The Absence of Core Fucose Up-regulates GnT-III and Wnt Target Genes: A POSSIBLE MECHANISM FOR AN ADAPTIVE RESPONSE IN TERMS OF GLYCAN FUNCTION*

Glycans play key roles in a variety of protein functions under normal and pathological conditions, but several glycosyltransferase-deficient mice exhibit no or only mild phenotypes due to redundancy or compensation of glycan functions. However, we have only a limited understanding of the underlying mechanism for these observations. Our previous studies indicated that 70% of Fut8-deficient (Fut8^−/−) mice that lack core fucose structure die within 3 days after birth, but the remainder survive for up to several weeks although they show growth retardation as well as emphysema. In this study, we show that, in mouse embryonic fibroblasts (MEFs) from Fut8^−/− mice, another N-glycan branching structure, bisecting GlcNAc, is specifically up-regulated by enhanced gene expression of the responsible enzyme N-acetylglucosaminyltransferase III (GnT-III). As candidate target glycoproteins for bisecting GlcNAc modification, we confirmed that level of bisecting GlcNAc on β1-integrin and N-cadherin was increased in Fut8^−/− MEFs. Moreover using mass spectrometry, glycan analysis of IgG₁ in Fut8^−/− mouse serum demonstrated that bisecting GlcNAc contents were also increased by Fut8 deficiency in vivo. As an underlying mechanism, we found that in Fut8^−/− MEFs Wnt/β-catenin signaling is up-regulated, and an inhibitor against Wnt signaling was found to abrogate GnT-III expression, indicating that Wnt/β-catenin is involved in GnT-III up-regulation. Furthermore, various oxidative stress-related genes were also increased in Fut8^−/− MEFs. These data suggest that Fut8^−/− mice adapted to oxidative stress, both ex vivo and in vivo, by inducing various genes including GnT-III, which may compensate for the loss of core fucose functions.     

Dissection of Hexosyl- and Sialyltransferase Domains in the Bifunctional Capsule Polymerases from Neisseria meningitidis W and Y Defines a New Sialyltransferase Family

Crucial virulence determinants of disease causing Neisseria meningitidis species are their extracellular polysaccharide capsules. In the serogroups W and Y, these are heteropolymers of the repeating units (→6)-α-d-Gal-(1→4)-α-Neu5Ac-(2→)_n in NmW and (→6)-α-d-Glc-(1→4)-α-Neu5Ac-(2→)_n in NmY. The capsule polymerases, SiaD_W and SiaD_Y, which synthesize these highly unusual polymers, are composed of two predicted GT-B fold domains separated by a large stretch of amino acids (aa 399–762). We recently showed that residues critical to the hexosyl- and sialyltransferase activity are found in the predicted N-terminal (aa 1–398) and C-terminal (aa 763–1037) GT-B fold domains, respectively. Here we use a mutational approach and synthetic fluorescent substrates to define the boundaries of the hexosyl- and sialyltransferase domains. Our results reveal that the active sialyltransferase domain extends well beyond the predicted C-terminal GT-B domain and defines a new glycosyltransferase family, GT97, in CAZy (Carbohydrate-Active enZYmes Database).     

Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides

Mammalian cell surfaces are all covered with bioactive oligosaccharides which play an important role in molecular recognition events such as immune recognition, cell-cell communication and initiation of microbial pathogenesis. Consequently, bioactive oligosaccharides have been recognized as a medicinally relevant class of biomolecules for which the interest is growing. For the preparation of complex and highly pure oligosaccharides, methods based on the application of glycosyltransferases are currently recognized as being the most effective. The present paper reviews the potential of glycosyltransferases as synthetic tools in oligosaccharide synthesis. Reaction mechanisms and selected characteristics of these enzymes are described in relation to the stereochemistry of the transfer reaction and the requirements of sugar nucleotide donors. For the application of glycosyltransferases, accepted substrate profiles are summarized and the whole-cell approach versus isolated enzyme methodology is compared. Sialyltransferase-catalyzed syntheses of gangliosides and other sialylated oligosaccharides are described in more detail in view of the prominent role of these compounds in biological recognition.     

The structure of sucrose synthase-1 from Arabidopsis thaliana and its functional implications

Sucrose transport is the central system for the allocation of carbon resources in vascular plants. During growth and development, plants control carbon distribution by coordinating sites of sucrose synthesis and cleavage in different plant organs and different cellular locations. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, provides a direct and reversible means to regulate sucrose flux. Depending on the metabolic environment, sucrose synthase alters its cellular location to participate in cellulose, callose, and starch biosynthesis through its interactions with membranes, organelles, and cytoskeletal actin. The x-ray crystal structure of sucrose synthase isoform 1 from Arabidopsis thaliana (AtSus1) has been determined as a complex with UDP-glucose and as a complex with UDP and fructose, at 2.8- and 2.85-Å resolutions, respectively. The AtSus1 structure provides insights into sucrose catalysis and cleavage, as well as the regulation of sucrose synthase and its interactions with cellular targets.     

