Hans Bakker

Galactose-extended glycans of antibodies produced by transgenic plants.

Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that β1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plant N-glycans into mammalian-like N-glycans. Here, the stable expression of human β1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bear N-glycans, of which about 15% exhibit terminal β1,4-galactose residues in addition to the specific plant N-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusual N-glycans with terminal β1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human β1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylated N-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in the in planta engineering of the N-glycosylation of recombinant antibodies.

Contribution of Galactofuranose to the Virulence of the Opportunistic Pathogen Aspergillus fumigatus

ABSTRACT The filamentous fungus Aspergillus fumigatus is responsible for a lethal disease called invasive aspergillosis that affects immunocompromised patients. This disease, like other human fungal diseases, is generally treated by compounds targeting the primary fungal cell membrane sterol. Recently, glucan synthesis inhibitors were added to the limited antifungal arsenal and encouraged the search for novel targets in cell wall biosynthesis. Although galactomannan is a major component of the A. fumigatus cell wall and extracellular matrix, the biosynthesis and role of galactomannan are currently unknown. By a targeted gene deletion approach, we demonstrate that UDP-galactopyranose mutase, a key enzyme of galactofuranose metabolism, controls the biosynthesis of galactomannan and galactofuranose containing glycoconjugates. The glfA deletion mutant generated in this study is devoid of galactofuranose and displays attenuated virulence in a low-dose mouse model of invasive aspergillosis that likely reflects the impaired growth of the mutant at mammalian body temperature. Furthermore, the absence of galactofuranose results in a thinner cell wall that correlates with an increased susceptibility to several antifungal agents. The UDP-galactopyranose mutase thus appears to be an appealing adjunct therapeutic target in combination with other drugs against A. fumigatus. Its absence from mammalian cells indeed offers a considerable advantage to achieve therapeutic selectivity.

Expression Cloning of a cDNA Encoding a Sulfotransferase Involved in the Biosynthesis of the HNK-1 Carbohydrate Epitope*

The HNK-1 carbohydrate epitope is expressed on several neural adhesion glycoproteins and as a glycolipid, and is involved in cell interactions. The structural element of the epitope common to glycoproteins and glycolipids has been determined to be sulfate-3-GlcAβ1→ 3Galβ1→4GlcNAc. The glucuronyltransferase and sulfotransferase are considered to be the key enzymes in the biosynthesis of this epitope because the rest of the structure occurs often in glycoconjugates. Here we describe the isolation of the rat sulfotransferase cDNA via an expression cloning strategy. The clone finally isolated predicts a protein of 356 amino acids, with characteristics of a type II transmembrane protein and with no sequence similarity to other known sulfotransferases. Both the enzyme expressed as a soluble fusion protein and homogenates of cells transfected with the full-length cDNA could transfer sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid.

Nucleotide sugar transporters: biological and functional aspects.

The Golgi apparatus serves as the major site of glycosylation reactions. Nucleotide sugars which are substrates of the Golgi localized glycosyltransferases are synthesized in the cytoplasm (cell nucleus in case of CMP-sialic acid) and must be transported into the compartment lumen. This transport function is carried out by nucleotide sugar transporters. The first genes were cloned in the year 1996 and revealed a family of structurally conserved multi-transmembrane-spanning proteins. Due to the high structural and functional conservation, the identification of many putative nucleotide sugar transporter sequences has become possible in the existing gene data bases and accelerates the increase in knowledge on structure-function-relationships. Recent developments in the nucleotide sugar transporter field are discussed in this article.

Identification and partial characterization of two eukaryotic UDP-galactopyranose mutases

Abstract Galactofuranose metabolism is valued as an important target for the development of new antituberculosis drugs. UDP-galactopyranose mutase, a central enzyme in galactofuranose biosynthesis, is essential for the growth and viability of mycobacteria. This enzyme catalyzes the conversion of UDP-galactopyranose into UDP-galactofuranose, the donor used by various galacto-furanosyltransferases. While D-galactofuranose residues are often found in important surface glycoconjugates of pathogenic bacteria, fungi and protozoan parasites, they are absent in the mammalian host, and thus their biosynthesis is an attractive target for the development of novel therapeutic strategies. In contrast to mycobacteria, the importance of galactofuranose for eukaryotic pathogens has not been ascertained because the enzymes involved in galactofuranose metabolism are unknown. Here, we report the identification and characterization of the first eukaryotic UDP-galactopyranose mutases. The genes encoding the enzymes were cloned from two different human pathogens: the parasite Leishmania major and the opportunistic fungus Aspergillus fumigatus. The newly identified eukaryotic enzymes exhibit 51% sequence identity, but are less than 20% identical to the prokaryotic counterparts. The sequence identity between pro- and eukaryotic enzymes is concentrated at amino acid residues that are involved in substrate and cofactor binding. Therefore, an inhibitor of UDP-galactopyranose mutase might be effective against a wide range of pathogenic organisms.

