Addition Of N-acetylglucosamine Research Articles

In Situ Imaging of O-Linked β-N-Acetylglucosamine Using On-Tissue Hydrolysis and MALDI Mass Spectrometry.

Post-translational O-glycosylation of proteins via the addition of N-acetylglucosamine (O-GlcNAc) is a regulator of many aspects of cellular physiology. Processes driven by perturbed dynamics of O-GlcNAcylation modification have been implicated in cancer development. Variability in O-GlcNAcylation is emerging as a metabolic biomarker of many cancers. Here, we evaluate the use of MALDI-mass spectrometry imaging (MSI) to visualize the location of O-GlcNAcylated proteins in tissue sections by mapping GlcNAc that has been released by the enzymatic hydrolysis of glycoproteins using an O-GlcNAc hydrolase. We use this strategy to monitor O-GlcNAc within hepatic VX2 tumor tissue. We show that increased O-GlcNAc is found within both viable tumor and tumor margin regions, implicating GlcNAc in tumor progression.

Multi-omics analysis of the specialised metabolism of two novel Pseudonocardia spp. isolated from the deep Southern Ocean

Multidrug-resistant pathogens have become a global threat. In this context, filamentous Actinobacteria has been proven to be an exceptional source of antimicrobial metabolites. In particular, rare actinomycetes isolated from marine environments have been proposed as a potential source of yet untapped specialised metabolites. In this study, two novel species, Pseudonocardia abyssalis sp. nov. and Pseudonocardia oceani sp. nov, isolated from deep Southern Ocean sediments are described, both in terms of their phenotypic and genomic characterization. Furthermore, the genomic architecture, with a focus on Biosynthetic Gene Cluster (BGC), across eight strains belonging to the two novel species were investigated. A total of 14 Gene Cluster Families (GCF) were identified, of which five GCFs comprise BGCs from both species, and nine were specific to each species. Moreover, a correlation of GCFs to phylogeny was observed. Following genome analysis, a comparative mass-spectrometry based metabolomics analysis was carried out with one strain from each new species, as well as Pseudonocardia ammonioxydans and Pseudonocardia sediminis, also of marine origin. The metabolomics profiles agreed with the GCF distribution, where a group of ubiquitous metabolites were produced by both new Pseudonocardia spp., while groups of species-specific metabolites were also detected. This metabolic-repertoire was found to be elicitated through the addition of N-acetyl glucosamine (GlcNAc), revealing chemically-inducible bioactivity against the fungi Candida albicas and multidrug-resistant Candida auris. These results showcase the power of a combined genomic-metabolomics approach to investigate rare-actinomycetes from understudied locations and have uncovered a wealth of both biosynthetic and chemical diversity for further investigation.

The Beginner's Guide to O-GlcNAc: From Nutrient Sensitive Pathway Regulation to Its Impact on the Immune System.

The addition of N-acetyl glucosamine (GlcNAc) on the hydroxy group of serine/threonine residues is known as O-GlcNAcylation (OGN). The dynamic cycling of this monosaccharide on and off substrates occurs via O-linked β-N-acetylglucosamine transferase (OGT) and O-linked β-N-acetylglucosaminase (OGA) respectively. These enzymes are found ubiquitously in eukaryotes and genetic knock outs of the ogt gene has been found to be lethal in embryonic mice. The substrate scope of these enzymes is vast, over 15,000 proteins across 43 species have been identified with O-GlcNAc. OGN has been known to play a key role in several cellular processes such as: transcription, translation, cell signaling, nutrient sensing, immune cell development and various steps of the cell cycle. However, its dysregulation is present in various diseases: cancer, neurodegenerative diseases, diabetes. O-GlcNAc is heavily involved in cross talk with other post-translational modifications (PTM), such as phosphorylation, acetylation, and ubiquitination, by regulating each other’s cycling enzymes or directly competing addition on the same substrate. This crosstalk between PTMs can affect gene expression, protein localization, and protein stability; therefore, regulating a multitude of cell signaling pathways. In this review the roles of OGN will be discussed. The effect O-GlcNAc exerts over protein-protein interactions, the various forms of crosstalk with other PTMs, and its role as a nutrient sensor will be highlighted. A summary of how these O-GlcNAc driven processes effect the immune system will also be included.

