Martine Malissard

Chemo-enzymatic synthesis of the galili epitope Galα(1→3)Galβ(1→4)GlcNAc on a homogeneously soluble peg polymer by a multi-enzyme system

Abstract The α-Gal trisaccharide Galα(1→3)Galβ(1→4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of β-1,4-galactosyltransferase (EC 2.4.1.38), α-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free α-Gal trisaccharide.

The yeast expression system for recombinant glycosyltransferases.

Glycosyltransferases are increasingly being used for in vitro synthesis of oligosaccharides. Since these enzymes are difficult to purify from natural sources, expression systems for soluble forms of the recombinant enzymes have been developed. This review focuses on the current state of development of yeast expression systems. Two yeast species have mainly been used, i.e. Saccharomyces cerevisiae and Pichia pastoris. Safety and ease of fermentation are well recognized for S. cerevisiae as a biotechnological expression system; however, even soluble forms of recombinant glycosyltransferases are not secreted. In some cases, hyperglycosylation may occur, P. pastoris, by contrast, secrete soluble orthoglycosylated forms to the supernatant where they can be recovered in a highly purified form.The review also covers some basic features of yeast fermentation and describes in some detail those glycosyltransferases that have successfully been expressed in yeasts. These include β1,4galactosyltransferase, α2,6sialyltransferase, α2,3sialyltransferase, α1,3fucosyltransferase III and VI and α1,2mannosyltransferase. Current efforts in introducing glycosylation systems of higher eukaryotes into yeasts are briefly addressed.

UDP-N-Acetyl-alpha-D-glucosamine as acceptor substrate of beta-1,4-galactosyltransferase. Enzymatic synthesis of UDP-N-acetyllactosamine.

The capacity of UDP-N-acetyl-α-D-glucosamine (UDP-GlcNAc) as an in vitro acceptor substrate for β-1,4-galactosyltransferase (β4GalT1, EC 2.4.1.38) from human and bovine milk and for recombinant human β4GalT1, expressed in Saccharomyces cerevisiae, was evaluated. It turned out that each of the enzymes is capable to transfer Gal from UDP-α-D-galactose (UDP-Gal) to UDP-GlcNAc, affording Gal(β1-4)GlcNAc(α1-UDP (UDP-LacNAc). Using β4GalT1 from human milk, a preparative enzymatic synthesis of UDP-LacNAc was carried out, and the product was characterized by fast-atom bombardment mass spectrometry and 1H and 13C NMR spectroscopy. Studies with all three β4GalTs in the presence of α-lactalbumin showed that the UDP-LacNAc synthesis is inhibited and that UDP-α-D-glucose is not an acceptor substrate. This is the first reported synthesis of a nucleotide-activated disaccharide, employing a Leloir glycosyltransferase with a nucleotide-activated monosaccharide as acceptor substrate. Interestingly, in these studies β4GalT1 accepts an α-glycosidated GlcNAc derivative. The results imply that β4GalT1 may be responsible for the biosynthesis of UDP-LacNAc, previously isolated from human milk.

Completely enzymic synthesis of the mucin-type sialyl Lewis x epitope, involved in the interaction between PSGL-1 and P-selectin

Sialyl Lewis x (sLex) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-α-D-galactosaminide (GalNAc(α1-pNp as core substrate, the sLex-oligosaccharide Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)[Gal(β1-3)]GalNAc(α1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(β1-3)GalNAc(α1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of β3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 β6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(β1-6)[Gal(β1-3)]GalNAc(α1-pNp in a yield of >85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk β4-galactosyltransferase 1 (EC 2.4.1.38) (yield of >85%), then sialylated using CMP-Neu5Ac and purified recombinant α3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human α3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 µmol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure.

Fed-batch production of a soluble β-1,4-galactosyltransferase with Saccharomyces cerevisiae

Abstract A feedback-controlled fed-batch process for the recombinant production of a soluble human β-1,4-galactosyltransferase (NdrGal-T) with Saccharomyces cerevisiae was developed and scaled up to the pilot scale. A 5000 U NdrGal-T fermentation run was performed on a 300 l scale. Indirect feedback control of the glucose feeding with RQ data at a set-point of RQ = 1.1 resulted in higher NdrGal-T activities (30 U/l) than direct feedback control of glucose with on-line flow-injection analysis (9 U/l). This increase in final activity of NdrGal-T by a factor of 42 compared to published data makes the fed-batch production of glycosyltransferases with a Saccharomyces cerevisiae expression system competitive with cell-culture systems.

Antibody recognition of epitopes on wild-type and mutant β-(1⃗4)-galactosyltransferase-1

The epitopes present on beta-(1-->4)-galactosyltransferase-1 (beta 4Gal-T1) have been explored using a panel of monoclonal antibodies (mAbs) raised against the soluble form of the human enzyme. Reactivity of the antibodies with site-specific and truncated mutants of human beta 4Gal-T1 suggests the presence of a major immunogenic epitope cluster consisting of four epitopes within the stem region and mapping between amino acids 42 and 115. The catalytic activity of the enzyme is increased in the presence of stem region-specific antibody. Two of the epitopes were further localized to a region between amino acids 42 and 77, sequences which are not shared with the recently cloned beta 4Gal-T2 and beta 4Gal-T3 enzymes. An epitope located close to or within the catalytic domain is also identified, and the mAb to this region binds synergistically with antibodies to the stem region.

Immunodetection of Glycosyltransferases: Prospects and Pitfalls

The key element to understand biosynthesis and function of the countless oligosaccharide structures expressed on cell surfaces and soluble glycoconjugates are the enzymes involved in their construction: the glycosyltransferases. Their investigation relies on three basic pillars: i) the biochemical which comprises methods for their purification and assays for enzymic activity; ii) the cell biological which is usually based on antibodies providing means to study their subcellular localization, trafficking and tissue distribution and iii) the approach based on recombinant DNA technology which provides insights in regulation of gene expression, structural data and approaches to test structure/function relationships of heterologously expressed enzymes.