Powerful new strategies based on mass spectrometry are revolutionizing the structural analysis and profiling of glycans and glycoconjugates. We survey here the major biosynthetic pathways that underlie the biological diversity in glycobiology, with emphasis on glycoproteins, and the approaches that can be used to address the resulting heterogeneity. Included among these are derivatizations, on- and off-line chromatography, electrospray and matrix-assisted laser desorption/ionization, and a variety of dissociation methods, the recently introduced electron-based techniques being of particular interest.
mass spectrometry; MS/MS; glycobiology; structural determinations; biosynthesis; chromatography; derivatives
The effects of varying the electron energy and cationizing agents on electron activated dissociation (ExD) of metal-adducted oligosaccharides were explored, using permethylated maltoheptaose as the model system. Across the examined range of electron energy, the metal-adducted oligosaccharide exhibited several fragmentation processes, including electron capture dissociation (ECD) at low energies, hot-ECD at intermediate energies, and electronic excitation dissociation (EED) at high energies. The dissociation threshold depended on the metal charge carrier(s), whereas the types and sequence spans of product ions were influenced by the metal-oligosaccharide binding pattern. Theoretical modeling contributed insight into the metal-dependent behavior of carbohydrates during low-energy ECD. When ExD was applied to a permethylated high mannose N-linked glycan, EED provided more structural information than either collision-induced dissociation (CID) or low-energy ECD, thus demonstrating its potential for oligosaccharide linkage analysis.
Structural characterization of highly sulfated glycosaminoglycans (GAGs) by collisionally activated dissociation (CAD) is challenging because of the extensive sulfate losses mediated by free protons. While removal of the free protons may be achieved through the use of derivatization, metal cation adducts, and/or electrospray supercharging reagents, these steps add complexity to the experimental workflow. It is therefore desirable to develop an analytical approach for GAG sequencing that does not require derivatization or addition of reagents to the electrospray solution. Electron detachment dissociation (EDD) can produce extensive and informative fragmentation for GAGs without the need to remove free protons from the precursor ions. However, EDD is an inefficient process, often requiring consumption of large sample quantities (typically several micrograms), particularly for highly sulfated GAG ions. Here, we report that with improved instrumentation, optimization of the ionization and ion transfer parameters, and enhanced EDD efficiency, it is possible to generate highly informative EDD spectra of highly sulfated GAGs on the liquid chromatography (LC) time-scale, with consumption of only a few nanograms of sample. We further show that negative electron transfer dissociation (NETD) is an even more effective fragmentation technique for GAG sequencing, producing fewer sulfate losses while consuming smaller amount of samples. Finally, a simple algorithm was developed for de novo HS sequencing based on their high resolution tandem mass spectra. These results demonstrate the potential of EDD and NETD as sensitive analytical tools for detailed, high-throughput, de novo structural analyses of highly sulfated GAGs.
Direct detection and quantification of protein/peptide palmitoylation by mass spectrometry (MS) is a challenging task because of the tendency of palmitoyl loss during sample preparation and tandem MS analysis. In addition, the large difference in hydrophobicity between the palmitoyl peptides and their unmodified counterparts could prevent their simultaneous analysis in a single liquid chromatography-MS experiment. Here, the stability of palmitoylation in several model palmitoyl peptides under different incubation and fragmentation conditions was investigated. It was found that the usual trypsin digestion protocol using dithiothreitol as the reducing agent in ammonium bicarbonate buffer could result in significant palmitoyl losses. Instead, it is recommended that sample preparation be performed in neutral Tris buffer with tris(2-carboxyethyl)phosphine as the reducing agent, conditions under which palmitoylation was largely preserved. For tandem MS analysis, collision-induced dissociation often led to facile palmitoyl loss, and electron capture dissociation frequently produced secondary side-chain losses remote from the backbone cleavage site, thus discouraging their use for accurate palmitoylation site determination. In contrast, the palmitoyl group was mostly preserved during electron transfer dissociation, which produced extensive inter-residue cleavage coverage, making it the ideal fragmentation method for palmitoyl peptide analysis. Finally, derivatization of the unmodified peptides with a perfluoroalkyl tag, N-[(3-perfluorooctyl)propyl] iodoacetamide, significantly increased their hydrophobicity, allowing them to be simultaneously analyzed with palmitoyl peptides for relative quantification of palmitoylation.
