Studies of lysosomal sialic acid metabolism: retention of sialic acid by Salla disease lysosomes

Purified rat liver lysosomes were incubated in 0.2 M sialic acid resulting in an increase in lysosomal free sialic acid of 3.8 +/- 1.5 nmol/unit beta hexosaminidase. Sialic acid loss by these lysosomes was stimulated 2-3 fold by 25 mM sodium phosphate. Loss of sialic acid by lysosomes from cultured human diploid fibroblasts was similar to that observed in rat liver lysosomes while loss of sialic acid by lysosomes from cultured fibroblasts from a patient with infantile Salla disease occurred much more slowly. Salla disease appears to be the consequence of defective lysosomal transport of sialic acid and is analogous to cystinosis, a disorder of lysosomal amino acid transport.     

Redirection of sialic acid metabolism in genetically engineered Escherichia coli.

Most microorganisms do not produce sialic acid (sialate), and those that do appear to use a biosynthetic mechanism distinct from mammals. Genetic hybrids of nonpathogenic, sialate-negative laboratory Escherichia coli K-12 strains designed for the de novo synthesis of the polysialic acid capsule from E. coli K1 proved useful in elucidating the genetics and biochemistry of capsule biosynthesis. In this article we propose a dynamic model of sialometabolism to investigate the effects of biosynthetic neu (N-acetylneuraminic acid) and catabolic nan (N-acylneuraminate) mutations on the flux of intermediates through the sialate synthetic pathway. Intracellular sialate concentrations were determined by high pH anion exchange chromatography with pulsed amperometric detection. The results indicated that a strain carrying a null defect in the gene encoding polysialyltransferase (neuS) accumulated > 50 times more CMP-sialic acid than the wild type when strains were grown in a minimal medium supplemented with glucose and casamino acids. Metabolic accumulation of CMP-sialic acid depended on a functional sialic acid synthase (neuB), as shown by the inability of a strain lacking this enzyme to accumulate a detectable endogenous sialate pool. The neuB mutant concentrated trace sialate from the medium, indicating its potential value for quantitative analysis of free sialic acids in complex biological samples. The function of the sialate aldolase (encoded by nanA) in limiting intermediate flux through the synthetic pathway was determined by analyzing free sialate accumulation in neuA (CMP-sialic acid synthetase) nanA double mutants. The combined results demonstrate how E. coli avoids a futile cycle in which biosynthetic sialate induces the system for its own degradation and indicate the feasibility of generating sialooligosaccharide precursors through targeted manipulation of sialate metabolism.     

Regulation of sialic acid metabolism in Escherichia coli: role of N-acylneuraminate pyruvate-lyase.

In Escherichia coli, synthesis of sialic acid is not regulated by allosteric inhibition mediated by cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuNAc). Evidence for the lack of metabolic control by feedback inhibition was demonstrated by measuring the intracellular level of sialic acid and CMP-NeuNAc in mutants defective in sialic acid polymerization and in CMP-NeuNAc synthesis. Polymerization-defective mutants could not synthesize the polysialic acid capsule and accumulated ca. 25-fold more CMP-NeuNAc than the wild type. Mutants unable to activate sialic acid because of a defect in CMP-NeuNAc synthetase accumulated ca. sevenfold more sialic acid than the wild type. An additional threefold increase in sialic acid levels occurred when a mutation resulting in loss of N-acylneuraminate pyruvate-lysase (sialic acid aldolase) was introduced into the CMP-NeuNAc synthetase-deficient mutant. The aldolase mutation could not be introduced into the polymerization-defective mutant, suggesting that any further increase in the intracellular CMP-NeuNAc concentration was toxic. These results show that sialic acid aldolase can regulate the intracellular concentration of sialic acid and therefore the concentration of CMP-NeuNAc. We conclude that regulation of aldolase, mediated by sialic acid induction, is necessary not only for dissimilating sialic acid (E.R. Vimr and F. A. Troy, J. Bacteriol. 164:845-853, 1985) but also for modulating the level of metabolic intermediates in the sialic acid pathway. In agreement with this conclusion, an increase in the intracellular sialic acid concentration was correlated with an increase in aldolase activity. Direct evidence for the central role of aldolase in regulating the metabolic flux of sialic adid in E. coli was provided by the finding that exogenous radiolabeled sialic acid was specifically incorporated into sialyl polymer in aldolase-negative strain but not in the wild type.     

