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Several pathogenic bacteria import and catabolize sialic acids as a source of carbon and nitrogen. Within the sialic acid catabolic pathway, the enzyme N-acetylmannosamine kinase (NanK) catalyzes the phosphorylation of N-acetylmannosamine to N-acetylmannosamine-6-phosphate. This kinase belongs to the ROK superfamily of enzymes, which generally contain a conserved zinc-finger (ZnF) motif that is important for their structure and function. Previous structural studies have shown that the ZnF motif is absent in NanK of Fusobacterium nucleatum (Fn-NanK), a Gram-negative bacterium that causes the gum disease gingivitis. However, the effect in loss of the ZnF motif on the kinase activity is unknown. Using kinetic and thermodynamic studies, we have studied the functional properties of Fn-NanK to its substrates ManNAc and ATP, compared its activity with other ZnF motif-containing NanK enzymes from closely related Gram-negative pathogenic bacteria Haemophilus influenzae (Hi-NanK), Pasteurella multocida (Pm-NanK), and Vibrio cholerae (Vc-NanK). Our studies show a 10-fold decrease in substrate binding affinity between Fn-NanK (apparent K-M approximate to 700 mu M) and ZnF motif-containing NanKs (apparent K-M 60 approximate to mu M). To understand the structural features that combat the loss of the ZnF motif in Fn-NanK, we solved the crystal structures of functionally homologous ZnF motif-containing NanKs from P. multocida and H. influenzae. Here, we report Pm-NanK:unliganded, Pm-NanK:AMPPNP, Pm-NanK:ManNAc, Hi-NanK:ManNAc, and Hi-NanK:ManNAc-6P:ADP crystal structures. Structural comparisons of Fn-NanK with Hi-NanK, Pm-NanK, and hMNK (human N-acetylmannosamine kinase domain of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, GNE) show that even though there is less sequence identity, they have high degree of structural similarity. Furthermore, our structural analyses highlight that the ZnF motif of Fn-NanK is substituted by a set of hydrophobic residues, which forms a hydrophobic cluster that helps the proper orientation of ManNac in the active site. In summary, ZnF-containing and ZnF-lacking NanK enzymes from different Gram-negative pathogenic bacteria are functionally very similar but differ in their metal requirement. Our structural studies unveil the structural modifications in Fn-NanK that compensate the loss of the ZnF motif in comparison to other NanK enzymes.

GNE myopathy is a rare, autosomal recessive, inborn error of sialic acid metabolism, caused by mutations in GNE, the gene encoding UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase. The disease manifests as an adult-onset myopathy characterized by progressive skeletal muscle weakness and atrophy. There is no medical therapy available for this debilitating disease. Hyposialylation of muscle glycoproteins likely contributes to the pathophysiology of this disease. N-acetyl-D-mannosamine (ManNAc), an uncharged monosaccharide and the first committed precursor in the sialic acid biosynthetic pathway, is a therapeutic candidate that prevents muscle weakness in the mouse model of GNE myopathy. We conducted a first-in-human, randomized, placebo-controlled, double-blind, single-ascending dose study to evaluate safety and pharmacokinetics of ManNAc in GNE myopathy subjects. Single doses of 3 and 6g of oral ManNAc were safe and well tolerated; 10g was associated with diarrhea likely due to unabsorbed ManNAc. Oral ManNAc was absorbed rapidly and exhibited a short half-life (~2.4h). Following administration of a single dose of ManNAc, there was a significant and sustained increase in plasma unconjugated free sialic acid (Neu5Ac) (Tmax of 8-11h). Neu5Ac levels remained above baseline 48h post-dose in subjects who received a dose of 6 or 10g. Given that Neu5Ac is known to have a short half-life, the prolonged elevation of Neu5Ac after a single dose of ManNAc suggests that intracellular biosynthesis of sialic acid was restored in subjects with GNE myopathy, including those homozygous for mutations in the kinase domain. Simulated plasma concentration-time profiles support a dosing regimen of 6g twice daily for future clinical trials.

Contains fulltext : 207220.pdf (Publisher’s version ) (Closed access) Contains fulltext : 207220po.pdf (Author’s version postprint ) (Open Access) Sialic acids cap the glycans of cell surface glycoproteins and glycolipids. They are involved in a multitude of biological processes and aberrant sialic acid expression is associated with several pathologies. Sialic acids modulate the characteristics and functions of glycoproteins and regulate cell-cell as well as cell-extracellular matrix interactions. Pathogens such as influenza virus use sialic acids to infect host cells and cancer cells exploit sialic acids to escape from the host's immune system. The introduction of unnatural sialic acids with different functionalities into surface glycans enables the study of the broad biological functions of these sugars and presents a therapeutic option to intervene with pathological processes involving sialic acids. Multiple chemically modified sialic acid analogs can be directly utilized by cells for sialoglycan synthesis. Alternatively, analogs of the natural sialic acid precursor sugar N-Acetylmannosamine (ManNAc) can be introduced into the sialic acid biosynthesis pathway resulting in the intracellular conversion into the corresponding sialic acid analog. Both, ManNAc and sialic acid analogs, have been employed successfully for a large variety of glycoengineering applications such as glycan imaging, targeting toxins to tumor cells, inhibiting pathogen binding, or altering immune cell activity. However, there are significant differences between ManNAc and sialic acid analogs with respect to their chemical modification potential and cellular metabolism that should be considered in sialic acid glycoengineering experiments.

Synthetically accessible C6-analogs of N-acetylmannosamine (ManNAc) were tested as potential inhibitors of the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE/MNK), the key enzyme of sialic acid biosynthesis. Enzymatic experiments revealed that the modification introduced at the C6 saccharide position strongly influences the inhibitory potency. A C6-ManNAc diselenide dimer showed the strongest kinase inhibition in the low μM range among all the substrates tested and successfully reduced cell surface sialylation in Jurkat cells.

Neisseria meningitidis serogroup A non-hydrolyzing uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase (NmSacA) catalyzes the interconversion between UDP-GlcNAc and uridine 5'-diphosphate-N-acetylmannosamine (UDP-ManNAc). It is a key enzyme involved in the biosynthesis of the capsular polysaccharide [-6ManNAcα1-phosphate-]n of N. meningitidis serogroup A, one of the six serogroups (A, B, C, W-135, X, and Y) that account for most cases of N. meningitidis-caused bacterial septicemia and meningitis. N. meningitidis serogroup A is responsible for large epidemics in the developing world, especially in Africa. Here we report that UDP-ManNAc could be used as a substrate for C-terminal His6-tagged recombinant NmSacA (NmSacA-His6) in the absence of UDP-GlcNAc. NmSacA-His6 was activated by UDP-GlcNAc and inhibited by 2-acetamidoglucal and UDP. Substrate specificity study showed that NmSacA-His6 could tolerate several chemoenzymatically synthesized UDP-ManNAc derivatives as substrates although its activity was much lower than non-modified UDP-ManNAc. Homology modeling and molecular docking revealed likely structural determinants of NmSacA substrate specificity. This is the first detailed study of N. meningitidis serogroup A UDP-GlcNAc 2-epimerase.