The identification of protein post-translational modifications (PTMs) is
an increasingly important component of proteomics and biomarker discovery, but
very few tools exist for performing fast and easy characterization of global PTM
changes and differential comparison of PTMs across groups of data obtained from
liquid chromatography-tandem mass spectrometry experiments. STRAP PTM (Software
Tool for Rapid Annotation of Proteins: Post-Translational Modification edition)
is a program that was developed to facilitate the characterization of PTMs using
spectral counting and a novel scoring algorithm to accelerate the identification
of differential PTMs from complex data sets. The software facilitates
multi-sample comparison by collating, scoring, and ranking PTMs and by
summarizing data visually. The freely available software (beta release) installs
on a PC and processes data in protXML format obtained from files parsed through
the Trans-Proteomic Pipeline. The easy-to-use interface allows examination of
results at protein, peptide, and PTM levels, and the overall design offers
tremendous flexibility that provides proteomics insight beyond simple assignment
Post-Translational Modifications; PTMs; proteomics; mass spectrometry; spectral counting; software; biomarkers
Activation of vascular endothelial growth factor receptor-2 (VEGFR-2), an endothelial cell receptor tyrosine kinase, promotes tumor angiogenesis and ocular neovascularization. We report the methylation of VEGFR-2 at multiple Lys and Arg residues, including Lys1041, a residue that is proximal to the activation loop of the kinase domain. Methylation of VEGFR-2 was independent of ligand binding and was not regulated by ligand stimulation. Methylation of Lys1041 enhanced tyrosine phosphorylation and kinase activity in response to ligands. Additionally, interfering with the methylation of VEGFR-2 by pharmacological inhibition or by site-directed mutagenesis revealed that methylation of Lys1041 was required for VEGFR-2–mediated angiogenesis in zebrafish and tumor growth in mice. We propose that methylation of Lys1041 promotes the activation of VEGFR-2 and that similar posttranslational modification could also regulate the activity of other receptor tyrosine kinases.
The structural complexity and diversity of glycans parallel their multilateral functions in living systems. To better understand the vital roles glycans play in biological processes, it is imperative to develop analytical tools that can provide detailed glycan structural information. This was conventionally achieved by multistage tandem mass spectrometry (MSn) analysis using collision-induced dissociation (CID) as the fragmentation method. However, the MSn approach lacks the sensitivity and throughput needed to analyze complex glycan mixtures from biological sources, often available in limited quantities. We define herein the critical parameters for a recently developed fragmentation technique, electronic excitation dissociation (EED), which can yield rich structurally informative fragment ions during liquid chromatographic (LC)-MS/MS analysis of glycans. We further demonstrate that permethylation, reducing end labeling and judicious selection of the metal charge carrier can greatly facilitate spectral interpretation. With its high sensitivity, throughput, and compatibility with on-line chromatographic separation techniques, EED appears to hold great promise for large-scale glycomics studies.
Complex mixtures of high molecular weight fractions of pooled neutral human milk oligosaccharides (obtained via gel permeation chromatography) have been investigated. The subfractions were each permethylated and analyzed by high-resolution mass spectrometry, using matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, in order to investigate their oligosaccharide compositions. The obtained spectra reveal that human milk contains more complex neutral oligosaccharides than have been described previously; the data show that these oligosaccharides can be highly fucosylated, and that their poly-N-acetyllactosamine cores are substituted with up to 10 fucose residues on a an oligosaccharide that has 7-N-acetyllactosamine units. This is the first report of the existence in human milk of this large range of highly fucosylated oligosaccharides which possess novel, potentially immunologically active structures.
