Metabolic Engineering Optimizes Bioorthogonal Glycan Labeling in Living Cells

Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac<sub>4</sub>GalNAlk and Ac<sub>4</sub>GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. Comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.

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Metabolic Engineering Optimizes Bioorthogonal Glycan Labeling in Living Cells

10.26434/chemrxiv.13514365 ◽

2021 ◽

Author(s):

Anna Cioce ◽

Ganka Bineva-Todd ◽

Anthony J. Agbay ◽

Junwon Choi ◽

Thomas M. Wood ◽

...

Keyword(s):

Metabolic Engineering ◽

Protein Glycosylation ◽

Side Reactions ◽

Metabolic Fate ◽

Nucleotide Sugar ◽

Magnitude Comparison ◽

Nucleotide Sugars ◽

Salvage Pathways ◽

Nucleotide Sugar Biosynthesis ◽

Impact Probe

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Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009132 ◽

2021 ◽

Vol 15 (2) ◽

pp. e0009132 ◽

Cited By ~ 1

Author(s):

Maria Lucia Sampaio Guther ◽

Alan R. Prescott ◽

Sabine Kuettel ◽

Michele Tinti ◽

Michael A. J. Ferguson

Keyword(s):

Trypanosoma Brucei ◽

Immunofluorescence Microscopy ◽

Subcellular Fractionation ◽

Bloodstream Form ◽

Nucleotide Sugar ◽

Procyclic Form ◽

Nucleotide Sugars ◽

Experimental Analyses ◽

Nucleotide Sugar Biosynthesis

In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages. The implications are discussed.

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The effect of increasing nucleotide-sugar concentrations on the incorporation of sugars into glycoconjugates in rat hepatocytes

Biochemical Journal ◽

10.1042/bj3050865 ◽

1995 ◽

Vol 305 (3) ◽

pp. 865-870 ◽

Cited By ~ 70

Author(s):

W R Pels Rijcken ◽

B Overdijk ◽

D H Van den Eijnden ◽

W Ferwerda

Keyword(s):

Specific Radioactivity ◽

Rat Hepatocytes ◽

Fold Increase ◽

Protein Glycosylation ◽

Nucleotide Sugar ◽

Nucleotide Sugars ◽

Pretreatment Conditions

Treatment of rat hepatocytes with 0.5 mM concentrations of uridine and cytidine results in increased cellular concentrations of UTP, UDP-sugars and CTP, whereas that of CMP-N-acetylneuraminate remained unchanged [Pels Rijcken, Overdijk, Van den Eijnden and Ferwerda (1993) Biochem. J. 293, 207-213]. The incorporation of radioactivity from 3H-labelled sugars into the cell-associated and secreted glycoconjugate fraction was influenced by these altered cellular concentrations of the nucleotides. For [3H]glucosamine, pretreatment with uridine resulted in a reduction of the glycosylation in both fractions. Increases in the secreted fractions were observed for fucose with both uridine and cytidine and for N-acetylglucosamine with uridine only. With [3H]N-acetylglucosamine, similar specific radioactivities for UDP-N-acetylhexosamine and CMP-N-acetylneuraminate were found, regardless of the pretreatment conditions. With [3H]N-acetylmannosamine, the specific radioactivity of CMP-N-acetylneuraminate showed an almost 2-fold increase on pretreatment. The latter increase did not result in an increased incorporation of radioactivity into the glycoconjugates. It was estimated that, in untreated cells, the ratio of radioactivity incorporated from [3H]glucosamine into glycoconjugate-bound N-acetylhexosamine and N-acetylneuraminate amounted to 2:3. In pretreated cells this ratio changed to approx. 2:1. Overall, the data show that pretreatment resulted in an increased incorporation of N-acetylhexosamine into cell-associated and secreted glycoconjugates, accompanied by a reduction in sialylation. It was concluded that an increased availability of UDP-N-acetylhexosamine caused the increased incorporation of N-acetylhexosamine. The elevated cytosolic level of UDP-N-acetylhexosamine (and of compounds like CMP) is suggested to impair the transport of CMP-acetylneuraminate to the Golgi, resulting in reduced sialylation. This study demonstrates that protein glycosylation can be regulated at the level of the availability of the various nucleotide-sugars in the Golgi lumen.