Proteomic identification of the UDP-GlcNAc: PI α1-6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei.

The first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis in all eukaryotes is the addition of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) which is catalysed by a UDP-GlcNAc: PI α1-6 GlcNAc-transferase, also known as GPI GnT. This enzyme has been shown to be a complex of seven subunits in mammalian cells and a similar complex of six homologous subunits has been postulated in yeast. Homologs of these mammalian and yeast subunits were identified in the Trypanosoma brucei predicted protein database. The putative catalytic subunit of the T. brucei complex, TbGPI3, was epitope tagged with three consecutive c-Myc sequences at its C-terminus. Immunoprecipitation of TbGPI3-3Myc followed by native polyacrylamide gel electrophoresis and anti-Myc Western blot showed that it is present in a ~240 kDa complex. Label-free quantitative proteomics were performed to compare anti-Myc pull-downs from lysates of TbGPI-3Myc expressing and wild type cell lines. TbGPI3-3Myc was the most highly enriched protein in the TbGPI3-3Myc lysate pull-down and the expected partner proteins TbGPI15, TbGPI19, TbGPI2, TbGPI1 and TbERI1 were also identified with significant enrichment. Our proteomics data also suggest that an Arv1-like protein (TbArv1) is a subunit of the T. brucei complex. Yeast and mammalian Arv1 have been previously implicated in GPI biosynthesis, but here we present the first experimental evidence for physical association of Arv1 with GPI biosynthetic machinery. A putative E2-ligase has also been tentatively identified as part of the T. brucei UDP-GlcNAc: PI α1-6 GlcNAc-transferase complex.

Quorum Sensing Activity and Hyphal Growth by External Stimuli in the Entomopathogenic Fungus Ophiocordyceps sinensis

The entomopathogenic fungus Ophiocordyceps sinensis is one of the best known and most precious medicines and health food in China. The blastospores-hyphae (dimorphism) transition of this fungus in host hemolymph is critical for the virulence and the mummification of host larvae. To regulate this transition, the effects of inoculum density and fifteen chemicals including fungal nutrients, fungal metabolites, quorum-sensing molecules (QSMs) and insect hormones on the dimorphism in O. sinensis were investigated in vitro. The blastospores tended to exhibit budding growth when inoculated at 107 blastospores per mL, and hyphal growth at concentrations lower than 106 blastospores per mL. At 105 blastospores per mL, the percentage of hyphal formation decreased with the addition of filtered spent medium containing 107 blastospores per mL, indicating the quorum-sensing effect. Blastospores-hyphae transition in this fungus by fifteen chemicals was varied from no response to dimorphic reversion. The addition of N-acetylglucosamine at three concentrations significantly stimulated hyphal formation while inhibiting budding growth. For the first time, insect hormone 20-hydroxyecdysone was found to be involved in the hyphal formation in fungi. These results open new possibilities to regulate the dimorphism, which would be beneficial for the cultivation of the Chinese cordyceps.

Glycan-dependent chikungunya viral infection divulged by antiviral activity of NAG specific chi-like lectin

Highly pathogenic alphaviruses display complex glycans on their surface. These glycans play a crucial role in viral pathogenesis by facilitating glycan-host interaction during viral entry which can be targeted. Various studies have reported antiviral activity of lectins that bind to the glycans present on the surface of enveloped viruses. This study evaluates the antiviral potential of a chitinase (chi)-like lectin from Tamarind (TCLL) having specificity for N-acetylglucosamine (NAG). Thus, TCLL might bind to N-glycan rich surface of alphavirus and inhibit the entry of virus into the host cells. The direct treatment of TCLL with virus reduced the virus infection. Remarkably, the addition of NAG to TCLL abolished antiviral activity confirming that NAG binding property of TCLL is accountable for its antiviral activity. Further, an ELISA assay confirmed the binding of TCLL to alphaviruses. Taken together, this study will prove to be beneficial in developing lectin therapeutics targeting alphavirus glycan.