carbohydrates; fucosylation; FT-ICR MS; MALDI; mixture analysis; sugars
For structural identification of glycans, the classic collision-induced dissociation (CID) spectra are dominated by product ions that derived from glycosidic cleavages, which provide only sequence information. The peaks from cross-ring fragmentation are often absent or have very low abundances in such spectra. Electron transfer dissociation (ETD) is being applied to structural identification of carbohydrates for the first time, and results in some new and detailed information for glycan structural studies. A series of linear milk sugars was analyzed by a variety of fragmentation techniques such as MS/MS by CID and ETD, and MS3 by sequential CID/CID, CID/ETD, and ETD/CID. In CID spectra, the detected peaks were mainly generated via glycosidic cleavages. By comparison, ETD generated various types of abundant cross-ring cleavage ions. These complementary cross-ring cleavages clarified the different linkage types and branching patterns of the representative milk sugar samples. The utilization of different MS3 techniques made it possible to verify initial assignments and to detect the presence of multiple components in isobaric peaks. Fragment ion structures and pathways could be proposed to facilitate the interpretation of carbohydrate ETD spectra and the main mechanisms were investigated. ETD should contribute substantially to confident structural analysis of a wide variety of oligosaccharides.
Although differentiation of the isomeric Asn deamidation products (Asp and isoAsp) at the peptide level by electron capture dissociation (ECD) has been well established, isoAsp identification at the intact protein level remains a challenging task. Here, a comprehensive top-down deamidation study is presented using the protein beta2-microglobulin (β2M) as the model system. Of the three deamidation sites identified in the aged β2M, isoAsp formation was detected at only one site by the top-down ECD analysis. The absence of diagnostic ions likely resulted from an increased number of competing fragmentation channels and a decreased likelihood of product ion separation in ECD of proteins. To overcome this difficulty, an MS3 approach was applied where a protein ion was first fragmented by collisionally activated dissociation (CAD) and a resulting product ion was isolated and further analyzed by ECD. IsoAsp formation at all three deamidation sites was successfully identified by this CAD-ECD approach. Further, the abundance of the isoAsp diagnostic ion was found to increase linearly with the extent of deamidation. These results demonstrated the potential of ECD in the detection and quantitative analysis of isoAsp formation using the top-down approach.
The aim of this study was to demonstrate, and to characterize by high resolution mass spectrometry, that it is possible to preferentially induce covalent cross-links in peptides by using high energy femtosecond UV laser pulses. The cross-link is readily formed only when aromatic amino acids are present in the peptide sequence.
Three peptides, xenopsin, angiotensin I, interleukin, individually or in combination, were exposed to high energy femtosecond UV laser pulses, either alone or in the presence of spin trapping molecules, the reaction products being characterized by high resolution mass spectrometry.
High resolution mass spectrometry and spin trapping strategies showed that cross-linking occurs readily, proceeds via a radical mechanism, and is the highly dominant reaction, proceeding without causing significant photo-damage in the investigated range of experimental parameters.
High energy femtosecond UV laser pulses can be used to induce covalent cross-links between aromatic amino acids in peptides, overcoming photo-oxidation processes, that predominate as the mean laser pulse intensity approaches illumination conditions achievable with conventional UV light sources.
Transthyretin (TTR) amyloidosis and hemoglobinopathies are the archetypes of molecular diseases where point mutation characterization is diagnostically critical. We have developed a Top-down analytical platform for variant and/or modified protein sequencing and are examining the feasibility of using this platform for the analysis of hemoglobin/TTR patient samples and evaluating the potential clinical applications. The platform is based on a commercial high resolution hybrid orbitrap mass spectrometer (LTQ-Orbitrap™) with automated sample introduction; automated data analysis is performed by our own software algorithm (BUPID topdown).