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Process-Orientated Metabolic Engineering: Cell Lines with New Properties in Nutrient Exploitation and Protein Glycosylation

Recombinant Protein Production with Prokaryotic and Eukaryotic Cells. A Comparative View on Host Physiology ◽

10.1007/978-94-015-9749-4_21 ◽

2001 ◽

pp. 289-302

Author(s):

R. Wagner

Keyword(s):

Metabolic Engineering ◽

Cell Lines ◽

Protein Glycosylation

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The Levels of Nucleotide-Sugar: Glycoprotein Sialyl- and N-Acetyl-glucosaminyltransferases in Normal and Pathological Human Sera

Canadian Journal of Biochemistry ◽

10.1139/o72-102 ◽

1972 ◽

Vol 50 (7) ◽

pp. 738-740 ◽

Cited By ~ 45

Author(s):

Sailen Mookerjea ◽

M. Alex Michaels ◽

Roger L. Hudgin ◽

Mario A. Moscarello ◽

Annie Chow ◽

...

Keyword(s):

Liver Diseases ◽

Serum Enzymes ◽

Nucleotide Sugar ◽

Acetylneuraminic Acid ◽

Data Support ◽

Human Sera ◽

Nucleotide Sugars

Enzymes which transfer N-acetylneuraminic acid and N-acetylglucosamine from their respective nucleotide-sugars to exogenously added glycoprotein acceptors are present in human sera. The levels of these enzymes have been determined in various pathological sera. Sialyltransferase and N-acetylglucosaminyltransferase levels were increased in a group of patients with various liver diseases. N-Acetylglucosaminyltransferase levels were also increased in a group of patients with a variety of infections. The data support the conclusion that these serum enzymes are derived at least in part from the liver.

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UDP-4-Keto-6-Deoxyglucose, a Transient Antifungal Metabolite, Weakens the Fungal Cell Wall Partly by Inhibition of UDP-Galactopyranose Mutase

mBio ◽

10.1128/mbio.01559-17 ◽

2017 ◽

Vol 8 (6) ◽

Cited By ~ 1

Author(s):

Liang Ma ◽

Omar Salas ◽

Kyle Bowler ◽

Maor Bar-Peled ◽

Amir Sharon

Keyword(s):