Identification of Glycosyltransferase 8 Family Members as Xylosyltransferases Acting on O-Glucosylated Notch Epidermal Growth Factor Repeats

The epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans. O-Fucosylation and elongation by the glycosyltransferase Fringe have been well studied and shown to be essential for proper Notch signaling. In contrast, biosynthesis of O-glucose and O-N-acetylglucosamine is less well understood. Recently, the isolation of the Drosophila mutant rumi has shown that absence of O-glucose impairs Notch function. O-Glucose is further extended by two contiguous α1,3-linked xylose residues. We have identified two enzymes of the human glycosyltransferase 8 family, now named GXYLT1 and GXYLT2 (glucoside xylosyltransferase), as UDP-d-xylose:β-d-glucoside α1,3-d-xylosyltransferases adding the first xylose. The enzymes are specific for β-glucose-terminating acceptors and UDP-xylose as donor substrate. Generation of the α1,3-linkage was confirmed by nuclear magnetic resonance. Activity on a natural acceptor could be shown by in vitro xylosylation of a Notch fragment expressed in a UDP-xylose-deficient cell line and in vivo by co-expression of the enzymes and the Notch fragment in insect cells followed by mass spectrometric analysis of peptide fragments.

Plant members of the α1→3/4‐fucosyltransferase gene family encode an α1→4‐fucosyltransferase, potentially involved in Lewisa biosynthesis, and two core α1→3‐fucosyltransferases1

Three putative α1→3/4‐fucosyltransferase (α1→3/4‐FucT) genes have been detected in the Arabidopsis thaliana genome. The products of two of these genes have been identified in vivo as core α1→3‐FucTs involved in N‐glycosylation. An orthologue of the third gene was isolated from a Beta vulgaris cDNA library. The encoded enzyme efficiently fucosylates Galβ1→3GlcNAcβ1→3Galβ1→4Glc. Analysis of the product by 400 MHz 1H‐nuclear magnetic resonance spectroscopy showed that the product is α1→4‐fucosylated at the N‐acetylglucosamine residue. In vitro, the recombinant B. vulgaris α1→4‐FucT acts efficiently only on neutral type 1 chain‐based glycan structures. In plants the enzyme is expected to be involved in Lewisa formation on N‐linked glycans.

Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated notch EGF repeats

The epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans. O-Fucosylation and elongation by the glycosyltransferase Fringe have been well studied and shown to be essential for proper Notch signaling. In contrast, biosynthesis of O-glucose and O-N-acetylglucosamine is less well understood. Recently, the isolation of the Drosophila mutant rumi has shown that absence of O-glucose impairs Notch function. O-Glucose is further extended by two contiguous α1,3-linked xylose residues. We have identified two enzymes of the human glycosyltransferase 8 family, now named GXYLT1 and GXYLT2 (glucoside xylosyltransferase), as UDP-d-xylose:β-d-glucoside α1,3-d-xylosyltransferases adding the first xylose. The enzymes are specific for β-glucose-terminating acceptors and UDP-xylose as donor substrate. Generation of the α1,3-linkage was confirmed by nuclear magnetic resonance. Activity on a natural acceptor could be shown by in vitro xylosylation of a Notch fragment expressed in a UDP-xylose-deficient cell line and in vivo by co-expression of the enzymes and the Notch fragment in insect cells followed by mass spectrometric analysis of peptide fragments.

Functional UDP-xylose Transport across the Endoplasmic Reticulum/Golgi Membrane in a Chinese Hamster Ovary Cell Mutant Defective in UDP-xylose Synthase *

In mammals, xylose is found as the first sugar residue of the tetrasaccharide GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, initiating the formation of the glycosaminoglycans heparin/heparan sulfate and chondroitin/dermatan sulfate. It is also found in the trisaccharide Xylα1-3Xylα1-3Glcβ1-O-Ser on epidermal growth factor repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes the formation of the UDP-xylose substrate for the different xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place in these organelles, no obvious requirement exists for membrane transport of UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has been documented, and we recently succeeded with the cloning of a human UDP-xylose transporter (SLC25B4). Here we provide new evidence for a functional role of UDP-xylose transport by characterization of a new Chinese hamster ovary cell mutant, designated pgsI-208, that lacks UXS activity. The mutant fails to initiate glycosaminoglycan synthesis and is not capable of xylosylating Notch. Complementation was achieved by expression of a cytoplasmic variant of UXS, which proves the existence of a functional Golgi UDP-xylose transporter. A ∼200 fold increase of UDP-glucuronic acid occurred in pgsI-208 cells, demonstrating a lack of UDP-xylose-mediated control of the cytoplasmically localized UDP-glucose dehydrogenase in the mutant. The data presented in this study suggest the bidirectional transport of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.

Negative Regulation of Notch Signaling by Xylose

The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14–20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16–20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16–20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16–20 of Notch.