31 P-MRS of healthy human brain: Measurement of guanosine diphosphate mannose at 7 T.

Guanosine diphosphate mannose (GDP-Man) is the donor substrate required for mannosylation in the synthesis of glycoproteins, glycolipids and the newly discovered glycoRNA. Normal GDP-Man biosynthesis plays a crucial role in support of a variety of cellular functions, including cell recognition, cell communication and immune responses against viruses. Here, we report the detection of GDP-Man in human brain for the first time, using 31 P MRS at 7 T. The presence of GDP-Man is evidenced by the detection of a weak 31 P doublet at -10.7 ppm that can be assigned to the phosphomannosyl group (Pß) of the GDP-Man molecule. This weak but well-resolved signal lies 0.9 ppm upfield of UDP(G) Pß-multiplet from a mixture of UDP-Glc, UDP-Gal, UDP-GlcNAc and UDP-GalNAc. In reference to ATP (2.8 mM), the concentration of GDP-Man in human brain was estimated to be 0.02 ± 0.01 mM, about 15-fold lower than the total concentration of UDP(G) (0.30 ± 0.04, N = 17) and consistent with previous reports of UDP-Man in cells and brain tissue extracts measured by high-performance liquid chromatography. The reproducibility of the measured GDP-Man between test and 2-week retest was 21% ± 15% compared with 5% ± 4% for UDP(G) (N = 7). The measured concentrations of GDP-Man and UDP(G) are linearly correlated ([UDP(G)] = 4.3 [GDP-Man] + 0.02, with R = 0.66 and p = 0.0043), likely reflecting the effect of shared sugar precursors, which may vary among individuals in response to variation in nutritional intake and consumption. Given that GDP-Man has another set of doublet (Pα) at -8.3 ppm that overlaps with NAD(H) and UDP(G)-Pα signals, the amount of GDP-Man could potentially interfere with the deconvolution of these mixed signals in composition analysis. Importantly, this new finding may be useful in advancing our understanding of glycosylation and its role in the development of cancer, as well as infectious and neurodegenerative diseases.

Sugar nucleotide quantification by liquid chromatography tandem mass spectrometry reveals a distinct profile in Plasmodium falciparum sexual stage parasites.

The obligate intracellular lifestyle of Plasmodium falciparum and the difficulties in obtaining sufficient amounts of biological material have hampered the study of specific metabolic pathways in the malaria parasite. Thus, for example, the pools of sugar nucleotides required to fuel glycosylation reactions have never been studied in-depth in well-synchronized asexual parasites or in other stages of its life cycle. These metabolites are of critical importance, especially considering the renewed interest in the presence of N-, O-, and other glycans in key parasite proteins. In this work, we adapted a liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on the use of porous graphitic carbon (PGC) columns and MS-friendly solvents to quantify sugar nucleotides in the malaria parasite. We report the thorough quantification of the pools of these metabolites throughout the intraerythrocytic cycle of P. falciparum The sensitivity of the method enabled, for the first time, the targeted analysis of these glycosylation precursors in gametocytes, the parasite sexual stages that are transmissible to the mosquito vector.

Nucleotides in sow colostrum and milk at different stages of lactation.