Interaction of N-acetyl-4-epi-D-neuraminic acid with key enzymes of sialic acid metabolism

In spite of the axially orientated hydroxy group at C-4, the benzyl alpha-glycoside of N-acetyl-4-epi-D-neuraminic acid (4-epi-NeuAc) is a substrate for sialidases from Vibrio cholerae, Clostridium perfringens, and Arthrobacter ureafaciens, although to an extent which differs depending on the enzyme. Surprisingly, V. cholerae sialidase is by far the slowest acting enzyme; this is in contrast to its usual behavior. Fowl plague virus sialidase and bovine testis sialidase also cleave this glycoside slowly. 4-Epi-NeuAc is not a substrate for N-acetylneuraminic acid aldolase from C. perfringens but reversibly inhibits the enzyme with a Ki = 2.3 mM. The N-acetylneuraminic acid analogue is not converted to the corresponding CMP-glycoside by CMP-sialic acid synthase from bovine brain; however, it is an effective reversible inhibitor of the enzyme. The kinetic properties were analyzed with an assay system at pH 9 as well as an assay system at pH 7.5. The results from Dixon and Hanes plots did not agree. Therefore, no conclusions about the mechanism of the inhibition could be reached. This is the first reported sialic acid analogue which can act as an inhibitor of CMP-sialic acid synthase.     

Rat colonic mucosal cell sialic acid metabolism in azoxymethane-induced tumours

Colonic tissue was examined from normal (control) rats and azoxymethane- (carcinogen-) treated animals. Tumour-bearing colons from azoxymethane-treated rats were divided into malignant and non-malignant areas. Mucosal cells were prepared from the three types of colonic tissue and then examined for DNA and protein content and for the activities of ten enzymes involved in sialic acid metabolism. Enzyme activities were related to either the protein or the DNA content of fractions. The DNA content of cell homogenates was significantly different between tumour and non-malignant tissue and between both these tissues and normal mucosa. The protein content of the 100000 X g membrane pellet and supernatant fraction did not vary significantly between normal and non-malignant material but both these tissues differed significantly from tumour tissue. Significant variation between normal control and tumour tissue was detected at all levels of sialic acid metabolism, including N-acetylhexosamine interconversion and phosphorylation, sialic acid formation and activation, CMP-NeuAc breakdown and transfer and sialic acid release from glycoconjugates. The results indicate that major changes at all levels of sialic acid metabolism are associated with malignancy in rat colonic mucosa. Some of these changes are apparent in non-malignant mucosa and may reflect a pre-malignant state.     

Diversity in the sialic acids

Fig. 1. The sialic acids. The nine-carbon backbone common toall sialic acids is shown. Natural substitutions described todate (at C₄, C₅, C₇ C₈, and C₉,) are indicated. Additional diversityis generated by various types of glycosidic linkage (at R₃)by generation of lactones (at C₁ ), by dehydro forms (eliminatingR₃), and anhydro forms diversity sialic acids neuraminic acids O-acetylation sialidase     

Versatile biosynthetic engineering of sialic acid in living cells using synthetic sialic acid analogues.

Sialic acids are critical components of many glycoconjugates involved in biologically important ligand-receptor interactions. Quantitative and structural variations of sialic acid residues can profoundly affect specific cell-cell, pathogen-cell, or drug-cell interactions, but manipulation of sialic acids in mammalian cells has been technically limited. We describe the finding of a previously unrecognized and efficient uptake and incorporation of sialic acid analogues in mammalian cells. We added 16 synthetic sialic acid analogues carrying distinct C-1, C-5, or C-9 substitutions individually to cell cultures of which 10 were readily taken up and incorporated. Uptake of C-5- and C-9-substituted sialic acids resulted in the structural modification of up to 95% of sialic acids on the cell surface. Functionally, binding of murine sialic acid-binding immunoglobulin-like lectin-2 (Siglec-2, CD22) to cells increased after N-glycolylneuraminic acid treatment, whereas 9-iodo-N-acetylneuraminic acid abolished binding. Furthermore, susceptibility to infection by the B-lymphotropic papovavirus via a sialylated receptor was markedly enhanced following pretreatment of host cells with selected sialic acid analogues including 9-iodo-N-acetylneuraminic acid. This novel experimental strategy allows for an efficient biosynthetic engineering of surface sialylation in living cells. It is versatile, extending the repertoire of modification sites at least to C-9 and enables detailed structure-function studies of sialic acid-dependent ligand-receptor interactions in their native context.     

Primer on genes encoding enzymes in sialic acid metabolism in mammals

Sialic acid, a nine-carbon sugar acid usually is present in the non-reducing terminal position of free oligosaccharides and glycoconjugates. Sialylated conjugates in mammals perform important roles in cellular recognition, signaling, host–pathogen interaction and neuronal development. Metabolism of sialylated conjugates involves a complex pathway consisting of enzymes distributed among the different compartments in the cell. These enzymes are encoded by 32 genes diversely distributed throughout the mammalian genome. Genetic variants in some of these genes are associated with embryonic lethality and abnormal phenotypes in mice and neuromuscular diseases, carcinomas and immune-mediated diseases in humans. In humans, the CMP-NeuAc-hydroxylase (CMAH) enzyme is inactivated due to a deletion mutation in the encoded enzyme. This lack of Neu5Gc phenotype makes humans unique among mammals. This review focuses on genes encoding enzymes in sialic acid metabolism pathways in mammalian cells with special emphasis on the human, mouse and cow.     

