Reduction of Arachidonate Is Associated With Increase in B-Cell Activation Marker in Infants: A Randomized Trial.

BACKGROUND: Infants who are not breast-fed benefit from formula with both docosahexaenoic acid (C22:6n3) and arachidonic acid (ARA; C20:4n6). The amount of ARA needed to support immune function is unknown. Infants who carry specific fatty acid desaturase (FADS) polymorphisms may require more dietary ARA to maintain adequate ARA status. OBJECTIVE: The aim of the study was to determine whether ARA intake or FADS polymorphisms alter ARA levels of lymphocytes, plasma, and red blood cells in term infants fed infant formula. METHODS: Infants (N = 89) were enrolled in this prospective, double-blind controlled study. Infants were randomized to consume formula containing 17 mg docosahexaenoic acid and 0, 25, or 34 mg ARA/100 kcal for 10 weeks. Fatty acid composition of plasma phosphatidylcholine and phosphatidylethanolamine, total fatty acids of lymphocytes and red blood cells, activation markers of lymphocytes, and polymorphisms in FADS1 and FADS2 were determined. RESULTS: Lymphocyte ARA was higher in the 25-ARA formula group than in the 0- or 34-ARA groups. In plasma, 16:0/20:4 and 18:0/20:4 species of phosphatidylcholine and phosphatidylethanolamine were highest and 16:0/18:2 and 18:0/18:2 were lowest in the 34-ARA formula group. In minor allele carriers of FADS1 and FADS2, plasma ARA content was elevated only at the highest level of ARA consumed. B-cell activation marker CD54 was elevated in infants who consumed formula containing no ARA. CONCLUSIONS: ARA level in plasma is reduced by low ARA consumption and by minor alleles in FADS. Dietary ARA may exert an immunoregulatory role on B-cell activation by decreasing 16:0/18:2 and 18:0/18:2 species of phospholipids. ARA intake from 25 to 34 mg/100 kcal is sufficient to maintain cell ARA level in infants across genotypes.

Kinetic stability of the streptavidin-biotin interaction enhanced in the gas phase.

Results of the first detailed study of the structure and kinetic stability of the model high-affinity protein-ligand interaction between biotin (B) and the homotetrameric protein complex streptavidin (S(4)) in the gas phase are described. Collision cross sections (Ω) measured for protonated gaseous ions of free and ligand-bound truncated (residues 13-139) wild-type (WT) streptavidin, i.e., S(4)(n+) and (S(4)+4B)(n+) at charge states n = 12-16, were found to be independent of charge state and in agreement (within 10%) with values estimated for crystal structures reported for S(4) and (S(4)+4B). These results suggest that significant structural changes do not occur upon transfer of the complexes from solution to the gas phase by electrospray ionization. Temperature-dependent rate constants were measured for the loss of B from the protonated (S(4)+4B)(n+) ions. Over the temperature range investigated, the kinetic stability increases with decreasing charge state, from n = 16 to 13, but is indistinguishable for n = 12 and 13. A comparison of the activation energies (E(a)) measured for the loss of B from the (S(4)+4B)(13+) ions composed of WT streptavidin and five binding site mutants (Trp79Phe, Trp108Phe, Trp120Phe, Ser27Ala, and Tyr43Ala) suggests that at least some of the specific intermolecular interactions are preserved in the gas phase. The results of molecular dynamics simulations performed on WT (S(4)+4B)(12+) ions with different charge configurations support this conclusion. The most significant finding of this study is that the gaseous WT (S(4)+4B)(n+) ions at n = 12-14, owing to a much larger E(a) (by as much as 13 kcal mol(-1)) for the loss of B, are dramatically more stable kinetically at 25 °C than the (S(4)+4B) complex in aqueous neutral solution. The differences in E(a) values measured for the gaseous (S(4)+4B)(n+) ions and solvated (S(4)+4B) complex can be largely accounted for by a late dissociative transition state and the rehydration of B and the protein binding cavity in solution.

Applications of a catch and release electrospray ionization mass spectrometry assay for carbohydrate library screening.

