CMP-N-acetylneuraminic acid hydroxylase activity determines the wheat germ agglutinin-binding phenotype in two mutants of the lymphoma cell line MDAY-D2

The dominant glycosylation mutants of MDAY-D2 mouse lymphoma cells, designated class 2 (D33W25 and D34W25) were selected for their resistance to the toxic effects of wheat germ agglutinin (WGA) and shown to express elevated levels of Neu5Gc. In accordance with this, the activity of CMP-Neu5Ac hydroxylase was found to be substantially higher in the mutant cells. The hydroxylase in the D33W25 mutant cells exhibited kinetic properties identical to those of the same enzyme from mouse liver. Growth rate experimentsin vivo andin vitro, where the mutant cells grew more slowly at low cell densities in serum-free medium and also formed slower growing tumours in syngeneic mice, indicate that CMP-Neu5Ac hydroxylase expression may be associated with altered growth of the mutant cells.Abbreviations WGA wheat germ agglutinin - Neu5Ac N-acetyl--d-neuraminic acid - Neu5Gc N-glycology--d-neuraminic acid - CMP-Neu5Ac cytidine-5-monophospho-N-acetylneuraminic acid - CMP-Neu5Gc cytidine-5-monophospho-N-glycoloylneuraminic acid - FACS fluorescence-activated cell sorting - buffer A triethylamine hydrogen carbonate, pH 7.6 (concentration given at appropriate points in the text) - SFM serum free medium - IMDM Iscove's modified Dulbecco's medium - CMP-Neu5Ac hydroxylase CMP-N-acetylneuraminate: NAD(P)H oxido-reductase (N-acetyl hydroxylating) (EC 1.14.99.18); CMP-sialate hydrolase (EC 3.1.4.40); sialic acid-pyruvate lyase (EC 4.1.3.3)     

Regulation of biosynthesis ofN-glycolylneuraminic acid-containing glycoconjugates: characterization of factors required for NADH-dependent cytidine 5′monophosphate-N-acetylneuraminic acid hydroxylation

The hydroxylation of CMP-NeuAc has been demonstrated to be carried out by several factors including the soluble form of cytochromeb ₅. In the present study, mouse liver cytosol was subjected to ammonium sulfate fractionation and cellulose phosphate column chromatography for the separation of two other essential fractions participating in the hydroxylation. One of the fractions, which bound to a cellulose phosphate column, was able to reduce the soluble cytochromeb ₅, using NADH as an electron donor. The other fraction, which flowed through the column, was assumed to contain the terminal enzyme which accepts electrons from cytochromeb ₅, activates oxygen, and catalyses the hydroxylation of CMP-NeuAc. Assay conditions for the quantitative determination of the terminal enzyme were established, and the activity of the enzyme in several tissues of mouse and rat was measured. The level of the terminal enzyme activity is associated with the expression ofN-glycolylneuraminic acid in these tissues, indicating that the expression of the terminal enzyme possibly regulates the overall velocity of CMP-NeuAc hydroxylation.Abbreviations CMP cytidine 5-monophosphate - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - DTT dithiothreitol     

Indications for the enzymatic synthesis of 9-O-lactoyl-N-acetylneuraminic acid in equine liver

Fractionation of horse liver homogenate by centrifugation into heavy membranes at 10 000 × g, microsomal fraction at 105 000 × g, and the supernatant revealed sialate 9-O-lactoyltransferase activity only in the latter fraction. For the enzyme assay, the various fractions were incubated with¹⁴C labelled CMP-N-acetylneuraminic acid,N-acetylneuraminic acid and glycoconjugate-boundN-acetylneuraminic acid. Lactoylation was identified in three different TLC systems after acid hydrolysis and purification of the sialic acids in the incubation mixtures. Enzyme activity was found only in the supernatant fraction. Glycoconjugate-boundN-acetylneuraminic acid was the best substrate tested, although some lactoylation was also found when using CMP-N-acetylneuraminic acid.     

