Fucosylation in prokaryotes and eukaryotes

Toxoplasma gondii is an intracellular parasite that causes disseminated infections in fetuses and immunocompromised individuals. Although gene regulation is important for parasite differentiation and pathogenesis, little is known about protein organization in the nucleus. Here we show that the fucose-binding Aleuria aurantia lectin (AAL) binds to numerous punctate structures in the nuclei of tachyzoites, bradyzoites, and sporozoites but not oocysts. AAL also binds to Hammondia and Neospora nuclei but not to more distantly related apicomplexans. Analyses of the AAL-enriched fraction indicate that AAL binds O-linked fucose added to Ser/Thr residues present in or adjacent to Ser-rich domains (SRDs). Sixty-nine Ser-rich proteins were reproducibly enriched with AAL, including nucleoporins, mRNA-processing enzymes, and cell-signaling proteins. Two endogenous SRDs-containing proteins and an SRD-YFP fusion localize with AAL to the nuclear membrane. Superresolution microscopy showed that the majority of the AAL signal localizes in proximity to nuclear pore complexes. Host cells modify secreted proteins with O-fucose; here we describe the O-fucosylation pathway in the nucleocytosol of a eukaryote. Furthermore, these results suggest O-fucosylation is a mechanism by which proteins involved in gene expression accumulate near the NPC.toxoplasma | fucose | nuclear glycosylation | nuclear pore complex T he apicomplexan parasite Toxoplasma gondii causes disseminated infections in humans, and these infections can lead to severe damage in immunocompromised individuals and fetuses (1, 2). There is no human vaccine against T. gondii, and recently the price of pyrimethamine, the drug used to treat toxoplasmosis in the United States, has increased more than 50-fold (2).T. gondii has a complex life cycle, and the parasite's ability to differentiate through its life stages in response to stresses and environmental conditions is fundamental for its pathogenicity and transmission (3). Transcriptome analyses have revealed that a large percentage of mRNAs show life stage-specific expression (4) and/or cell cycle regulation (5). Recent studies have increased our understanding of gene expression in T. gondii by identifying the AP2 family of transcription factors (6-8) and by describing posttranslational modifications (PTMs) of histones and some of the enzymes responsible for them (9-11). However, little is known about protein organization at the nuclear periphery, a subnuclear compartment that plays a critical role in transcriptional regulation in many eukaryotes. In particular, the gene-gating model (12) suggests that the nuclear pore complex (NPC) has a role in transcriptional regulation and chromatin organization as well as in protein and mRNA transport (13,14).In T. gondii chromodomain protein 1 localizes with heterochromatin at the nuclear periphery (15), and centromeres sequester to an apical nuclear region (16). Although the nuclear localization signal (NLS) and importin-α system are present, key nuclear import and export molecules are...

Section: Resultsmentioning

confidence: 99%

O -fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii

Bandini

Haserick

Motari

et al. 2016

“…Specifically, UDP/TDP-sugars (predominate in microbial natural product biosynthetic pathways) (5,20), CDP-sugars (important to the biosynthesis of pathogenic bacteria antigens) (21,22), GDP-sugars (used in a range of glycobiological processes) (5,23), and ADPsugars (important to intracellular trafficking, posttranslational modification of proteins, DNA repair, programmed cell death, and glycogen metabolism) (24)(25)(26) were targeted in this study. While UDP or TDP with 2-chloro-4-nitrophenyl glucoside were previously recognized as excellent substrates in the OleD-catalyzed sugar nucleotide syntheses reactions, ADP and GDP were identified as relatively poor substrates (i.e., seven-and 50-fold decrease in conversion for ADP and GDP, respectively, compared with equivalent reactions with UDP), while CDP was not recognized as a substrate (19).…”

Section: Resultsmentioning

confidence: 99%

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

Gantt

Peltier‐Pain

Singh

et al. 2013

We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in k cat /K m of >400-fold and >15-fold for formation of NDP-glucoses and UDP-sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP-sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.carbohydrate | enzyme | glycobiology | protein engineering T he lack of accessibility and availability of uncommon and uniquely functionalized sugar nucleotides (NDP-sugars) continues to restrict research focused upon understanding the regulation, biosynthesis, and/or role of glycosylated macromolecules and glycosylated small molecules in biology or therapeutic development (1-7). Although there are many reported chemical, enzymatic, and chemoenzymatic strategies for NDP-sugar synthesis, those that extend beyond the reach of common biological sugars (e.g., Dglucose, D-galactose, etc.) nearly all suffer from long reaction times (>16 h), relatively low yields, and difficulties associated with product purification and/or stability (3,4,8,9). Thus, the development of robust methods for sugar nucleotide synthesis directly compatible to the downstream biological processes to be studied may be advantageous.From a traditional viewpoint, NDP-sugars are used as donors by Leloir glycosyltransferases (sugar nucleotide-dependent enzymes) for formation of glycosidic bonds. However, many glycosyltransferase (GT)-catalyzed reactions are known to be readily reversible, enabling the "pirating" of unique sugars from natural products or alternative donors (resulting in generation of the respective sugar nucleotide) and one-pot sugar exchange reactions between unique natural products (4, 10-13). This general reaction feature, in conjunction with availability of highly permissive glycosyltransferases (14-18) and simple donors designed to fundamentally alter the reaction thermodynamics, recently enabled a unique platform for NDP-sugar synthesis and a high throughput colorimetric screen for NDP-sugar formation and utilization (19). While the prior platform proof-of-concept study highlighted the syntheses of 22 natural and nonnatural TDP/UDP-sugars from 11 distinct 2-chloro-4-nitrophenyl glycoside donors using a single GT catalyst (Fig. 1A) (19), the substrate specificity of the glycosyltransferase used ...