Applications of a catch and release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against target proteins are described. Direct ESI-MS measurements were performed on solutions containing a target protein (a single chain antibody, an antigen binding fragment, or a fragment of a bacterial toxin) and a library of carbohydrates containing multiple specific ligands with affinities in the 10(3) to 10(6) M(-1) range. Ligands with moderate affinity (10(4) to 10(6) M(-1)) were successfully detected from mixtures containing >200 carbohydrates (at concentrations as low as 0.25 µM each). Additionally, the absolute affinities were estimated from the abundance of free and ligand-bound protein ions determined from the ESI mass spectrum. Multiple low affinity ligands (~10(3) M(-1)) were successfully detected in mixtures containing >20 carbohydrates (at concentrations of ~10 µM each). However, identification of specific interactions required the use of the reference protein method to correct the mass spectrum for the occurrence of nonspecific carbohydrate-protein binding during the ESI process. The release of the carbohydrate ligands, as ions, was successfully demonstrated using collision-induced dissociation performed on the deprotonated ions of the protein-carbohydrate complexes. The use of ion mobility separation, performed on deprotonated carbohydrate ions following their release from the complex, allowed for the positive identification of isomeric ligands.

Norwalk virus assembly and stability monitored by mass spectrometry.

Viral capsid assembly, in which viral proteins self-assemble into complexes of well defined architecture, is a fascinating biological process. Although viral structure and assembly processes have been the subject of many excellent structural biology studies in the past, questions still remain regarding the intricate mechanisms that underlie viral structure, stability, and assembly. Here we used native mass spectrometry-based techniques to study the structure, stability, and assembly of Norwalk virus-like particles. Although detailed structural information on the fully assembled capsid exists, less information is available on potential capsid (dis)assembly intermediates, largely because of the inherent heterogeneity and complexity of the disassembly pathways. We used native mass spectrometry and atomic force microscopy to investigate the (dis)assembly of the Norwalk virus-like particles as a function of solution pH, ionic strength, and VP1 protein concentration. Native MS analysis at physiological pH revealed the presence of the complete capsid (T = 3) consisting of 180 copies of VP1. The mass of these capsid particles extends over 10 million Da, ranking them among the largest protein complexes ever analyzed by native MS. Although very stable under acidic conditions, the capsid was found to be sensitive to alkaline treatment. At elevated pH, intermediate structures consisting of 2, 4, 6, 18, 40, 60, and 80 copies of VP1 were observed with the VP1(60) (3.36-MDa) and VP1(80) (4.48-MDa) species being most abundant. Atomic force microscopy imaging and ion mobility mass spectrometry confirmed the formation of these latter midsize spherical particles at elevated pH. All these VP1 oligomers could be reversely assembled into the original capsid (VP1(180)). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T = 3 VP1(180) particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP1(60) and VP1(80) species.

Prestress strengthens the shell of Norwalk virus nanoparticles.

We investigated the influence of the protruding domain of Norwalk virus-like particles (NVLP) on its overall structural and mechanical stability. Deletion of the protruding domain yields smooth mutant particles and our AFM nanoindentation measurements show a surprisingly altered indentation response of these particles. Notably, the brittle behavior of the NVLP as compared to the plastic behavior of the mutant reveals that the protruding domain drastically changes the capsid's material properties. We conclude that the protruding domain introduces prestress, thereby increasing the stiffness of the NVLP and effectively stabilizing the viral nanoparticles. Our results exemplify the variety of methods that nature has explored to improve the mechanical properties of viral capsids, which in turn provides new insights for developing rationally designed, self-assembled nanodevices.

Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases.

