NeuA sialic acid O-acetylesterase activity modulates O-acetylation of capsular polysaccharide in group B Streptococcus

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) was enhanced by CTP and Mg(2+), the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac(2) followed by CMP activation of Neu5Ac or activation of Neu5,9Ac(2) followed by de-O-acetylation of CMP-Neu5,9Ac(2). Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity.     

NeuA O-acetylesterase activity is specific for CMP-activated O-acetyl sialic acid in Streptococcus suis serotype 2

Several bacteria causing meningitis, such as Escherichia coli K1, Streptococcus suis, Neisseria meningitidis, and group B Streptococci (GBS), produce sialic acid (Neu5Ac)-containing capsular polysaccharide (CPS). Biosynthesis of the Neu5Ac-containing CPS requires CMP-Neu5Ac as substrate, which is synthesized by CMP-Neu5Ac synthetase from CTP and Neu5Ac. In E. coli or GBS, the NeuA protein encoded by the neuA gene has been known encoding a bifunctional enzyme that possesses both CMP-Neu5Ac synthetase and O-acetylesterase activity. In this report, we found that the S. suis NeuA (SsNeuA) was also a bifunctional CMP-Neu5Ac synthetase/O-acetylesterase. Biochemical analyses revealed that the SsNeuA strictly de-O-acetylated CMP-O-acetyl-Neu5Ac, whereas the E. coli NeuA (EcNeuA) preferentially de-O-acetylated CMP-O-acetyl-Neu5Ac. E. coli devoid of NeuA O-acetylesterase activity was unable to produce capsule and only CMP-Neu5Ac synthetase activity of the EcNeuA or SsNeuA could not restore its ability to produce capsule. These results suggest that the O-acetylesterase is essential for the synthesis of capsular Neu5Ac in E. coli, probably in S. suis and GBS as well. Our findings are key to understanding the biosynthesis of capsular Neu5Ac in E. coli, S. suis and GBS.     

Characterization of CMP-N-acetylneuraminic acid synthetase of group B streptococci.

The capsular polysaccharide is a critical virulence factor for group B streptococci associated with human infections, yet little is known about capsule biosynthesis. We detected CMP-Neu5Ac synthetase, the enzyme which activates N-acetylneuraminic acid (Neu5Ac, or sialic acid) for transfer to the nascent capsular polysaccharide, in multiple group B streptococcus serotypes, all of which elaborate capsules containing Neu5Ac. CMP-Neu5Ac synthetase isolated from a high-producing type Ib strain was purified 87-fold. The enzyme had apparent Km values of 7.6 for Neu5Ac and 1.4 for CTP and a pH optimum of 8.3 to 9.4, required magnesium, and was stimulated by dithiothreitol. This is the first characterization of an enzyme involved in group B streptococcus capsular polysaccharide biosynthesis.     

High production of polysialic acid [Neu5Ac alpha(2-8)-Neu5Ac alpha(2-9)]n by Escherichia coli K92 grown in a chemically defined medium. Regulation of temperature

The capsular polysaccharide of Escherichia coli K92 consists of a linear polymer of Neu5Ac with alternating alpha(2-8) and alpha(2-9) linkages. It accumulates when the bacterium is grown at 37 degrees C in a defined medium containing D-xylose and L-asparagine as carbon and nitrogen sources. Release of the capsular polymer into the medium was maximal (450 micrograms x ml-1) in the stationary phase of growth (76 h). This medium could be useful for obtaining sufficient polymer to develop effective vaccines. The enzyme, CMP-Neu5Ac synthetase, was not detected in cells grown at 20 degrees C. The lack of this enzyme explains the absence of polymer biosynthesis when the bacterium was grown at 20 degrees C.     

Biochemical conditions for the production of polysialic acid by Pasteurella haemolytica A2

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with (2–8) linkages. When the bacterium was grown at 37°C for 90 h in 250 ml shake flasks at 200 rpm in Brain heart infusion broth (BHIB), it accumulated, attaining a level of 60 g/ml. Release of this polymer was strictly regulated by the growth temperature, and above 40° no production was detected. The pathway for the biosynthesis of this sialic acid capsular polymer was also examined in P. haemolytica A2 and was seen to involve the sequential presence of three enzymatic activities: Neu5Ac lyase activity, which synthesizes Neu5Ac by condensation of N-acetyl-D-mannosamine and pyruvate with apparent Km values of 91 mM and 73 mM, respectively; a CMP-Neu5Ac synthetase, which catalyzes the production of CMP-Neu5Ac from Neu5Ac and CTP with apparent Km values of 2 mM and 0.5 mM, respectively, and finally a membrane-associated polysialyltransferase, which catalyzes the incorporation of sialic acid from CMP-Neu5Ac into polymeric products with an apparent CMP-Neu5Ac Km of 250 M.     

Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides

We have previously described a microbiological process for the conversion of lactose into 3'sialyllactose and other ganglioside sugars by living Escherichia coli cells expressing the appropriate recombinant glycosyltransferase genes. In this system the activated sialic acid donor (CMP-Neu5Ac) was generated from exogenous sialic acid, which was transported into the cells by the permease NanT. Since sialic acid is an expensive compound, a more economical process has now been developed by genetically engineering E. coli K12 to be capable of generating CMP-Neu5Ac using its own internal metabolism. Mutant strains devoid of Neu5Ac aldolase and of ManNAc kinase were shown to efficiently produce 3'sialyllactose by coexpressing the alpha-2,3-sialyltransferase gene from Neisseria meningitidis with the neuC, neuB and neuACampylobacter jejuni genes encoding N-acetylglucosamine-6-phosphate-epimerase, sialic acid synthase and CMP-Neu5Ac synthetase, respectively. A sialyllactose concentration of 25 g l(-1) was obtained in long-term high cell density culture with a continuous lactose feed. This high concentration and low cost of fermentation medium should make possible to use sialylated oligosaccharides in new fields such as the food industry.     

A CMP-N-acetylneuraminic acid synthetase purified from a marine bacterium, Photobacterium leiognathi JT-SHIZ-145

A cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase was found in a crude extract prepared from Photobacterium leiognathi JT-SHIZ-145, a marine bacterium that also produces a β-galactoside α2,6-sialyltransferase. The CMP-Neu5Ac synthetase was purified from the crude extract of the cells by a combination of anion-exchange and gel filtration column chromatography. The purified enzyme migrated as a single band (60 kDa) on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The activity of the enzyme was maximal at 35 °C at pH 9.0, and the synthetase required Mg(2+) for activity. Although these properties are similar to those of other CMP-Neu5Ac synthetases isolated from bacteria, this synthetase produced not only CMP-Neu5Ac from cytidine triphosphate and Neu5Ac, but also CMP-N-glycolylneuraminic acid from cytidine triphosphate and N-glycolylneuraminic acid, unlike CMP-Neu5Ac synthetase purified from Escherichia coli.     

Regulation of capsular polysialic acid biosynthesis by temperature in Pasteurella haemolytica A2

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with alpha(2-8) linkages. The production of this polymer is strictly regulated by the growth temperature and above 40 degrees C no production is detected. Analysis of the enzymatic activities directly involved in its biosynthesis reveals that Neu5Ac lyase, CMP-Neu5Ac synthetase and polysialyltransferase are involved in this regulation. Very low activities were found in P. haemolytica grown at 43 degrees C (at least 25 times lower than those observed when the growth temperature was 37 degrees C). The synthesis of these enzymes increased rapidly when bacteria grown at 43 degrees C were transferred to 37 degrees C and decreased dramatically when cells grown at 37 degrees C were transferred to 43 degrees C. These findings indicate that the cellular growth temperature regulates the synthesis of these enzymes and hence the concentration of the intermediates necessary for capsular polysaccharide genesis in P. haemolytica A2.     

Nuclear localization signal of murine CMP-Neu5Ac synthetase includes residues required for both nuclear targeting and enzymatic activity