Different reactivity of two Brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAcα2-3Galβ1-3GalNAc α(2–6)sialyltransferase

We have studied the amino-acid residues involved in the catalytic activity of two distinct brain sialyltransferases acting on fetuin and asialofetuin. These two enzymes were strongly inhibited byN-bromosuccinimide, a specific blocking reagent for tryptophan residues. This result suggests the involvement of such residues in the catalytic process of the two sialytransferases. Furthermore, chemical modifications by various sulfhydryl reagents led to a strong inhibition of the fetuin sialyltransferase while the asialofetuin sialyltransferase was only slightly inhibited. For a more thorough understanding of the thiol inactivation mechanism of the fetuin sialyltransferase, we studied in more detail the reactivity of this enzyme with NEM (N-ethylmaleimide), an irreversible reagent. The time-dependent inactivation followed first-order kinetics and these kinetic data afforded presumptive evidence for the binding of 1 mol NEM per mol of enzyme. Only CMP-NeuAc protected the enzyme against NEM inactivation effectively. MnCl₂ did not enhance the protective effect of CMP-NeuAc. The modifications of the fetuin sialyltransferase kinetic parameters by NEM showed a competitive mechanism between NEM and CMP-NeuAc. The results suggest the involvement of a sulfhydryl residue in or near the nucleotide-sugar binding may induce a change in conformation of the protein, leading to a decreased accessibility of this thiol group located near the nucleotide-sugar binding site). This SH group, is essential to the enzyme activity, which is not the case for the asialofetuin sialyltransferase.Abbreviations p-CMB p-chloromercuribenzoic acid - CPDS 6,6-dithiodinicotinic acid carboxypyridine disulfide - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - DTT dithiothreitol - Mes 2-(N-morpholino)ethane sulfonic acid - NeuAc N-acetylneuraminic acid     

Low incidence of N-glycolylneuraminic acid in birds and reptiles and its absence in the platypus

The sialic acids of the platypus, birds, and reptiles were investigated with regard to the occurrence of N-glycolylneuraminic (Neu5Gc) acid. They were released from tissues, eggs, or salivary mucin samples by acid hydrolysis, and purified and analyzed by thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry. In muscle and liver of the platypus only N-acetylneuraminic (Neu5Ac) acid was found. The nine bird species studied also did not express N-glycolylneuraminic acid with the exception of an egg, but not tissues, from the budgerigar and traces in poultry. Among nine reptiles, including one turtle, N-glycolylneuraminic acid was only found in the egg and an adult basilisk, but not in a freshly hatched animal. BLAST analysis of the genomes of the platypus, the chicken, and zebra finch against the CMP-N-acetylneuraminic acid hydroxylase did not reveal the existence of a similar protein structure. Apparently monotremes (platypus) and sauropsids (birds and reptiles) cannot synthesize Neu5Gc. The few animals where Neu5Gc was found, especially in eggs, may have acquired this from the diet or by an alternative pathway. Since Neu5Gc is antigenic to man, the observation that this monosaccharide does not or at least only rarely occur in birds and reptiles, may be of nutritional and clinical significance.     

Hydroxylation of CMP-NeuAc controls the expression of N-glycolylneuraminic acid in GM3 ganglioside of the small intestine of inbred rats

An enzymatic activity responsible for the hydroxylation of CMP-NeuAc into CMP-N-glycolylneuraminic acid (CMP-NeuGc) was found in the cytosolic fraction after cellular fractionation of the mucosa of rat small intestine. It was maximum in the presence of NADPH or NADH, but it was reduced by 50% by addition of 1 mM EDTA. The Km value for CMP-NeuAc was 0.6 microM. The CMP-NeuAc hydroxylase activity paralleled the expression of the GM3 (NeuGc) phenotype in the epithelium of the small intestine and was not measurable in the mutant rats BN and SHR that only expressed GM3 (NeuAc). Furthermore, the only form of CMP-sialic acid present in the intestinal mucosa of the mutants was CMP-NeuAc, whereas in the other strains CMP-NeuGc accounted for 70-85% of the native CMP-sialic acids. Wild-type and CMP-NeuAc hydroxylase-deficient inbred rats were mated. Individuals of F1 and backcross generations were typed for the phenotypes GM3(NeuGc)/GM3(NeuAc) and the activity of CMP-NeuAc hydroxylase in the small intestine. It was found that the expression of NeuGc in GM3 depends on a single autosomal dominant gene and correlates with the activity of CMP-NeuAc hydroxylase. Two tissues other than small intestine, kidney and spleen, which expressed GM3(NeuGc) in BN and SHR, also expressed the CMP-NeuAc hydroxylase activity, as in the other strains. It was concluded that the key enzyme responsible for the presence of NeuGc in GM3 is a CMP-NeuAc hydroxylase and that mutant rats carry a defect that is specific to intestine. The comparative analysis of the respective contribution of NeuGc and NeuAc to the glycoprotein sialic acids of the small intestine showed that CMP-NeuAc hydroxylase is also responsible for part of the NeuGc present in the glycoproteins. However, the occurrence of 20-30% of NeuGc in the intestinal glycoproteins of the CMP-NeuAc hydroxylase-deficient rats indicated that there is another enzyme providing intestinal glycoproteins with NeuGc and operating under a different genetic control.     

