisted purification.

Oligo(ethylene glycol)-linked light fluorous tags have been found to be optimal for conjugating to glycans for both high-yield enzymatic glycosylation reactions using one-pot multienzyme (OPME) systems and quick product purification using fluorous solid-phase extraction (FSPE) cartridges. The combination of OPME glycosylation systems and the FSPE cartridge purification scheme provides a highly effective strategy for facile synthesis and purification of glycans.

One-pot multienzyme synthesis of Lewis x and sialyl Lewis x antigens.

L-Fucose has been found abundantly in human milk oligosaccharides, bacterial lipopolysaccharides, glycolipids, and many N- and O-linked glycans produced by mammalian cells. Fucose-containing carbohydrates have important biological functions. Alterations in the expression of fucosylated oligosaccharides have been observed in several pathological processes such as cancer and atherosclerosis. Chemical formation of fucosidic bonds is challenging due to its acid lability. Enzymatic construction of fucosidic bonds by fucosyltransferases is highly efficient and selective but requires the expensive sugar nucleotide donor guanosine 5'- diphosphate-L-fucose (GDP-Fuc). Here, we describe a protocol for applying a one-pot three-enzyme system in synthesizing structurally defined fucose-containing oligosaccharides from free L-fucose. In this system, GDP-Fuc is generated from L-fucose, adenosine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP) by a bifunctional L-fucokinase/GDP-fucose pyrophosphorylase (FKP). An inorganic pyrophosphatase (PpA) is used to degrade the by-product pyrophosphate (PPi) to drive the reaction towards the formation of GDP-Fuc. In situ generated GDP-Fuc is then used by a suitable fucosyltransferase for the formation of fucosides. The three-enzyme reactions are carried out in one pot without the need for high cost sugar nucleotide or isolation of intermediates. The time for the synthesis is 4-24 hours. Purification and characterization of products can be completed in 2-3 days.

A sialyltransferase mutant with decreased donor hydrolysis and reduced sialidase activities for directly sialylating LewisX.

Glycosyltransferases are important catalysts for enzymatic and chemoenzymatic synthesis of complex carbohydrates and glycoconjugates. The glycosylation efficiencies of wild-type glycosyltransferases vary considerably when different acceptor substrates are used. Using a multifunctional Pasteurella multocida sialyltransferase 1 (PmST1) as an example, we show here that the sugar nucleotide donor hydrolysis activity of glycosyltransferases contributes significantly to the low yield of glycosylation when a poor acceptor substrate is used. With a protein crystal structure-based rational design, we generated a single mutant (PmST1 M144D) with decreased donor hydrolysis activity without significantly affecting its α2-3-sialylation activity when a poor fucose-containing acceptor substrate was used. The single mutant also has a drastically decreased α2-3-sialidase activity. X-ray and NMR structural studies revealed that unlike the wild-type PmST1, which changes to a closed conformation once a donor binds, the M144D mutant structure adopts an open conformation even in the presence of the donor substrate. The PmST1 M144D mutant with decreased donor hydrolysis and reduced sialidase activity has been used as a powerful catalyst for efficient chemoenzymatic synthesis of complex sialyl Lewis(x) antigens containing different sialic acid forms. This work sheds new light on the effect of donor hydrolysis activity of glycosyltransferases on glycosyltransferase-catalyzed reactions and provides a novel strategy to improve glycosyltransferase substrate promiscuity by decreasing its donor hydrolysis activity.

Decreasing the sialidase activity of multifunctional Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) by site-directed mutagenesis.

Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2-6-sialyltransferase, α2-3-sialidase, and α2-3-trans-sialidase activities in addition to its major α2-3-sialyltransferase activity. The presence of the α2-3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2-3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2-3-sialidase activity of PmST1 can be significantly decreased by protein crystal structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2-3-sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2-3-linked sialosides.

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies.

Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in the sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. A low cellular expression level of the human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of the human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty p-nitrophenol (pNP)-tagged α2-3- or α2-6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.