The analytical strategy consists of iterative data capture, first recording a mass profile of the protein(s). The presence of a variant is revealed by a mass shift consistent with the amino acid substitution. Nozzle-skimmer dissociation (NSD) of the protein(s) yields a wide variety of sequence-defining fragment ions. The fragment ion containing the amino acid substitution or modification can be identified by searching for a peak exhibiting the mass shift observed in the protein mass profile. This fragment ion can then be selected for MS/MS analysis in the ion trap to yield sequence information permitting the identification of the variant. Substantial sequence coverage has been obtained in this manner. This strategy allows for a stepwise MS/MS analysis of the protein structure. The sequence information obtained can be supplemented with whole protein NSD fragmentation and MS/MS analysis of specific protein charge states. The analyses of variant forms of TTR and hemoglobin are presented to illustrate the potential of the method.
Top-down; proteins; variants; transthyretin; hemoglobin; post-translational modifications
The MIRAGE (minimum information required for a glycomics experiment) initiative was founded in Seattle, WA, in November 2011 in order to develop guidelines for reporting the qualitative and quantitative results obtained by diverse types of glycomics analyses, including the conditions and techniques that were applied to prepare the glycans for analysis and generate the primary data along with the tools and parameters that were used to process and annotate this data. These guidelines must address a broad range of issues, as glycomics data are inherently complex and are generated using diverse methods, including mass spectrometry (MS), chromatography, glycan array-binding assays, nuclear magnetic resonance (NMR) and other rapidly developing technologies. The acceptance of these guidelines by scientists conducting research on biological systems in which glycans have a significant role will facilitate the evaluation and reproduction of glycomics experiments and data that is reported in scientific journals and uploaded to glycomics databases. As a first step, MIRAGE guidelines for glycan analysis by MS have been recently published (Kolarich D, Rapp E, Struwe WB, Haslam SM, Zaia J., et al. 2013. The minimum information required for a glycomics experiment (MIRAGE) project – Improving the standards for reporting mass spectrometry-based glycoanalytic data. Mol. Cell Proteomics. 12:991–995), allowing them to be implemented and evaluated in the context of real-world glycobiology research. In this paper, we set out the historical context, organization structure and overarching objectives of the MIRAGE initiative.
Periodontitis is an inflammatory disease initiated by host-parasite interactions which contributes to connective tissue destruction and alveolar bone resorption. Porphyromonas gingivalis (P.g.), a black-pigmented Gram-negative anaerobic bacterium, is a major pathogen in the development and progression of periodontitis. To characterize the role that Porphyromonas gingivalis and its cell surface components play in disease processes, we investigated the differential expression of proteins induced by live P.g., P.g LPS and P.g FimA, using two dimensional gel electrophoresis in combination with mass spectrometry. We have tested whether, at the level of protein expression, unique signaling pathways are differentially induced by the bacterial components P.g. LPS and P.g. FimA, as compared to live P.g..
We found that P.g. LPS stimulation of THP-1 up-regulated the expression of a set of proteins compared to control: deoxyribonuclease, actin, carbonic anhydrase 2, alpha enolase, adenylyl cyclase-associated protein (CAP1), protein disulfide isomerase (PDI), glucose regulated protein (grp78) and 70-kDa heat shock protein (HSP70), whereas FimA treatment did not result in statistically significant changes to protein levels versus the control. Live P.g. stimulation resulted in 12 differentially expressed proteins: CAP1, tubulin beta-2 chain, ATP synthase beta chain, tubulin alpha-6 chain, PDI, vimentin, 60-kDa heat shock protein and nucleolin were found to be up-regulated, while carbonic anhydrase II, beta-actin and HSP70 were down-regulated relative to control. These differential changes by the bacteria and its components are interpreted as preferential signal pathway activation in host immune/inflammatory responses to P.g. infection.