Rosmarinic acid synthase is a new member of the superfamily of BAHD acyltransferases

Purification of rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase) from suspension cells of Coleus blumei Benth. (Lamiaceae) by fractionated ammonium sulphate precipitation, hydrophobic interaction chromatography and two affinity chromatography steps led to the identification of peptide sequences, which enabled a PCR-based approach to isolate the full-length cDNA encoding this enzyme. The open reading frame of the cDNA had a length of 1290 base pairs encoding a protein of 430 amino acid residues with a molecular mass of 47,932 Da with typical characteristics of an acyltransferase of the BAHD superfamily. The cDNA was heterologously expressed in Escherichia coli. The enzyme displayed the activity of rosmarinic acid synthase using 4-coumaroyl- and caffeoyl-coenzyme A and 4-hydroxyphenyllactate as well as 3.4-dihydroxyphenyllactate as substrates. Shikimic acid and quinic acid were not able to serve as hydroxycinnamoyl acceptors. This therefore is the first report of the cDNA-cloning of a rosmarinic acid synthase.     

Molecular cloning of cDNA encoding N-acetylglucosaminyltransferase II from Arabidopsis thaliana

N-acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi enzyme involved in the biosynthesis of glycoprotein-bound N-linked oligosaccharides, catalysing an essential step in the conversion of oligomannose-type to complex N-glycans. GnTII activity has been detected in both animals and plants. However, while cDNAs encoding the enzyme have already been cloned from several mammalian sources no GnTII homologue has been cloned from plants so far. Here we report the molecular cloning of an Arabidopsis thalianaGnTII cDNA with striking homology to its animal counterparts. The predicted domain structure of A. thalianaGnTII indicates a type II transmembrane protein topology as it has been established for the mammalian variants of the enzyme. Upon expression of A. thalianaGnTII cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited GnTII activity.     

大肠杆菌CMP-唾液酸合成酶最小活性域的研究

比较大肠杆菌与脑膜炎奈瑟氏球菌的CMP－唾液酸合成酶的氨基酸序列,发现大肠杆菌CMP－唾液酸合成酶的保守区域主要位于N－端,其C－末端似乎对其催化活性没有作用。通过PCR方法,对大肠杆菌CMP－唾液酸合成酶的C－末端进行了一系列截短,将得到的产物连接至表达载体pET-15b中,在大肠杆菌BL21（DE3）pLysS中表达。经IPTG诱导,发现从C－末端截去189个氨基酸酶仍有催化活性,说明大肠杆菌CMP－唾液酸合成酶的最小活性域主要集中在N－不端的229个氨基酸。在催化活性的C－端缺失突变合成酶的比活,最适pH及热稳定性发生变化,提示被截去的C－端氨基酸残基虽不直接参与构成酶的催化活性中心,但可影响催化活性域的构象,从而对酶的催化活性与稳定性产生影响。     

Molecular cloning of BPL1, a pectate lyase-like gene in Brassica napus

Pectate lyase (EC 4.2.2.2) is an enzyme involved in the maceration and soft rotting of plant tissue via degradation of cell wall in organisms. Plants as well as bacteria and fungi are capable of producing pectate lyases. Here we report the cloning of a novel full-length cDNA of pectate lyase gene, designated BPL1, from Brassica napus by rapid amplification of cDNA ends. BPL1 cDNA is 1787 bp containing a 1503 bp ORF encoding a 500 amino acid protein precursor. The protein precursor has a potential signal peptide with 22 amino acids. Alignment of sequences shows that there are some extremely conserved amino acids among pectate lyase-like proteins from different plant species, and novel C-terminal domains are found in Arabidopsis and Brassica. Phylogenetic analysis of 50 pectate lyase-like proteins from various species demonstrates the obvious distinction among pectate lyase-like proteins from plants, bacteria and fungi, which are subsequently clustered into three groups. The cloning of BPL1 enables us to explore its diverse roles in higher plants and potential application in crop improvement.     