An experiment was conducted with the objective of measuring the concentrations of total milk solids (TMS), CP, and 5'monophosphate nucleotides in sow colostrum and milk. Twelve multiparous sows (Landrace x Yorkshire x Duroc) were used. Litter size was standardized at 11 piglets for all sows at farrowing. Sows were fed an 18.45% CP corn-soybean meal-based diet throughout lactation. The experimental period was the initial 28 d of lactation, with colostrum collected within 12 h of farrowing and milk collected on d 3, 7, 14, 21, and 28. Colostrum and milk samples were analyzed for TMS, CP, adenosine 5'monophosphate (5'AMP), cytidine 5'monophosphate (5'CMP), guanosine 5'monophosphate (5'GMP), inosine 5'monophosphate (5'IMP), and uridine 5'monophosphate (5'UMP). Total milk solids decreased (P < 0.05) from 26.7% on d 0 to 23.1% on d 3. The TMS further decreased (P < 0.05) to 19.3% on d 7, but remained relatively constant thereafter at 18.2, 18.8, and 19.2% on d 14, 21, and 28, respectively. The concentration of CP decreased from 16.6% in colostrum to 7.7, 6.2, 5.5, 5.7, and 6.3% in milk collected on d 3, 7, 14, 21, and 28, respectively (linear and quadratic effect; P < 0.05). Concentrations of 5'AMP, 5'CMP, 5'GMP, and 5'IMP increased from d 0 to d 3 and d 7, and then decreased during the remaining lactation period (quadratic effect; P < 0.05). The concentration of 5'UMP decreased from d 0 to 28 of lactation (linear and quadratic effects; P < 0.05). In colostrum, 5'UMP represented 98% of all 5'monophosphate nucleotides, and in milk, 5'UMP accounted for 86 to 90% of all nucleotides, regardless of day of lactation. The results of this experiment suggest that the concentrations of TMS and CP in sow mammary secretions changed during the first week of lactation, but were constant thereafter. Likewise, the concentrations of 5'monophosphate nucleotides changed during the initial week postpartum, but during the last 2 wk of a 4-wk lactation period, the concentrations were constant.

Overexpression of the gene encoding GTP:mannose-1-phosphate guanyltransferase, mpg1, increases cellular GDP-mannose levels and protein mannosylation in Trichoderma reesei.

To elucidate the regulation and limiting factors in the glycosylation of secreted proteins, the mpg1 and dpm1 genes from Trichoderma reesei (Hypocrea jecorina) encoding GTP:alpha-D-mannose-1-phosphate guanyltransferase and dolichyl phosphate mannose synthase (DPMS), respectively, were overexpressed in T. reesei. No significant increases were observed in DPMS activity or protein secretion in dpm1-overexpressing transformants, whereas overexpression of mpg1 led to a twofold increase in GDP-mannose (GDPMan) levels. GDPMan was effectively utilized by mannnosyltransferases and resulted in hypermannosylation of secreted proteins in both N and O glycosylation. Overexpression of the mpg1 gene also increased the transcription of the dpm1 gene and DPMS activity. Our data indicate that the level of cellular GDPMan can play a major regulatory role in protein glycosylation in T. reesei.

Two time-resolved fluorometric high-throughput assays for quantitation of GDP-L-fucose.

Two rapid and simple procedures for the quantitative analysis of GDP-l-fucose (GDP-Fuc) are described. The methods are based on time-resolved fluorescence and microplate assay technology. The first assay relies on measuring the enzyme activity of alpha1, 3-fucosyltransferase. In this assay, transfer of fucose from GDP-Fuc converts sialyllactosamine to sialyl Lewis x tetrasaccharide, which is detected and quantified by relevant antibodies on a microplate. The formation of the reaction product is directly dependent on the presence of GDP-Fuc in the concentration range of 10-10,000 nM. In the second method GDP-Fuc inhibits the binding of fucose-specific Aleuria aurantia lectin to fucosylated glycan on a microwell. The lectin-based assay is less sensitive than the enzyme assay, but it is cheaper and faster. We used these assays in monitoring the amount of GDP-Fuc in crude lysates of transgenic yeast, which expresses the enzymes producing GDP-Fuc. The newly developed assays are versatile and applicable to measure also other nucleotide sugars or glycosyltransferase activities in a high-throughput manner.

The VIG9 gene products from the human pathogenic fungi Candida albicans and Candida glabrata encode GDP-mannose pyrophosphorylase.