The first comparative thermodynamic study of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB), interacting with donor substrates, donor and acceptor analogs, and trisaccharide products in vitro is reported. The binding constants, measured at 24 degrees C with the direct electrospray ionization mass spectrometry (ES-MS) assay, provide new insights into these model GTs and their interactions with substrate and product. Notably, the recombinant forms of GTA and GTB used in this study are shown to exist as homodimers, stabilized by noncovalent interactions at neutral pH. In the absence of divalent metal ion, neither GTA nor GTB exhibits any appreciable affinity for its native donors (UDP-GalNAc, UDP-Gal). Upon introduction of Mn(2+), both donors undergo enzyme-catalyzed hydrolysis in the presence of either GTA or GTB. Hydrolysis of UDP-GalNAc in the presence of GTA proceeds very rapidly under the solution conditions investigated and a binding constant could not be directly measured. In contrast, the rate of hydrolysis of UDP-Gal in the presence of GTB is significantly slower and, utilizing a modified approach to analyze the ES-MS data, a binding constant of 2 x 10(4) M(-1) was established. GTA and GTB bind the donor analogs UDP-GlcNAc, UDP-Glc with affinities similar to those measured for UDP-Gal and UDP-GalNAc (GTB only), suggesting that the native donors and donor analogs bind to the GTA and GTB through similar interactions. The binding constant determined for GTA and UDP-GlcNAc (approximately 1 x 10(4) M(-1)), therefore, provides an estimate for the binding constant for GTA and UDP-GalNAc. Binding of GTA and GTB with the A and B trisaccharide products was also investigated for the first time. In the absence of UDP and Mn(2+), both GTA and GTB recognize their respective trisaccharide products but with a low affinity approximately 10(3) M(-1); the presence of UDP and Mn(2+) has no effect on A trisaccharide binding but precludes B-trisaccharide binding.

Temperature-dependent cooperativity in donor-acceptor substrate binding to the human blood group glycosyltransferases.

Affinities of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB) for their common acceptor substrate alpha-l-Fucp-(1-->2)-beta-d-Galp-O(CH2)(7)CH3 (1), in the absence and presence of bound uridine 5'-diphosphate (UDP) and Mn2+ were determined using temperature-controlled electrospray ionization mass spectrometry. The presence of bound UDP and Mn(2+) in the donor binding site has a marked influence on the thermodynamic parameters for the association of 1 with GTA and GTB. Both the enthalpy and entropy of association (DeltaH(a), DeltaS(a)) decrease significantly. However, the free energy of association (DeltaG(a)) is unchanged at physiological temperature. The differences in the DeltaH(a) and DeltaS(a) values determined in the presence and absence of bound UDP are attributed to structural changes in the glycosyltransferases induced by the simultaneous binding of 1 and UDP.

Ganglioside Intake Increases Plasma Ganglioside Content in Human Participants.

BACKGROUND: Preclinical studies reveal associations between intestinal ganglioside content and inflammatory bowel disease (IBD). Since a low level of ganglioside is associated with higher production of proinflammatory signals in the intestine, it is important to determine safety and bioavailability of dietary ganglioside for application as a potential therapeutic agent. MATERIALS AND METHODS: Healthy volunteers (HVs; n = 18) completed an 8-week supplementation study to demonstrate safety and bioavailabity of ganglioside consumption. HVs were randomized to consume a milk fat fraction containing 43 mg/d ganglioside or placebo, and patients with IBD (n = 5) consumed ganglioside supplement in a small pilot study. Plasma gangliosides were characterized using reverse-phase liquid chromatography-QQQ mass spectrometry. Intestinal permeability was assessed by oral lactulose/mannitol, and quality of life was assessed by quality of life in the IBD questionnaire. RESULTS: There were no adverse events associated with dietary ganglioside intake. Ganglioside consumption increased ( P < .05) plasma content of total GD3 by 35% over 8 weeks. HVs consuming ganglioside exhibited a 19% decrease in intestinal permeability ( P = .04). Consumption of ganglioside was associated with a 39% increase ( P < .01) in emotional health and a 36% improvement ( P < .02) in systemic symptoms in patients with IBD. CONCLUSION: Impaired intestinal integrity characteristic of IBD results in increased permeability to bacterial antigens and decreased nutrient absorption. Intestinal integrity may be improved by dietary treatment with specific species of ganglioside. Ganglioside is a safe, bioavailable dietary compound that can be consumed to potentially improve quality of life in patients with IBD and treat other disorders involving altered ganglioside metabolism. This study was registered at clinicaltrials.gov as NCT02139709.

Increased catabolism and decreased unsaturation of ganglioside in patients with inflammatory bowel disease.