Lipopolysaccharide; mass spectrometry; monocytes/macrophages; Porphyromonas gingivalis; Toll-like receptors; proteomics
Myelin protein zero (P0), a glycosylated single-pass transmembrane protein, is essential in the formation and maintenance of peripheral nervous system (PNS) compact myelin. P0 in Xenopus (xP0) exists primarily as a dimeric form that remains stable after various physical and chemical treatments. In exploring the nature of the interactions underlying the dimer stability, we found that xP0 dimer dissociated into monomer during continuous elution gel electrophoresis and conventional SDS-PAGE, indicating that the dimer is stabilized by non-covalent interactions. Furthermore, as some of the gel-purified monomer re-associated into dimer on SDS-PAGE gels, there is likely a dynamic equilibrium between xP0 dimer and monomer in vivo. Because the carbohydrate and fatty acyl moieties may be crucial for the adhesion role of P0, we used sensitive mass spectrometry approaches to elucidate the detailed N-glycosylation and S-acylation profiles of xP0. Asn92 was determined to be the single, fully-occupied glycosylation site of xP0, and a total of 12 glycans was detected that exhibited new structural features compared with those observed from P0 in other species: (1) the neutral glycans were composed mainly of high mannose and hybrid types; (2) five of twelve were acidic glycans, among which three were sialylated and the other two were sulfated; (3) none of the glycans had core fucosylation; and (4) no glucuronic acid, hence no HNK-1 epitope, was detected. The drastically different carbohydrate structures observed here support the concept of the species-specific variation in N-glycosylation of P0. Cys152 was found to be acylated with stearoyl (C18:0), whereas palmitoyl (C16:0) is the corresponding predominant fatty acyl group on P0 from higher vertebrates. We propose that the unique glycosylation and acylation patterns of Xenopus P0 may underlie its unusual dimerization behaviour. Our results should shed light on the understanding of the phylogenetic development of P0’s adhesion role in PNS compact myelin.
De novo N-linked glycan sequencing; mass spectrometry; species-specific N-glycosylation
Autoantigen presentation by HLA-DR molecules is thought to be a central component of many autoimmune diseases, but uncovering disease-relevant autoantigens has been a difficult challenge. Our goal was to identify autoantigens in patients with antibiotic-refractory Lyme arthritis, which is thought to result from infection-induced autoimmunity.
Using tandem mass spectrometry, naturally presented HLA-DR self-peptides from a patient’s synovium were identified, synthesized and reacted with his peripheral blood mononuclear cells (PBMC). Immunoreactive peptides and their source proteins were then tested for T and B cell responses using large numbers of patients’ cells or sera.
Of 120 HLA-DR-presented self-peptides identified from one patient, one peptide derived from endothelial cell growth factor (ECGF) caused his PBMC to proliferate. We then found that T and B cell responses to ECGF occurred systemically in about 10–30% of patients with early or late manifestations of Lyme disease, primarily in those with refractory arthritis-associated HLA-DR alleles, such as DRB1*0101 and 0401. Compared with patients with antibiotic-responsive arthritis, those with antibiotic-refractory arthritis had significantly higher concentrations of ECGF in synovial fluid (P<0.0001) and more often had ECGF antibody reactivity. In non-antibiotic-treated historic patients who developed arthritis, 26% had ECGF reactivity, which often developed before the onset of arthritis and was associated with significantly longer courses of arthritis.
T and B cell responses to ECGF occur in a subset of patients with Lyme disease, particularly in those with antibiotic-refractory arthritis, providing the first direct evidence for autoimmune T and B cell responses in this illness.
The glycosphingolipid GM1 binds cholera toxin (CT) on host cells and carries it retrograde from the plasma membrane (PM) through endosomes, the trans-Golgi (TGN), and the endoplasmic reticulum (ER) to induce toxicity. To elucidate how a membrane lipid can specify trafficking in these pathways, we synthesized GM1 isoforms with alternate ceramide domains and imaged their trafficking in live cells. Only GM1 with unsaturated acyl chains sorted efficiently from PM to TGN and ER. Toxin binding, which effectively crosslinks GM1 lipids, was dispensable, but membrane cholesterol and the lipid raft-associated proteins actin and flotillin were required. The results implicate a protein-dependent mechanism of lipid-sorting by ceramide structure and provide a molecular explanation for the diversity and specificity of retrograde trafficking by CT in host cells.