Cloning and expression of antiviral/ribosome-inactivating protein from <Emphasis Type="Italic">Bougainvillea</Emphasis> x<Emphasis Type="Italic">buttiana</Emphasis>

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP)from the leaves of Bougainvillea x buttiana was isolated.The cDNA consisted of 1364 nucleotides with an open reading frame (ORF)of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids.The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species.The deduced protein has been designated BBAP1 (Bougainvillea x buttiana antiviral protein1).The ORF was cloned into an expression vector and expressed in E.coli as a fusion protein of approximately 78 kDa.The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity,and imparted a high level of resistance against the tobacco mosaic virus (TMV).     

Changes in the activity of the multifunctional β-glycosyl hydrolase (Cel44C-Man26A) from Paenibacillus polymyxa by removal of the C-terminal region to minimum size

Paenibacillus polymyxa GS01 secretes Cel44C-Man26A as a multifunctional enzyme with cellulase, xylanase, lichenase, and mannanase activities. Cel44C-Man26A consists of 1,352 amino acids in which present a catalytic domain (CD) of the glycosyl hydrolase family 44 (GH44), fibronectin domain type 3 (Fn3), catalytic domain of glycosyl hydrolase family 26 (GH26), and a cellulose-binding module type 3 (CBM3). A truncated Cel44C-Man26A protein, consisting of 549 amino acid residues, reacted as a multifunctional mature enzyme despite the absence of the 10 amino acids containing GH44, Fn3, GH26, and CBM3. However, the multifunctional activity was not found in the mature Cel44C-Man26A protein truncated to less than 548 amino acids. The truncated Cel44C-Man26A proteins showed the optimum pH for the lichenase activity was pH 7.0, pH 6.0 for the xylanase and mannanase, and pH 5.0 for the cellulase. The truncated Cel44C-Man26A proteins exhibited enzymatic activity 40–120% higher than the full-length Cel44C.     

青花菜谷胱甘肽-S-转移酶基因克隆及其表达分析

为探讨青花菜在模拟酸雨胁迫下谷胱甘肽-S-转移酶的表达变化,克隆了青花菜谷胱甘肽-S-转移酶基因(glutathione-S-transferase,GST)的cDNA序列全长,并进行了生物信息学和表达分析。结果表明:青花菜GST基因cDNA全长为915bp,开放阅读框为642bp,编码213个氨基酸,推测分子式为C1091H1719N289O306S5,分子量为23 940.7,没有跨膜螺旋区域和信号肽。系统进化树分析表明,该青花菜基因GST与芥菜的GST聚类关系最近。实时荧光定量PCR结果显示,在模拟酸雨胁迫下,GST基因的表达量在胁迫初期显著增大,随时间延长开始下降,表明其参与了青花菜抗酸雨的应答反应。     

Comparison of theN-glycoloylneuraminic andN-acetylneuraminic acid content of platelets and their precursors using high performance anion exchange chromatography

N-Acetylneuraminic acid (Neu5Ac) andN-glycoloylneuraminic acid (Neu5Gc) are distributed widely in nature. Using a Carbopac PA-1 anion exchange column, we have determined the ratios of Neu5Ac and Neu5Gc in hydrolysates of platelets and their precursors: a rat promegakaryoblastic (RPM) cell line and a human megakaryoblastic leukemia cell line (MEG-01). The ratio of Neu5Gc:Neu5Ac in cultured RPM cells is 16:1, whereas in platelet rich plasma and cultured MEG-01 cells it is 1:38 and 1:28, respectively. The nature of these sialic acids from RPM cells was verified using thin layer chromatography and liquid secondary ion mass spectrometry. The relevance of increased Neu5Gc levels in early stages of development is discussed.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - RPM rat promegakaryoblast - MEG-01 human megakaryoblastic leukaemia cell line - PAD pulsed amperometric detection - WGA wheat germ agglutinin - FCS foetal calf serum - PPEADF phosphatidylethanolamine dipalmitoyl - LSIMS liquid secondary ion mass spectrometry - HPAEC high performance anion exchange chromatography - TBA thiobarbituric acid     

Mutanase from a Paenibacillus isolate: Nucleotide sequence of the gene and properties of recombinant enzymes