Borrelia burgdorferi, the causative agent of Lyme disease, lacks the ability to biosynthesize many essential nutrients de novo, including N-acetylglucosamine (GlcNAc). This amino sugar is required for cell wall synthesis, and is a component of the complex growth medium used for in vitro propagation. When cultured without free GlcNAc, B. burgdorferi cells exhibit a unique biphasic growth pattern. We hypothesized that genes involved in the GlcNAc starvation response would be differentially expressed when compared to cells cultured in complete medium, and investigated this using transcriptomics. Twenty-one genes were differentially regulated in wild-type and starvation-adapted cells cultured without GlcNAc compared to wild-type cells cultured with GlcNAc. Of those, three genes involved in carbohydrate utilization were upregulated: bbb04 (chbC) encoding a subunit of the chitobiose transporter, bb0629 (fruA-2) encoding a putative carbohydrate transporter and bb0644 (nanE) encoding a putative GlcNAc-6-phosphate-2-epimerase predicted to catalyze the conversion of N-acetylmannosamine-6-phosphate (ManNAc-6-P) to GlcNAc-6-P. Quantitative RT-PCR was used to confirm differential expression of select genes, and substitution of free GlcNAc with free ManNAc resulted in growth to high cell density, suggesting B. burgdorferi cells can utilize free ManNAc for cell wall synthesis and energy production.

Sialic acids have a special place in vertebrate glycobiology, where they constitute the dominant terminal saccharides on many cell surface glycans. From early studies that identified sialoglycans as receptors for important pathogens and toxins to more recent discoveries of sialic acid-binding proteins essential for immune system (and other) functions in humans, sialic acids and sialoglycans have become cornerstones in understanding vertebrate glycobiology and pathology. During a remarkable 3-year period in the late 1950s, a newly minted postdoctoral fellow (Donald G. Comb) and his young mentor (Saul Roseman) made a surprising series of discoveries that put sialic acid research on sound chemical and biochemical footing. A detailed personal letter written by Dr. Roseman that describes this period of intense sialic acid discovery, complete with inserted figures, was given to one of us (Y.C.L.) several years later. The text and figures of this letter provide a look back at the enthusiasm, rigor and serendipity that led to their important findings through the eyes of one of the key figures in sialic acid research.

Distal myopathy with rimmed vacuoles/hereditary inclusion body myopathy (DMRV/hIBM), characterized by progressive muscle atrophy, weakness, and degeneration, is due to mutations in GNE, a gene encoding a bifunctional enzyme critical in sialic acid biosynthesis. In the DMRV/hIBM mouse model, which exhibits hyposialylation in various tissues in addition to muscle atrophy, weakness, and degeneration, we recently have demonstrated that the myopathic phenotype was prevented by oral administration of N-acetylneuraminic acid, N-acetylmannosamine, and sialyllactose, underscoring the crucial role of hyposialylation in the disease pathomechanism. The choice for the preferred molecule, however, was limited probably by the complex pharmacokinetics of sialic acids and the lack of biomarkers that could clearly show dose response. To address these issues, we screened several synthetic sugar compounds that could increase sialylation more remarkably and allow demonstration of measurable effects in the DMRV/hIBM mice. In this study, we found that tetra-O-acetylated N-acetylmannosamine increased cell sialylation most efficiently, and in vivo evaluation in DMRV/hIBM mice revealed a more dramatic, measurable effect and improvement in muscle phenotype, enabling us to establish analysis of protein biomarkers that can be used for assessing response to treatment. Our results provide a proof of concept in sialic acid-related molecular therapy with synthetic monosaccharides.

Sialylation (e.g. expression of sialic acid) plays a crucial role for function and stability of most glycoproteins. The key enzyme for the biosynthesis of sialic acid is the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE). Mutations in the binding site of the feedback inhibitor CMP-sialic acid of the GNE leads to sialuria, a disease in which patients produce sialic acid in gram scale. Here, we report on the use in biotechnology of sialuria-mutated GNE. Expression of the sialuria-mutated GNE in CHO-cells leads to increased sialylation of recombinant expressed erythropoietin (EPO). Our data show that sialuria-mutated-GNE over-expressing cells are the perfect platform to express highly sialylated therapeutic proteins, such as EPO.

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme of the biosynthesis of sialic acids, terminal components of glycoconjugates associated with a variety of cellular processes. Two novel isoforms of human GNE, namely GNE2 and GNE3, which possess extended and deleted N-termini, respectively, were characterized. GNE2 was also found in other species like apes, rodents, chicken or fish, whereas GNE3 seems to be restricted to primates. Both, GNE2 and GNE3, displayed tissue specific expression patterns, therefore may contribute to the complex regulation of sialic acid metabolism.

Peracetylated N-alpha-azidoacetylmannosamine (Ac(4)ManNAz) is metabolized by cells to CMP-azidosialic acid. It has been demonstrated previously that in this way azidosialic acid-containing glycoproteins are formed that can be labeled on the cell surface by a modified Staudinger ligation. Here, we first demonstrate that the same procedure also results in the formation of azidosialic acid-containing gangliosides. Deoxymannojirimycin, an inhibitor of N-glycan processing in proteins, decreases the total cell surface labeling in Jurkat cells by approximately 25%. Inhibition of ganglioside biosynthesis with N-[5-(adamantan-1-yl-methoxy)-pentyl]1-deoxynojirimycin reduces cell surface labeling by approximately 75%. In conclusion, exposure of cells to Ac(4)ManNAz allows in vivo chemical tagging of gangliosides.

N-Acetylneuraminic acid (Neu5Ac or NANA) is the most predominant sialic acid in mammals. As a terminal component in many glycoproteins and glycolipids, sialic acid is believed to be an important biomarker related to various diseases. Its precursor, N-acetylmannosamine (ManNAc), is being investigated as a potential treatment for GNE myopathy. In this work, we developed two highly sensitive and selective liquid chromatography–tandem mass spectrometry (LC-MS/MS) methods for the quantitation of ManNAc and free Neu5Ac in human plasma. A fit-for-purpose approach was adopted during method validation and sample analysis. To measure the endogenous compounds and overcome the interference from plasma samples, a surrogate matrix that contained 5% bovine serum albumin (BSA) was used for the preparation of calibration standards and certain levels of quality control (QC) samples. QC samples at higher concentrations were prepared in the authentic matrix (human plasma) to best mimic incurred samples. For both methods, an Ostro 96-well phospholipid removal plate was used for sample extraction, which efficiently removed the phospholipids from the plasma samples prior to LC injection, eliminated matrix effect, and improved sensitivity. Chromatographic separation was achieved using hydrophilic interaction chromatography (HILIC) and gradient elution in order to retain the two polar compounds. The lower limit of quantitation (LLOQ) for ManNAc and Neu5Ac was 10.0 and 25.0 ng/mL, respectively. The overall accuracy of the two assays was no less than 91.7% based on three levels of QC samples. Inter- and intra-run precision (coefficient of variation [%CV]) across three analytical runs was less than 6.7% for ManNAc and less than 10.8% for Neu5Ac. These methods have been validated to support clinical studies.