N-Acetyl-glucosamine influences the biofilm formation of Escherichia coli

BackgroundThe intestinal mucous layer is a physical barrier that limits the contact between bacteria and host epithelial cells. There is growing evidence that microbiota-produced metabolites can also be specifically sensed by gut pathogens as signals to induce or repress virulence genes. Many E. coli, including adherent and invasive (AIEC) strains, can form biofilm. This property can promote their intestinal colonization and resistance to immune mechanisms. We sought to evaluate the impact of mucus-derived sugars on biofilm formation of E. coli.ResultsWe showed that the mucin sugar N-acetyl-glucosamine (NAG) can reduce biofilm formation of AIEC strain LF82. We demonstrated that the inactivation of the regulatory protein NagC, by addition of NAG or by mutation of nagC gene, reduced the biofilm formation of LF82 in static condition. Interestingly, real-time monitoring of biofilm formation of LF82 using microfluidic system showed that the mutation of nagC impairs the early process of biofilm development of LF82. Thus, NAG sensor NagC is involved in the early steps of biofilm formation of AIEC strain LF82 under both static and dynamic conditions. Its implication is partly due to the activation of type 1 fimbriae. NAG can also influence biofilm formation of other intestinal E. coli strains.ConclusionsThis study highlights how catabolism can be involved in biofilm formation of E. coli. Mucus-derived sugars can influence virulence properties of pathogenic E. coli and this study will help us better understand the mechanisms used to prevent colonization of the intestinal mucosa by pathogens.

Keratan Sulfate Phenotype in the β-1,3-N-Acetylglucosaminyltransferase-7-Null Mouse Cornea.

PurposeSynthesis of keratan sulfate (KS) relies on coordinated action of multiple enzymes, including the N-acetylglucosamine–transferring enzyme, β-1,3-N-acetylglucosaminyltransferase-7 (β3GnT7). A mouse model deficient in β3GnT7 was developed to explore structural changes in KS and the extracellular matrix (ECM; i.e., the corneal stroma), elucidate the KS biosynthesis mechanism, and understand its role in corneal organization.MethodsA knockout vector for the β3GnT7-encoding gene, B3gnt7, was created to develop heterozygous- (htz) and homozygous-null (null) knockouts. Epithelial, stromal, and whole cornea thicknesses were measured from each group. Proteoglycans were stained with cupromeronic blue for visualization by electron microscopy, and Western blot analyses were conducted on the KS core protein, lumican. Corneal sections were labelled fluorescently for KS and chondroitin sulfate/dermatan sulfate (CS/DS) using monoclonal antibodies 1B4 or 2B6, respectively.ResultsWild-type (WT) and htz corneas were of similar stromal thickness, whereas null specimens measured relatively thin. Electron micrographs revealed that WT and htz samples contained comparable levels of KS- and CS/DS-PGs. Null corneas, however, lacked detectable KS and featured uncharacteristically elongated electron dense PG filaments, which were susceptible to chondroitinase ABC digestion. Western blotting revealed lumican in the null corneas was substituted with low-molecular-weight KS, relative to WT or htz tissue. KS was not immunohistochemically detectable in the null cornea, whereas CS/DS content appeared increased.ConclusionsAddition of N-acetylglucosamine via β3GnT7 to KS glycosaminoglycans is necessary for their biosynthesis. Without β3GnT7, murine corneal stromas lack KS and appear to compensate for this loss with upregulation of chondroitinase ABC-sensitive PGs.

Lack of O-GlcNAcylation enhances exercise-dependent glucose utilization potentially through AMP-activated protein kinase activation in skeletal muscle