Coccidia are protozoan parasites that cause significant human disease and are of major agricultural importance. Cryptosporidium spp. cause diarrhea in humans and animals, while Toxoplasma causes disseminated infections in fetuses and untreated AIDS patients. Eimeria is a major pathogen of commercial chickens. Oocysts, which are the infectious form of Cryptosporidium and Eimeria and one of two infectious forms of Toxoplasma (the other is tissue cysts in undercooked meat), have a multilayered wall. Recently we showed that the inner layer of the oocyst walls of Toxoplasma and Eimeria is a porous scaffold of fibers of β-1,3-glucan, which are also present in fungal walls but are absent from Cryptosporidium oocyst walls. Here we present evidence for a structural role for lipids in the oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. Briefly, oocyst walls of each organism label with acid-fast stains that bind to lipids in the walls of mycobacteria. Polyketide synthases similar to those that make mycobacterial wall lipids are abundant in oocysts of Toxoplasma and Eimeria and are predicted in Cryptosporidium. The outer layer of oocyst wall of Eimeria and the entire oocyst wall of Cryptosporidium are dissolved by organic solvents. Oocyst wall lipids are complex mixtures of triglycerides, some of which contain polyhydroxy fatty acyl chains like those present in plant cutin or elongated fatty acyl chains like mycolic acids. We propose a two-layered model of the oocyst wall (glucan and acid-fast lipids) that resembles the two-layered walls of mycobacteria (peptidoglycan and acid-fast lipids) and plants (cellulose and cutin).
Oocysts, which are essential for the fecal-oral spread of coccidia, have a wall that is thought responsible for their survival in the environment and for their transit through the stomach and small intestine. While oocyst walls of Toxoplasma and Eimeria are strengthened by a porous scaffold of fibrils of β-1,3-glucan and by proteins cross-linked by dityrosines, both are absent from walls of Cryptosporidium. We show here that all oocyst walls are acid fast, have a rigid bilayer, dissolve in organic solvents, and contain a complex set of triglycerides rich in polyhydroxy and long fatty acyl chains that might be synthesized by an abundant polyketide synthase. These results suggest the possibility that coccidia build a waxy coat of acid-fast lipids in the oocyst wall that makes them resistant to environmental stress.
The selective capture of target peptides poses a great challenge to modern chemists and biologists, especially when enriching them from proteome samples possessing extremes in concentration dynamic range and sequence diversity. While approaches based on traditional techniques such as biotin-avidin pairing offer versatile tools to design strategies for selective enrichment, problems are still encountered due to sample loss or poor selectivity of enrichment. Here we show that the recently introduced fluorous chemistry approach has attractive properties as an alternative method for selective enrichment. Through appending a perfluorine group to the target peptide, it is possible to dramatically increase the peptide's hydrophobicity and thus enable facile separation of labeled from non-labeled peptides. Use of reversed-phase chromatography allowed for improved peptide recovery in comparison with results obtained using the formerly reported fluorous bonded phase methods. Furthermore, this approach also allowed for on-line separation and identification of both labeled and unlabeled peptides in a single experiment. The net result is an increase in the confidence of protein identification by tandem mass spectrometry (MS2) as all peptides and subsequent information are retained. Successful off-line and on-line enrichment of cysteine-containing peptides was obtained, and high quality MS2 spectra were obtained by tandem mass spectrometry due to the stability of the tag, allowing for facile identification via standard database searching. We believe that this strategy holds great promise for selective enrichment and identification of low abundance target proteins or peptides.
