Glen K. Shoemaker

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.

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 VP160 (3.36-MDa) and VP180 (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 (VP1180). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T = 3 VP1180 particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP160 and VP180 species.

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.

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 capsids 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.

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.

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.

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 Crohns 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.

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.

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.