O-GlcNAcylation is a post-translational modification that is characterized by the addition of N-acetylglucosamine (GlcNAc) to proteins by O-GlcNAc transferase (Ogt). The degree of O-GlcNAcylation is thought to be associated with glucotoxicity and diabetic complications, because GlcNAc is produced by a branch of the glycolytic pathway. However, its role in skeletal muscle has not been fully elucidated. In this study, we created skeletal muscle-specific Ogt knockout (Ogt-MKO) mice and analyzed their glucose metabolism. During an intraperitoneal glucose tolerance test, blood glucose was slightly lower in Ogt-MKO mice than in control Ogt-flox mice. High fat diet-induced obesity and insulin resistance were reversed in Ogt-MKO mice. In addition, 12-month-old Ogt-MKO mice had lower adipose and body mass. A single bout of exercise significantly reduced blood glucose in Ogt-MKO mice, probably because of higher AMP-activated protein kinase α (AMPKα) protein expression. Furthermore, intraperitoneal injection of 5-aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, resulted in a more marked decrease in blood glucose levels in Ogt-MKO mice than in controls. Finally, Ogt knockdown by siRNA in C2C12 myotubes significantly increased protein expression of AMPKα, glucose uptake and oxidation. In conclusion, loss of O-GlcNAcylation facilitates glucose utilization in skeletal muscle, potentially through AMPK activation. The inhibition of O-GlcNAcylation in skeletal muscle may have an anti-diabetic effect, through an enhancement of glucose utilization during exercise.

Effect of light emutting diode (LED) lamps and N-acetylglucosamine (NAG) on organogenesis in protocorm-like bodies (PLBs) of a Cymbidium hybrid cultured in vitro

Abstract not available
 Plant Tissue Cult. & Biotech. 24(2): 273-277, 2014 (December)

Triggering effect of N-acetylglucosamine on retarded drug release from a lectin-anchored chitosan nanoparticles-in-microparticles system

The aim of this study was to investigate the use of N-acetylglucosamine (NAG) to accelerate drug release from a lectin-modified carrier. A wheat germ agglutinin (WGA)-anchored salmeterol xinafoate (SalX)-loaded nanoparticles-in-microparticles system (NiMS) was prepared with an ionotropic gelation technique combined with a spray drying method. The formulated microparticles were spherical, with diameters ranging mainly from 2 to 8 μm; the drug entrapment efficiency was >70% (w/w), and the loading capacity was approximately 8% (w/w). Drug release from WGA-SalX-NiMS, within the first 4h, was approximately 30% less than that from SalX-NiMS, indicating an effect of lectin-modification to retard drug release from the NiMS. Due to "sugar-lectin" interactions, drug release from WGA-SalX-NiMS was substantially increased after the addition of NAG to the release medium. However, no significant influence of NAG was observed on the drug release profile of SalX-NiMS without WGA anchorage. The characteristics of NAG-WGA interaction may provide valuable insights into the "triggering-effects" of specific sugars on drug release from lectin-anchored carriers. These results suggest that it is possible to control drug release from a lectin-anchored drug delivery system using a specific sugar, and that the designed novel WGA-SalX-NiMS may be a suitable formulation for chronotherapy of asthma.

Grazing resistant freshwater bacteria profit from chitin and cell‐wall‐derived organic carbon

The rise of grazing resistant planktonic bacteria in freshwater lakes during vernal phytoplankton blooms is favoured by predation of heterotrophic nanoflagellates (HNF). The spring period is also characterized by increased availability of organic carbon species that are in parts derived from cellular debris generated during bacterivory or viral lysis, such as peptidoglycan, chitin and their subunit N-acetylglucosamine (NAG). We tested the hypothesis that two dominant grazing resistant bacterial taxa, the ac1 tribe of Actinobacteria (ac1) and filamentous bacteria from the LD2 lineage (Saprospiraceae), profit from such carbon sources during periods of intense HNF predation. The abundances of ac1 and LD2 rose in parallel with HNF, and disproportionally high fractions of cells from both lineages were involved in NAG uptake. Members of ac1 and LD2 were significantly more enriched after NAG addition to lake water. However, highest growth rates of both bacterial lineages were found on chitin and peptidoglycan. Moreover, the direct or indirect transfer of organic carbon from peptidoglycan to LD2 filaments could be demonstrated. We thus provide evidence that these taxa may benefit twofold from protistan predation: by removal of their competitors, and by specific physiological adaptations to utilize carbon sources that are released during grazing or viral lysis.