5-N-Acetylneuraminic acid (Neu5Ac) is the major sialic acid derivative found in animal cells. As a component of cell surface glycoconjugates, Neu5Ac is pivotal to numerous cellular recognition and communication processes including host-parasite interactions. A prerequisite for the synthesis of sialylated glycoconjugates is the activation of Neu5Ac to cytidine-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). The reaction is catalyzed by CMP-Neu5Ac-synthetase (syn), which, for unknown reasons, resides in the nucleus. Sequence analysis of the cloned murine CMP-Neu5Ac synthetase identified three clusters of basic amino acids (BC1-BC3) that might function as nuclear localization signals (NLS). In the present study chimeric protein and mutagenesis strategies were used to show that BC1 and BC2 are active NLS sequences when attached to the green fluorescent protein (enhanced GFP), but only BC2 is necessary and sufficient to mediate the nuclear import of CMP-Neu5Ac synthetase. Site-directed mutations identified the residues K(198)RXR to be essential for nuclear transport and Arg(202) to be necessary to complete the transport process. Cytoplasmic forms of CMP-Neu5Ac synthetase generated by single site mutations in BC2 demonstrated that (i) enzyme activity is independent of nuclear localization, and (ii) Arg(199) and Arg(202) are involved in both nuclear transport and synthetase activity. Comparison of all known and predicted CMP-sialic acid synthetases reveals Arg(202) and Gln(203) as highly conserved in evolution and critically important for optimal synthetase activity but not for nuclear localization. Combined, the data demonstrate that nuclear transport and enzyme activity are independent functions that share some common amino acid requirements in CMP-Neu5Ac synthetase.     

CpsK of Streptococcus agalactiae exhibits alpha2,3-sialyltransferase activity in Haemophilus ducreyi

Streptococcus agalactiae (GBS) is a major cause of serious newborn bacterial infections. Crucial to GBS evasion of host immunity is the production of a capsular polysaccharide (CPS) decorated with sialic acid, which inactivates the alternative complement pathway. The CPS operons of serotypes Ia and III GBS have been described, but the CPS sialyltransferase gene was not identified. We identified cpsK, an open reading frame in the CPS operon of most serotypes, which was homologous to the lipooligosaccharide (LOS) sialyltransferase gene, lst, of Haemophilus ducreyi. To determine if cpsK might encode a sialyltransferase, we complemented a H. ducreyi lst mutant with cpsK. CpsK was expressed in H. ducreyi and LOS was isolated and analysed for sialic acid content by SDS-PAGE and high-performance liquid chromatography (HPLC). Sialo-LOS was seen in the wild-type, cpsK- or lst-complemented mutant strains, but not in the mutant without cpsK. Addition of Neu5Ac to the LOS was confirmed by mass spectroscopy. Lectin binding studies detected terminal Neu5Ac(alpha 2-->3)Gal(beta 1- on LOS produced by the wild-type, cpsK or lst-complemented mutant strain LOS, compared with the mutant alone. Our data characterize the first sialyltransferase gene from a Gram- positive bacterium and provide compelling evidence that its product catalyses the alpha2,3 addition of Neu5Ac to H. ducreyi LOS and therefore the terminal side-chain of GBS CPS. Phylogenetic studies further indicated that lst and cpsK are related but distinct from sialyltransferases of most other bacteria and, along with their similar codon usage bias and G + C content, suggests acquisition by lateral transfer from an ancestral low G + C organism.     

Engineering intracellular CMP-sialic acid metabolism into insect cells and methods to enhance its generation

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

High-level expression of recombinant Neisseria CMP-sialic acid synthetase in Escherichia coli

The CMP-sialic acid synthetase (CMP-Neu5Ac, synthetase) is responsible for the synthesis of CMP-Neu5Ac, which is the donor used by sialyltransferases to attach sialic acid to acceptor hydroxyl groups in various polysaccharides, glycolipids, and glycoproteins. Since CMP-Neu5Ac is unstable and relatively expensive, the CMP-Neu5Ac synthetase is valuable for the preparative enzymatic synthesis of sialylated oligosaccharides. We made a construct to over-express the Neisseria meningitidis CMP-Neu5Ac synthetase in Escherichia coli. The recombinant enzyme was expressed at very high level (over 70,000 U/L) in a soluble form. It was purified by a sequence of anion-exchange chromatography and gel filtration with an overall yield of 23% (specific activity 220 U/mg). The purified CMP-Neu5Ac synthetase was used in the gram-scale synthesis of CMP-Neu5Ac.     

Penicillin-binding proteins in Streptococcus agalactiae: a novel mechanism for evasion of immune clearance

Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia and meningitis in the USA despite CDC-recommended chemoprophylaxis strategies for preventing infection. To cause infection pathogens such as GBS must evade recognition and clearance by the host's immune system. Strategies for avoidance of opsonization and phagocytic killing include elaboration of antiopsonophagocytic capsules and surface proteins. During screening for mutants of GBS that were attenuated for virulence in a neonatal rat sepsis model, we identified a mutant with a transposon insertion in the ponA gene. ponA encodes an extra-cytoplasmic penicillin-binding protein PBP1a, a newly identified virulence trait for GBS that promotes resistance to phagocytic killing independent of capsular polysaccharide. Complementation analysis in vivo and in vitro confirmed that the altered phenotypes observed in the mutant were due to the transposon insertion in ponA. Deletion of PBP1a does not affect C3 deposition on GBS suggesting that mechanism by which PBP1a protects GBS from phagocytic killing is distinct from the antiopsonic activity of capsular polysaccharide. This is the first report describing expression of an antiphagocytic surface protein by GBS and represents a novel mechanism for evasion of immune recognition and clearance that may explain the decreased virulence observed in Gram-positive bacterial species for penicillin-binding protein mutants.     

Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis.

The activity of the cytoplasmic CMP-2-keto-3-deoxyoctulosonic acid synthetase (CMP-KDO synthetase), which is low in Escherichia coli rough strains such as E. coli K-12 and in uncapsulated strains such as E. coli O111, was significantly elevated in encapsulated E. coli O10:K5 and O18:K5. This enzyme activity was even higher in an E. coli clone expressing the K5 capsule. This and the following findings suggest a correlation between elevated CMP-KDO synthetase activity and the biosynthesis of the capsular K5 polysaccharide. (i) Expression of the K5 polysaccharide and elevated CMP-KDO synthetase activity were observed with bacteria grown at 37 degrees C but not with cells grown at 20 degrees C or below. (ii) The recovery kinetics of capsule expression of intact bacteria, in vitro K5 polysaccharide-synthesizing activity of bacteria, and CMP-KDO synthetase activity of bacteria after temperature upshift from 18 to 37 degrees C were the same. (iii) Chemicals which inhibit capsule (polysaccharide) expression also inhibited the elevation of CMP-KDO synthetase activity. The chromosomal location of the gene responsible for the elevation of this enzyme activity was narrowed down to the distal segment of the transport region of the K5 expression genes.     

A study on the regulation of N-glycoloylneuraminic acid biosynthesis and utilization in rat and mouse liver

The relative contribution of N-glycoloyl-beta-D-neuraminic acid (Neu5Gc) to total sialic acids expressed in mouse and rat liver glycoconjugates was found to be 95% and 11%, respectively. This considerable difference in sialic acid composition made these two tissues suitable models for a comparative investigation into the regulation of Neu5Gc biosynthesis and utilization. An examination of the CMP-glycoside specificity of Golgi-associated sialyltransferases using CMP-N-acetyl-beta-D-neuraminic acid (CMP-Neu5Ac) and CMP-Neu5Gc revealed no significant tissue-dependent differences. The Golgi membrane CMP-sialic acid transport system from rat liver did, however, exhibit a slightly higher internalisation rate for CMP-Neu5Ac, though no preferential affinity for this sugar nucleotide over CMP-Neu5Gc was observed. In experiments, where Golgi membrane preparations were incubated with an equimolar mixture of labelled CMP-Neu5Ac and CMP-Neu5Gc, no significant tissue-dependent differences in [14C]sialic acid composition were observed, either in the luminal soluble sialic acid fraction or in the precipitable sialic acid fraction, results which are consistent with the above observations. From this experiment, evidence was also obtained for the presence of a Golgi-lumen-associated CMP--sialic acid hydrolase which exhibited no apparent specificity for either CMP-Neu5Ac or CMP-Neu5Gc. The specific activity of the CMP-Neu5Ac hydroxylase, the enzyme responsible for the biosynthesis of Neu5Gc, was found to be 28-fold greater in high-speed supernatants of mouse liver than of rat liver. No hydroxylase activity was detected in the Golgi membrane preparations. It is therefore proposed that the cytoplasmic ratio of CMP-Neu5Ac and CMP-Neu5Gc produced by the hydroxylase, remains largely unmodified after CMP-glycoside uptake into the Golgi apparatus and transfer on to growing glycoconjugate glycan chains. The close relationship between the total sialic acid composition and the sialic acid pattern in the CMP-glycoside pools of the tissues lends considerable weight to this hypothesis.     

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.     

Cloning and expression of a membrane-bound CMP-N-acetylneuraminic acid hydroxylase from the starfish Asterias rubens.

