Molecular cloning and expression of the mouse N-acetylneuraminic acid 9-phosphate synthase which does not have deaminoneuraminic acid (KDN) 9-phosphate synthase activity

A cDNA of the mouse homologue of Escherichia coli N-acetylneuraminic acid (Neu5Ac) synthase (neuB gene product) was cloned by the PCR-based method. The mouse homologue consists of 359 amino acids, and the cDNA sequence displays 33% identity to that of the E. coli Neu5Ac synthase. The recombinant mouse homologue which is transiently expressed in HeLa cells does not exhibit the Neu5Ac synthase activity, which catalyzes condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc) to synthesize Neu5Ac, but the Neu5Ac 9-phosphate (Neu5Ac-9-P) synthase activity, which catalyzes condensation of PEP and ManNAc 6-phosphate (ManNAc-6-P) to synthesize Neu5Ac-9-P. Thus, the mouse homologue of E. coli Neu5Ac synthase is the Neu5Ac-9-P synthase. The Neu5Ac-9-P synthase is a cytosolic enzyme and ubiquitously distributed in mouse various tissues. Notably, the Neu5Ac-9-P synthase can not catalyze the synthesis of deaminoneuraminic acid (KDN) or KDN-9-P from PEP and Man or ManNAc-6-P, thus suggesting that the enzyme is not involved in the synthesis of KDN. This is consistent with the previous observation that only a very low activity to synthesize KDN is found in mouse B16 cells [Angata, T., et al. (1999) Biochem. Biophys. Res. Commun. 261, 326-331].     

Elimination of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activity from human N-acetylneuraminic acid 9-phosphate synthase by a single mutation

The most commonly occurring sialic acid Neu5Ac (N-acetylneuraminic acid) and its deaminated form, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid), participate in many biological functions. The human Neu5Ac-9-P (Neu5Ac 9-phosphate) synthase has the unique ability to catalyse the synthesis of not only Neu5Ac-9-P but also KDN-9-P (KDN 9-phosphate). Both reactions are catalysed by the mechanism of aldol condensation of PEP (phosphoenolpyruvate) with sugar substrates, ManNAc-6-P (N-acetylmannosamine 6-phosphate) or Man-6-P (mannose 6-phosphate). Mouse and putative rat Neu5Ac-9-P synthases, however, do not show KDN-9-P synthase activity, despite sharing high sequence identity (>95%) with the human enzyme. Here, we demonstrate that a single mutation, M42T, in human Neu5Ac-9-P synthase can abolish the KDN-9-P synthase activity completely without compromising the Neu5Ac-9-P synthase activity. Saturation mutagenesis of Met42 of the human Neu5Ac-9-P synthase showed that the substitution with all amino acids except leucine retains only the Neu5Ac-9-P synthase activity at levels comparable with the wild-type enzyme. The M42L mutant, like the wild-type enzyme, showed the additional KDN-9-P synthase activity. In the homology model of human Neu5Ac-9-P synthase, Met42 is located 22 A (1 A=0.1 nm) away from the substrate-binding site and the impact of this distant residue on the enzyme functions is discussed.     

Identification of the sequence encoding N-acetylneuraminate-9-phosphate phosphatase

The synthesis of N-acetylneuraminate (Neu5Ac), the main form of sialic acid, proceeds in vertebrates through the condensation of N-acetylmannosamine 6-phosphate and phosphoenolpyruvate to Neu5Ac-9-phosphate, followed by the dephosphorylation of the latter by a specific phosphatase. The sequence encoding Neu5Ac-9-phosphate phosphatase (Neu5Ac-9-Pase; E.C. 3.1.3.29) has not been determined until now. In this work, we have purified Neu5Ac-9-Pase more than 1000-fold from rat liver. Its dependency on Mg2+ and the fact that it was inhibited by vanadate and Ca2+ suggested that it belonged to the haloacid dehalogenase family of phosphatases. Trypsin digestion and mass spectrometry analysis of a polypeptide of about 30 kDa that co-eluted with the activity in the last purification step indicated the presence of a protein designated "haloacid dehalogenase-like hydrolase domain containing 4." The human ortholog of this protein is encoded by a 2-exon gene present on chromosome 20p11. The human protein was overexpressed in Escherichia coli as a fusion protein with a polyHis tag and purified to homogeneity. The recombinant enzyme displayed a >230-fold higher catalytic efficiency on Neu5Ac-9-phosphate than on its second best substrate. Its properties were similar to those of the enzyme purified from rat liver. Neu5Ac inhibited the enzymatic activity by 50% at 15 mM, indicating that no significant inhibition is exerted at physiological concentrations of Neu5Ac. The identification of the gene encoding Neu5Ac-9-Pase will facilitate studies aimed at testing its potential implication in unexplained forms of glycosylation deficiency.     