Inducible colitis-associated glycome capable of stimulating the proliferation of memory CD4+ T cells

Immune responses are modified by a diverse and abundant repertoire of carbohydrate structures on the cell surface, which is known as the glycome. In this study, we propose that a unique glycome that can be identified through the binding of galectin-4 is created on local, but not systemic, memory CD4+ T cells under diverse intestinal inflammatory conditions, but not in the healthy state. The colitis-associated glycome (CAG) represents an immature core 1-expressing O-glycan. Development of CAG may be mediated by down-regulation of the expression of core-2 β1,6-N-acetylglucosaminyltransferase (C2GnT) 1, a key enzyme responsible for the production of core-2 O-glycan branch through addition of N-acetylglucosamine (GlcNAc) to a core-1 O-glycan structure. Mechanistically, the CAG seems to contribute to super raft formation associated with the immunological synapse on colonic memory CD4+ T cells and to the consequent stabilization of protein kinase C θ activation, resulting in the stimulation of memory CD4+ T cell expansion in the inflamed intestine. Functionally, CAG-mediated CD4+ T cell expansion contributes to the exacerbation of T cell-mediated experimental intestinal inflammations. Therefore, the CAG may be an attractive therapeutic target to specifically suppress the expansion of effector memory CD4+ T cells in intestinal inflammation such as that seen in inflammatory bowel disease.

O-GlcNAc processing enzymes: catalytic mechanisms, substrate specificity, and enzyme regulation

The addition of N-acetylglucosamine (GlcNAc) O-linked to serine and threonine residues of proteins is known as O-GlcNAc. This post-translational modification is found within multicellular eukaryotes on hundreds of nuclear and cytoplasmic proteins. O-GlcNAc transferase (OGT) installs O-GlcNAc onto target proteins and O-GlcNAcase (OGA) removes O-GlcNAc. Their combined action makes O-GlcNAc reversible and serves to regulate cellular O-GlcNAc levels. Here I review select recent literature on the catalytic mechanism of these enzymes and studies on the molecular basis by which these enzymes identify and process their substrates. Molecular level understanding of how these enzymes work, and the basis for their specificity, should aid understanding how O-GlcNAc contributes to diverse cellular processes ranging from cellular signaling through to transcriptional regulation.

GlcNAc in Place of Glucose

Addition of N-acetylglucosamine to the medium can substitute for glucose in maintaining cell growth, but not proliferation.

Melanin externalization in Candida albicans depends on cell wall chitin structures.

The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Delta and chs2Delta chs3Delta mutants but were fully externalized in chs8Delta and chs2Delta chs8Delta mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.

The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants

BackgroundSoil bacteria collectively known as Rhizobium, characterized by their ability to establish beneficial symbiosis with legumes, share several common characteristics with pathogenic bacteria when infecting the host plant. Recently, it was demonstrated that a fadD mutant of Sinorhizobium meliloti is altered in the control of swarming, a type of co-ordinated movement previously associated with pathogenicity, and is also impaired in nodulation efficiency on alfalfa roots. In the phytopathogen Xanthomonas campestris, a fadD homolog (rpfB) forms part of a cluster of genes involved in the regulation of pathogenicity factors. In this work, we have investigated the role in swarming and symbiosis of SMc02161, a S. meliloti fadD-linked gene.ResultsThe SMc02161 locus in S. meliloti shows similarities with members of the Major Facilitator Superfamily (MFS) of transporters. A S. meliloti null-mutant shows increased sensitivity to chloramphenicol. This indication led us to rename the locus tep1 for transmembrane efflux protein. The lack of tep1 does not affect the appearance of swarming motility. Interestingly, nodule formation efficiency on alfalfa plants is improved in the tep1 mutant during the first days of the interaction though nod gene expression is lower than in the wild type strain. Curiously, a nodC mutation or the addition of N-acetyl glucosamine to the wild type strain lead to similar reductions in nod gene expression as in the tep1 mutant. Moreover, aminosugar precursors of Nod factors inhibit nodulation.Conclusiontep1 putatively encodes a transmembrane protein which can confer chloramphenicol resistance in S. meliloti by expelling the antibiotic outside the bacteria. The improved nodulation of alfalfa but reduced nod gene expression observed in the tep1 mutant suggests that Tep1 transports compounds which influence nodulation. In contrast to Bradyrhizobium japonicum, we show that in S. meliloti there is no feedback regulation of nodulation genes. Moreover, the Nod factor precursor, N-acetyl glucosamine reduces nod gene expression and nodulation efficiency when present at millimolar concentrations. A role for Tep1 in the efflux of Nod factor precursors could explain the phenotypes associated with tep1 inactivation.