GlcNAc 2-epimerase can serve a catabolic role in sialic acid metabolism

Sialic acid is a major determinant of carbohydrate-receptor interactions in many systems pertinent to human health and disease. N-Acetylmannosamine (ManNAc) is the first committed intermediate in the sialic acid biosynthetic pathway; thus, the mechanisms that control intracellular ManNAc levels are important regulators of sialic acid production. UDP-GlcNAc 2-epimerase and GlcNAc 2-epimerase are two enzymes capable of generating ManNAc from UDP-GlcNAc and GlcNAc, respectively. Whereas the former enzyme has been shown to direct metabolic flux toward sialic acid in vivo, the function of the latter enzyme is unclear. Here we study the effects of GlcNAc 2-epimerase expression on sialic acid production in cells. A key tool we developed for this study is a cell-permeable, small molecule inhibitor of GlcNAc 2-epimerase designed based on mechanistic principles. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes biosynthesis of sialic acid, GlcNAc 2-epimerase can serve a catabolic role, diverting metabolic flux away from the sialic acid pathway.     

Inhibition of alkaline phosphatase by sialic acid.

The interaction of human organ alkaline phosphatases (orthophosphoric-monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1) with sugars was studied. Hexosamines, N-acetylneuraminic acid (NANA or sialic acid), N-acetylmuramic acid and N-acetylglycolylneuraminic acid inhibited human organ alkaline phosphatase activities. Of these, sialic acid was the most effective inhibitor. The pH profiles for the enzymes in the absence and presence of sialic acid were similar. The sialic acid - enzyme complex was more heat stable than the free enzyme between 20 and 45 degrees C. Lineweaver-Burk plots of 1/v versus 1/S at various concentrations of sialic acid showed intersecting straight lines indicating that the mechanism of inhibition was a mixed type. The Ki value obtained from the plots of 1/v versus the square of sialic acid concentration was 0.07 mM for the hepatic, sialidase-treated hepatic, and intestinal alkaline phosphatases. The respective Hill coefficients varied somewhat with the alkaline phosphatase isoenzyme. Hyperbolic curves were obtained when the percentage of remaining activity was plotted against the substrate concentration at different concentrations of sialic acid. The Hill coefficient was lowered in the presence of sialic acid. The sialidase-treated hepatic enzymes used gave the most effective conversion. Partial denaturation of the enzyme with urea, or pronase digestion had a little if any effect on the sialic acid inhibition with constant time.     

Induction of microbial secondary metabolism.

Precursors often stimulate production of secondary metabolites either by increasing the amount of a limiting precursor, by inducing a biosynthetic enzyme (synthase) or both. These are usually amino acids but other small molecules also function as inducers. The most well-known are the auto-inducers which include gamma-butyrolactones (butanolides) of the actinomycetes, N-acylhomoserine lactones of Gram-negative bacteria, oligopeptides of Gram-positive bacteria, and B-factor (3'-[1-butylphosphoryl] adenosine) of Amycolatopsis mediterranei. The actinomycete butanolides exert their effects via receptor proteins which normally repress chemical and morphological differentiation (secondary metabolism and differentiation into aerial mycelia and spores respectively) but, when complexed with the butanolide, can no longer function. Homoserine lactones of Gram-negative bacteria function at high cell density and are structurally related to the butanolides. They turn on plant and animal virulence, light emission, plasmid transfer, and production of pigments, cyanide and beta-lactam antibiotics. They are made by enzymes homologous to Lux1, excreted by the cell, enter other cells at high density, bind to a LuxR homologue, the complex then binding to DNA upstream of genes controlled by "quorum sensing" and turning on their expression. Quorum sensing also operates in the case of the peptide pheromones of the Gram-positive bacteria. Here, secretion is accomplished by an ATP binding casette (ABC transporter), the secreted pheromone being recognized by a sensor component of a two-component signal transduction system. The pheromone often induces its own synthesis as well as those proteins involved in protein/peptide antibiotic (including bacteriocins and lantibiotics) production, virulence and genetic competence. The B-factor of A. mediterranei is an inducer of ansamycin (rifamycin) formation.     

Engineering microbial fatty acid metabolism for biofuels and biochemicals

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Developmental sialic acid modifications in rat organs.

The changes in expression of sialic acids in Sprague-Dawley rats in the prenatal and early postnatal time period have been examined in multiple organs, both visceral and non-visceral. In all organs examined, there is a dramatic increase in both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) shortly after birth. The bulk of the sialic acid is present in the ganglioside fraction in all tissues examined. As total amounts of sialic acid present in gangliosides decrease, the proportion present in the low molecular weight cytosolic fraction increases. A curious observation is that Neu5Ac hydroxylase activity is present at the time of the increase in sialic acid, but its activity does not correlate with Neu5Gc expression after the early postnatal period. This implies that Neu5Gc expression has another level of regulation besides CMP-Neu5Ac hydroxylase activity.