Oral sodium phenyl butyrate (SPB) is currently under investigation as a histone deacetylation (HDAC) inhibitor in Huntington disease (HD). Ongoing studies indicate that symptoms related to HD genetic abnormalities decrease with SPB therapy. In a recently reported safety and tolerability study of SPB in HD, we analyzed overall chromatographic patterns from a method that employs gradient Liquid Chromatography with series Electrochemical array, UV and Fluorescence (LCECA/UV/F) for measuring SPB and its metabolite phenylacetate (PA). We found that plasma and urine from SPB-treated patients yielded individual-specific patterns of ca. 20 metabolites which may provide a means for the selection of subjects for extended trials of SPB. The structural identification of these metabolites is of critical importance, since their characterization will facilitate understanding the mechanisms of drug action and possible side effects. We have now developed an iterative process with LCECA, parallel LCECA/LCMS, and high performance tandem MS, for metabolite characterization. We report here the details of this method and its use for identification of 10 plasma and urinary metabolites in treated subjects, including indole species in urine that are not themselves metabolites of SPB. This approach thus contributes to understanding metabolic pathways that differ among HD individuals being treated with SPB.
Sodium phenyl butyrate; Huntington disease; LCECA; metabolites; parallel LC-EC-array/LC-MS; tandem mass spectrometry
Tularemia is a severe infectious disease in humans caused by the gram-negative bacterium Francisella tularensis (Ft). Due to its low infectious dose, high mortality rate, and the threat of its large-scale dissemination in weaponized form, development of vaccines and immunotherapeutics against Ft is essential. Ft lipopolysaccharide (LPS), which contains the linear graded-length saccharide component O-antigen (OAg) attached to a core oligosaccharide, has been reported as a protective antigen. Purification of LPS saccharides of defined length and composition is necessary to reveal the epitopes targeted by protective antibodies. In this study, we purified saccharides from LPS preparations from both the Ft subspecies holarctica live vaccine strain (LVS) and the virulent Ft subspecies tularensis SchuS4 strain using liquid chromatography. We then characterized the fractions using high resolution mass spectrometry and tandem mass spectrometry. Three types of saccharides were observed in both the LVS and SchuS4 preparations: two consisting of OAg tetrasaccharide repeats attached to one of two core-oligosaccharide variants, and one consisting of tetrasaccharide repeats only (coreless). The coreless OAg oligosaccharides were shown to contain Qui4NFm (4,6-dideoxy-4-formamido-D-glucose) at the non-reducing end and QuiNAc (2-acetamido-2,6-dideoxy-O-D-glucose) at the reducing end. Purified homogeneous preparations of saccharides of each type will allow mapping of protective epitopes in Ft LPS.
Little is known about the antigen specificity of CD1d-restricted T cells, except that they frequently recognize CD1d-expressing antigen-presenting cells in the absence of exogenous antigen. We previously demonstrated that the 24.8.A iNKT cell hybridoma was broadly reactive with CD1d-transfected cell lines and recognized the polar lipid fraction of a tumor cell extract. In the present study, the antigen recognized by the 24.8.A iNKT cell hybridoma was purified to homogeneity and identified as palmitoyl-oleoyl-sn-glycero-3-phosphoethanolamine (16:0–18:1 PE). The 24.8.A iNKT cell hybridoma recognized synthetic 16:0-18:1[cis] PE, confirming that this phospholipid is antigenic. Recognition correlated with the degree of unsaturation of the acyl chains. Using a panel of synthetic PEs, the 24.8.A iNKT cell hybridoma was shown to be activated by PEs that contained at least one unsaturated acyl chain. The configuration of the double bonds was important, as the 24.8.A iNKT cell hybridoma recognized unsaturated acyl chains in the cis, but not the trans, configuration. PEs with multiple double bonds were recognized better than those with a single double bond, and increasing acyl chain unsaturation correlated with increased binding of PE to CD1d. These data illustrate the potential importance of the acyl chain structure for phospholipid antigen binding to CD1d.