AIM: To investigate whether accelerated catabolism of ganglioside and decreased ganglioside content contribute to the etiology of pro-inflammatory intestinal disease. METHODS: Intestinal mucosa from terminal ileum or colon was obtained from patients with ulcerative colitis or inflammatory Crohn's disease (n = 11) undergoing bowel resection and compared to control samples of normal intestine from patients with benign colon polyps (n = 6) and colorectal cancer (n = 12) in this observational case-control study. Gangliosides and phospholipids of intestinal mucosa were characterized by class and ceramide or fatty acid composition using liquid chromatography triple-quad mass spectrometry. Content and composition of ganglioside classes GM1, GM3, GD3, GD1a, GT1 and GT3 were compared among subject groups. Content and composition of phospholipid classes phosphatidylcholine (PC) and phosphatidylethanolamine were compared among subject groups. Unsaturation index of individual ganglioside and phospholipid classes was computed and compared among subject groups. Ganglioside catabolism enzymes beta-hexosaminidase A (HEXA) and sialidase-3 (NEU3) were measured in intestinal mucosa using western blot and compared among subject groups. RESULTS: Relative GM3 ganglioside content was 2-fold higher (P < 0.05) in intestine from patients with inflammatory bowel disease (IBD) compared to control intestine. The quantity of GM3 and ratio of GM3/GD3 was also higher in IBD intestine than control tissue (P < 0.05). Control intestine exhibited 3-fold higher (P < 0.01) relative GD1a ganglioside content than IBD intestine. GD3 and GD1a species of ganglioside containing three unsaturated bonds were present in control intestine, but were not detected in IBD intestine. The relative content of PC containing more than two unsaturated bonds was 30% lower in IBD intestine than control intestine (P < 0.05). The relative content of HEXA in IBD intestine was increased 1.7-fold (P < 0.05) and NEU3 was increased 8.3-fold (P < 0.01) compared to normal intestine. Intestinal mucosa in IBD is characterized by increased GM3 content, decreased GD1a, and a reduction in polyunsaturated fatty acid constituents in GD3, GD1a and PC. CONCLUSION: This study suggests a new paradigm by proposing that IBD occurs as a consequence of increased metabolism of specific gangliosides.

Interrogating viral capsid assembly with ion mobility-mass spectrometry.

Most proteins fulfil their function as part of large protein complexes. Surprisingly, little is known about the pathways and regulation of protein assembly. Several viral coat proteins can spontaneously assemble into capsids in vitro with morphologies identical to the native virion and thus resemble ideal model systems for studying protein complex formation. Even for these systems, the mechanism for self-assembly is still poorly understood, although it is generally thought that smaller oligomeric structures form key intermediates. This assembly nucleus and larger viral assembly intermediates are typically low abundant and difficult to monitor. Here, we characterised small oligomers of Hepatitis B virus (HBV) and norovirus under equilibrium conditions using native ion mobility mass spectrometry. This data in conjunction with computational modelling enabled us to elucidate structural features of these oligomers. Instead of more globular shapes, the intermediates exhibit sheet-like structures suggesting that they are assembly competent. We propose pathways for the formation of both capsids.

Ligand specificity of CS-35, a monoclonal antibody that recognizes mycobacterial lipoarabinomannan: a model system for oligofuranoside-protein recognition.

The CS-35 antibody is widely used in the characterization of glycans containing D-arabinofuranose residues, in particular polysaccharides present in the mycobacterial cell wall. A detailed understanding of the combining site of this antibody and the measurement of its binding to different ligands is of interest as this knowledge will have implications in the characterization of arabinofuranose-containing glycoconjugates that are increasingly recognized as important biological molecules. Of even greater significance is that an in-depth study of this carbohydrate-protein interaction will provide insights into the mechanisms by which oligosaccharides containing furanose rings are bound by proteins, an area that has, to date, received little attention. This system has been refractory to X-ray crystallography, and thus we report here a study of the interaction of CS-35 with its ligands using a combination of chemical synthesis, mass spectrometry, titration microcalorimetry, and NMR spectroscopy. Through these investigations we have established that the binding pocket recognizes, as a minimum epitope, a linear tetrasaccharide motif and that the residues at the reducing and non-reducing end of the oligosaccharide are essential for tight binding. The residue at the non-reducing end appears to be bound in an aliphatic pocket, whereas the rest of the tetrasaccharide interacts more strongly with aromatic amino acids.