The N-acetyl-D-galactosamine (GalNAc) transport system of Escherichia coli K92 was studied when the bacterium was grown in a chemically defined medium containing GalNAc as a carbon source. Kinetic measurements were carried out in vivo at 37 degrees C in 25 mM phosphate buffer, pH 7.0. Under these conditions, the uptake rate was linear for at least 3 min and the calculated Km for GalNAc was 3 microM. The transport system was strongly inhibited by sodium arsenate (70%), potassium cyanide (62%) and 2,4-dinitrophenol (75%). Analysis of bacterial GalNAc phosphotransferase activity revealed in vitro GalNAc phosphorylation activity only when phosphoenolpyruvate was present. These results strongly support the notion that GalNAc uptake depends on a specific phosphotransferase system. Study of activity regulation showed that N-acetylglucosamine and mannosamine specifically inhibit the transport of GalNAc in this bacterium. Analysis of expression revealed that the GalNAc transport system is specifically induced by GalNAc but not by N-acetylglucosamine (GlcNAc) or N-acetylmannosamine (ManNAc), two intimately related sugars. Moreover, full induction of GalNAc transport required the presence of both cAMP and GalNAc. Comparative studies revealed that E. coli K92 has developed a regulation mechanism that specifically induces the appropriate permease based on the presence of each respective phospho-amino sugar (GlcNAc, ManNAc and GalNAc). In this regulation system, GlcNAc is the preferred amino sugar as the carbon source. Finally, when E. coli K92 was grown using GalNAc, capsular polysialic acid production was strongly affected. The presence of intracellular phosphoderivative acetylamino sugars, generated by the action of the phosphotransferase transport system, can be responsible for this effect.

Sialic acids are expressed as terminal sugars in many glycoconjugates and play an important role during development and regeneration, as they are involved as polysialic acid in a variety of cell–cell interactions mediated by the neural cell adhesion molecule NCAM. The key enzyme for the biosynthesis of sialic acid is the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE). Mutations in the binding site of the feedback inhibitor CMP-sialic acid of the GNE leads to sialuria, a disease in which patients produce sialic acid in gram scale. Here, we report on the consequences after expression of a sialuria-mutated GNE. Expression of the sialuria-mutated GNE leads to a dramatic increase of both cellular sialic acid and polysialic acid on NCAM. This could also be achieved by application of the sialic acid precursor N-acetylmannosamine. Our data suggest that biosynthesis of sialic acid regulates and limits the synthesis of polysialic acid.

A variety of pathogens or commensals use at least one of four distinct mechanisms for decorating their surfaces with sialic acid as a strategy to avoid, subvert or inhibit host innate immunity. The metabolism of sialic acid thus is central to a range of host-pathogen interactions. The first committed step in this process, the production of free N-acetylmannosamine (ManNAc), has not been defined. Here we show that ManNAc-6-phosphate (ManNAc-6-P) is not an obligate sialate precursor in Escherichia coli K1. This conclusion was supported by 31P NMR spectroscopy of E. coli K1 derivatives engineered with different combinations of mutations in nanA (sialate aldolase or lyase), nanK (ManNAc kinase), nanE (ManNAc-6-P 2-epimerase), neuS (polysialyltransferase) and neuB (sialate synthase). The product specificities for purified NanK and NanE were determined by chromatographic analyses. Direct biochemical analysis showed that ManNAc-6-P was stable in a nanE mutant extract. The combined results indicate that neither ManNAc-6-P nor specific or non-specific phosphatase are necessary to generate the requisite ManNAc for sialate biosynthesis. Our results imply that the neuC gene product encodes an UDP-N-acetylglucosamine 2-epimerase that generates ManNAc directly from the dinucleotide-sugar precursor despite detection of only this enzyme's UDP-GlcNAc hydrolase activity. This study describes the first use of NMR for analysing intermediate flux within the sialate biosynthetic pathway.

Due to its position at the outermost of glycans, sialic acid is involved in a myriad of physiological and pathophysiological cell functions such as host-pathogen interactions, immune regulation, and tumor evasion. Inhibitors of cell surface sialylation could be a useful tool in cancer, immune, antibiotic, or antiviral therapy. In this work, four different C-3 modified N-acetylmannosamine analogs were tested as potential inhibitors of cell surface sialylation. Peracetylated 2-acetylamino-2-deoxy-3-O-methyl-d-mannose decreases cell surface sialylation in Jurkat cells in a dose-dependent manner up to 80%, quantified by flow cytometry and enzyme-linked lectin assays. High-performance liquid chromatography experiments revealed that not only the concentration of membrane bound but also of cytosolic sialic acid is reduced in treated cells. We have strong evidence that the observed reduction of sialic acid expression in cells is caused by the inhibition of the bifunctional enzyme UDP-GlcNAc-2-epimerase/ManNAc kinase. 2-Acetylamino-2-deoxy-3-O-methyl-d-mannose inhibits the human ManNAc kinase domain of the UDP-GlcNAc-2-epimerase/ManNAc kinase. Binding kinetics of the inhibitor and human N-acetylmannosamine kinase were evaluated using surface plasmon resonance. Specificity studies with human N-acetylglucosamine kinase and hexokinase IV indicated a high specificity of 2-acetylamino-2-deoxy-3-O-methyl-d-mannose for MNK. This substance represents a novel class of inhibitors of sialic acid expression in cells, targeting the key enzyme of sialic acid de novo biosynthesis.

The N-acetyl-D-mannosamine (ManNAc) transport system of Escherichia coli K92 was studied when this bacterium was grown in a chemically defined medium containing ManNAc as carbon source. Kinetic measurements were carried out in vivo at 37 degrees C in 25 mM phosphate buffer, pH 7.5. Under these conditions, the uptake rate was linear for at least 15 min and the calculated Km for ManNAc was 280 microM. The transport system was strongly inhibited by sodium arsenate (97%), potassium cyanide (84%) and 2,4-dinitrophenol (88%) added at final concentrations of 1 mM (each). Analysis of bacterial ManNAc phosphotransferase activity revealed in vitro ManNAc phosphorylation activity only when phosphoenolpyruvate was present. These results strongly support the notion that ManNAc uptake depends on a specific phosphotransferase system. Study of specificities showed that N-acetylglucosamine and mannosamine specifically inhibited the transport of ManNAc in this bacterium. Analysis of expression revealed that the ManNAc transport system was induced by ManNAc, glucosamine, galactosamine, mannosamine and mannose but not by N-acetylglucosamine or N-acetylgalactosamine. Moreover, ManNAc permease was subject to glucose repression and cAMP stimulation. Full induction of the ManNAc transport system required the simultaneous presence of both cAMP and ManNAc.