PMID: 12963715 CAMSID: cams2348
The walls of infectious pathogens, which are essential for transmission, pathogenesis, and diagnosis, contain sugar polymers that are defining structural features, e.g., β-1,3-glucan and chitin in fungi, chitin in Entamoeba cysts, β-1,3-GalNAc in Giardia cysts, and peptidoglycans in bacteria. The goal here was to determine in which of three walled forms of Toxoplasma gondii (oocyst, sporocyst, or tissue cyst) is β-1,3-glucan, the product of glucan synthases and glucan hydrolases predicted by whole-genome sequences of the parasite. The three most important discoveries were as follows. (i) β-1,3-glucan is present in oocyst walls of Toxoplasma and Eimeria (a chicken parasite that is a model for intestinal stages of Toxoplasma) but is absent from sporocyst and tissue cyst walls. (ii) Fibrils of β-1,3-glucan are part of a trabecular scaffold in the inner layer of the oocyst wall, which also includes a glucan hydrolase that has a novel glucan-binding domain. (iii) Echinocandins, which target the glucan synthase and kill fungi, arrest development of the Eimeria oocyst wall and prevent release of the parasites into the intestinal lumen. In summary, β-1,3-glucan, which can be targeted by drugs, is an important component of oocyst walls of Toxoplasma but is not a component of sporocyst and tissue cyst walls.
We show here that walls of Toxoplasma oocysts, the infectious stage shed by cats, contain β-1,3-glucan, a sugar polymer that is a major component of fungal walls. In contrast to fungi, β-1,3-glucan is part of a trabecular scaffold in the inner layer of the oocyst wall that is independent of the permeability barrier formed by the outer layer of the wall. While glucan synthase inhibitors kill fungi, these inhibitors arrest the development of the oocyst walls of Eimeria (an important chicken pathogen that is a surrogate for Toxoplasma) and block release of oocysts into the intestinal lumen. The absence of β-1,3-glucan in tissue cysts of Toxoplasma suggests that drugs targeted at the glucan synthase might be used to treat Eimeria in chickens but not to treat Toxoplasma in people.
Heparan Sulfate (HS) and heparin are linear, heterogeneous carbohydrates of the glycosaminoglycan (GAG) family that are modified by N-acetylation, N-sulfation, O-sulfation, and uronic acid epimerization. HS interacts with growth factors in the extracellular matrix, thereby modulating signaling pathways that govern cell growth, development, differentiation, proliferation, and adhesion. HPLC-chip-based hydrophilic interaction liquid chromatography/mass spectrometry has emerged as a method for analyzing the domain structure of GAGs. However, analysis of highly sulfated GAG structures decasaccharide or larger in size has been limited by spray instability in the negative-ion mode. This report demonstrates that addition of post-column make-up flow to the amide-HPLC-chip configuration permits robust and reproducible analysis of extended GAG domains (up to degree of polymerization 18) from HS and heparin. This platform provides quantitative information regarding oligosaccharide profile, degree of sulfation, and non-reducing chain termini. It is expected that this technology will enable quantitative, comparative glycomics profiling of extended GAG oligosaccharide domains of functional interest.
Sirtuin-1 (SIRT1) is an NAD+-dependent protein deacetylase that is sensitive to oxidative signals. Our purpose was to determine whether SIRT1 activity is sensitive to the low molecular weight nitrosothiol, S-nitrosoglutathione (GSNO), which can transduce oxidative signals into physiological responses. SIRT1 formed mixed disulfides with GSNO-Sepharose, and mass spectrometry identified several cysteines that are modified by GSNO, including Cys-67 which was S-glutathiolated. GSNO had no effect on basal SIRT1deacetylase activity, but inhibited stimulation of activity by resveratrol (RSV) with an IC50 of 69 μM. These observations indicate that S-glutathiolation of SIRT1 by low concentrations of reactive glutathione can modulate its enzymatic activity.
Antioxid. Redox Signal. 13, 1023–1032.