Multiple sampling in single-cell enzyme assays using CE-laser-induced fluorescence to monitor reaction progress.

A novel method for assaying enzymes from a single cell or small cell populations is described. The key advantage of this method is the ability to repeatedly sample a single cell enzyme reaction. Whereas multiple sampling has been achieved for larger cell types with a diameter of 1 mm, we report a technique by which single cell enzyme assays of small cells (15 microm in diameter) can be repeatedly carried out. Individual cells were isolated using an in-house-built micromanipulator and placed in nanoliter-scale reaction vessels. The cells were lysed with solution containing substrate, and enzyme activity was assayed by removing 5-nL aliquots with a recently developed nanopipettor. The reaction aliquot was then analyzed using capillary electrophoresis with laser-induced fluorescence detection to quantitate enzyme activity. Sf9 cells were assayed at the single cell level and found to be highly heterogeneous with respect to alpha-glucosidase II activity. Since only 5 nL of the single cell reaction was removed, multiple sampling was possible, allowing triplicate analysis of enzyme activity for each individual cell. Multiple sampling also permitted a single cell reaction to be monitored over time. The sensitivity of this method was demonstrated in the analysis of a low-abundance enzyme, alpha1,3-N-acetylgalactosaminyltransferase, from single HT29 cells. Detecting the product of this enzyme reaction required minimizing the dilution of cellular contents. To demonstrate the potential applications of this methodology in small scale biochemical analyses, single Arabidopsis knf embryos lacking the alpha-glucosidase I encoding KNOPF gene were assayed. Mutant embryos demonstrated insignificant conversion of a triglucose substrate, as compared to wild type, confirming the deletion of alpha-glucosidase I. Embryos were simultaneously assayed for a second enzyme, beta-galactosidase, illustrating that the mutants were viable except for their lack of alpha-glucosidase I activity.

Quantitative nanopipettor for miniaturized chemical and biochemical reaction sampling.

A novel and readily available pipettor capable of nanoliter-sized volume manipulation was developed to improve and increase the flexibility of small-scale reaction processing. The volume delivery was found to be reproducible, with typical relative standard deviations of 1-5%, and easily tunable over a range of nanoliter-sized aliquots. The nanopipettor was combined with capillary electrophoresis using laser-induced fluorescence detection to monitor a small-scale enzyme reaction (beta-galactosidase) using a tetramethylrhodamine-labeled substrate. The results were in good agreement with a standard enzyme assay using a micropipet, thus demonstrating the nanopipettor's potential in developing new nanoscale utrasensitive enzyme assays.

Crystallization of beta-galactosidase does not reduce the range of activity of individual molecules.

By use of a capillary electrophoresis-based procedure, it is possible to measure the activity of individual molecules of beta-galactosidase. Molecules from the crystallized enzyme as well as the original enzyme preparation used to grow the crystals both displayed a range of activity of 20-fold or greater. beta-Galactosidase molecules obtained from two different crystals had indistinguishable activity distributions of 31,600 +/- 1100 and 31,800 +/- 1100 reactions min(-1) (enzyme molecule)(-1). This activity was found to be significantly different from that of the enzyme used to grow the crystals, which showed an activity distribution of 38,500 +/- 900 reactions min(-1) (enzyme molecule)(-1).

Differences in the average single molecule activities of E. coli beta-galactosidase: effect of source, enzyme molecule age and temperature of induction.

Using a capillary electrophoresis-based method, single enzyme molecule assays were performed on E. coli beta-galactosidase from three different sets of samples. The first set consisted of lysates of induced cells from five different strains of the bacteria, as well as two different commercial preparations of the enzyme. These samples were found to have substantially different distributions of single molecule activities. For the second set of samples, beta-galactosidase expression was induced for 1.5 hr, followed by further incubation where expression was repressed. Assays were performed on the lysates of the preinduction and on the lysates from aliquots taken set times postinduction. The recently induced enzyme had a 25% higher average single molecule activity than the basally expressed enzyme. This average activity returned to the basal value 3.5 hr postinduction and remained unchanged thereafter. Finally, beta-galactosidase was induced at 26 and 42 degrees C. The enzyme was assayed before and after partial thermal denaturation. The samples were found to be indistinguishable with respect to their average single molecule activities.