Synthesis of unnatural sugar nucleotides and their evaluation as donor substrates in glycosyltransferase-catalyzed reactions

New unnatural sugar nucleotides, UDP-Fuc and CDP-Fuc were synthesized from fucose-beta-1-phosphate and nucleotide monophosphates activated as morpholidates. Furthermore, a nucleotide analogue was prepared by phosphorylation of 1-(beta-D-ribofuranosyl)cyanuric acid, itself obtained as a protected derivative by condensation of the persilylated derivative of cyanuric acid with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose in 74% yield. This phosphate activated according to the same procedure was condensed with fucose-beta-1-phosphate, affording a new sugar nucleotide conjugate (NDP-Fuc) which was evaluated together with UDP-Fuc, CDP-Fuc and ADP-Fuc, as fucose donors in alpha-(1-->4/3)-fucosyltransferase (FucT-III) catalyzed reaction. Fucose transfer could be observed with each of the donors and kinetic parameters were determined using a fluorescent acceptor substrate. Efficiency of the four analogues towards FucT-III was in the following order: UDP-Fuc=ADP-Fuc>NDP-Fuc>CDP-Fuc. According to the same strategy ADP-GlcNAc was prepared from AMP-morpholidate and N-acetylglucosamine-alpha-1-phosphate; tested as a glucosaminyl donor towards Neisseria meningitidis N-acetylglucosaminyl transferase (LgtA), ADP-GlcNAc was recognized with 0.1% efficiency as compared with UDP-GlcNAc, the natural donor substrate.     

Loss of LARGE2 Disrupts Functional Glycosylation of α-Dystroglycan in Prostate Cancer

Dystroglycan (DG) is a cell surface receptor for extracellular matrix proteins and is involved in cell polarity, matrix organization, and mechanical stability of tissues. Previous studies documented loss of DG protein expression and glycosylation in a variety of cancer types, but the underlying mechanisms and the functional consequences with respect to cancer progression remain unclear. Here, we show that the level of expression of the βDG subunit as well as the glycosylation status of the αDG subunit inversely correlate with the Gleason scores of prostate cancers; furthermore, we show that the functional glycosylation of αDG is substantially reduced in prostate cancer metastases. Additionally, we demonstrate that LARGE2 (GYLTL1B), a gene not previously implicated in cancer, regulates functional αDG glycosylation in prostate cancer cell lines; knockdown of LARGE2 resulted in hypoglycosylation of αDG and loss of its ability to bind laminin-111 while overexpression restored ligand binding and diminished growth and migration of an aggressive prostate cancer cell line. Finally, our analysis of LARGE2 expression in human cancer specimens reveals that LARGE2 is significantly down-regulated in the context of prostate cancer, and that its reduction correlates with disease progression. Our results describe a novel molecular mechanism to account for the commonly observed hypoglycosylation of αDG in prostate cancer.     

N-Acetylglucosaminylation of Serine-Aspartate Repeat Proteins Promotes Staphylococcus aureus Bloodstream Infection

Staphylococcus aureus secretes products that convert host fibrinogen to fibrin and promote its agglutination with fibrin fibrils, thereby shielding bacteria from immune defenses. The agglutination reaction involves ClfA (clumping factor A), a surface protein with serine-aspartate (SD) repeats that captures fibrin fibrils and fibrinogen. Pathogenic staphylococci express several different SD proteins that are modified by two glycosyltransferases, SdgA and SdgB. Here, we characterized three genes of S. aureus, aggA, aggB (sdgA), and aggC (sdgB), and show that aggA and aggC contribute to staphylococcal agglutination with fibrin fibrils in human plasma. We demonstrate that aggB (sdgA) and aggC (sdgB) are involved in GlcNAc modification of the ClfA SD repeats. However, only sdgB is essential for GlcNAc modification, and an sdgB mutant is defective in the pathogenesis of sepsis in mice. Thus, GlcNAc modification of proteins promotes S. aureus replication in the bloodstream of mammalian hosts.     

Novel Regulation of Skp1 by the Dictyostelium AgtA α-Galactosyltransferase Involves the Skp1-binding Activity of Its WD40 Repeat Domain

The role of Skp1 as an adaptor protein that links Cullin-1 to F-box proteins in E3 Skp1/Cullin-1/F-box protein (SCF) ubiquitin ligases is well characterized. In the social amoeba Dictyostelium and probably many other unicellular eukaryotes, Skp1 is modified by a pentasaccharide attached to a hydroxyproline near its C terminus. This modification is important for oxygen-sensing during Dictyostelium development and is mediated by a HIF-α type prolyl 4-hydroxylase and five sequentially acting cytoplasmic glycosyltransferase activities. Gene disruption studies show that AgtA, the enzyme responsible for addition of the final two galactose residues, in α-linkages to the Skp1 core trisaccharide, is unexpectedly critical for oxygen-dependent terminal development. AgtA possesses a WD40 repeat domain C-terminal to its single catalytic domain and, by use of domain deletions, binding studies, and enzyme assays, we find that the WD40 repeats confer a salt-sensitive second-site binding interaction with Skp1 that mediates novel catalytic activation in addition to simple substrate recognition. In addition, AgtA binds similarly well to precursor isoforms of Skp1 by a salt-sensitive mechanism that competes with binding to an F-box protein and recognition by early modification enzymes, and the effect of binding is diminished when AgtA modifies Skp1. Genetic studies show that loss of AgtA is more severe when an earlier glycosylation step is blocked, and overexpressed AgtA is deleterious if catalytically inactivated. Together, the findings suggest that AgtA mediates non-enzymatic control of unmodified and substrate precursor forms of Skp1 by a binding mechanism that is normally relieved by switch-like activation of its glycosylation function.