N -Acetylglucosamine (GlcNAc) and N -acetylneuraminic acid (NANA) are good carbon sources for Escherichia coli K-12, whereas N -acetylmannosamine (ManNAc) is metabolized very slowly. The isolation of regulatory mutations which enhanced utilization of ManNAc allowed us to elucidate the pathway of its degradation. ManNAc is transported by the manXYZ -encoded phosphoenolpyruvate-dependent phosphotransferase system (PTS) transporter producing intracellular ManNAc-6-P. This phosphorylated hexosamine is subsequently converted to GlcNAc-6-P, which is further metabolized by the nagBA -encoded deacetylase and deaminase of the GlcNAc-6-P degradation pathway. Two independent mutations are necessary for good growth on ManNAc. One mutation maps to mlc , and mutations in this gene are known to enhance the expression of manXYZ . The second regulatory mutation was mapped to the nanAT operon, which encodes the NANA transporter and NANA lyase. The combined action of the nanAT gene products converts extracellular NANA to intracellular ManNAc. The second regulatory mutation defines an open reading frame (ORF), called yhcK , as the gene for the repressor of the nan operon ( nanR ). Mutations in the repressor enhance expression of the nanAT genes and, presumably, three distal, previously unidentified genes, yhcJIH . Expression of just one of these downstream ORFs, yhcJ , is necessary for growth on ManNAc in the presence of an mlc mutation. The yhcJ gene appears to encode a ManNAc-6-P-to-GlcNAc-6-P epimerase ( nanE ). Another putative gene in the nan operon, yhcI , likely encodes ManNAc kinase ( nanK ), which should phosphorylate the ManNAc liberated from NANA by the NanA protein. Use of NANA as carbon source by E. coli also requires the nagBA gene products. The existence of a ManNAc kinase and epimerase within the nan operon allows us to propose that the pathways for dissimilation of the three amino sugars GlcNAc, ManNAc, and NANA, all converge at the step of GlcNAc-6-P.

Because the presence of sialic acid can extend circulatory lifetime, a high degree of sialylation is often a desirable feature of therapeutic glycoproteins. In this study, the incomplete intracellular sialylation of inter- feron-g (IFN-g), produced by Chinese hamster ovary cell culture, was minimized by supplementing the culture medium with N-acetylmannosamine (ManNAc), a direct intracellular precursor for sialic acid synthesis. By intro- ducing 20 mM ManNAc into the culture medium, incom- pletely sialylated biantennary glycan structures were re- duced from 35% to 20% at the Asn 97 glycosylation site. This effect was achieved without affecting cell growth or product yield. The intracellular pool of CMP-sialic acid, the nucleotide sugar substrate for sialyltransferase, was also extracted and quantified by HPLC. Feeding of 20 mM ManNAc increased this intracellular pool of CMP-sialic acid by nearly thirtyfold compared with unsupplemented medium. When radiolabeled ManNAc was used to trace the incorporation of the precursor, it was found that supplemental ManNAc was exclusively incorporated into IFN-g as sialic acid and that, at 20 mM ManNAc feeding, nearly 100% of product sialylation originated from the supplemental precursor. © 1998 John Wiley & Sons, Inc. Bio- technol Bioeng 58: 642-648, 1998.

N-Acetylneuraminic acid (Neu5Ac) is the precursor of sialic acids, a group of important molecules in biological recognition systems. Biosynthesis of Neu5Ac is initiated and regulated by its key enzyme, UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC 5.1. 3.14)/N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60) in rat liver (Hinderlich, S., Stasche, R., Zeitler, R., and Reutter, W. (1997) J. Biol. Chem. 272, 24313-24318). In the present paper we report the isolation and characterization of a cDNA clone encoding this bifunctional enzyme. An open reading frame of 2166 base pairs encodes 722 amino acids with a predicted molecular mass of 79 kDa. The deduced amino acid sequence contains exact matches of the sequences of five peptides derived from tryptic cleavage of the enzyme. The recombinant bifunctional enzyme was expressed in COS7 cells, where it displayed both epimerase and kinase activity. Distribution of UDP-GlcNAc 2-epimerase/ManNAc kinase in the cytosol of several rat tissues was investigated by determining both specific enzyme activities. Secreting organs (liver, salivary glands, and intestinal mucosa) showed high specific activities of UDP-GlcNAc 2-epimerase/ManNAc kinase, whereas significant levels of these activities were absent from other organs (lung, kidney, spleen, brain, heart, skeletal muscle, and testis). Northern blot analysis revealed no UDP-GlcNAc 2-epimerase/ManNAc kinase mRNA in the non-secreting tissues.

Biosynthesis ofN-acetylneuraminic acid (Neu5Ac), a prominent component of glycoconjugates, is initiated by the action of UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC 5.1.3.14) and N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60). We demonstrate for the first time that the two activities are parts of one bifunctional enzyme in rat liver. The enzyme was purified to homogeneity from rat liver cytosol using salmine sulfate precipitation and chromatography on phenyl-Sepharose, ATP-agarose, and Mono Q. The purification resulted in one polypeptide with an apparent molecular mass of 75 kDa. Immunoprecipitation with a polyclonal antibody against the polypeptide reduced both enzyme activities in equal amounts. Gel filtration analysis of purified UDP-GlcNAc 2-epimerase/ManNAc kinase showed that the polypeptide self-associates as a dimer and as a hexamer with apparent molecular masses of 150 and 450 kDa, respectively. The hexamer was fully active for both enzyme activities, whereas the dimer catalyzed only the phosphorylation ofN-acetylmannosamine (ManNAc). Incubation of the dimer with UDP-N-acetylglucosamine led to reassembly of the fully active hexamer; maximal quantities of the hexamer were produced after incubation for 3 h. Kinetic analysis of purified hexameric and dimeric enzyme revealed significantly lower Michaelis constants (93 ± 3 to 121 ± 15 μm for ManNAc and 1.18 ± 0.13 to 1.67 ± 0.20 mm for ATP) and higher cooperativity (Hill coefficients of 1.42 ± 0.16 to 1.17 ± 0.06 for ManNAc and 1.30 ± 0.09 to 1.05 ± 0.14 for ATP) for the hexamer for both substrates of ManNAc kinase. The Michaelis constant of UDP-GlcNAc 2-epimerase for its substrate was 11 ± 2 μm. The Hill coefficient of 0.45 ± 0.07 represents strongly negative cooperativity in substrate binding. UDP-GlcNAc 2-epimerase was feedback inhibited by CMP-Neu5Ac. Complete inhibition was achieved with 60 μmCMP-Neu5Ac, and highly positive cooperativity (Hill coefficient of 4.1) was found for inhibitor binding.