We have identified two genomic DNA fragments from the human pathogenic fungi, Candida albicans (CaVIG9) and Candida glabrata (CgVIG9) that encode GDP-mannose pyrophosphorylase, a key enzyme for protein glycosylation. The VIG9 homologues of CaVIG9 and CgVIG9 complement an identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae. The nucleotide sequences of the ORFs, which are 83 and 90% identical to that of the ScVIG9 protein, respectively, showed a predicted gene product homologous to S. cerevisiae GDP-mannose pyrophosphorylase. We examined the enzyme activity of a glutathione S-transferase fusion of each VIG9 gene to synthesize GDP mannose in the cell extracts of a heterologous Escherichia coli expression system. We also developed a method for detecting the enzyme activity using a non-radioactive substrate that would be applicable to high throughput screening.

GDP-mannose dehydrogenase is the key regulatory enzyme in alginate biosynthesis in Pseudomonas aeruginosa: evidence from metabolite studies.

The Pseudomonas aeruginosa enzyme GDP-mannose dehydrogenase (GMD) is encoded by the algD gene, and previous genetic studies have indicated that it is a key regulatory and committal step in the biosynthesis of the polysaccharide alginate. In the present study the algD gene has been cloned into the broad-host-range expression vector pMMB66EH and GMD overexpressed in mucoid and genetically-related non-mucoid strains of P. aeruginosa. The metabolic approach of P. J. Tatnell, N. J. Russell & P. Gacesa (1993), J Gen Microbiol 139, 119-127, has been used to investigate the subsequent effect of GMD overexpression on the intracellular concentrations of the key metabolites GDP-mannose and GDP-mannuronate, which have been related to GMD activity and total alginate production. The overexpression of algD in mucoid and non-mucoid strains resulted in elevated GMD activities compared to wild-type strains; there was a concomitant reduction in GDP-mannose concentrations and greatly increased GDP-mannuronate concentrations. However, significantly, alginate biosynthesis was detected only in mucoid strains and GMD overexpression resulted in only a marginal increase in exopolysaccharide production. The GDP-mannuronate concentrations in mucoid strains which overexpressed GMD were always significantly greater than those of GDP-mannose, indicating that GMD was no longer the major kinetic control point in the biosynthesis of alginate by these genetically-manipulated strains. The small but significant increase in alginate production by such strains together with the increased GDP-mannuronate concentrations is interpreted as meaning that a later enzyme of the alginate pathway has become the major kinetic control point and now determines the extent of alginate production. This study has provided direct metabolic evidence that GMD is the key regulatory enzyme in alginate biosynthesis in P. aeruginosa.

Evidence that the enzyme catalyzing the conversion of guanosine diphosphate D-mannose to a 4-keto sugar nucleotide intermediate requires nicotinamide adenine dinucleotide phosphate.

The first enzyme in the formation of GDP-L-fucose from GDP-D-mannose, which forms a GDP-4-keto sugar intermediate, was purified to homogeneity from cell extracts of Klebsiella pneumoniae. During purification, the enzyme was found to be highly activated by NADP. It was proven that the pyridine nucleotide coenzyme of the enzyme was NADP, not NAD, which differs from previously accepted information. NAD had no effect on enzyme activity. The product of the enzyme reaction with NADP as coenzyme was separated from other nucleotides by high-performance liquid chromatography, and using ion spray liquid chromatography/mass spectrometry the mass was determined for the first time, as 587, which is same as the calculated mass of GDP-4-keto-6-deoxy-D-mannose.

A metabolic study of the activity of GDP-mannose dehydrogenase and concentrations of activated intermediates of alginate biosynthesis in Pseudomonas aeruginosa.

GDP-mannose dehydrogenase (GMD) is a key regulatory enzyme and the committal step in alginate biosynthesis. In this study, a metabolic approach has been used to investigate GMD activity in non-mucoid and isogenically related mucoid strains of Pseudomonas aeruginosa. Intracellular concentrations of GDP-mannose and GDP-mannuronate have been quantified using HPLC separation methods, and their concentrations have been related to GMD activity and total alginate production. In all strains of P. aeruginosa tested, GDP-mannose accumulated particularly during the exponential phase of growth in batch culture; the GDP-mannose concentrations in mucoid strains were significantly lower compared with isogenic non-mucoid strains. The product of GMD activity, GDP-mannuronate, was detectable only in mucoid strains, albeit at low but relatively constant levels irrespective of growth phase. The GDP-mannose concentrations in mucoid strains were always significantly greater than those of GDP-mannuronate, indicating that GMD is a rate-limiting enzyme in the biosynthesis of alginate. Significant GMD activity and extracellular alginate production were detected only in mucoid strains. The metabolic data reported here, together with previous genetic studies, provide strong evidence that GMD is the key regulatory enzyme controlling alginate biosynthesis in mucoid strains of P. aeruginosa.