A mutanase (α-1,3-glucanase)-producing microorganism was isolated from a soil sample and was identified as a relative of Paenibacillus sp. The mutanase was purified to homogeneity from culture, and its molecular mass was around 57 kDa. The gene for the mutanase was cloned by PCR using primers based on the N-terminal amino acid sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of 3651-bp open reading frame that encoded a predicted 1217-amino acid polypeptide including a 43-amino acid signal peptide. The mature enzyme showed similarity to mutanases RM1 of Bacillus sp. strain RM1 and KA-304 of Bacillus circulans with 65.6% and 62.7% identity, respectively. The predicted molecular mass of the mutanase was 123 kDa. Thus, the enzyme purified from the isolate appears to be truncated by proteolysis. The genes for the full-length and truncated mutanases were expressed in Bacillus subtilis cells, and the corresponding recombinant enzymes were purified to homogeneity. The molecular masses of the two enzymes were 116 and 57 kDa, respectively. The specific activity was 10-fold higher for the full-length enzyme than for the truncated enzyme. The optimal pH and temperature for both recombinant enzymes was pH 6.4 in citrate buffer and 45 °C to 50 °C. Amongst several tested polysaccharides, the recombinant full-length enzyme specifically hydrolyzed mutan.     

New stabilized FastPrep-CLEAs for sialic acid synthesis

N-acetyl-d-neuraminic acid aldolase, a key enzyme in the biotechnological production of N-acetyl-d-neuraminic acid (sialic acid) from N-acetyl-d-mannosamine and pyruvate, was immobilized as cross-linked enzyme aggregates (CLEAs) by precipitation with 90% ammonium sulfate and crosslinking with 1% glutaraldehyde. Because dispersion in a reciprocating disruptor (FastPrep) was only able to recover 40% of the activity, improved CLEAs were then prepared by co-aggregation of the enzyme with 10 mg/mL bovine serum albumin followed by a sodium borohydride treatment and final disruption by FastPrep (FastPrep-CLEAs). This produced a twofold increase in activity up to 86%, which is a 30% more than that reported for this aldolase in cross-linked inclusion bodies (CLIBs). In addition, these FastPrep-CLEAs presented remarkable biotechnological features for Neu5Ac synthesis, including, good activity and stability at alkaline pHs, a high K_M for ManNAc (lower for pyruvate) and good operational stability. These results reinforce the practicability of using FastPrep-CLEAs in biocatalysis, thus reducing production costs and favoring reusability.     

Preparation ofN-acetylneuraminic acid from delipidated egg yolk

Egg yolk, a large proportion of the egg, was studied for the preparation ofN-acetylneuraminic acid (Neu5Ac). The delipidated hen egg yolk (DEY; 500 kg containing 0.2% w/w, Neu5Ac) was hydrolysed with HCl (pH 1.4) at 80 °C and neutralized with NaOH (pH 6.0). The mixture was filtered and electrodialysed until the conductivity was 240 µS cm^–1. The filtrate was applied on a column of Dowex HCR-W2 (20–50 mesh), followed by a column of Dowex 1-X8 (200–400 mesh). The latter column was washed with water, and then eluted with a linear gradient of HCO₂H (0–2m). The eluates containing Neu5Ac were concentrated using a reverse osmosis membrane and, finally, rotary evaporated at 40 °C. The residue was then lyophilized to yield 500 g Neu5Ac. The purity of Neu5Ac was >98% (TBA method). HPLC, NMR spectroscopy and TLC chromatography of the product obtained from the DEY showed that Neu5Ac was the sole derivative present in egg yolk. The DEY, a byproduct from egg processing plants, was found to be an excellent source for the large-scale preparation of Neu5Ac.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycolylneuraminic acid - DEY delipidated egg yolk - HPLC high performance liquid chromatography - TLC thin layer chromatography - NMR nuclear magnetic resonance - IR infrared spectroscopy Presented at the 11th International Symposium on Glycoconjugates, Toronto, Canada.     

Cloning and expression of a porcine UDP-GalNAc: polypeptideN-acetylgalactosaminyl transferase

By employing a bovine UDP-N-acetylgalactosamine: polypeptideN-acetylgalactosaminyl transferase (O-GalNAc transferase) cDNA as a probe, we isolated four overlapping cDNAs from a porcine lung cDNA library. Both the nucleotide sequence of the porcine cDNA and the predicted primary structure of the protein (559 amino acids) proved to be very similar to those of the bovine enzyme (95% and 99% identity, respectively). Transient expression of the clone in COS-7 cells, followed by enzymatic activity assays, demonstrated that this cDNA sequence encodes a porcine O-GalNAc transferase. The intracellular O-GalNAc transferase activity was increased approximately 100-fold by transfecting cells with the porcine cDNA.Abbreviations O-GalNAc transferase UDP-N-acetylgalactosamine: polypeptideN-acetylgalactosaminyltransferase - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - GnT-III UDP-N-acetylglucosamine: -mannoside -1,4N-acetylglucosaminyltransferase III