Sialic acids may modulate cell proliferation and gene expression, particularly in neural cells in vitro. However, the function of sialic acids in the central nervous system has not previously been examined. We examined whether N-acetylmannosamine (ManNAc) could improve object recognition and hippocampal cell proliferations in middle-aged mice. C56BL/6J mice aged 52 weeks were treated with ManNAc for 4 weeks. Their cognitive-ability was assessed with a place and object recognition test. ManNAc, but not N-acetylglucosamine or N-acetylneuraminic acid, improved the index score in the place recognition task at a dosage of 5.0 mg/mL in drinking water. Additionally, ManNAc enhanced the hippocampal cell proliferation, which was evaluated by a bromodeoxyuridine assay and the number of Ki67-immunoreactive cells. We could demonstrate that ManNAc had positive effects on the age-related brain dysfunction. These findings suggest that the use of ManNAc or related compounds may be a new approach for the treatment of human dementia.

Sialic acids are essential components of membrane glycoconjugates. They are responsible for the interaction, structure, and functionality of all deuterostome cells and have major functions in cellular processes in health and diseases. The key enzyme of the biosynthesis of sialic acid is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase that transforms UDP-N-acetylglucosamine to N-acetylmannosamine (ManNAc) followed by its phosphorylation to ManNAc 6-phosphate and has a direct impact on the sialylation of cell surface components. Here, we present the crystal structures of the human N-acetylmannosamine kinase (MNK) domain of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase in complexes with ManNAc at 1.64 Å resolution, MNK·ManNAc·ADP (1.82 Å) and MNK·ManNAc 6-phosphate · ADP (2.10 Å). Our findings offer detailed insights in the active center of MNK and serve as a structural basis to design inhibitors. We synthesized a novel inhibitor, 6-O-acetyl-ManNAc, which is more potent than those previously tested. Specific inhibitors of sialic acid biosynthesis may serve to further study biological functions of sialic acid.

Terminal sialic acid residues often mediate the interactions of cell surface glycoconjugates. Sialic acid-dependent interactions typically exhibit rapid dissociation rates, precluding the use of traditional biological techniques for complex isolation. To stabilize these transient interactions, we employ a targeted photo-cross-linking approach in which a diazirine photo-cross-linker is incorporated into cell surface sialylated glycoconjugates through the use of metabolic oligosaccharide engineering. We describe three diazirine-modified N-acetylmannosamine (ManNAc) analogues in which the length of the linker between the pyranose ring and the diazirine was varied. These analogues were each metabolized to their respective sialic acid counterparts, which were added to both glycoproteins and glycolipids. Diazirine-modified sialic acid analogues could be incorporated into both α2-3 and α2-6 linkages. Upon exposure to UV irradiation, diazirine-modified glycoconjugates were covalently cross-linked to their interaction partners. We demonstrate that all three diazirine-modified analogues were capable of competing with endogeneous sialic acid, albeit to varying degrees. We found that larger analogues were less efficiently metabolized, yet could still function as effective cross-linkers. Notably, the addition of the diazirine substituent interferes with metabolism of ManNAc analogues to glycans other than sialosides, providing fidelity to selectively incorporate the cross-linker into sialylated molecules. These compounds are nontoxic and display only minimal growth inhibition at the concentrations required for cross-linking studies. This report provides essential information for the deployment of photo-cross-linking analogues to capture and study ephemeral, yet essential, sialic acid-mediated interactions.

There have been considerable strides made in the characterization of the dispensability of teichoic acid biosynthesis genes in recent years. A notable omission thus far has been an early gene in teichoic acid synthesis encoding the N -acetylmannosamine transferase ( tagA in Bacillus subtilis ; tarA in Staphylococcus aureus ), which adds N -acetylmannosamine to complete the synthesis of undecaprenol pyrophosphate-linked disaccharide. Here, we show that the N -acetylmannosamine transferases are dispensable for growth in vitro, making this biosynthetic enzyme the last dispensable gene in the pathway, suggesting that tagA (or tarA ) encodes the first committed step in wall teichoic acid synthesis.

The sialic acids are a family of nine carbon alpha-keto acids that play a wide variety of biological roles in nature. In mammals, they are found at the distal ends of cell surface glycoconjugates, and thus are major determinants of cellular recognition and adhesion events. In certain strains of pathogenic bacteria, they are found in capsular polysaccharides that mask the organism from the immune system by mimicking the exterior of a mammalian cell. This review outlines recent developments in the understanding of the two main enzymes responsible for the biosynthesis of the sialic acid, N-acetylneuraminic acid. The first, a hydrolyzing UDP-N-acetylglucosamine 2-epimerase, generates N-acetylmannosamine and UDP from UDP-N-acetylglucosamine. The second, sialic acid synthase, generates either N-acetylneuraminic acid (bacteria) or N-acetylneuraminic acid 9-phosphate (mammals) in a condensation reaction with phosphoenolpyruvate. An emphasis is placed on an understanding of the mechanistic and structural features of these enzymes.

Metabolic substrate-based sialic acid engineering techniques, where exogenously supplied N-acetylmannosamine (ManNAc) analogues are utilized by the sialic acid biosynthetic pathway, allow the cell surface to be endowed with novel physical and chemical properties and show promise for increasing the quality of recombinant glycoproteins. The in vitro toxicity of many ManNAc analogues, however, hinders the large-scale adoption of this technology. In this study, we used a selection strategy where cells were subjected to progressively higher levels of ManNAc analogues to establish novel cell lines that showed decreased sensitivity to analogue-induced in vitro toxicity. The decreased sensitivity to sugar analogue-induced apoptosis, demonstrated by the Annexin V-FITC detection method and DNA fragmentation assays, corresponded to increased sialic acid production in the resistant cell lines. The ManNAc analogue-resistant cell lines exhibited cross-resistance to apoptosis induced by staurosporine and an apoptosis-activating Fas antibody. We propose that the selection strategy employed to develop these novel cell lines, which serve as superior hosts for substrate-based sialic acid engineering applications, will generally apply to the development of host cell lines for biotechnology applications.

