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A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility.

ABSTRACT: Vertebrate CMP-sialic acid synthetase (CSS), which catalyzes the synthesis of CMP-sialic acid (CMP-Sia), consists of a 28 kDa-N-domain and a 20 kDa-C-domain. The N-domain is known to be a catalytic domain; however, the significance of the C-domain still remains unknown. To elucidate the function of the C-domain at the organism level, we screened the medaka TILLING library and obtained medaka with non-synonymous mutations (t911a), or single amino acid substitutions of CSS, L304Q, in the C-domain. Prominently, most L304Q medaka was lethal within 19 days post-fertilization (dpf). L304Q young fry displayed free Sia accumulation, and impairment of sialylation, up to 8 dpf. At 8 dpf, a marked abnormality in ventricular contraction and skeletal myogenesis was observed. To gain insight into the mechanism of L304Q-induced abnormalities, L304Q was biochemically characterized. Although bacterially expressed soluble L304Q and WT showed the similar V_max/K_m values, very few soluble L304Q was detected when expressed in CHO cells in sharp contrast to the WT. Additionally, the thermostability of various mutations of L304 greatly decreased, except for WT and L304I. These results suggest that L304 is important for the stability of CSS, and that an appropriate level of expression of soluble CSS is significant for animal survival.

SUBMITTER: Wu D

PROVIDER: S-EPMC8636478 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.

Project description:CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission.

| S-EPMC5086458 | biostudies-literature

Synthesis of sulfone-based nucleotide isosteres: identification of CMP-sialic acid synthetase inhibitors.

Project description:A modular replacement approach to the synthesis of sulfo-nucleotide analogs prepared from condensation of nucleoside aldehydes with bis phosphonate Horner-Wadsworth-Emmons reagents is disclosed. These analogs were shown to be inhibitors of Neisseria meningitidis CSS (NmCSS), which is a key enzyme in the biosynthesis of the capsular polysaccharides required for bacterial infection.

| S-EPMC4505548 | biostudies-literature

Inhibition of CMP-sialic acid transport by endogenous 5-methyl CMP.

Project description:Nucleotide-sugar transporters (NSTs) transport nucleotide-sugar conjugates into the Golgi lumen where they are then used in the synthesis of glycans. We previously reported crystal structures of a mammalian NST, the CMP-sialic acid transporter (CST) (Ahuja and Whorton 2019). These structures elucidated many aspects of substrate recognition, selectivity, and transport; however, one fundamental unaddressed question is how the transport activity of NSTs might be physiologically regulated as a means to produce the vast diversity of observed glycan structures. Here, we describe the discovery that an endogenous methylated form of cytidine monophosphate (m5CMP) binds and inhibits CST. The presence of m5CMP in cells results from the degradation of RNA that has had its cytosine bases post-transcriptionally methylated through epigenetic processes. Therefore, this work not only demonstrates that m5CMP represents a novel physiological regulator of CST, but it also establishes a link between epigenetic control of gene expression and regulation of glycosylation.

| S-EPMC8174729 | biostudies-literature

Catalytic Cycle of Neisseria meningitidis CMP-Sialic Acid Synthetase Illustrated by High-Resolution Protein Crystallography.

Project description:Cytidine 5'-monophosphate (CMP)-sialic acid synthetase (CSS) is an essential enzyme involved in the biosynthesis of carbohydrates and glycoconjugates containing sialic acids, a class of ?-keto acids that are generally terminal key recognition residues by many proteins that play important biological and pathological roles. The CSS from Neisseria meningitidis (NmCSS) has been commonly used with other enzymes such as sialic acid aldolase and/or sialyltransferase in synthesizing a diverse array of compounds containing sialic acid or its naturally occurring and non-natural derivatives. To better understand its catalytic mechanism and substrate promiscuity, four NmCSS crystal structures trapped at various stages of the catalytic cycle with bound substrates, substrate analogues, and products have been obtained and are presented here. These structures suggest a mechanism for an "open" and "closed" conformational transition that occurs as sialic acid binds to the NmCSS/cytidine-5'-triphosphate (CTP) complex. The closed conformation positions critical residues to help facilitate the nucleophilic attack of sialic acid C2-OH to the ?-phosphate of CTP, which is also aided by two observed divalent cations. Product formation drives the active site opening, promoting the release of products.

| S-EPMC7814854 | biostudies-literature

A mutation in human CMP-sialic acid hydroxylase occurred after the Homo-Pan divergence.

Project description:Sialic acids are important cell-surface molecules of animals in the deuterostome lineage. Although humans do not express easily detectable amounts of N-glycolylneuraminic acid (Neu5Gc, a hydroxylated form of the common sialic acid N-acetylneuraminic acid, Neu5Ac), it is a major component in great ape tissues, except in the brain. This difference correlates with lack of the hydroxylase activity that converts CMP-Neu5Ac to CMP-Neu5Gc. Here we report cloning of human and chimpanzee hydroxylase cDNAs. Although this chimpanzee cDNA is similar to the murine homologue, the human cDNA contains a 92-bp deletion resulting in a frameshift mutation. The isolated human gene also shows evidence for this deletion. Genomic PCR analysis indicates that this deletion does not occur in any of the African great apes. The gene is localized to 6p22-p23 in both humans and great apes, which does not correspond to known chromosomal rearrangements that occurred during hominoid evolution. Thus, the lineage leading to modern humans suffered a mutation sometime after the common ancestor with the chimpanzee and bonobo, potentially affecting recognition by a variety of endogenous and exogenous sialic acid-binding lectins. Also, the expression of Neu5Gc previously reported in human fetuses and tumors as well as the traces detected in some normal adult humans must be mediated by an alternate pathway.

| S-EPMC21712 | biostudies-literature

Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase.