“…A 10-fold dilution of the culture was grown for 2 h before induction with 0.02% L-arabinose. Following induction, growth was continued for an additional 2 h, after which the cells were harvested by centrifugation, washed with 100 mM potassium phosphate (KP i ; pH 7.0) and 10 mM MgSO 4 , and adjusted to an absorbance at 420 nm (A 420 ) of 15 (∼1.0 mg/mL of protein) for transport measurements. Transport was carried out with L- [5, H]fucose (10 mCi/mmol) at a final concentration of 40 μM at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, L-fucose, which is present in bacterial polysaccharides, is needed for adhesion and localization and is also used by a variety of microorganisms as a carbon and energy source (3,4). In Escherichia coli, transport of L-fucose is mediated by the transmembrane protein FucP, which is encoded by fucP, one of the genes of the fuc regulon that is responsible for the catabolism of L-fucose (5)(6)(7).…”

mentioning

confidence: 99%

Dynamics of the l -fucose/H ⁺ symporter revealed by fluorescence spectroscopy

Sugihara

Sun

Yan

et al. 2012

FucP of Escherichia coli catalyzes L-fucose/H + symport, and a crystal structure in an outward-facing conformation has been reported. However, nothing is known about FucP conformational dynamics. Here, we show that addition of L-fucose to purified FucP in detergent induces ∼20% quenching of Trp fluorescence in a concentration-dependent manner without a shift in λ max . Quenching is essentially abolished when both Trp38 and Trp278, which are positioned on opposing faces of the outward-facing cavity walls, are replaced with Tyr or Phe, and reduced quenching is observed when either Trp is mutated. Therefore, both Trp residues are involved in the phenomenon. Furthermore, replacement of either Trp38 or Trp278, predominantly Trp38, causes decreased quenching, decreased apparent affinity for L-fucose, and significant inhibition of active L-fucose transport, indicating that the two residues are likely involved directly in sugar binding. It is proposed that sugar binding induces a conformational change in which the outward-facing cavity in FucP closes, thereby bringing Trp38 and Trp278 into close proximity around the bound sugar to form an "occluded" intermediate. The location of these two Trp residues provides a unique method for analyzing structural dynamics in FucP.membrane proteins | membranes | protein dynamics | tryptophan A naturally occurring methylpentose, L-fucose (6-deoxy-Lgalactose) is found in mammalian cells as a major component of N-and O-linked glycans and glycolipids (1, 2). In addition, L-fucose, which is present in bacterial polysaccharides, is needed for adhesion and localization and is also used by a variety of microorganisms as a carbon and energy source (3, 4). In Escherichia coli, transport of L-fucose is mediated by the transmembrane protein FucP, which is encoded by fucP, one of the genes of the fuc regulon that is responsible for the catabolism of L-fucose (5-7). FucP consists of 438 amino acids (47,773 Da) and is an L-fucose/H + symporter belonging to the fucose-galactose-glucose/H + symporter (FGHS) subfamily of the major facilitator superfamily (MFS) (8-10). FucP catalyzes symport (cotransport) of L-fucose and H + across the cytoplasmic membrane by transducing free energy stored in the electrochemical H + gradient into a substrate concentration gradient (5,8,9).FucP shares limited sequence homology with other sugar/H + symporters (9), but the X-ray crystal structure reveals many characteristics observed in other MFS transporters (11). Thus, FucP contains 12 transmembrane α-helical domains arranged into two pseudosymmetrical 6-helix bundles surrounding a deep water-filled cavity with the N and C termini on the cytoplasmic side of the membrane. However, unlike other MFS transporters, FucP is in an outward-facing conformation; the cavity is open on the periplasmic side and tightly sealed on the cytoplasmic side. Although it is likely that FucP functions by an alternating access mechanism like LacY (reviewed in ref. 12), nothing is known about its functional dynamics.Out of a total of six Trp ...