Expression of dolichol-linked saccharide intermediate synthesis during the development of B lymphocytes.

There are large developmental increases in the rates of dolichol-linked oligosaccharide synthesis and protein N-glycosylation when resting murine splenic B lymphocytes are activated by bacterial lipopolysaccharide (LPS). These in vivo and in vitro studies were carried out to investigate the underlying biochemical mechanisms involved in the dramatic increase in the rate of oligosaccharide-lipid biosynthesis in LPS-stimulated B cells. Metabolic labelling experiments showed that the rate of synthesis of N-acetyl-glucosaminylpyrophosphoryldolichol (GlcNAc-P-P-Dol), mannosylphosphoryldolichol (Man-P-Dol) and glucosylphosphoryldolichol (Glc-P-Dol) increased 4- to 15-fold between 20 and 40 h after exposure to LPS. When the glycosyltransferase activities catalysing the formation of the three dolichol-bound monosaccharides were assayed in vitro with endoplasmic reticulum (ER)-enriched fractions, the initial rates were found to be elevated 4-fold prior to the major increases in oligosaccharide-lipid intermediate biosynthesis observed in vivo. Based on kinetic analyses, the higher enzyme activities were due to an increase in the amount of the three glycosyltransferases in activated cells. The time courses for elevated cellular content and rate of synthesis of guanosine-diphosphomannose (GDP)-Man corresponded to the developmental increase in oligosaccharide-lipid synthesis. The kinetics and magnitude of the induction of oligosaccharide-lipid synthesis were similar whether the initial rates were calculated on the basis of [2-3H]mannose-labelling or the specific activity of the GDP-[2-3H]mannose pool.(ABSTRACT TRUNCATED AT 250 WORDS)

Topological orientation of mitochondrial GDPmannose: dolichyl-monophosphate mannosyltransferase in the outer membrane.

Previous studies have shown the existence of an autonomous mitochondrial GDPmannose:dolichylmonophosphate mannosyltransferase, located in mitochondrial outer membrane of liver cells. As nothing is known about glycosylation sites in mitochondria, we have investigated the topological orientation of this enzyme in intact mitochondria, using controlled proteolysis with trypsin. Mitochondria were purified sequentially by mild ultrasonic treatment and sucrose density gradient. Purity and homogeneity of mitochondrial fraction were assessed by electron microscopy and specific marker enzymes measures. Our data provide evidence for a mitochondrial GDPmannose:dolichylmonophosphate mannosyltransferase facing the cytoplasmic side of the outer membrane. However, the exposure of this enzyme to the water phase has been shown to be dependent on the ionic strength of the environment.

Acid-soluble nucleotides of human milk at different stages of lactation.

The acid-soluble ribonucleotides of human milk were measured by enzymic and ion-exchange chromatographic procedures at different stages of lactation. Human colostrum and milk contained CMP, AMP, GMP, UMP, GDP-mannose, UDP-N-acetyl-glucosamine, UDP-N-acetyl-galactosamine, UDP-glucose, UDP-galactose, UDP and other minor nucleotides. Cytidine and adenosine derivatives were present in relatively higher amounts than in milk from ruminant species. The nucleotide concentration decreased with advancing lactation. Human milk at 3 months of lactation contained about 10 mumol/100 ml nucleotides, which represented about 75% of the amount of nucleotides present in human colostrum. Human milk did not contain orotic acid. The UDP-glucose/UDP-galactose ratio was constant during lactation and was similar to that of milks from ruminant species.