Project description:BackgroundIn whole-cell bio-catalysis, the biosystems engineering paradigm shifts from the global reconfiguration of cellular metabolism as in fermentation to a more focused, and more easily modularized, optimization of comparably short cascade reactions. Human milk oligosaccharides (HMO) constitute an important field for the synthetic application of cascade bio-catalysis in resting or non-living cells. Here, we analyzed the central catalytic module for synthesis of HMO-type sialo-oligosaccharides, comprised of CMP-sialic acid synthetase (CSS) and sialyltransferase (SiaT), with the specific aim of coordinated enzyme co-expression in E. coli for reaction flux optimization in whole cell conversions producing 3'-sialyllactose (3SL).ResultsDifference in enzyme specific activity (CSS from Neisseria meningitidis: 36 U/mg; α2,3-SiaT from Pasteurella dagmatis: 5.7 U/mg) was compensated by differential protein co-expression from tailored plasmid constructs, giving balance between the individual activities at a high level of both (α2,3-SiaT: 9.4 × 102 U/g cell dry mass; CSS: 3.4 × 102 U/g cell dry mass). Finally, plasmid selection was guided by kinetic modeling of the coupled CSS-SiaT reactions in combination with comprehensive analytical tracking of the multistep conversion (lactose, N-acetyl neuraminic acid (Neu5Ac), cytidine 5'-triphosphate; each up to 100 mM). The half-life of SiaT in permeabilized cells (≤ 4 h) determined the efficiency of 3SL production at 37 °C. Reaction at 25 °C gave 3SL (40 ± 4 g/L) in ∼ 70% yield within 3 h, reaching a cell dry mass-specific productivity of ∼ 3 g/(g h) and avoiding intermediary CMP-Neu5Ac accumulation.ConclusionsCollectively, balanced co-expression of CSS and SiaT yields an efficient (high-flux) sialylation module to support flexible development of E. coli whole-cell catalysts for sialo-oligosaccharide production.

| S-EPMC10683312 | biostudies-literature

Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase.

Project description:Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12-->Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in k(cat), K(m) and K(s) for CTP, which implicates Arg-12 in catalysis and substrate binding.

| S-EPMC1220567 | biostudies-other

Enhanced phagocytosis and complement-mediated killing of Mannheimia haemolytica serotype 1 following in-frame CMP-sialic acid synthetase (neuA) gene deletion.

Project description:ImportanceThe Gram-negative coccobacillus Mannheimia haemolytica is a natural inhabitant of the upper respiratory tract in ruminants and the most common bacterial agent involved in bovine respiratory disease complex development. Key virulence factors harbored by M. haemolytica are leukotoxin, lipopolysaccharide, capsule, adhesins, and neuraminidase which are involved in evading innate and adaptive immune responses. In this study, we have shown that CMP-sialic acid synthetase (neuA) is necessary for the incorporation of sialic acid onto the membrane, and inactivation of neuA results in increased phagocytosis and complement-mediated killing of M. haemolytica, thus demonstrating that sialylation contributes to the virulence of M. haemolytica.

| S-EPMC10714724 | biostudies-literature

Encephalopathy caused by novel mutations in the CMP-sialic acid transporter, SLC35A1.

Project description:Transport of activated nucleotide-sugars into the Golgi is critical for proper glycosylation and mutations in these transporters cause a group of rare genetic disorders termed congenital disorders of glycosylation. We performed exome sequencing on an individual with a profound neurological presentation and identified rare compound heterozygous mutations, p.Thr156Arg and p.Glu196Lys, in the CMP-sialic acid transporter, SLC35A1. Patient primary fibroblasts and serum showed a considerable decrease in the amount of N- and O-glycans terminating in sialic acid. Direct measurement of CMP-sialic acid transport into the Golgi showed a substantial decrease in overall rate of transport. Here we report the identification of the third patient with CMP-sialic acid transporter deficiency, who presented with severe neurological phenotype, but without hematological abnormalities.

| S-EPMC5650519 | biostudies-literature

Expression cloning of the Golgi CMP-sialic acid transporter.

Project description:Translocation of nucleotide sugars across the membrane of the Golgi apparatus is a prerequisite for the synthesis of complex carbohydrate structures. While specific transport systems for different nucleotide sugars have been identified biochemically in isolated microsomes and Golgi vesicles, none of these transport proteins has been characterized at the molecular level. Chinese hamster ovary (CHO) mutants of the complementation group Lec2 exhibit a strong reduction in sialylation of glycoproteins and glycolipids due to a defect in the CMP-sialic acid transport system. By complementation cloning in the mutant 6B2, belonging to the Lec2 complementation group, we were able to isolate a cDNA encoding the putative murine Golgi CMP-sialic acid transporter. The cloned cDNA encodes a highly hydrophobic, multiple membrane spanning protein of 36.4 kDa, with structural similarity to the recently cloned ammonium transporters. Transfection of a hemagglutinin-tagged fusion protein into the mutant 6B2 led to Golgi localization of the hemagglutinin epitope. Our results, together with the observation that the cloned gene shares structural similarities to other recently cloned transporter proteins, strongly suggest that the isolated cDNA encodes the CMP-sialic acid transporter.

| S-EPMC38787 | biostudies-other

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