Construction of a functional CMP-sialic acid biosynthesis pathway in Arabidopsis

Previous studies have reported that plants contain negligible amounts of free or protein-bound N-acetylneuraminic acid (Neu5Ac). This is a major disadvantage for the use of plants as a biopharmaceutical expression system, since N-glycans with terminal Neu5Ac residues are important for the biological activities and half-lives of recombinant therapeutic glycoproteins in humans. For the synthesis of Neu5Ac-containing N-glycans, plants have to acquire the ability to synthesize Neu5Ac and its nucleotide-activated derivative, cytidine monophospho-N-acetylneuraminic acid. In this study, we have generated transgenic Arabidopsis (Arabidopsis thaliana) plants expressing three key enzymes of the mammalian Neu5Ac biosynthesis pathway: UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, N-acetylneuraminic acid phosphate synthase, and CMP-N-acetylneuraminic acid synthetase. Simultaneous expression of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase and N-acetylneuraminic acid phosphate synthase resulted in the generation of significant Neu5Ac amounts (1,275 nmol g(-1) fresh weight in leaves) in planta, which could be further converted to cytidine monophospho-N-acetylneuraminic acid (2.4 nmol g(-1) fresh weight in leaves) by coexpression of CMP-N-acetylneuraminic acid synthetase. These findings are a major step toward the production of Neu5Ac-containing glycoproteins in plants.     

Cloning and expression of the human N-acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero- D-galacto-nononic acid biosynthetic ability

Sialic acids participate in many important biological recognition events, yet eukaryotic sialic acid biosynthetic genes are not well characterized. In this study, we have identified a novel human gene based on homology to the Escherichia coli sialic acid synthase gene (neuB). The human gene is ubiquitously expressed and encodes a 40-kDa enzyme. The gene partially restores sialic acid synthase activity in a neuB-negative mutant of E. coli and results in N-acetylneuraminic acid (Neu5Ac) and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) production in insect cells upon recombinant baculovirus infection. In vitro the human enzyme uses N-acetylmannosamine 6-phosphate and mannose 6-phosphate as substrates to generate phosphorylated forms of Neu5Ac and KDN, respectively, but exhibits much higher activity toward the Neu5Ac phosphate product.     

Multiple co-regulatory elements and IHF are necessary for the control of fimB expression in response to sialic acid and N-acetylglucosamine in Escherichia coli K-12

Expression of the FimB recombinase, and hence the OFF-to-ON switching of type 1 fimbriation in Escherichia coli, is inhibited by sialic acid (Neu(5)Ac) and by GlcNAc. NanR (Neu(5)Ac-responsive) and NagC (GlcNAc-6P-responsive) activate fimB expression by binding to operators (O(NR) and O(NC1) respectively) located more than 600 bp upstream of the fimB promoter within the large (1.4 kb) nanC-fimB intergenic region. Here it is demonstrated that NagC binding to a second site (O(NC2)), located 212 bp closer to fimB, also controls fimB expression, and that integration host factor (IHF), which binds midway between O(NC1) and O(NC2), facilitates NagC binding to its two operator sites. In contrast, IHF does not enhance the ability of NanR to activate fimB expression in the wild-type background. Neither sequences up to 820 bp upstream of O(NR), nor those 270 bp downstream of O(NC2), are required for activation by NanR and NagC. However, placing the NanR, IHF and NagC binding sites closer to the fimB promoter enhances the ability of the regulators to activate fimB expression. These results support a refined model for how two potentially key indicators of host inflammation, Neu(5)Ac and GlcNAc, regulate type 1 fimbriation.     