Chikungunya, l'épidémie que l'on n'attendait pas

Highly pathogenic alphaviruses display complex glycans on their surface. These glycans play a crucial role in viral pathogenesis by facilitating glycan-host interaction during viral entry which can be targeted. Various studies have reported antiviral activity of lectins that bind to the glycans present on the surface of enveloped viruses. This study evaluates the antiviral potential of a chitinase (chi)-like lectin from Tamarind (TCLL) having specificity for N-acetylglucosamine (NAG). Thus, TCLL might bind to N-glycan rich surface of alphavirus and inhibit the entry of virus into the host cells. The direct treatment of TCLL with virus reduced the virus infection. Remarkably, the addition of NAG to TCLL abolished antiviral activity confirming that NAG binding property of TCLL is accountable for its antiviral activity. Further, an ELISA assay confirmed the binding of TCLL to alphaviruses. Taken together, this study will prove to be beneficial in developing lectin therapeutics targeting alphavirus glycan.

Properties of yeast expressed Aspergillus nidulans chitin synthase B which is essential for hyphal growth1

A complementary DNA of the Aspergillus nidulans chsB gene encoding chitin synthase, an essential gene for hyphal growth, was obtained by RT-PCR and expressed in Saccharomyces cerevisiae by using the GAL1 promoter in a multicopy plasmid. The biochemical characteristics of chitin synthase B (ChsB) expressed in S. cerevisiae were examined. The chitin synthase B produced in galactose medium showed zymogenicity due to activation by trypsin treatment and required Mg2+ ion to exert maximal activity. It was competitively inhibited by polyoxin D. The Ki value of the inhibitor was 10 microM, and the K(m) for the substrate was 1.6 mM. The activity was enhanced by the addition of N-acetylglucosamine. The optimal pH is 7.5 when Mg2+ is used. These characteristics are the same as those of other chitin synthases.

Engineering Intracellular CMP-Sialic Acid Metabolism into Insect Cells and Methods to Enhance Its Generation

Previous studies have reported that insect cell lines lack the capacity to generate endogenously the nucleotide sugar, CMP-Neu5Ac, required for sialylation of glycoconjugates. In this study, the biosynthesis of this activated form of sialic acid completely from endogenous metabolites is demonstrated for the first time in insect cells by expressing the mammalian genes required for the multistep conversion of endogenous UDP-GlcNAc to CMP-Neu5Ac. The genes for UDP-GlcNAc-2-epimerase/ManNAc kinase (EK), sialic acid 9-phosphate synthase (SAS), and CMP-sialic acid synthetase (CSAS) were coexpressed in insect cells using baculovirus expression vectors, but the CMP-Neu5Ac and precursor Neu5Ac levels synthesized were found to be lower than those achieved with ManNAc supplementation due to feedback inhibition of the EK enzyme by CMP-Neu5Ac. When sialuria-like mutant EK genes, in which the site for feedback regulation has been mutated, were used, CMP-Neu5Ac was synthesized at levels more than 4 times higher than that achieved with the wild-type EK and 2.5 times higher than that achieved with ManNAc feeding. Addition of N-acetylglucosamine (GlcNAc), a precursor for UDP-GlcNAc, to the media increased the levels of CMP-Neu5Ac even more to a level 7.5 times higher than that achieved with ManNAc supplementation, creating a bottleneck in the conversion of Neu5Ac to CMP-Neu5Ac at higher levels of UDP-GlcNAc. The present study provides a useful biochemical strategy to synthesize and enhance the levels of the sialylation donor molecule, CMP-Neu5Ac, a critical limiting substrate for the generation of complex glycoproteins in insect cells and other cell culture systems.