"Sialic acid engineering" refers to the strategy where cell surface carbohydrates are modified by the biosynthetic incorporation of metabolic intermediates, such as non-natural N-acetylmannosamine (ManNAc) analogues, into cellular glycoconjugates. While this technology has promising research, biomedical, and biotechnological applications due to its ability to endow the cell surface with novel physical and chemical properties, its adoption on a large scale is hindered by the inefficient metabolic utilization of ManNAc analogues. We address this limitation by proposing the use of acetylated ManNAc analogues for sialic acid engineering applications. In this paper, the metabolic flux of these "second-generation" compounds into a cell, and, subsequently, into the target sialic acid biosynthetic pathway is characterized in detail. We show that acetylated ManNAc analogues are metabolized up to 900-fold more efficiently than their natural counterparts. The acetylated compounds, however, decrease cell viability under certain culture conditions. To determine if these toxic side effects can be avoided, we developed an assay to measure the cellular uptake of acetylated ManNAc from the culture medium and its subsequent flux into sialic acid biosynthetic pathway. This assay shows that the majority ( > 80%) of acetylated ManNAc is stored in a cellular "reservoir" capable of safely sequestering this analogue. These results provide conditions that, from a practical perspective, enable the acetylated analogues to be used safely and efficaciously and therefore offer a general strategy to facilitate metabolic substrate-based carbohydrate engineering efforts. In addition, these results provide fundamental new insights into the metabolic processing of non-natural monosaccharides.

N-Acetyl-D-mannosamine (ManNAc) and N-acetyl-D-glucosamine (GlcNAc) are the essential precursors of N-acetylneuraminic acid (NeuAc), the specific monomer of polysialic acid (PA), a bacterial pathogenic determinant. Escherichia coli K1 uses both amino sugars as carbon sources and uptake takes place through the mannose phosphotransferase system transporter, a phosphoenolpyruvate-dependent phosphotransferase system that shows a broad range of specificity. Glucose, mannose, fructose, and glucosamine strongly inhibited the transport of these amino-acetylated sugars and GlcNAc and ManNAc strongly affected ManNAc and GlcNAc uptake, respectively. The ManNAc and the GlcNAc phosphorylation that occurs during uptake affected NeuAc synthesis in vitro. These findings account for the low in vivo PA production observed when E. coli K1 uses ManNAc or GlcNAc as a carbon source for growth.

The degree of sialylation is a major issue in the development of therapeutic glycoproteins. In this study, the feeding of N-acetylmannosamine (ManNAc) to Chinese hamster ovary (CHO) cell cultures was investigated as a method of improving sialylation profile, using a model glycoprotein we call, “Glycoprotein X”. For the purpose, moreover, a simplified 4-step method for sialylation profile analysis was developed without the use of special or expensive equipment. The addition of ManNAc into the culture medium largely improved sialylation of Glycoprotein X in a dose-dependent manner up to 20 mM ManNAc.

Sialic acids are prominent termini of mammalian glycoconjugates and are key binding determinants for cell-cell recog-nition lectins. Binding of the sialic acid-dependent lectin, myelin-associated glycoprotein (MAG), to nerve cells is implicated in the inhibition of nerve regeneration after injury. Therefore, blocking MAG binding to nerve cell sialoglycoconjugates might enhance nerve regeneration. Previously, we reported that certain sialoglycoconjugates bearing N-acetylneuraminic acid (NeuAc) but not N-glycolylneuraminic acid (NeuGc) support MAG binding (Collins et al., 1997a). We now report highly efficient conversion of sialic acids on living neural cells from exclusively NeuAc to predominantly NeuGc using a novel synthetic metabolic precursor, N-glycolylmannosamine pentaacetate (Man-NGc-PA). When NG108-15 neuroblastoma-glioma hybrid cells, which normally express only NeuAc (and bind to MAG), were cultured in the presence of 1 mM ManNGcPA, they expressed 80-90% of their sialic acid precursor pool as NeuGc within 24 h. Within 5 days, 80% of their ganglioside-associated sialic acids and 70% of their glycoprotein-associated sialic acids were converted to NeuGc. Consistent with this result, treatment of NG108-15 cells with ManNGcPA resulted in nearly complete abrogation of MAG binding. These results demonstrate that ManNGcPA treatment efficiently alters the sialic acid structures on living cells, with a commensurate change in recognition by a physiologically important lectin.

Modification of cell surface molecules with sialic acid is crucial for their function in many biological processes, including cell adhesion and signal transduction. Uridine diphosphate-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) is an enzyme that catalyzes an early, rate-limiting step in the sialic acid biosynthetic pathway. UDP-GlcNAc 2-epimerase was found to be a major determinant of cell surface sialylation in human hematopoietic cell lines and a critical regulator of the function of specific cell surface adhesion molecules.

Ammonia in animal cell cultures has been shown to specifically inhibit terminal sialylation of N- and O-linked oligosaccharides of glycoproteins. For example, we have previously shown that as little as 2.5 mM NH 4 Cl can decrease neural cell adhesion molecule (NCAM) polysialylation in both small cell lung cancer (SCLC) and Chinese hamster ovary (CHO) cells. Besides its potential involvement in SCLC metastasis, polysialic acid (PolySia) is a sensitive marker for measuring changes in sialylation. The role of UDP-N-acetylglucosamine (UDP-GlcNAc) in ammonia's inhibition of NCAM polysialylation was examined by adding glucosamine (GlcN) and uridine (Urd) to the cultures. This bypassed feedback inhibition of GlcN-6-P synthase and increased UDP-GlcNAc content by 25-fold in SCLC cells. After 3 days, PolySia levels were reduced to 10% of control with little effect on NCAM protein content. The extensive decrease in PolySia was confirmed in CHO cells. The effects of GlcN or Urd alone were less extensive, lending support to a specific role for UDP-GlcNAc in inhibition by ammonia. By comparison, 20 mM NH 4 Cl decreased PolySia content by 45% and increased UDP-GlcNAc in SCLC cells by 2-fold. The discrepancy between the {GlcN+Urd} and NH4Cl effects on UDP-GlcNAc and PolySia suggests that accumulation of UDP-GlcNAc is only partially responsible for decreased polysialylation in response to NH 4 Cl. In an attempt to increase NCAM polysialylation, N-acetylmannosamine and cytidine were added to cultures in order to circumvent the feedback inhibition of CMP-sialic acid synthesis. However, this only slightly increased PolySia levels and failed to counter ammonia's inhibition of NCAM polysialylation.

An enzyme which catalyzes the conversion of GlcNAc-PP-undecaprenol into ManNAc(beta 1----4)GlcNAc-PP-undecaprenol, a key lipid intermediate in the de novo synthesis of various teichoic acids, was partially purified from the 20,000 x g supernatant fraction of Bacillus subtilis AHU 1035 cell homogenate. By means of ammonium sulfate precipitation, gel chromatography, and ion-exchange chromatography, the enzyme was purified about 70-fold, giving a preparation virtually free from substances obstructive to measurement of the N-acetylmannosaminyltransferase reaction. The enzyme was shown to be specific to UDP-ManNAc. The Km value for UDP-ManNAc was 4.4 microM, and the optimum pH was 7.3. The enzyme required 10 mM MgCl2, 0.3 M KCl, 25% glycerol, and 0.1% Nonidet P-40 to function at full activity.