The sialic acid N-glycolylneuraminic acid (Neu5Gc) is synthesized by the action of CMP-Neu5Ac hydroxylase. The enzyme from various mammals has been purified, characterized and sequenced by cDNA cloning. Although functional sequence motifs can be postulated from comparisons with several enzymes, no global homologies to any other proteins have been found. The unusual characteristics of this hydroxylase raise questions about its evolution. As echinoderms are phylogenetically the oldest organisms possessing Neu5Gc, they represent a starting point for investigations on the origin of this enzyme. Despite many similarities with its mammalian counterpart, CMP-Neu5Ac hydroxylase from the starfish A. rubens exhibits fundamental differences, most notably its association with a membrane and a requirement for high ionic strength. In order to shed light on the structural basis for these differences, the primary structure of CMP-Neu5Ac hydroxylase from A. rubens has been determined by PCR and cDNA-cloning techniques, using initial sequence information from the mouse enzyme. The complete assembled cDNA contained an ORF coding for a protein of 653 amino acids with a molecular mass of 75 kDa. The deduced amino-acid sequence exhibited a high degree of homology with the mammalian enzyme, although the C-terminus was some 60 residues longer. This extension consists of a terminal hydrophobic region, which may mediate membrane-binding, and a preceding hydrophilic sequence which probably serves as a hinge or linker. The identity of the ORF was confirmed by expression of active CMP-Neu5Ac hydroxylase in E. coli at low temperatures.     

Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver.

The finding that N-glycoloylneuraminic acid (Neu5Gc) in pig submandibular gland is synthesized by hydroxylation of the sugar nucleotide CMP-Neu5Ac [Shaw & Schauer (1988) Biol. Chem. Hoppe-Seyler 369, 477-486] prompted us to investigate further the biosynthesis of this sialic acid in mouse liver. Free [14C]Neu5Ac, CMP-[14C]Neu5Ac and [14C]Neu5Ac glycosidically bound by Gal alpha 2-3- and Gal alpha 2-6-GlcNAc beta 1-4 linkages to fetuin were employed as potential substrates in experiments with fractionated mouse liver homogenates. The only substrate to be hydroxylated was the CMP-Neu5Ac glycoside. The product of the reaction was identified by chemical and enzymic methods as CMP-Neu5Gc. All of the CMP-Neu5Ac hydroxylase activity was detected in the high-speed supernatant fraction. The hydroxylase required a reduced nicotinamide nucleotide [NAD(P)H] coenzyme and molecular oxygen for activity. Furthermore, the activity of this enzyme was enhanced by exogenously added Fe2+ or Fe3+ ions, all other metal salts tested having a negligible or inhibitory influence. This hydroxylase is therefore tentatively classified as a monooxygenase. The cofactor requirement and CMP-Neu5Ac substrate specificity are identical to those of the enzyme in high-speed supernatants of pig submandibular gland, suggesting that this is a common route of Neu5Gc biosynthesis. The relevance of these results to the regulation of Neu5Gc expression in sialoglycoconjugates is discussed.     

Identification, expression and tissue distribution of cytidine 5′-monophosphate N-acetylneuraminic acid synthetase activity in the rat

We report the postnatal developmental profiles of N-acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase) in different rat tissues. This enzyme, which catalyses the activation of NeuAc to CMP-Neu5Ac, was detected in brain, kidney, heart, spleen, liver, stomach, intestine, lung, thymus, prostate and urinary bladder but not in skeletal muscle. Comparative analysis of the different specific activity profiles obtained shows that the expression of CMP Neu5Ac synthetase is tissue-dependent and does not seem to be embryologically determined. Changes in the level of sialylation during development were also found to be intimately related to variations in the expression of this enzyme, at least in brain, heart, kidney, stomach, intestine and lung.     

Gene Expression and Pathway Analysis of Effects of the CMAH Deactivation on Mouse Lung,Kidney and Heart

BackgroundN-glycolylneuraminic acid (Neu5Gc) is generated by hydroxylation of CMP-Neu5Ac to CMP-Neu5Gc, catalyzed by CMP-Neu5Ac hydroxylase (CMAH). However, humans lack this common mammalian cell surface molecule, Neu5Gc, due to inactivation of the CMAH gene during evolution. CMAH is one of several human-specific genes whose function has been lost by disruption or deletion of the coding frame. It has been suggested that CMAH inactivation has resulted in biochemical or physiological characteristics that have resulted in human-specific diseases.Conclusions/SignificanceMice bearing a human-like deletion of the Cmah gene serve as an important model for the study of abnormal pathogenesis and/or metabolism caused by the evolutionary loss of Neu5Gc synthesis in humans.