Cell Wall ◽

Structural Similarity ◽

Intermediate Compound ◽

Antifungal Drugs ◽

Nucleotide Sugar ◽

Undetermined Function ◽

Nucleotide Sugars ◽

Toxic Potential ◽

Additional Nucleotide ◽

Surprising Finding

ABSTRACT Can accumulation of a normally transient metabolite affect fungal biology? UDP-4-keto-6-deoxyglucose (UDP-KDG) represents an intermediate stage in conversion of UDP-glucose to UDP-rhamnose. Normally, UDP-KDG is not detected in living cells, because it is quickly converted to UDP-rhamnose by the enzyme UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase (ER). We previously found that deletion of the er gene in Botrytis cinerea resulted in accumulation of UDP-KDG to levels that were toxic to the fungus due to destabilization of the cell wall. Here we show that these negative effects are at least partly due to inhibition by UDP-KDG of the enzyme UDP-galactopyranose mutase (UGM), which reversibly converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). An enzymatic activity assay showed that UDP-KDG inhibits the B. cinerea UGM enzyme with a K i of 221.9 µM. Deletion of the ugm gene resulted in strains with weakened cell walls and phenotypes that were similar to those of the er deletion strain, which accumulates UDP-KDG. Galf residue levels were completely abolished in the Δugm strain and reduced in the Δer strain, while overexpression of the ugm gene in the background of a Δer strain restored Galf levels and alleviated the phenotypes. Collectively, our results show that the antifungal activity of UDP-KDG is due to inhibition of UGM and possibly other nucleotide sugar-modifying enzymes and that the rhamnose metabolic pathway serves as a shunt that prevents accumulation of UDP-KDG to toxic levels. These findings, together with the fact that there is no Galf in mammals, support the possibility of developing UDP-KDG or its derivatives as antifungal drugs. IMPORTANCE Nucleotide sugars are donors for the sugars in fungal wall polymers. We showed that production of the minor sugar rhamnose is used primarily to neutralize the toxic intermediate compound UDP-KDG. This surprising finding highlights a completely new role for minor sugars and other secondary metabolites with undetermined function. Furthermore, the toxic potential of predicted transition metabolites that never accumulate in cells under natural conditions are highlighted. We demonstrate that UDP-KDG inhibits the UDP-galactopyranose mutase enzyme, thereby affecting production of Galf, which is one of the components of cell wall glycans. Given the structural similarity, UDP-KDG likely inhibits additional nucleotide sugar-utilizing enzymes, a hypothesis that is also supported by our findings. Our results suggest that UDP-KDG could serve as a template to develop antifungal drugs. IMPORTANCE Nucleotide sugars are donors for the sugars in fungal wall polymers. We showed that production of the minor sugar rhamnose is used primarily to neutralize the toxic intermediate compound UDP-KDG. This surprising finding highlights a completely new role for minor sugars and other secondary metabolites with undetermined function. Furthermore, the toxic potential of predicted transition metabolites that never accumulate in cells under natural conditions are highlighted. We demonstrate that UDP-KDG inhibits the UDP-galactopyranose mutase enzyme, thereby affecting production of Galf, which is one of the components of cell wall glycans. Given the structural similarity, UDP-KDG likely inhibits additional nucleotide sugar-utilizing enzymes, a hypothesis that is also supported by our findings. Our results suggest that UDP-KDG could serve as a template to develop antifungal drugs.

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Biosynthesis and Transport of Nucleotide Sugars for Plant Hemicellulose

Frontiers in Plant Science ◽

10.3389/fpls.2021.723128 ◽

2021 ◽

Vol 12 ◽

Author(s):

Wenjuan Zhang ◽

Wenqi Qin ◽

Huiling Li ◽

Ai-min Wu

Keyword(s):

Cell Wall ◽

Hydrogen Bonds ◽

Secondary Wall ◽

Vital Role ◽

Regulatory Mechanisms ◽

Nucleotide Sugar ◽

Number Of Genes ◽

Substrate Level ◽

Nucleotide Sugars ◽

Specific Nucleotide

Hemicellulose is entangled with cellulose through hydrogen bonds and meanwhile acts as a bridge for the deposition of lignin monomer in the secondary wall. Therefore, hemicellulose plays a vital role in the utilization of cell wall biomass. Many advances in hemicellulose research have recently been made, and a large number of genes and their functions have been identified and verified. However, due to the diversity and complexity of hemicellulose, the biosynthesis and regulatory mechanisms are yet unknown. In this review, we summarized the types of plant hemicellulose, hemicellulose-specific nucleotide sugar substrates, key transporters, and biosynthesis pathways. This review will contribute to a better understanding of substrate-level regulation of hemicellulose synthesis.

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Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012362 ◽

2020 ◽

Vol 295 (48) ◽

pp. 16445-16463 ◽

Cited By ~ 1

Author(s):

Bozena Szulc ◽

Paulina Sosicka ◽

Dorota Maszczak-Seneczko ◽

Edyta Skurska ◽

Auhen Shauchuk ◽

...

Keyword(s):

Golgi Apparatus ◽

Hepg2 Cells ◽

Double Knockout ◽

Nucleotide Sugar ◽

Sugar Transporters ◽

Nucleotide Sugars ◽

Nucleotide Sugar Transporters ◽

Hepg2 Cell Lines ◽

Nucleotide Sugar Transporter ◽

Qualitative Changes

Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.

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