The labeling pattern of the major individual gangliosides from the microsomal and synaptosomal fractions of rat brain was determined following intracerebral injection of the radioactive sialic acid precursor, N-acetylmannosamine. Microsomal gangliosides initially had a higher specific radioactivity than synaptosomal gangliosides, with both fractions reaching similar specific radioactivities 18 h after precursor injection. In both subcellular fractions, the polysialogangliosides GT1b and GQ1b were initially more highly labeled than all other gangliosides. With the establishment of the labeling pattern, the effect of the convulsant pentylenetetrazol on brain gangliosides was examined in detail. Significant decreases in radioactive label were noted in the polysialogangliosides, GT1b and GQ1b, from the synaptosomal and microsomal fractions of the convulsed animals. The decreases may be due to activation of the membrane-bound neuraminidase present with the gangliosides in neuronal tissue. Prior to experimentation, a methodology was developed to insure quantitative isolation of small amounts of ganglioside free of other lipids and water-soluble contaminants. Combination of this isolation procedure with quantitative densitometry of thin-layer chromatograms permits accurate distributional analyses for individual gangliosides. In applications involving radioactive gangliosides, the method allows the determination of both radioactivity and sialic acid distributions from the same thin-layer chromatogram.

A mixture of hexosaminitols obtained by reducing N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine with sodium borohydride was resolved by borate complex anion-exchange chromatography. This procedure yielded a complete separation of N-acetylglucosaminitol, N-acetylgalactosaminitol, and N-acetylmannosaminitol and provided a rapid and accurate means for identifying and measuring N-acetylhexosamines in biological samples. This method was applied to studies on N-acetylneuraminic acid metabolism in human skin fibroblasts. It was used to identify reaction products in two enzymatic reactions: the conversion of UDP-N-acetylglucosamine to N-acetylmannosamine and UDP by UDP-N-acetylglucosamine 2-epimerase and the conversion of N-acetylneuraminic acid to N-acetylmannosamine and pyruvate by N-acetylneuraminate pyruvate-lyase. It was also used to identify the free 3H-labeled N-acetylhexosamines found in fibroblasts cultured in the presence of N-[3H]acetylmannosamine.

Skin fibroblasts from normal individuals and a patient with the infantile form of sialidosis were cultured for up to 72 h in medium containing [3H]-N-acetylmannosamine. The sialidosis fibroblasts consistently accumulated more labeled compound(s) than the control cells, i.e. 37-88% more cpm per mg protein. Precipitation of sonicates of these cells with 10% trichloracetic acid, TCA, demonstrated that the excess radioactivity in the sialidosis fibroblasts was in one or more TCA soluble compounds. There was no detectable difference in the amount of label in the TCA insoluble material. The TCA soluble, labeled, material from the sialidosis and the control fibroblasts was separated, isolated and purified on AG1-X8, QAE Sephadex A-25 and Bio-Gel P-4 chromatography columns. Analysis of the isolated material showed the excess radioactivity in the sialidosis fibroblasts to be due to increased levels of [3H]sialic acid covalently bound to a variety of anionic sialyl conjugates. These compounds have been separated and partially purified. Finally, acid hydrolysis and chromatographic analysis of the TCA insoluble fractions showed that greater than 80% of the label in this material was also due to [3H]sialic acid. There was no detectable difference between the control and the sialidosis patient in the amount of label in this fraction.

To study the site of incorporation of sialic acid residues into glycoproteins in hepatocytes, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of [3H]N-acetylmannosamine (8 mCi) and then sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N-acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Concurrent biochemical experiments were carried out to test the specificity of injected [3H]N-acetylmannosamine as a precursor for sialic acid residues of glycoproteins. In radioautographs from rats and mice sacrificed 10 min after injection, grain counts showed that over 69% of the silver grains occurred over the Golgi region. The majority of these grains were localized over the trans face of the Golgi stack, as well as over associated secretory vesicles and possibly GERL. In rats, the proportion of grains over the Golgi region decreased with time to 37% at 1 h, 11% at 4 h, and 6% at 24 h. Meanwhile, the proportion of grains over the plasma membrane increased from 4% at 10 min to 29% at 1 h and over 55% at 4 and 24 h; two-thirds of these grains lay over the sinusoidal membrane, and the remainder were equally divided over the lateral and bile canalicular membranes. Many silver grains also appeared over lysosomes at the 4- and 24-h time intervals, accounting for 15-17% of the total. At 3 and 9 d after injection, light microscope radioautographs revealed a grain distribution similar to that seen at 24 h, with a progressive decrease in the intensity of labeling such that by 9 d only a very light reaction remained. Because our biochemical findings indicated that [3H]N-acetylmannosamine is a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids), the interpretation of these results is that sialic acid is incorporated into these molecules in the Golgi apparatus and that the latter then migrate to secretion products, to the plasma membrane, and to lysosomes in a process of continuous renewal. It is possible that some of the label seen in lysosomes at later time intervals may have been derived from the plasma membrane or from material arising outside the cells.

SUMMARY: Addition of N-acetylmannosamine to a basal culture medium inoculated with Vibrio cholerae strain 4Z stimulated the production of neuraminidase. N-Acetylglueosamine and N-acetylgalactosamine under similar circumstances were inactive. N-Acetylmannosamine, added to medium inoculated with V. cholerae, persisted for at least 12 hr; under similar conditions N-acetylglucosamine was rapidly metabolized. Both D-mannosamine HCl and D-glucosamine HCl resisted enzymic attack by 561V. cholerae. The former compound severely inhibited growth of the organisms; the latter had no such effect. These findings are discussed, particularly in their relationship to the discovery that N-acetylneuraminic acid, which is a condensation product between N-acetylmannosamine and pyruvate, also induces neuraminidase.

Two analogs of N-acetylmannosamine, 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-mannopyranose (Ac4-NAcMan) and the 2-trifluoroacetamido derivative (AC4F3-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of N-[3H]acetylamannosamine into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac4F3-NAcMan and Ac4-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac4-NAcMan was relatively reversible, whereas that produced by Ac4-F3-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of N-[3H]acetylmannosamine into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac4F3-NAcMan caused accumulation of radioactivity from N-[3H]acetylmannosamine in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound 3H caused by this agent. In contrast, similar exposure to Ac4-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from N-[3H]acetylmannosamine in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with 3H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac4-NAcMan labeled with 14C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.

