Ricardo Gutiérrez Gallego

Chemoenzymatic Synthesis of Biotinylated Nucleotide Sugars as Substrates for Glycosyltransferases

The enzymatic oxidation of uridine 5′‐diphospho‐α‐D‐galactose (UDP‐Gal) and uridine 5′‐diphospho‐N‐acetyl‐α‐D‐galactosamine (UDP‐GalNAc) with galactose oxidase was combined with a chemical biotinylation step involving biotin‐ε‐amidocaproylhydrazide in a one‐pot synthesis. The novel nucleotide sugar derivatives uridine 5′‐diphospho‐6‐biotin‐ε‐amidocaproylhydrazino‐α‐D‐galactose (UDP‐6‐biotinyl‐Gal) and uridine 5′‐diphospho‐6‐biotin‐ε‐amidocaproylhydrazino‐N‐acetyl‐α‐D‐galactosamine (UDP‐6‐biotinyl‐GalNAc) were synthesized on a 100‐mg scale and characterized by mass spectrometry (fast atom bombardment and matrix‐assisted laser desorption/ionization time of flight) and one/two dimensional NMR spectroscopy. It could be demonstrated for the first time, by use of UDP‐6‐biotinyl‐Gal as a donor substrate, that the human recombinant galactosyltransferases β3Gal‐T5, β4Gal‐T1, and β4Gal‐T4 mediate biotinylation of the neoglycoconjugate bovine serum albumin–p‐aminophenyl N‐acetyl‐β‐D‐glucosaminide (BSA–(GlcNAc)17) and ovalbumin. The detection of the biotin tag transferred by β3Gal‐T5 onto BSA–(GlcNAc)17 with streptavidin–enzyme conjugates gave detection limits of 150 pmol of tagged GlcNAc in a Western blot analysis and 1 pmol of tagged GlcNAc in a microtiter plate assay. The degree of Gal‐biotin tag transfer onto agalactosylated hybrid N‐glycans present at the single glycosylation site of ovalbumin was dependent on the Gal‐T used (either β3Gal‐T5, β4Gal‐T4, or β4Gal‐T1), which indicates that the acceptor specificity may direct the transfer of the Gal‐biotin tag. The potential of this biotinylated UDP‐Gal as a novel donor substrate for human galactosyltransferases lies in the targeting of distinct acceptor structures, for example, under‐galactosylated glycoconjugates, which are related to diseases, or in the quality control of glycosylation of recombinant and native glycoproteins.

Synthesis of nucleotide-activated oligosaccharides by beta-galactosidase from Bacillus circulans.

Abstract The enzymatic access to nucleotideactivated oligosaccharides by a glycosidasecatalyzed transglycosylation reaction was explored. The nucleotide sugars UDPGlcNAc and UDPGlc were tested as acceptor substrates for ?galactosidase from Bacillus circulans using lactose as donor substrate. The UDPdisaccharides Gal(?1-4)GlcNAc(?1-UDP) (UDPLacNAc) and Gal(?1 4) Glc(?1-UDP) (UDPLac) and the UDPtrisaccharides Gal(?1-4)Gal(?1-4)GlcNAc(?1- UDP and Gal(?1 4 ) Gal(?1 4 ) Glc(?1-UDP) were formed stereo and regioselectively. Their chemical structures were characterized by [1]H and [13]C NMR spectroscopy and fast atom bombardment mass spectrometry. The synthesis in frozen solution at -5 C instead of 30 C gave significantly higher product yields with respect to the acceptor substrates. This was due to a remarkably higher product stability in the small liquid phase of the frozen reaction mixture. Under optimized conditions, at 5 C and pH 4.5 with 500 mM lactose and 100 mM UDPGlcNAc, an overall yield of 8.2% (81.8 mol, 62.8 mg with 100% purity) for Gal(b1 4 ) GlcNAc(?1-UDP) and 3.6% (36.1 mol, 35 mg with 96% purity) for Gal(?1-4)Gal(?1-4)GlcNAc(?1- UDP) was obtained. UDPGlc as acceptor gave an overall yield of 5.0% (41.3 mol, 32.3 mg with 93% purity) for Gal(?1-4)Glc(?1-UDP) and 1.6% (13.0 mol, 12.2 mg with 95% purity) for Gal(?1-4)Gal(?1- 4)Glc(?1-UDP). The analysis of other nucleotide sugars revealed UDPGal, UDPGalNAc, UDPXyl and dTDP, CDP, ADP and GDPGlc as further acceptor substrates for ?galactosidase from Bacillus circulans.

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.

Structural characterization of the N-glycans of gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus

Gp273, a glycoprotein of the egg extracellular coats of the mollusc bivalve Unio elongatulus, is the ligand molecule for sperm-egg interaction during fertilization. In this study we have analyzed the N-glycans from gp273. N-glycans were enzymatically released by PNGase F digestion and their structures were elucidated by normal phase HPLC profiling of the 2-aminobenzamide-labeled N-glycans, MALDI-TOF mass spectrometry and 1H NMR spectroscopy. The combined data revealed that the N-glycans of gp273 consist of Glc1Man9GlcNAc2 and Man9GlcNAc2. In Unio, the presence of noncomplex-type N-glycans parallels the inefficacy of these glycans in the ligand function. Their role in the protection of the polypeptide chain from proteolytic attack is suggested by the electrophoretic patterns obtained after enzymatic digestion of the native and the N-deglycosylated protein. These results are discussed in the light of the evolution of the recognition and adhesion properties of oligosaccharide chains in the fertilization process.

Isolation of oligosaccharides from a partial-acid hydrolysate of pneumococcal type 3 polysaccharide for use in conjugate vaccines

A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry. The oligosaccharides can be used to produce neoglycoprotein vaccines with a defined carbohydrate part.

In situ generated O-glycan core 1 structure as substrate for Gal(β1-3)GalNAc β-1,6-GlcNAc transferase

β-Galactosidase from bovine testes was used in a one pot reaction together with a recombinant β-1,6-GlcNAc transferase for the synthesis of GlcNAc(β1–6)GalNAc(α1-OBn) (core 6-Bn). The galactosidase, which reversibly links galactose via a (β1–3) linkage to N-acetylgalactosamine, provides the substrate for the GlcNAc transferase in situ. The synthesis was carried out with a yield >90%.