Structure and binding analysis of Polyporus squamosus lectin in complex with the Neu5Ac{alpha}2-6Gal{beta}1-4GlcNAc human-type influenza receptor

Glycan chains that terminate in sialic acid (Neu5Ac) are frequently the receptors targeted by pathogens for initial adhesion. Carbohydrate-binding proteins (lectins) with specificity for Neu5Ac are particularly useful in the detection and isolation of sialylated glycoconjugates, such as those associated with pathogen adhesion as well as those characteristic of several diseases including cancer. Structural studies of lectins are essential in order to understand the origin of their specificity, which is particularly important when employing such reagents as diagnostic tools. Here, we report a crystallographic and molecular dynamics (MD) analysis of a lectin from Polyporus squamosus (PSL) that is specific for glycans terminating with the sequence Neu5Acα2-6Galβ. Because of its importance as a histological reagent, the PSL structure was solved (to 1.7??) in complex with a trisaccharide, whose sequence (Neu5Acα2-6Galβ1-4GlcNAc) is exploited by influenza A hemagglutinin for viral adhesion to human tissue. The structural data illuminate the origin of the high specificity of PSL for the Neu5Acα2-6Gal sequence. Theoretical binding free energies derived from the MD data confirm the key interactions identified crystallographically and provide additional insight into the relative contributions from each amino acid, as well as estimates of the importance of entropic and enthalpic contributions to binding.     

Engineering sialic acid synthetic ability into insect cells: identifying metabolic bottlenecks and devising strategies to overcome them

Previous studies have indicated negligible levels of both sialylation and the precursor N-acetylneuraminic acid (Neu5Ac) in a number of insect cell lines grown in serum-free medium. The overexpression of the human sialic acid 9-phosphate synthase (SAS) in combination with N-acetylmannosamine (ManNAc) feeding has been shown to overcome this limitation. In this study we evaluated the potential bottlenecks in the sialic acid synthesis pathway in a Spodoptera frugiperda (Sf9) insect cell line and devised strategies to overcome them by overexpression of the enzymatic pathway enzymes combined with appropriate substrate feeding. Coexpression of SAS and UDP-GlcNAc 2-epimerase/ManNAc kinase, the bifunctional enzyme initiating sialic acid biosynthesis in mammals, resulted in Neu5Ac synthesis without use of any external media supplementation to demonstrate that Neu5Ac could be generated intracellularly in Sf9 cells using natural metabolic precursors. N-Acetylglucosamine (GlcNAc) feeding in combination with this coexpression resulted in much higher levels of Neu5Ac compared to levels obtained with ManNAc feeding with SAS expression alone. The lower Neu5Ac levels obtained with ManNAc feeding suggested limitations in the transport and phosphorylation of ManNAc. The bottleneck in phosphorylation was likely due to utilization of GlcNAc kinase for phosphorylation of ManNAc in insect cells and was overcome by expression of ManNAc kinase. The transport limitation was addressed by the addition of tetra-O-acetylated ManNAc, which is easily taken up by the cells. An alternative sialic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), could also be generated in insect cells, suggesting the potential for controlling not only the production of sialic acids but also the type of sialic acid generated. The levels of KDN could be increased with virtually no Neu5Ac generation when Sf9 cells were fed excess GlcNAc. The results of these studies may be used to enhance the sialylation of target glycoproteins in insect and other eukaryotic expression systems.     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

Biosynthesis of N-glycolyneuraminic acid. The primary site of hydroxylation of N-acetylneuraminic acid is the cytosolic sugar nucleotide pool