Human skin fibroblasts incubated for 72 h in medium containing 10 mM N- acetyl - d - mannosamine accumulate material that yields a chromophore in the presence of thiobarbituric acid. This material was tentatively identified as free (unbound) sialic acid due to its reactivity with thiobarbituric acid prior to acid hydrolysis, its solubility in 10% trichloroacetic acid, its chromatographic properties on an anion-exchange column and its enzymatic susceptibility to acylneuraminate pyruvate-lyase. Mass spectrometry analysis established that the accumulated material was, in fact, N -acetylneuraminic acid. Loading studies demonstrated a linear relationship between the amount of N -acetylmannosamine in the medium and the level of sialic acid accumulating within the cells. Cells grown in the absence of N -acetylmannosamine contained an average of 5 nmol free sialic acid/mg protein, while cells cultured for 72 h in 20 mM amounts of this material contained an average of 156.3 nmol free sialic acid/mg protein. When the cells were removed from the N -acetylmannosamine-enriched medium and incubated in regular medium, more than 80% of the accumulated, intracellular sialic acid disappeared within the first 96 h. It was concluded from these data that normal fibroblasts cultured in medium enriched with N -acetylmannosamine store large amounts of N -acetylneuraminic acid and can thus serve as an excellent model for the study of both normal and abnormal sialic acid metabolism, transport, storage and/or metabolic (feedback) regulation in human tissue.

Incorporation of two glycoconjugate precursors, [3H]fucose and [3H]N-acetylmannosamine, in the arcuate nucleus of the rat hypothalamus is compared 30 min after intraventricular administration. Electron microscopic autoradiographs were analyzed by a sampling technique which provides information about (i) the distribution of radioactivity in the tissue, (ii) the relative volumes, and (iii) the amount of radioactivity per unit volume of the various tissue compartments. With both precursors, the highest incorporation was found in glial cell bodies, being about five to six times that of neurons. This holds true for all three types of glia. The concentration of radioactivity in the neuropil consisting of neuronal and glial processes was exceedingly low, although, due to its large volume, it contained the highest fraction of total tissue radioactivity. The limitations imposed on the interpretation of the data in terms of synthesis of fucosyl- and sialoglycoconjugates are discussed. It is proposed that the observations support the concept that high rates of synthesis for glycoproteins (and possibly gangliosides) are a characteristic metabolic property of glial cells.

Adult male rats were injected intraventricularly with N-[3H]acetylmannosamine. After different time intervals the rats were killed and free sialic acid, CMP-sialic acid, lipid- and protein-bound sialic acid were isolated from brain and the specific radioactivities determined. Maximal specific radioactivity was reached after approximately 4 h for CMP-sialic acid, after 10-12 h for free sialic acid and after approximately 42 h for lipid- and protein-bound sialic acid. After some days the specific radioactivities of all four pools were the same and decreased equally, with a calculated turnover rate of approximately 3.5 weeks. The conclusion was that this phenomenon was the result of reutilisation of sialic acid and/or precursors. Therefore, the calculated turnover is not the turnover of bound sialic acid, but merely the rate of leakage of sialic acid and/or precursors out of the brain, so that no real turnover can be measured by this method. The first few hours after injection the specific radioactivity of CMP-sialic acid rose above that of free sialic acid. It is supposed that a compartmentalization exists of free sialic acid. The newly synthesised sialic acid molecules are not secreted into the cytoplasmic pool but are preferentially used for the synthesis of CMP-sialic acid. The results and conclusions are discussed in view of the general problems concerning turnover measurements of glycoconjugates.

N-Acetyl-D-[6-3H]mannosamine was injected into the 9th dorsal root ganglion of Xenopus laevis and the intra-axonal transport of chloroform/methanol-soluble radioactivity was analyzed using thin-layer chromatography coupled with fluorography. Three radioactive groups were distinct in consecutive segments of the sciatic nerve. The first is due to N-acetyl-D-mannosamine itself which labels the nerve uniformly, but does not seem to migrate within axons. The second group, representing most probably CMP-sialic acid, migrates at about 8 mm day at 15°C. The third is a species of ganglioside uniquely present in the frog nerve, and this migrates at 1–3 mm day . Our demonstration of the intra-axonal transport of CMP-sialic acid affords direct support to the contention that sialosylation of the ganglioside can occur in axon terminals.

The ganglioside content of rat hepatocytes increases several-fold during the first 6 days in monolayer culture. To correlate increased levels with rates of de novo synthesis, the incorporation of N-acetyl-[6-3H]D-mannosamine into individual gangliosides was determined. The calculation of synthetic rates was made possible by the simultaneous measurement of the specific radioactivity of the immediate sialic-acid donor, CMP-Neu5Ac. The CMP-Neu5Ac content of hepatocytes was found by HPLC analysis to be 30.5 nmol/g of plated cells. The specific radioactivity of this precursor pool reached a constant plateau 5 h after addition of the labeled N-acetyl-mannosamine and remained constant for at least 70 h. The incorporation into individual gangliosides was measured in primary cultures of rat hepatocytes between 72 and 144 h after seeding. During this period, the increase in ganglioside levels was greatest. The highest rates of incorporation were seen in GD1a followed by GM3, GM1, GD3 and the polysialylated compounds. The following rates of synthesis (nmol per 60 h and mg of protein) were calculated: GD1a 0.68, GM3 0.59, GM1 0.36, GD3 0.13 and GT1 0.02. These values are compared with the net increase of the gangliosides as measured by the resorcinol reaction.

The incorporation of N-acetylmannosamine ([3H]ManNAc) and N-acetylglucosamine ([3H]GlcNAc and [14C]GlcNAc) has been studied in rat thyroid hemilobes and homogenates. The radioactive label is incorporated into thyroglobulin (TG), [3H]ManNAc showing a time course more rapid than [3H]GlcNAc. [3H]ManNAc is incorporated directly into 19 S thyroglobulin showing no lower molecular weight-labeled precursors of TG either in the soluble or in the solubilized proteins; [3H]GlcNAc is incorporated into 19 S TG and into precursors both in soluble and solubilized proteins. [3H]ManNAc is converted only into N-acetylneuraminic acid (sialic acid). [3H]GlcNAc and [14C]GlcNAc are incorporated mainly as GlcNAc, and an additional small proportion is converted into sialic acid. Both labeled GlcNAc and sialic acid are found in soluble TG but only GlcNAc is found in membrane-bound TG. [3H]ManNAc is particularly useful to study the last step of carbohydrate incorporation while [3H]GlcNAc is a better tool to study the initiation of carbohydrate incorporation. The presence of the label as sialic acid only in soluble TG suggests that sialic acid is incorporated at the time of TG release from endoplasmic membranes. It may, therefore, serve as an indicator of TG secretion.