N-Glycolylneuraminic acid (Neu5Gc) is an oncofetal antigen in humans and is developmentally regulated in rodents. We have explored the biology of N-acetylneuraminic acid hydroxylase, the enzyme responsible for conversion of the parent sialic acid, N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. We show that the major sialic acid in all compartments of murine myeloma cell lines is Neu5Gc. Pulse-chase analysis in these cells with the sialic acid precursor [6-3H]N-acetylmannosamine demonstrates that most of the newly synthesized Neu5Gc appears initially in the cytosolic low-molecular weight pool bound to CMP. The percentage of Neu5Gc on membrane-bound sialic acids closely parallels that in the CMP-bound pool at various times of chase, whereas that in the free sialic acid pool is very low initially, and rises only later during the chase. This implies that conversion from Neu5Ac to Neu5Gc occurs primarily while Neu5Ac is in its sugar nucleotide form. In support of this, the hydroxylase enzyme from a variety of tissues and cells converted CMP-Neu5Ac to CMP-Neu5Gc, but showed no activity towards free or alpha-glycosidically bound Neu5Ac. Furthermore, the majority of the enzyme activity is found in the cytosol. Studies with isolated intact Golgi vesicles indicate that CMP-Neu5Gc can be transported and utilized for transfer of Neu5Gc to glycoconjugates. The general properties of the enzyme have also been investigated. The Km for CMP-Neu5Ac is in the range of 0.6-2.5 microM. No activity can be detected against the beta-methylglycoside of Neu5Ac. On the other hand, inhibition studies suggest that the enzyme recognizes both the 5'-phosphate group and the pyrimidine base of the substrate. Taken together, the data allow us to propose pathways for the biosynthesis and reutilization of Neu5Gc, with initial conversion from Neu5Ac occurring primarily at the level of the sugar nucleotide. Subsequent release and reutilization of Neu5Gc could then account for the higher steady-state level of Neu5Gc found in all of the sialic acid pools of the cell.     

Repression and induction of the nag regulon of Escherichia coll K-12: the roles of nagC and nagA in maintenance of the uninduced state

The nag regulon located at 15.5 min on the Escherichia coli chromosome consists of two divergent operons, nagE and nagBACD, encoding genes involved in the uptake and metabolism of N-acetylglucosamine. Null mutations have been created in each of the genes by insertion of antibiotic resistance cartridges. The phenotypes of the strains carrying the insertions in nagE, B and A were consistent with the previous identification of gene products: nagE, EII(Nag), the N-acetylglucosamine specific transporter of the phosphotransferase system and nagB and nagA, the two enzymes necessary for the degradation of N-acetylglucosamine. Insertions in the nagC result in derepression of the nag genes, which is consistent with earlier observations that the nagC gene encodes the repressor of the regulon. Insertions in nagA also provoke a derepression, implying that nagA has a role in the regulation of the expression of the nag regulon as well as in the degradation of the amino-sugars. N-acetylglucosamine-6-phosphate, the intracellular product of N-acetylglucosamine transport and the substrate of the nagA gene product, is shown to be an inducer of the regulon and this suggests how nagA mutations result in derepression: the absence of N-acetylglucosamine-6-phosphate deacetylase allows N-acetylglucosamine-6-phosphate to accumulate and induce the regulon.     

Purification and characterization of N-acetylneuraminic acid-9-phosphate synthase from rat liver

Sialic acids are a group of carboxylated amino sugars important for a variety of cellular functions. N-Acetylneuraminic acid (Neu5Ac) is the predominant sialic acid in nature. Neu5Ac-9-phosphate synthase catalyzes the formation of Neu5Ac-9-phosphate from N-acetylmannosamine-6-phosphate and phosphoenolpyruvate. Neu5Ac-9-phosphate synthase was purified 11,700-fold from rat liver cytosol to apparent homogeneity by ammonium sulfate precipitation, chromatography on hydroxylapatite, phenyl-Sepharose, MonoQ, and finally gel filtration. SDS-PAGE and gel filtration chromatography indicated that the enzyme is a dimer composed of 37-kDa subunits. Analysis of trypic peptides by MALDI-TOF MS verified a high sequence similarity to the corresponding murine enzyme. The K(m) values of Neu5Ac-9-phosphate synthase were 35 microM for N-acetylmannosamine-6-phosphate and 100 microM for phosphoenolpyruvate. The enzyme displayed an absolute requirement for divalent cations, Mn(2+), Fe(2+), and Mg(2+) being the most effective. In contrast to human Neu5Ac-9-phosphate synthase, the rat enzyme did not utilize mannose-6-phosphate in the synthesis of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid 9-phosphate. Neu5Ac-9-phosphate synthase was inactivated by the sulfhydryl modifying reagents, 5,5'-dithio-bis (2-nitrobenzoic acid) and N-ethylmaleimide, and protected from inactivation by the presence of the substrate phosphoenolpyruvate, but not by the presence of N-acetylmannosamine-6-phosphate, showing that at least one cysteine residue is located in the active site of the enzyme.